Page 1

CANCER THERAPY

Volume 3 Number 1 June, 2005


CANCER THERAPY FREE ACCESS www.cancer-therapy.org

!!!!!!!!!!!!!!!!!!!!!!!! ! Editor

Teni Boulikas Ph. D., CEO Regulon Inc. 715 North Shoreline Blvd. Mountain View, California, 94043 USA Tel: 650-968-1129 Fax: 650-567-9082 E-mail: teni@regulon.org

Teni Boulikas Ph. D., CEO, Regulon AE. Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9853849 Fax: +30-210-9858453 E-mail: teni@regulon.org

!!!!!!!!!!!!!!!!!!!!!!!! ! Assistant to the Editor Maria Vougiouka B.Sc., Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9858454 Fax: +30-210-9858453 E-mail: maria@cancer-therapy.org

!!!!!!!!!!!!!!!!!!!!!!!! ! Editorial Board

Ablin, Richard J., Ph.D., Arizona Cancer Center, University of Arizona, USA Armand, Jean Pierre, M.D. Ph.D., European Organization for Research and Treatment of Cancer (EORTC), Belgium Aurelian, Laure, Ph.D., University of Maryland School of Medicine, USA Berdel, Wolfgang E, M.D., University Hospitals, Germany Bertino, Joseph R., M.D., Cancer Institute of New Jersey, USA Beyan Cengiz, M.D.,!Gulhane Military Medical Academy, Turkey Bottomley, Andrew, Ph.D., European Organization for Research and Treatment of Cancer Data Center (EORTC), Belgium Bouros, Demosthenes, M.D., University Hospital of Alexandroupolis. Greece Cabanillas, Fernando, M.D, The University of Texas M. D. Anderson Cancer Center, USA Castiglione, Monica, MHA, SIAK/IBCSG Coordinating Center, Switzerland Chou, Kuo-Chen, Ph.D., D.Sc., Pharmacia Upjohn, USA Chu, Kent-Man, M.D., University of Hong Kong Medical Center, Queen Mary Hospital, Hong Kong, China Chung, Leland W.K, Ph.D., Winship Cancer Institute,

USA Coukos, George, M.D., Ph.D., Hospital of the University of Pennsylvania, USA Darzynkiewicz, Zbigniew, M.D., Ph.D., New York Medical College, USA Devarajan, Prasad M.D., Cincinnati Children's Hospital, USA Der Channing, J. Ph.D, Lineberger Comprehensive Cancer Center, USA Dritschilo, Anatoly, M.D., Georgetown University Hospital, USA Duesberg, Peter H., Ph.D, University of California at Berkeley, USA El-Deiry, Wafik S. M.D., Ph.D., Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, USA Federico, Massimo, M.D. Università di Modena e Reggio Emilia, Italy Fiebig, Heiner H, Albert-Ludwigs-Universität, Germany Fine, Howard A., M.D., National Cancer Institute, USA Frustaci, Sergio, M.D., Centro di Riferimento Oncologico di Aviano, Italy Georgoulias, Vassilis, M.D., Ph.D., University General Hospital of Heraklion, Greece Giordano, Antonio, M.D., Ph.D., Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, USA Greene, Frederick Leslie, M.D., Carolinas Medical


Center, USA Gridelli, Cesare M.D., Azienda Ospedaliera, "S.G.Moscati", Italy Hengge, Ulrich, M.D., Heinrich-Heine-University Duesseldorf, Germany Huber, Christian M.D., Johannes-GutenbergUniversity, Germany Hunt, Kelly, M.D., The University of Texas M. D. Anderson Cancer Center, USA Kamen, Barton A., M.D. Ph.D, Cancer Institute of New Jersey, USA Kaptan, Kürsat, M.D., Gülhane Military Medicine Academy, Turkey Kazuma, Ohyashiki, M.D., Ph.D., Tokyo Medical University, Japan Kinsella, Timothy J. M.D., The research Institute of University Hospitals in Cleveland, USA Kmiec, Eric B, Ph.D., University of Delaware, USA Kosmidis Paris, M.D., "Hygeia" Hospital, Athens, Greece Koukourakis Michael, M.D., Democritus University of Thrace, Greece Kroemer, Guido, M.D. Ph.D., Institut Gustave Roussy, France Kurzrock, Razelle, M.D., F.A.C.P., M. D. Anderson Cancer Center, USA Leung, Thomas Wai-Tong M.D., Chinese University of Hong Kong, China Levin, Mark M.D., Sister Regina Lynch Regional Cancer Center, Holy Name Hospital, USA Lichtor, Terry M.D., Ph.D., Rush Medical College, USA Liebermann, Dan A., Ph.D., Temple Univ. School of Medicine, USA Lipps, Hans J, Ph.D., Universität Witten/Herdecke, Germany Lokeshwar, Balakrishna L., Ph.D., University of Miami School of Medicine, USA Mackiewicz, Andrzej, M.D., Ph.D., University School of Medical Sciences (USOMS) at Great Poland Cancer Center, Poland Marin, Jose J. G., Ph.D., University of Salamanca, Spain McMasters, Kelly M., M.D., Ph.D., University of Louisville, J. Graham Brown Cancer Center, USA Morishita, Ryuichi, M.D., Ph.D., Osaka University, Japan Mukhtar, Hasan Ph.D., University of Wisconsin, USA Norris, James Scott, Ph.D., Medical University of South Carolina, USA Palu, Giorgio, M.D., University of Padova, Medical School, Italy

Park, Jae-Gahb, M.D., Ph.D., Seoul National University College of Medicine, Korea Perez-Soler, Roman M.D., The Albert Einstein Cancer Center, USA Peters, Godefridus J., Ph.D., VU University Medical Center (VUMC), The Netherlands Poon, Ronnie Tung-Ping, M.D., Queen Mary Hospital, Hong Kong, China Possinger, Kurt-Werner, M.D., Humboldt University, Germany Rainov G Nikolai M.D., D.Sc., The University of Liverpool. UK Randall, E Harris, M.D., Ph.D., The Ohio State University, USA Ravaioli Alberto, M.D. Ospedale Infermi, Italy Remick, Scot, C. M.D., University Hospitals of Cleveland, USA Rhim, Johng S M.D., Uniformed Services University of Health Sciences, USA Schadendorf, Dirk, M.D., Universitäts-Hautklinik Mannheim, Germany Schmitt, Manfred, Ph.D., Universität München, Klinikum rechts der Isar, Germany Schuller, Hildegard M., D.V.M., Ph.D., University of Tennessee, USA Slaga, Thomas J., Ph.D., AMC Cancer Research Center (UICC International Directory of Cancer Institutes and Organisations), USA Soloway, Mark S., M.D., University of Miami School of Medicine, USA Srivastava, Sudhir, Ph.D., MPH, MS, Division of Cancer Prevention, National Cancer Institute, USA Stefanadis, Christodoulos, M.D., University of Athens, Medical School, Greece, Stein, Gary S Ph.D., University Of Massachusetts, USA Tirelli, Umberto, National Cancer Institute, Italy Todo, Tomoki, M.D., Ph.D., The University of Tokyo, Japan van der Burg, Sjoerd H, Leiden University Medical Center, The Netherlands Wadhwa Renu, Ph. D., Nat. Inst. of Advan. Indust. Sci. and Technol. (AIST), Japan Waldman, Scott A. M.D., Ph.D., USA Walker, Todd Ph.D., Charles Sturt University, Australia Watson, Dennis K. Ph.D., Medical University of South Carolina, Hollings Cancer Center, USA Waxman, David J., Ph.D., Boston University, USA Weinstein, Bernard I., M.D., D.Sci (Hon.), Columbia University, USA

!!!!!!!!!!!!!!!!!!!!!!!! ! Associate Board Members

Chen, Zhong, M.D, Ph.D, National Institute of Deafness and other Communication Disorders, National Institutes of Health, USA Dietrich Pierre Yves, Hopitaux Universitaires de GenFve Switzerland Jeschke Marc G, M.D., Ph.D. Universität Erlangen-Nürnberg. Germany Limacher Jean-Marc, MD Hôpitaux Universitaires de Strasbourg, France Los Marek J, M.D., Ph.D. University of Manitoba, USA Mazda Osam, M.D., Ph.D. Kyoto Prefectural University of Medicine, Japan Merlin Jean-Louis, Ph.D Centre Alexis Vautrin, National Cancer Institute University Henri Poincaré France Okada Takashi, M.D., Ph.D. Jichi Medical School Japan Pisa Pavel, M.D, Ph.D. Karolinska Hospital, Sweden


Squiban Patrick, MD Transgene SA France Tsuchida Masanori, M.D, Ph.D Niigata University Graduate School of Medical and Dental Sciences Japan Ulutin, Cuneyt, M.D., Gulhane Military Medicine Academy, Turkey Xu Ruian, Ph.D., The University of Hong Kong, Hong Kong

!!!!!!!!!!!!!!!!!!!!!!!! ! For submission of manuscripts and inquiries: Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org


Instructions to authors: Cancer Therapy FREE ACCESS www.cancer-therapy.org

Scope This journal, bridging various fields is one of the most rapid with free access at www.cancer-therapy.org. The scope of Cancer Therapy is to rapidly publish original and in-depth review articles on cancer embracing all fields from molecular mechanisms to results on clinical trials. Articles (both invited and submitted) review or report novel findings of importance to a general audience in cancer therapy, molecular medicine, gene discovery, and molecular biology with emphasis to molecular mechanisms and clinical applications. The journal will accept papers on all aspects of cancer, at the clinical, preclinical or cell culture stage on chemotherapy and new experimental drugs, gene discovery, cancer immunotherapy, DNA vaccines, use of DNA regulatory elements in gene transfer, cell therapy and drug discovery related to cancer therapy. The authors are encouraged to elaborate on the molecular mechanisms that govern a cancer therapy approach. To make the publication attractive authors are encouraged to include color figures. Type of articles Both review articles and original research articles will be considered. Original research articles should contain a generous introduction in addition to experimental data. The articles contain information important to a general audience as the volume is addressed to researches outside the field. There is no limit on the length of the articles provided that the subject is interesting to a general audience and covers exhaustively a field. The typical length of each manuscript is 12-60 manuscript pages (approximately 420 printed pages) plus Figures and Tables. Free of Charge publication, Complimentary reprints & Subscriptions There are no charges for color figures or page numbers. Corresponding authors get a one-year free subscription (hard copy) plus 25 reprints free of charge. The free subscription can be renewed for additional years by having one paper per year accepted for publication. Sections of the manuscript Each manuscript should have a Title, Authors, Affiliation, Corresponding Author (with Tel, Fax, and Email), Summary, and Introduction; review articles are subdivided into headings I, II, III, etc. (starting with I. Introduction) and subdivided into A, B, C, etc. You can further subdivide into 1, 2, 3, etc. Research articles are divided into Summary; I. Introduction; II. Results; III Discussion; Acknowledgments IV. Materials and Methods and References. Please include in your text citations the name of authors and year in parenthesis; for three or more authors use: (name of first author et al, with year); for two authors please use both names. Please delete hidden text for references. In the reference list, please, type references with year and Journal in boldface and provide full title of the article such as:


Buschle M, Schmidt W, Berger M, Schaffner G, Kurzbauer R, Killisch I, Tiedemann J-K, Trska B, Kirlappos H, Mechtler K, Schilcher F, Gabler C, and Birnstiel ML (1998) Chemically defined, cell-free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination. Gene Ther Mol Biol 1, 309-321. Please use Microsoft Word, font “Times” (Mac users) or “Times New Roman” (PC users) and insert Greek or other characters using the “Insert/Symbol” function in the Microsoft Word rather than simple conversion to font “Symbol”. Please boldface Figure 1, 2, 3 etc. as well as Table 1, 2, etc. throughout the text. Please provide the highest quality of prints of your Figures; whenever possible, please provide in addition an electronic version of your figures (optional). Corresponding authors are kindly requested to provide a color (or black/white) head photo of themselves (preferably 4x5 cm or any size), as we shall include these in the publication. Submission and reviewing Peer reviewing is by members of the Editorial Board and external referees. Please suggest 2-3 reviewers providing their electronic addresses, mailing addresses and telephone/fax numbers. Authors are being sent page proofs. Cancer Therapy (Volume 1, 2003) is published on high quality paper with excellent reproduction of color figures and electronically. Reviewing is completed within 5-15 days from receiving the manuscript. Articles accepted without revisions (i.e., review articles) will be published online (www.cancertherapy.org) in approximately 1 month following submission. Please submit an electronic version of full text and figures preferably in jpeg format. The electronic version of the figures will be used for the rapid reviewing process. High quality prints or photograph of the figures and the original with one copy should be sent via express mail to the Editorial Office. Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org The free electronic access to articles published in "Cancer Therapy" to a big general audience, the attractive journal title, the speed of the reviewing process, the no-charges for page numbers or color figure reproduction, the 25 complimentary reprints, the rapid electronic publication, the embracing of many fields in cancer, the anticipated high quality in depth reviews and first rate research articles and most important, the eminent members of the Editorial Board being assembled are prognostic factors of a big success for the newly established journal.


Table of contents Cancer Therapy Vol 3 Number 1, June 2005

Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

1-4

Case Report

5-28

Review Article

Cardiac metastasis from carcinoma of the cervix C-Kit signal transduction and involvement in cancer

29-30

Case Report

31-40

Research Article

Nadereh Behtash, Haleh Ayatollahi, Fereshteh Fakor, Morteza M Dini Johan Lennartsson, Olexandr Voytyuk, Elke Heiss, Christina Sundberg, Jianmin Sun and Lars Rönnstrand Malihe Hasanzadeh and Hasan Malekoti Eberhard Stoeckle, Jean-Michel Coindre, Guy Kantor, Laurence Thomas, Antoine Avril, Philippe Lagarde, Michèle Kind, Binh Nguyen Bui

41-56

Review Article

57-64

Review Article

65-67

Review Article

77-84

Review Article

85-94

Review Article

95-100

Review Article

101-104

Review Article

105-130

Review Article

131-138

Review

Miliary tuberculosis peritonitis mimicking advanced ovarian cancer Quality of surgery in soft tissue sarcoma: a single centre experience with the French Sarcoma Group (FSG) surgical system Current role of erythropoietin in the management of patients with haematological and solid malignancies Targeting of cytostatic bile acid derivatives toward tumours of the enterohepatic circuit Thalidomide and its use in renal and prostate cancer Genetic predisposition in preinvasive lesions of the breast Head and neck cancer in elderly patients

Radiotherapy and surgery in the management of non-small cell lung cancer in the elderly patients, a review of the recent literature P-glyco protein (multi drug resistance protein product) does not predict the response of laryngeal and hypopharyhgeal cancer to chemotherapy Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? Micro and nano drug delivery systems

Max Mano, Priska Butzberger, Anne Reid, Alan Rodger, Richard Soutar, John Welsh Jose J.G. Marin, Marta R. Romero, Marta Vallejo, Maria J. Monte Simon Pridgeon, Marcus Drake George C. Zografos, Flora Zagouri, Costantinos Fotiadis, John Bramis Daniele Bernardi, Domenico Errante, Luigi Barzan, Giovanni Franchin, Luigi Salvagno, Antonio Bianco, Luca Balestreri, Umberto Tirelli and Emanuela Vaccher H. Cuneyt Ulutin and Gorkem Aksu

Avi Khafif, Elizabeth Gillis, Jesus E. Medina

Daila S. Gridley, Jerry R. Williams and James M. Slater Gorka Orive, Rosa Mar£a Hern!ndez,


in cancer therapy Transvaginal color Doppler in the assessment of cervical carcinoma Outcomes of hysteroscopy and hysterectomy in breast cancer patients

139-146

Article Review Article

147-152

Research Article

153-158

Case Report

159-166

Review Article

167-176

Research Article

177-184

Research Article

Tumor-specific human monoclonal antibody GAH recognizes non-muscle myosin heavy chain type A as a cell surface antigen

185-188

Research Article

189-192

Research Article

193-200

Research Article

201-218

Research Article

219-226

Review Article

227-230

Research Article

Radical parametrectomy in the treatment of invasive cervical cancer after simple hysterectomy A summary on lamivudine primary prophylaxis of hepatitis B virus reactivation in chronic HBsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy Meat consumption and risk of colorectal cancer: a case-control study in Uruguay FDG PET and PET-CT in uterine cancers Prevention of tumor progression as the ultimate goal of cancer therapy Outcome of subsequent pregnancy in patients with gestational trophoblastic disease

231-236

Review Article Research

237-242

Ovarian mixed germ cell tumor presenting as tuberculosis Role of somatostatin analogues in the treatment of androgen ablationrefractory prostate adenocarcinoma HER-2/neu overexpression in Croatian breast cancer patients: results of one-year multicentric prospective study

Cervical cancer screening Phase II trial of celecoxib plus

Alicia R. Gasc"n, José Luis Pedraz Juan Luis Alc!zar Pedro T. Ramirez, Charlotte C. Sun, Claudia I. Vidal, Veronica Schimp, Brian Slomovitz, Michael W. Bevers, and Diane C. Bodurka Fatemeh Ghaemmaghami, Azam Sadat Moosavi, Malihe Hasanzadeh Alessandro Sciarra, Gianna Mariotti, Anna Maria Autran Gomez, Franco Di Silverio Jasminka Jakic-Razumovic, Jadranka Bozikov, Bozena Sarcevic, Viktor Separovic, Smiljana Kosanovic, Nives Jonjic, Elvira Mustac, Snjezana Tomic, Josko Bezic, Bozo Kruslin, Majda Vucic, Davor Tomas, Branko Dmitrovic, Valerija Blazicevic V, Tratincica Jakovina, Drazen Svagelj, Igor Boric, Zdenko Njiric, Vesna Stitic, Mira Mlinac-Lucijanic, Nada RestekSamarzija, Hrvoje Predrijevac, Milan Gosev Yoko Hirakawa, Yoshiko Yoshiyama, Hisae Niki, Shinsuke Ooike, Jun Kondo, Saiko Hosokawa, Kazuhiro Takahashi, Kazuhiro Nagaike, Hideo Nakamura, Makoto Tsurufuji and Toshiaki Tagawa Nadereh Behtash, Haleh Ayatollahi, Fatemeh Ghaemmaghami,Malihe Hasanzadeh, Fatemeh Esfehani Viroj Wiwanitkit

Hugo Deneo-Pellegrini, Paolo Boffetta, Eduardo De Stefani, Alvaro L. Ronco, Pelayo Correa and Mar£a Mendilaharsu Tarik Z Belhocine and Perry W Grigsby Futoshi Okada and Junichi Fujii Azam Sadat Mousavi, Afsaneh Tehranian, Nadereh Behtash, Fatemeh Ghaemmaghami, Mitra Modares, Roudabeh Pourghorban, Zahra Samizadeh Malihe Hasanzadeh and Nadereh Behtash Frank E. Mott, Christian T. Cable, Jon


Article

243-248 249-266

Review Article Review Article

267-284

Review Article

285-292

Case Report

293-298

Review Article Review Article

299-320

321-324 325-340 341-346 347-356

357-358 359-364

carboplatin and gemcitabine for firstline therapy in stage IIIB/IV nonsmall cell lung cancer-a negative study Update on cervical cancer The Oncofetal H19 RNA in human cancer, from the bench to the patient Adenoviral vectors for prostate cancer gene therapy AML1-MTG16 gene rearrangement in a pediatric therapy related AML after Ewing sarcoma: a case discussion and review of literature Placental site trophoblastic tumor

Management of peritoneal carcinomatosis from colon cancer, gastric cancer and appendix malignancy Review Photodynamic therapy for Article nasopharyngeal cancer Research Glioma cell integrin expression and Article their interactions with integrin antagonists Review Radiation esophagitis, treatment Article strategies and prevention Research Association between MUC1mucin, EArticle cadherin and nm23-H1expression and clinicopathological features in patients with invasive carcinoma of the cervix Case report Improper simple hysterectomy in invasive cervical cancer Research Selective tumor cell growth on tissue Article culture polystyrene and PrimariaTM: a preliminary study

Herrington, Joel Marcus, Rebecca Griggs, Melissa Ainslie Aaron C Han, Maria Merzouk, Richard Z Belch Imad Matouk, Patricia Ohana, Suhail Ayesh, Ami Sidi, Abraham Czerniak, Nathan de Groot, Abraham Hochberg Shawn E. Lupold and Ronald Rodriguez Emanuela Frascella, Claudia Zampieron, Laura Sainati, Letizia Casula, Francesco Pasquali, Rossella Mura, Emanuela Maserati, Martina Pigazzi, Monica Spinelli, Silvia Disar‰, Pier Francesco Biddau, Giuseppe Basso Nadereh Behtash, Malihe Hasanzadeh Paulo Goldstein, Rodrigo Gomes da Silva, Jacobo Cabanas, Paul H. Sugarbaker Viroj Wiwanitkit Ralph-Heiko Mattern, Susana B. Read, Michael D. Pierschbacher, Chun-I Sze, Brian P. Eliceiri, Carol A. Kruse Gorkem Aksu, Hakan Bakkal, Merdan Fayda, Binnaz Celebioglu Sarper Veronica S. Jeyadoss, Chetlur Srinivasan Vijayalakshmi, Balaraman Nair M, Halagowder Devaraj, Niranjali Devaraj, Prabha Balaram Fatemeh Ghaemmaghami and Malihe Hasanzadeh Shamim A. Faruqi, Okeychukwu A. Ibeanu, Tariq Ali, Harvey b. Spector, Joel S. Noumoff


Cancer Therapy Vol 3, page 1 Cancer Therapy Vol 3, 1-4, 2005

Cardiac metastasis from carcinoma of the cervix Case Report

Nadereh Behtash*1, Haleh Ayatollahi1, Fereshteh Fakor1, Morteza M Dini2 1

Gynecology Oncology Department, Vali –Asr Hospital, Tehran University of Medical Sciences, Keshavarz Blvd., Tehran 14194,Iran 2 Gynecologic Oncology, Advocate Illinois Masonic,Medical Center,Rush Medical College, 836 Wellington Chicago IL 60657

__________________________________________________________________________________ *Correspondence: Nadereh Behtash, Associate Professor, Gynecologist Oncologist, Tehran University of Medical Sciences. Gynecology Oncology Department, Vali-e-Asr Hospital, Imam Khomeini Hospital Complex, Keshavarz Blvd., Tehran 14194, Iran. Phone: #98-21-6939320, Fax: #98-21-6937321, E-mail: valrec2@yahoo.com, nadbehtash@yahoo.com Key words: Cervical carcinoma, Heart metastasis Abbreviations: patients, (pts); squamous cell carcinoma, (SCC); Received: 24 December 2004; Accepted: 14 January 2005; electronically published: January 2005

Summary The presence of cardiac metastasis from cervical carcinoma is extremely rare. Most of the cases were diagnosed postrmortem. There are a few cases of premortem diagnosis and it is believed that in these cases, the prognosis is extremely poor. We present the only case of premortem diagnosis of cardiac metastasis of more than 500 cases of cervical carcinoma in our center during the last 10 years. We present a 59 years-old woman with multiple ventricular metastasis from cervical carcinoma, 2.5 years after the primary diagnosis. She died 10 days after echocardiographic diagnosis of multiple metastatic lesions in heart, following a massive DIC. The rapidly progressive DIC, and death, in the presenting case, confirms the previous finding of poor prognosis of patients (pts) with premortem diagnosis of cardiac metastasis of cervical carcinoma. diagnosis.

I. Introduction Cervical carcinoma is the 3rd most common cancers in women throughout the world (Disaia and Creasman, 2002). In contrast to the industerialized world, cancer of the cervix remains the primary cancer killer of women in third–world countries (Disaia and Creasman, 2002). The incidence of cardiac metastases at autopsy ranges from 15% to 20% (mean 6%) in patients with malignant diseases (Abraham, 1990; MacGee, 1991; Lam et al, 1993). Carcinoma of the lung and breast, malignant melanoma, lymphoma, and leukemias rank among the most common tumors associated with this condition (Nelson and Rose, 1993; Shulman et al, 1997). The most common sites of extrapelvic metastasis in cervical carcinoma are lung, bone, cervical or supracervical lymph node (Brenner, 1982). The presence of cardiac metastases from cervical carcinoma is rare (1.8-3%) (Disaia and Creasman, 2002). Only with a high index of suspicious, it is possible to make a diagnosis antemortem. It is believed that, when present, cardiac metastasis from cervical carcinoma represents a rapidly fatal condition (Jamshed et al, 1996). Presented here is a case of right ventricular metastases from a stage IIa cervical carcinoma, who died rapidly after

II. Case A 59 year–old female, Gravida 8 Para 7 had been referred to Gynecology Oncologic service in Vali Asr University Hospital in January 1999. She had postmenopausal bleeding for almost 2 years. In pelvic exam, there was a large firm endophytic tumoral cervical mass measured 45mm diameter. Biopsy showed SCC (squamous cell carcinoma). She underwent a class III Rutledge Radical Hysterectomy and pelvic and paraaortic lymphadenectomy and upper vaginectomy in February /99 following 3 courses of neoadjuvant chemotherapy (Bulky stage IIa) with vincristin plus cisplatin. Preoperative metastatic work up revealed no abnormal findings in pelvic and abdominal CT scan, and chest XR. Pathologic report showed involvement of one iliac node and lower segment of uterus, vaginal margine and parametrium were free of tumor. She had normal pelvic exam, pap smear ,chest XR and CT , and pelvic CT in follow-up period of 24 months. In early postoperative visits, she had some uninary retention symptoms, responded to antibiotic therapy. In June 2001, she was hospitalized with intractable cough, Chest XR and bronchoscopy showed no abnormal finding. 1


Behtash et al: Cardiac metastasis from carcinoma of the cervix Her respiratory symptoms diminished after medical management for asthma. The patient did well until she started to develop dyspnea, weakness and lower limb edema in early September 2001. Again admitted to the hospital, abdominal and pelvic CT showed free fluid in pelvis. Echocardiography revealed multiple right ventricular tumors (Figure 1).This was believed to be consistent with a myocardial metastasis. A few days later, the patient, deteriorated rapidly and developed DIC, cerebral hemorrhage and coma, she died on September 2001 one week from diagnosis of cardiac metastasis.

or retrograde lymphatic flow (Murphy et al, 1986; Kountz, 1993). The occurrences of cardiac metastasis in general are a relatively rare event. Prichard suggested four factors possibly contributing to the low incidence of cardiac metastasis: 1-.The strong kneading action of the myocardium, 2- metabolic pecularitites of striated muscle 3- the rapid flow of blood through the heart and 4- lymph flow normally moving away from the heart (Prichard, 1951). The incidence of heart metastases in two most large series, was 10.7%-11.6% in all kinds of human malignancies (Burke and Virmari, 1996; Senzaki et al, 1999). The literature lists breast, lung, lymphoma, lukemia and melanoma as the most frequent primary sites (Nelson and Rose, 1993; Shulman et al, 1997). The most common location of cardiac involvement is the pericardium and an endocardium site is very rare (Burke and Virmari, 1996). The right side of the heart is more commonly involved than the left side, probably due to direct seeding of the heart by microemboli from lymphatics and venous return (Yanuck et al, 1991). One of cardinal feature involving diagnosis of metastatic carcinoma to the heart is that there is no early symptom, this was the same as for our patient, she had respiratory problems and intractable cough for more than 4 months. As the condition progresses, however,patients will typically complain of dyspnea. Other symptoms include: pericardial effusion, pericarditis, cardiac tamponade, arrythmias and sudden death (Yanuck et al, 1991; Kountz, 1993; Okamoto et al, 1993). The diagnosis is usually made only by a strong index of suspicion.

III. Discussion Cardiac metastasis from malignant neoplasm was first described by Bonet in 1700, but the first antemortem diagnosis of cardiac metastasis was not made until 1924 (Hanfling, 1960). Of all malignancies involving the heart, more than 96% are metastatic compared to primary (Lam et al, 1993; Nelson and Rose, 1993). The incidence of cardiac metastasis from carcinoma has increased from 1% in 1891 to 12% in 1953 and has remained stable at about 10-15% since that time (Prichard, 1951; Murphy et al, 1986). It seems the more sophisticated diagnostic techniques, are the main cause of this increasing incidence. Because Patient live longer from their primary malignancy with metastatic disease in other sites controlled, less common sites of metastatic involvement have become evident. A number of theories have been advanced to explain routes of involvement of the heart by metastatic carcinoma. These include embolic tumor emptying into right side of the heart, other mechanisms include direct invasion through either direct extension through the lymphatics of the heart and mediastinal nodes

Figure 1. Echocardiography showing right ventricular tumor

2


Cancer Therapy Vol 3, page 3 Table 1. Cases of reported premortem diagnosis of cardiac metastasis from cervical carcinoma Year

Age (Years)

Stage

Charles et al, 1977 Richter and Yon, 1979 Krivokapich et al, 1981 Itoh et al, 1984 Yanuck et al, 1991 Okamoto et al, 1993 Lee and Fisher, 1989 Lee and Fisher, 1989 Hsuj et al, 1992 Kountz, 1993 Nelson and Rose, 1993 Nelson and Rose, 1993 Jamshed et al, 1996 Ando et al, 1997 Lemus et al, 1998 Lemus et al, 1998 Senzaki et al, 1999 Chiou et al, 1999 Harvey et al, 2000 Iwaki et al, 2001 Yasuda et al, 2002 Inamura et al 2004 Behtash et al 2004

46 33 32 64 43 49 42 37 36 28 61 51 57 41 53 49 28 56 44 49 38 58 59

IIIb IIb IIb IIa Ib IIa IIIb IIIb Ib IIb IIIb IV Ib IIb Ib Ib Ib III Ib IIa IIb Ib IIb

Histology & type SCC SCCII SCCIII SCCI SCCIII SCCIII SCCII SCC SCC SCCI SCC SCC SCCIII SCCI SCCIII SCC SCCII SCC SCC SCC SCC SCC SCC

Chest X- ray finding, can include pleural effusion and cardiomegaly, though the latter can be absent in cases of endomyocardial metastases, as with our patient. Other helpful diagnostic clues are electrocardiographic changes, echocardiography, cardiac catheterization, CT scanning, MRI scanning and transvenous biopsy (Hanfling, 1960; Cacciapuot et al, 1988; Lee and Fisher, 1989; Shulman et al, 1997). One report of 1100 gynecologic cancer patients revealed only 6 cases of cardiac metastases (Greenwald, 1980). The first antemortem diagnosis for cervical carcinoma was made by Charles et al, (1977).Badib and colleauges reported cases of cardiac metastasis from 278 consecutive autopsy cases with the carcinoma of the uterine cervix (Badib et al, 1968). Antemortem diagnosis of cervical carcinoma metastatic to the heart is, however exteremely rare, with only a few cases reported (Table 1). Interestingly, in many cases of heart metastases from cervical carcinoma, the tumor metastasizes only to the right ventricle, and no other site (including the lung) just the same as our case. A review of the English language literature revealed that 22 casesof antemortem diagnosis of cardiac metastasis from cervival carcinoma have been described (Table 1). The mean age of these patients is 45 years (range 2864). The clinical stage was Ib in 7cases, IIa in 3 cases, IIb is 6 cases, III is 5 cases, IV in 2 cases. The stage of the disease at initial presentation dose not predict the future development of cardiac metestasis. The common symptom was shortness of breath and dyspnea (78%). The prognosis of these cases was poor. Patient died

Symptom

Prognosis

Dyspnea Shortness of breath Dyspnea Shortness of breath Chest pain Dyspnea Dyspnea Couph and dyspnea Shortness of breath Ileus Shortness of breath Dyspnea Couphand dyspnea Abdominal pain Shortness of breath Dyspnea Dyspnea Couph and dyspnea Shortness of breath Couph and low grade Fever Dyspnea Dyspnea Couph and dyspnea

8m 15d 9m 10d 5m 9m 5d 3m 9m 3m 12m 4m 5m 17m 1m 7m 18m Died suddenly 8m 2m 2m 4m Died suddenly

on average of 5/5 month after diagnosis. In conclusion we report a case of stage IIa squamous cell carcinoma of the cervix, in a 59 year-old woman. She underwent a radical hysterectomy after 3 courses of neoadjuvant chemotherapy and presented with a long standing intractable cough and finally dyspnea 30 months after primary diagnosis. She died rapidly after echocardiographic diagnosis. Imaging investigations showed no other site of metastasis (Ando et al, 1997).

References Abraham JM (1990) Neoplasms metastatic to the heart, Review of 3314 consecutive autopsies. Am J Cardiovasc Pathol 3, 195-8. Ando KO, Kashilara KI, Haraola M et al (1997) Carcinoma of the uterine cervix with myocardial metastasis Gynecol Oncol 65, 169-172, Badib AO, Kurohara SS et al (1968) Metastasis to organs in carcinoma of the uterine cervix, Influence of treatment on incidence and distribution. Cancer 21, 434-439. Brenner D (1982) Carcinoma of the cervix, A review. Am J Med Sci 289, 31-48. Burke A, Virmari R (1996) Tumors of the heart and blood vessels. Athlas of tumor pathology, fascicle 16, series 3. Armed Forces Institute of Pathology, Washington DC. Cacciapuot F, Arpino GD, D’ Avino M (1988) Reliability of echocardiography in the detection of metastatic malignant pericardial masses. Int J Cardiol 18, 109-112. Charles EH, Condori J, Sall S (1977) Metastasis to the pericardium from squamous cell carcinoma of the cervix. Am J Obstet Gynecol 129, 349-351. Chiou JS, Wang PH, Yuan CC et al (1999) Intracavitary cardiac tumor secoundry to carcinoma of the cervix a case report.

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Behtash et al: Cardiac metastasis from carcinoma of the cervix Zhonghua Yi Xue Za ZhI (Taipei) 62, 828-831. Disaia PJ, Creasman WT (2002) Germ cell, stromal and other ovarian tumors. In: DiSaia PJ, Creasman WT, editors. Clinical Gynecologic Oncology, 5th ed. St Louis: Mosby Year Book ,351-74 Greenwald EF (1980) Cardiac metastasis associated with gynecologic malignancies. Gynecol Oncol 10, 75-83. Hands ME, Lloyd Bl, Hopkins BE (1986) Carcinoma of uterine cervix with myocardial metastasis associated with chest pain and asystolic arrest. Int J Cardiol 11, 132-135. Hanfling SM (1960) Metastasis cancer to the heart review of the literature and report of 127cases. Circulation 23, 174-183, Harvey RL, Mychaskiw G, Sachdev V et al (2000) Isolated cardiac metastasis of cervival carcinoma presenting as disseminated intravascular coagulopathy.A case report. J Reprod Med 45, 603-606. Hsuj J, Chang TC, Hsueh S et al (1992) Cardiac tamponade resulting from recurrent small-cell carcinoma of the uterine cervix temporarily responding to CE/CAV chemotherapy. J Formosan Med Assoc 91, 828-830. Itoh KI, Mastsubara T, Yanagisawa K et al (1984) Right ventricular metastasis of cervical squamous cell carcinoma. Am Heart J 108, 1369-1370. Iwaki T, Kanaya H, Namura M et al (2001) Right ventricular metastasis from a primary cervical carcinoma. Jpn Circ J 65, 761-763. Jamshed A, Khafaga Y, EL-Husseiny G et al (1996) Pericardial metastasis in carcinoma of the uterine cervix. Gynecol Oncol 61, 451-53. Klatt EC, Heitz DR (1990) Cardiac Metastasis. Cancer 65, 14561459. Kountz DS (1993) Isolated cardiac metastasis from cervical carcinoma, presentation as acute anteroseptal myocardial infarction. South Med J 86, 228-230. Krivokapich J, Warren SE, Child JS et al (1981) M -Mode and cross -sectional echocardiographic diagnosis or right ventricular cavity masses. J Clin Ultrasound 9, 5-10. Lam KY, Dickens P, Chan ACL (1993) Tumor of the heart, A 20 year experience with a review of 12.485 consecutive autopsies. Arch Pathol Lab Med 117, 1027-31, Lee R, Fisher MR (1989) MR imaging of cardiac Metastasis from malignant fibrous histiocytoma. J Comput Assist Tomogr 13, 126-8.

Lemus JF, Abdulhay G et al (1998) Cardiac metastast from carcinoma of the cervix, report of two cases. Gynecol Oncol 69, 264-268. MacGee W (1991) Metastatic and invasive tumors involving the heart in a geriatric population, A necropsy study. Virchows Arch A Pathol Anat Histopathol 419, 183-189. Malriva VK, Casselberry JM, Perekh N et al (1990) Pericardial metastasis in squamous cell cancer of the cervix. J Reprod Med 35, 49-52. Murphy P, Alexander P, Kirkham N, Fleming J, Taylor I (1986) Pattern of spread of blood born tumor. Br J Surg 73, 829-34. Nelson BE, Rose PG (1993) Malignant pericardial effusion from squamous cell carcinoma of the cervix. J Surg Oncol 1993 52, 203-6. Okamoto H, Shinkai T, Yamakido M, Saijo N (1993) Cardiac tamponade caused by primary lung cancer and the management of pericardial effusion. Cancer 71, 93-98. Prichard RW (1951) Tumor of the heart, Review of the subject and report of one hundred and fifty cases. Arch Pathol, 51, 98-128. Richter N, Yon JL (1979) Squamous cell carcinoma of the cervix metastatic to the heart. Gynecol Oncol 7, 394-400. Rieke JW, Kapp DS (1988) Successful manangment of malignant pericardial effusion in metastatic squamous cell carcinoma of the uterine cervix. Gynecol Oncol 31, 338-351. Senzaki H, Uemura Y, Yamamoto D et al (1999) Right intra ventricular metastasis of squamous cell carcinoma of the uterine cervix, An autopsy case and literature review. Pathol Int 49, 447-52. Shulman LN, Braunwald E, Rosenthal DS (1997) Cardiac manifestations of neoplastic disease. In, Braunwald E, editor. Heart disease, A text book of cardiovascular medicine, 5th edn. Hematological disorders and heart disease. Philadelphia, saunders, 1794-1799. Yanuck MD, Kaufman RH, Woods KV, Adler Storthz K (1991) Cervical carcinoma metastatic to the skull, heart, and lungs, analysis for human papilomavirus DNA. Gynecol Oncol 42, 94-97. Yasuda N, Ishiki R, Agetsuma H (2002) Single large metastatic tumor growing progressively and occupying right ventricular cavity. Heart 87, 328.

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Cancer Therapy Vol 3, page 5 Cancer Therapy Vol 3, 5-28, 2005

C-Kit signal transduction and involvement in cancer Review Article

Johan Lennartsson1, Olexandr Voytyuk2, Elke Heiss2, Christina Sundberg2, Jianmin Sun2 and Lars Rönnstrand2,* 1

Ludwig Institute for Cancer Research, Uppsala University, Box 595, SE-751 24 Uppsala, Sweden Experimental Clinical Chemistry, Lund University, Wallenberg Laboratory, Malmö University Hospital, SE-205 02 Malmö, Sweden 2

__________________________________________________________________________________ *Correspondence: Lars Rönnstrand; Experimental Clinical Chemistry, Lund University, Wallenberg Laboratory, Malmö University Hospital, SE-205 02 Malmö, Sweden; Tel. +46 40 33 72 22; Fax +46 40 92 90 23; Email Lars.Ronnstrand@expklkemi.mas.lu.se Key words: c-Kit signal transduction, cancer, Dimerization, Internalization, degradation, Ras/Erk pathway, PI3-kinase, Phospholipase C-!, tyrosine kinases, JAK/STAT pathway, Adapter proteins, Transcription factors, Protein tyrosine phosphatases, homeostasis, Fertility, Hematopoiesis, Pigmentation, Gastrointestinal tract, Nervous system, Mastocytosis, Melanoma, Small-cell lung cancer Abbreviations: acute myeloid leukemia (AML); basic Helix-Loop-Helix Leucine Zipper (bHLHZip); Bruton tyrosine kinase (Btk); diacylglycerol (DAG); epidermal growth factor (EGF); Extracellular Regulated Kinase (Erk); gastrointestinal stromal tumors (GIST); Hematopoietic stem cells (HSC); inositol-1, 4, 5-trisphosphate (IP3); Interstitial cells of Cajal (ICC); Janus kinases (JAKs); mitogen activated protein kinase (MAPK); phorbol myristate acetate (PMA); phosphatidylinositol-3,4,5-trisphosphate (PIP3); phosphoinositide 3’-kinase (PI3-kinase); phospholipase C-! (PLC-!); phospholipase D (PLD); phosphotyrosine binding (PTB); pleckstrin homology (PH); protein inhibitor of activated Stat3 (PIAS3); protein kinase C (PKC); protein tyrosine phosphatases (PTPs); small cell lung cancer (SCLC); Src family kinase (SFK); Src homology 2 (SH2); stem cell factor (SCF); suppressors of cytokine signaling (SOCS); Tec homology (TH); truncated form of c-Kit (tr-Kit) Received: 17 January 2005; Accepted: 20 January 2005; electronically published: January 2005

Summary Receptor tyrosine kinases, such as c-Kit, are proteins whose function it is to transduce signals from the environment into the cell leading to complex behaviors such as proliferation, migration, survival and differentiation. Many of these behaviors are deregulated in cancer, which is characterized by uncontrolled proliferation, insensitivity towards death stimuli, migration of tumor cells away from the primary tumor site and in some cases also block of cellular differentiation leaving the cell in an immature proliferative state. To be able to target these processes it is vital to have a detailed understanding of the receptor function and the downstream pathways activated. In this article we will review the mechanisms by which c-Kit induces signal transduction as well as describing tumors in which c-Kit function is perturbed. kinase activity of the receptor. Mutations in the so-called Steel (Sl) locus in mice, that encodes the ligand for c-Kit, stem cell factor (SCF), give rise to a phenotype very similar to mutations in c-Kit (Copeland et al, 1990; Williams et al, 1990). For a review on W and Sl mutations, see Lev et al, 1994.

I. Introduction A. c-Kit and Stem cell factor (SCF) The viral oncogene v-Kit was identified in 1986 as the transforming gene of the Hardy-Zuckerman 4 feline sarcoma virus (Besmer et al, 1986) and shortly thereafter its cellular homolog, c-Kit, was cloned and sequenced (Yarden et al, 1987). Within a few years time, c-Kit was found to be allelic with the dominant white spotting locus (W) of mice (Chabot et al, 1988; Geissler et al, 1988). Numerous naturally occurring loss-of-function mutations in c-Kit have been found in both mice and humans. Complete loss of c-Kit expression leads to death in utero or perinatally, most likely due to severe anemia. Heterozygous animals display anemia, reduced fertility and defects in pigmentation. The severity of the phenotype has been found to inversely correlate with the tyrosine

B. c-Kit alternative splicing Alternative mRNA splicing leads to the occurrence of four isoforms of c-Kit in humans and two in mice. In both mice and humans, two isoforms are characterized by the presence or absence of a tetrapeptide sequence (GNNK) in the extracellular part of the juxtamembrane region (Reith et al, 1991; Crosier et al, 1993; Zhu et al, 1994). This alternative splicing occurs due to alternate use of 5’ splice donor sites (Hayashi et al, 1991). In addition, splice variants exist that differ in the presence or absence 5


Lennartsson et al: c-Kit signal transduction of a single serine residue in the kinase insert region of human c-Kit, due to alternative splice acceptor site usage (Crosier et al, 1993). Furthermore, post meiotic germ cells of the testis have been found to express a shorter transcript of c-Kit. This transcript encodes a truncated version of cKit (tr-Kit) consisting only of the second part of the kinase domain. Thus, extracellular and transmembrane domains as well as the first part of the kinase domain are missing which results in a non-functional tyrosine kinase activity (Rossi et al, 1992). Nonetheless, tr-kit is able to signal. Microinjection of tr-kit into mouse eggs, triggers metaphase-to-anaphase transition by the sequential activation of the Src family kinase (SFK) Fyn and phospholipase C-!1 (PLC-!1), and their association with Sam68 (Paronetto et al, 2003). Interestingly, the truncated isoform of c-Kit has been found to be frequently expressed in prostate carcinoma, in particular in more advanced stages (Paronetto et al, 2004), where it induces activation of SFK’s. The two GNNK+ and GNNK- isoforms of c-Kit (also denoted Kit and KitA, respectively) are co-expressed in most tissues (Reith et al, 1991; Crosier et al, 1993; Zhu et al, 1994) with the GNNK- form predominating. Expression of the two isoforms has been studied in human acute myeloid leukemia (AML). It was shown that among various AML cell lines the ratio of the two isoforms varied from as low as 1.3 to as high as 12 (Piao et al, 1994). In contrast, the ratio in normal bone marrow was around 4.45.5. However, no correlation was found between the expression of either isoform and the response to therapy or other clinical parameters. Despite these findings, NIH3T3 cells expressing the different isoforms were shown to possess distinct transforming activity (Caruana et al, 1999). In the presence of the ligand SCF, the GNNK- form induced loss of contact inhibition, anchorage-independent growth and tumorigenicity, with no difference in ligand affinity between the two isoforms. Upon ligand stimulation, the GNNK- isoform was more highly tyrosine-phosphorylated, more rapidly internalized, and activated Extracellular Regulated Kinase (Erk) more strongly than the GNNK+ isoform. In a recent study, it was shown that the kinetics of phosphorylation of the adapter protein ShcA, previously demonstrated to be phosphorylated by SFKs downstream of c-Kit, was stronger and more rapid in the GNNK- form (Voytyuk et al, 2003). However, no functional evidence for a role of enhanced ShcA phosphorylation in mediating the increased activation of Erks has been reported. There is also a possibility that other docking proteins, such as Gab1 or Gab-2, that are known to be phosphorylated by SFKs might contribute to the activation of Erk. Inhibition of SFKs by treatment with the selective inhibitor SU6656 altered the kinetics of activation of the GNNK- form of cKit so that they resembled those of the GNNK+ form. Thus, to summarize, a very minor difference in amino acid sequence in a region with no apparent enzymatic function or substrate binding ability, appears to lead to dramatic differences in signaling. Alternative splicing has been described in several tyrosine kinases and interestingly, several of these isoforms differ in the extracellular part of the juxtamembrane region, as is the case with c-Kit.

Recently, Leibiger and colleagues described differences in the signal transduction pathways activated and the repertoire of gene expression induced upon stimulation of the two splice forms of the insulin receptor, INSR-A and INSR-B. The INSR-A isoform was shown to induce expression of the insulin gene, whereas the INSR-B isoform mediated induction of the glucokinase gene (Leibiger et al, 2001). Through differential use of exon 11, the two splice forms of the insulin receptor differ in only 12 amino acids in the C terminus of the a-subunit. Furthermore, splice variants involving deletion of parts of the juxtamembrane region of the extracellular domain have been described for both ErbB2 and ErbB4. In the case of ErbB2, a deletion of sixteen amino acids in the extracellular juxtamembrane region caused a stronger kinase activity and transforming ability of ErbB2 (Kwong and Hung, 1998).

II. Signal transduction through c-Kit Signaling downstream of c-Kit has been studied extensively in a variety of cell systems with mast cells, which express endogenous c-Kit, being the most commonly used model system. Several studies have used transient transfection systems that lead to strong overexpression, such as COS cells or HEK293. In particular in the early studies, investigators have used chimeras, i. e. the extracellular part of another receptor, e. g. the epidermal growth factor (EGF) receptor fused to the intracellular part of c-Kit. These differences in experimental setup make it difficult to directly compare the results obtained in different laboratories. Discrepancies in the literature will be discussed here. In this review the individual tyrosine phosphorylation sites of c-Kit are for simplicity, coherence and for the purpose of avoiding confusion numbered according to the human c-Kit sequence, even in cases where the murine c-Kit was studied.

A. Activation of c-Kit through ligand induced oligomerization Binding of SCF to c-Kit leads to oligomerization of the receptors followed by activation of its intrinsic tyrosine kinase activity (Blume-Jensen et al, 1991). It is hypothesized that oligomerization is driven by the simultaneous binding of a dimeric SCF molecule to two receptor monomers (Philo et al, 1996; Lemmon et al, 1997). The activated receptor becomes autophosphorylated on a number of tyrosine residues (Figure 1). These tyrosine residues are mainly located outside the kinase domain, and serve as docking sites for signal transduction molecules containing Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains (for review, see Pawson, 2004). The c-Kit ligand, SCF, is expressed as a glycosylated transmembrane protein. Alternative splicing leads to two isoforms of SCF that differ in the absence or presence of a particular proteolytic cleavage site (Huang et al, 1992). The isoform containing the cleavage site undergoes proteolysis and becomes soluble upon release from the plasma membrane, whereas the isoform lacking the

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Cancer Therapy Vol 3, page 7 become ubiquitinated upon ligand-stimulation (BlumeJensen et al, 1991). It was thought that RTKs become polyubiquitinated and degraded in the proteasomes, but recently it was shown that in the case of RTKs, monoubiqutination rather than polyubiqutination takes place (Haglund et al, 2003) and targets the receptors for internalization and degradation in the lysosomes. This is in contrast to polyubiquitination of cytosolic proteins that targets them for degradation in the proteasomes. Furthermore, other investigators have found that c-Kit is degraded both in the lysosomes and the proteasomes (Zeng et al, 2004). Important components of the ubiquitination machinery are the ubiquitin E3 ligases that covalently attach ubiquitin moieties to target proteins. In RTK signaling, one of the important E3 ligases is the adapter protein Cbl, and its close family members Cbl-B and Cblc, which bind to activated receptors and other tyrosine phosphorylated proteins via its SH2 domain (Joazeiro et al, 1999). Phosphorylation by SFKs leads to activation of Cbl (Yokouchi et al, 2001). Cbl is able to bind to activated c-Kit through the adapter proteins CrkL (Wisniewski et al, 1996; Sattler et al, 1997) and APS (Wollberg et al, 2003) and is phosphorylated in response to SCF stimulation. Several studies have shown that internalization of cKit is dependent on the activity of SFKs (Broudy et al, 1999; Jahn et al, 2002; Voytyuk et al, 2003). Given the role of monoubiquitination in internalization, it is likely that SFK-dependent activation of Cbl and subsequent monoubiquitination regulates internalization of c-Kit. Recently, Zeng et al, could demonstrate that degradation of c-Kit was partly mediated by the proteasomal and lysosomal pathway (Zeng et al, 2004).

Figure 1. Signal transduction molecules binding to the activated c-Kit receptor. Upon ligand-binding, c-Kit dimerizes and its intrinsic tyrosine kinase activity isactivated, leading to phosphorylation of key residues. These residues constitute high affinity binding sites for signal transduction molecules. The numbers refer to tyrosineresidues phosporylated in c-Kit and the corresponding signal transduction molecule isdepicted.

C. The Ras/Erk pathway Numerous studies have demonstrated the critical importance of the Ras/Erk pathway in cell division, survival and transformation (Lewis et al, 1998). Ras is a small G-protein that can alternate between an active GTPbound form and an inactive GDP-bound form. Although Ras can activate a number of signal transduction molecules such as PI3-kinase and Rac (Qiu et al, 1995; Rodriguez-Viciana et al, 1997), its role in the Ras/Erk cascade is the most well characterized. RTKs activate Ras through association with Sos, a guanine nucleotide exchange factor that facilitates exchange of GDP for GTP leading to activation of Ras (Buday and Downward, 1993). In the cell, Sos exists in a preformed complex with the adapter protein Grb2, which in turn associates via its SH2 domain to phosphorylated tyrosine residues within the consensus sequence pYXN. These tyrosine residues exist either in the receptor or in downstream signal transduction molecules such as the protein tyrosine phosphatase SHP-2, the adapter proteins ShcA (Lowenstein et al, 1992; Rozakis-Adcock et al, 1992; Li et al, 1994) Gab-1 or Gab-2 (Nishida et al, 1999). Thus, the Grb2-Sos complex is recruited to the vicinity of the plasma membrane, where it can act on Ras. Activated GTP-bound Ras has the ability to interact with the serine/threonine kinase Raf-1 leading to its activation. Raf-1 kinase phosphorylates and activates the dual-

cleavage site remains cell-associated. The two isoforms are not equivalent in their signaling capabilities. They each have specific abilities to transmit signals. Stimulation with the soluble isoform leads to a rapid and transient activation and autophosphorylation of c-Kit, followed by a fast degradation, whereas stimulation with the membraneassociated isoform leads to a more sustained activation of c-Kit (Miyazawa et al, 1995). Differences exist also in signaling downstream of c-Kit. The membrane-bound ligand induced a more persistent activation of Erk1/2 and p38 mitogen activated protein kinase (MAPK), when compared to the soluble ligand (Kapur et al, 2002). It is thought that the differences in signaling might in part arise from the fact that membrane anchoring of the ligand might prevent internalization of the receptor-ligand complex. Using immobilized agonistic anti-Kit monoclonal antibodies to induce receptor dimerization in the absence of internalization, Kurosawa et al. could mimic the action of membrane-bound SCF (Kurosawa et al, 1996).

B. Internalization and degradation of cKit Ligand-induced down-regulation of RTKs is an important means of modulating signaling in the normal physiology of cell surface receptors. RTKs including c-Kit 7


Lennartsson et al: c-Kit signal transduction specificity kinases Mek1 and Mek2 (Kyriakis et al, 1992). The serine/threonine kinases Erk1 and Erk2 are activated through phosphorylation by Mek1/2 (Crews and Erikson, 1992). Activated Erks dimerize and are translocated to the nucleus (Khokhlatchev et al, 1998) where transcription factors are phosphorylated. Phosphorylation of transcription factors, such as c-Fos, regulates their activity and thereby influences gene transcription (Murphy et al, 2002). A number of studies have demonstrated the ability of SCF to activate the Ras-Erk pathway. The adapter protein Grb2 can directly associate with c-Kit (Tauchi et al, 1994a) through phosphorylated Y703 and Y936 in c-Kit (Thömmes et al, 1999). In addition, Grb2 can associate with SHP-2, ShcA, Gab-1 and Gab-2 following SCF stimulation (Nishida et al, 1999; Tauchi et al, 1994a,b). The adaptor protein Gab-2 can link to the Ras/Erk pathway through association with SHP-2 (Dorsey et al, 2002). Several studies have suggested an important role for SCF-induced activation of SFKs in the activation of the Ras/Erk cascade (Bondzi et al, 2000; Ueda et al, 2002; Kimura et al, 2004; Lennartsson et al, 1999;). However, others have shown no effect for SFK inhibition on the activity of Erk (Timokhina et al, 1998). In the same study, an independence of PI3-kinase for activation of Erk in mast cells was also demonstrated. However, under certain conditions activation of Erk has been implicated to be dependent on the activity of PI3-kinase. Recently, Wandzioch et al. showed that inhibition of PI3-kinase with the pharmacological agent LY294002 effectively inhibited Erk phosphorylation in a hematopoietic progenitor cell line (Wandzioch et al, 2004). Moreover, it has been demonstrated that GM-CSF and SCF synergistically activated the Ras/Erk pathway in Mo7e cells in a PI3kinase dependent manner (Lennartsson et al, 2004). However, in these cells Erk phosphorylation by SCF or GM-CSF alone was independent on PI3-kinase.

with the adapter proteins such as CrkII, CrkL and Cbl, respectively (Hartley et al, 1995; Sattler et al, 1997). The activation of PI3-kinase by c-Kit has been linked to mitogenesis, differentiation, adhesion, secretion, survival, and actin cytoskeletal reorganization (Serve et al, 1995; Vosseller et al, 1997; Blume-Jensen et al, 1998; Kubota et al, 1998; Timokhina et al, 1998). In c-Kit, Y721 was found to directly interact with PI3-kinase (Serve et al, 1995). c-Kit promotes survival via PI3-kinase dependent activation of Akt and phosphorylation of Bad, a proapoptotic molecule, at S136 in vivo. Furthermore, mutation of S741 and S746 in c-Kit, the two serine sites involved in negative regulation by PKC, led to increased mitogenic response and increased activation of PI3-kinase, as well as enhanced Akt activation, Bad phosphorylation and survival (Blume-Jensen et al, 1998). Recently Jin et al. showed that in cultured primordial follicles of the ovary, stimulation of c-Kit led to prevention of apoptosis through a PI3-kinase mediated pathway (Jin et al, 2005). SCF stimulation led to upregulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL, while it induced a downregulation of the expression of the pro-apoptotic factor Bax. Inhibition of PI3-kinase reversed the regulation of Bcl-xL and Bcl-2, but not Bax. An alternative pathway for c-Kit mediated survival is through Akt-mediated phosphorylation and inactivation of the forkhead transcription factor FoxO3a (Engström et al, 2003). Whether FoxO transcription factors or Bad are involved in the anti-apoptotic signaling induced by c-Kit seems to be cell type dependent. A number of studies have implicated the importance of PI3-kinase activity in transforming ability of mutated cKit. Using immortalized murine progenitor cells transduced with the Y721F mutant D816V c-Kit, Chian et al. showed that transformation by this constitutively active form of c-Kit is dependent on PI3-kinase (Chian et al, 2001). In another study, mutants with the cytoplasmic tyrosines of c-Kit individually mutated to phenylalanine residues were used to assess the role of individual signal transduction pathways in D816V transformation (Hashimoto et al, 2003). Two mutants, Y721F and a deletion mutant in which the two most distal tyrosines in the carboxyterminal tail were mutated, severely impaired receptor activation. The deletion included Y936, that previously was shown to be a docking site for Grb2, Grb7 and APS (Thömmes et al, 1999; Wollberg et al, 2003). Interestingly, in contrast to their effect on transformation, these mutants showed no effect on normal ligand-induced activation of c-Kit. The physiological role of c-Kit-mediated activation of PI3-kinase was demonstrated in two studies using transgenic mice expressing c-Kit with a Y721F mutation. Blume-Jensen et al. showed that c-Kit-induced activation of PI3-kinase was essential for male fertility, while in another study Kissel et al. also could demonstrate an effect on female fertility ((Blume-Jensen et al, 2000; Kissel et al, 2000). Thus, loss of c-Kit mediated PI3-kinase signaling may be compensated for in a number of physiological processes, but appears to be critical in gametogenesis. These findings assume that Y721 solely serves as the docking site for PI3-kinase and for no other signal

D. PI3-kinase PI3-kinase is a class of lipid kinases that phosphorylate the 3’-hydroxyl group of phosphoinositides. Although phosphatidylinositol can be phosphorylated by PI3-kinase in vitro, phosphatidylinositol-4,5-bisphosphate (PIP2) is the physiologically relevant substrate (for review, see Foster et al, 2003). The resulting product, phosphatidylinositol-3,4,5-trisphosphate (PIP3), is able to physically associate with proteins containing a pleckstrin homology (PH) domain, leading to their recruitment to plasma membrane where they can be activated. A majority of the published work on PI3-kinase has been focused on the classical class I PI3-kinases. The regulatory p85 subunit of PI3-kinase contains two SH2 domains by which it binds to target proteins. Upon binding, conformational changes are induced and PI3kinase enzyme is activated (Carpenter et al, 1993). The p110 subunit of PI3-kinase contains the enzymatic activity to phosphorylate phosphoinositides. The two subunits exist in the cell as a preformed complex. Interestingly, there is a stoichiometric excess of p85 subunit within the cell (Ueki et al, 2002), suggesting other functions of p85. It is well known that p85 also has the ability to associate 8


Cancer Therapy Vol 3, page 9 transduction molecule. This, however, remains to be proven. One of the PI3-kinase association sites present in the closely related PDGF "-receptor, Y751, has also been known to bind to the adapter protein Nck# (Nishimura et al, 1993). Thus, some of the found Y721F phenotypic outcomes might be due to blocking of additional, hitherto unknown interactions and signal transduction pathways. Bone-marrow-derived mast cells from mice with a targeted deletion of the p85# subunit of PI3-kinase demonstrated a dramatically reduced SCF-mediated proliferative response compared to wild-type cells, further emphasizing the role of PI3-kinase in c-Kit signaling (Fukao et al, 2002). This effect paralleled a reduction of SCF-induced activation of JNK in the p85#-deficient mast cells. Interestingly, SCF-stimulated activation of Akt was only partially impaired in p85#-deficient bone marrow derived mast cells, and no effect on c-Kit mediated survival was observed. Therefore additional, PI3-kinase independent signal transduction pathways may contribute to c-Kit-mediated survival. In addition, SCF-mediated chemotaxis has also been demonstrated to be dependent on p85# (Tan et al, 2003). It should be noted that discrepancies do exist in results obtained from p85#-deficient cells and Y721F mutant c-Kit. The reason for these differences could be that Y721 is able to dock to signal transduction molecules other than PI3-kinase, as mentioned above. Furthermore, p85# is known to bind to proteins other than the p110 subunit of PI3-kinase. In an elegant study, Ali and colleagues generated transgenic mice expressing a kinase inactive form of the p110$ subunit of PI3-kinase (Ali et al, 2004). Thus, the lipid kinase activity of PI3-kinase was targeted, while other possible functions of the p85 subunits were intact. It was shown that the p110$ isoform of PI3kinase plays an essential role in allergic response. Inactivation of p110$ in mast cells led to defective SCFmediated in vitro proliferation, adhesion and migration, as well as impaired allergen-IgE-induced degranulation and cytokine release. Furthermore, mice carrying an inactivated version of p110$ were protected against anaphylactic allergic responses. Apart from the classical type I forms of PI3-kinase, the type II isoform PI3KC2" was shown to physically associate with activated c-Kit and mediate part of the SCFdependent activation of Akt in small lung carcinoma cells (Arcaro et al, 2002). Interestingly, in contrast to the type I PI3-kinases, PI3KC2b association with c-Kit was ligandindependent and constitutive. However, ligand stimulation of c-Kit led to tyrosine phosphorylation of PI3KC2". The site of interaction with c-Kit is not known, although given that the association to c-Kit seems constitutive, and that other receptors previously shown to interact with the classical isoforms of PI3-kinase did not interact with PI3KC2", it appears likely that the classical PI3-kinase association site is not involved.

is ubiquitously expressed, PLC-!2 is mainly expressed in the hematopoietic system (for review, see Carpenter and Ji, 1999). The substrate upon which PLC acts is the phosphoinositide PIP2, whereby the second messengers DAG and inositol-1, 4, 5-trisphosphate (IP 3) are generated. DAG is an activator of the classical and novel forms of PKC, while IP3 binds to specific calcium channels present on the endoplasmic reticulum, triggering release of Ca2+ from internal stores. The intracellular concentration of free Ca2+ regulates a number of cellular processes (for review, see Berridge et al, 2003). Some studies have demonstrated association with and activation of PLC-! by c-Kit, while others have failed to do so. It has been claimed that Y730 is the site of association of PLC-!1. Using overexpression of EGFR-cKit chimeras and PLC-!1 in HEK293 cells, Herbst et al, demonstrated ligand-induced tyrosine phosphorylation of PLC-!1, although weaker than that seen with the EGF receptor (Herbst et al, 1991). In a later study, the same authors saw no association between PLC-!1 and the EGFR-c-Kit chimera unless they overexpressed PLC-!1 (Herbst et al, 1995). However, those studies were performed using chimeric receptors with the extracellular domain of the EGF receptor fused to the intracellular part of c-Kit. In addition, association was only seen when the receptor was overexpressed together with overexpressed PLC-!1. In a recent study, Gommerman et al. studied the differential signaling of various c-Kit mutants induced by membrane-bound and soluble SCF, respectively, using retrovirally transduced 32D cells (Gommerman et al, 2000). Using soluble SCF, a weak SCF-stimulated tyrosine phosphorylation of PLC-!1 was shown in wildtype murine c-Kit expressing cells, but in cells expressing the Y730F mutant, no phosphorylation of PLC-!1 was seen. Furthermore, cells expressing the Y730F mutant cKit did not respond with calcium release following treatment with SCF. Interestingly, PLC-!1 phosphorylation was much stronger in bone-marrowderived mast cells than in 32D infectants. Likewise, Trieselmann et al. showed that stimulation of mast cells by membrane-bound, but not soluble SCF, was dependent on PLC-! activation (Trieselmann et al, 2003). They also demonstrated ligand-induced tyrosine phosphorylation of PLC-!2. Other studies have shown that activation of PLC! by c-Kit might be involved in the SCF-mediated protection against apoptosis induced by radiation and chemotherapy (Plo et al, 2001; Maddens et al, 2002). Also studying bone marrow mast cells, Huber et al. detected a robust and sustained SCF-stimulated tyrosine phosphorylation of PLC-!2 (Huber et al, 1998). Others, however, failed to detect c-Kit mediated activation of PLC-!, but were able to detect SCF-dependent activation of phospholipase D (PLD; Koike et al, 1993). These data were further confirmed by Kozawa et al. who also could inhibit SCF-stimulated PLD activity with the PI3-kinase inhibitor LY294002 (Kozawa et al, 1997). There are several plausible explanations to these discrepancies. It is possible that some of the differences in the findings as to whether PLC-! is activated or not might arise from different expression levels of the two isoforms

E. Phospholipase C-! Two isoforms of PLC-! exists, PLC-!1 and PLC-!2, and they both consist of two SH2 domains, one SH3 domain, one PH domain and a catalytic domain. While -!1

9


Lennartsson et al: c-Kit signal transduction of PLC-! in different cell types, with the cell types expressing PLC-!2 showing stronger activation of PLC. Another possible explanation for differences in the data on activation of PLC-!1 might be the differential signaling abilities of alternative splice forms of c-Kit. It is known that the two alternative splice forms denoted GNNK+ and GNNK- do signal at quantitatively and qualitatively different levels (Caruana et al, 1999; Voytyuk et al, 2003). Thus, expression of various splice forms of c-Kit might influence the outcome of studies on PLC-! activation. A direct physical interaction between c-Kit and PLC-! isoforms has not been demonstrated, except in cells overexpressing the receptor and PLC-!1. Thus, it is possible that activation of PLC-! might be a result of activation of other tyrosine kinases downstream of c-Kit that might not necessarily require a direct physical association between PLC-! and c-Kit. One such candidate kinase is the Src family of tyrosine kinases.

pathway. This is in agreement with previous findings that SFKs play an important role in phosphorylating ShcA, thereby recruiting the Grb2-Sos complex, leading to activation of Ras (Lennartsson et al, 1999; Bondzi et al, 2000). In addition, SCF-induced activation of other signal transduction molecules such as Rac and JNK were shown to be restored by adding back Y568 and Y570. The function of SFKs in a more physiological context was addressed by Agosti et al, (2004) who generated transgenic mice carrying c-Kit with a Y568F mutation. They found that mutation of Y568, the primary binding site of SFKs in c-Kit, led to a block in pro T cell and pro B cell development, in contrast to the Y721F mutant (affecting PI3-kinase activation) that had no effect on hematopoiesis. These data suggest that SFKs mediate a critical signal for lymphocyte development. However, one of the difficulties in interpreting these data lies in the probability that additional signal transduction molecules apart from SFKs might be involved. For example, the protein tyrosine phosphatase SHP-2 (Kozlowski et al, 1998), the tyrosine kinase CHK (Jhun et al, 1995) and the adaptor protein APS (Wollberg et al, 2003) have also been shown to bind to phosphorylated Y568. Using a similar approach, Kimura et al. generated mice carrying the Y568F and Y570 of c-Kit. These tyrosine residues were shown to be crucial for the function of c-Kit in melanogenesis and mast cell development. In contrast, they were dispensable for the normal development of erythroid cells, interstitital cells of Cajal and germ cells. They also displayed splenomegaly, dysregulation of B-cell and megakaryocyte development (Kimura et al, 2004). However, since Y568 has been demonstrated to bind to the protein tyrosine phosphatase SHP-2 (Kozlowski et al, 1998) and the adapter protein APS (Wollberg et al, 2003), and Y570 has been shown to be associating with the protein tyrosine phosphatase SHP1 (Kozlowski et al, 1998), the phenotype of the Y568F/Y570F mice might not necessarily solely reflect a function of the SFK, but also the function of other signal transduction molecules.

F. The Src family of tyrosine kinases The SFK is named after its prototypic family member c-Src, the cellular homolog of the transforming protein of Rous sarcoma virus, v-Src. Some members, like Src, Yes and Fyn, are ubiquitously expressed, while others, such as Lck, Hck, Fgr, Lyn and Blk, display a more restricted expression pattern and are mainly expressed in cells of the hematopoietic lineages (Abram and Courtneidge, 2000). They consist of an N-terminal sequence which directs myristoylation and in some cases palmitoylation. These lipid modifications serve to anchor the kinases to the plasma membrane. They also contain an SH3 domain, an SH2 domain and a tyrosine kinase domain. They have been implicated in a number of cellular functions, including survival, chemotaxis, adhesion, proliferation and protein trafficking. Ligand-induced activation of c-Kit leads to a rapid increase in SFK activity (Linnekin et al, 1997; Krystal et al, 1998). A number of investigators have shown that SFKs associate primarily with phosphorylated Y568 in cKit, while Y570 contributes to the overall affinity of binding by acting as an acidic determinant (Price et al, 1997; Timokhina et al, 1998; Lennartsson et al, 1999). SCF-induced chemotaxis of Mo7e cells was dependent on SFK activity (Broudy et al, 1999). In another study, overexpression of a dominant negative form of Lyn in either primary hematopoietic progenitor cells or bone marrow derived mast cells led to inhibition of both SCFmediated proliferation and chemotaxis (O'LaughlinBunner et al, 2001). In Mo7e cells, activation of the SFK Lyn was demonstrated to occur during the late G1 phase of SCF-stimulated cell cycle progression (Mou and Linnekin, 1999). Using an approach where 32D cells were transfected with chimeric c-Kit containing the extracellular domain of the M-CSF receptor, and by mutating seven tyrosine residues of the intracellular part of c-Kit, Hong et al. demonstrated a complete loss of mitogenic response of 32D cells (Hong et al, 2004). However, by adding back Y568 and Y570 to this mutant, the mitogenic response was restored, as well as survival and migration. Furthermore, restoration of the Src binding sites also lead to restored activation of the Ras/Erk

G. Other tyrosine kinases: Tec, CHK, Fer and Fes Tec belongs to a family of tyrosine kinases that also includes the Bruton tyrosine kinase (Btk), Bmx, Itsk/Tsk, and Rlk/Txk (Smith et al, 2001). They each contain a pleckstrin homology (PH) domain, and a Tec homology (TH) domain in the amino-terminus followed by SH3, SH2 and tyrosine kinase domains. In contrast to the SFKs, they lack membrane targeting myristoylation site, but are recruited to the plasma membrane through the PH domain interacting with membrane-bound PIP3. Activation of Tec family kinases is thought to be mediated by members of the Src family. Tec is phosphorylated on tyrosine residues and activated upon ligand stimulation of c-Kit (Tang et al, 1994). More recent studies have shown that Tec forms multiprotein complexes with Lyn and the adapter protein Dok-1 (van Dijk et al, 2000; Liang et al, 2002). Phosphorylation of Tec and Dok-1 was dependent on their recruitment to the plasma membrane through activation of 10


Cancer Therapy Vol 3, page 11 PI3-kinase (van Dijk et al, 2000). Both Lyn and Tec were capable of phosphorylating Dok-1, but using cells derived from animals with a targeted deletion of Lyn (Liang et al, 2002) it was shown that Lyn was required for SCFdependent phosphorylation of Dok-1. CHK (for Csk Homologous Kinase, also known as MATK) displays an about 50 % sequence identity with Csk, and like Csk it phosphorylates and inactivates SFKs. Similarly to SFKs, CHK has been shown to associate to the phosphorylated Y568 in the juxtamembrane region of ligand-stimulated c-Kit (Jhun et al, 1995). The cytoplasmic tyrosine kinases Fer and Fes belong to a separate family of tyrosine kinases. They both contain an SH2 domain immediately aminoterminal to the kinase domain. Following SCF-stimulation Fer associates with cKit and becomes phosphorylated on tyrosine residues (Kim and Wong, 1995). Using mast cells derived from transgenic mice carrying a kinase inactivating mutation of Fer, Craig and Greer found a requirement for Fer kinase activity for sustained p38 kinase activation and maximal chemotactic response to SCF (Craig and Greer, 2002). Fes has been found to bind to c-Kit (Masuhara et al, 2000), although its role in c-Kit signaling remains to be shown.

constitutively active D816H mutant of c-Kit in order to be tumorigenic (Ning et al, 2001). However, as is the case with the activation of PLC-!, there are discrepancies in the literature. Some investigators have failed to detect activation of the JAK/STAT pathway by c-Kit (Jacobs-Helber et al, 1997; O'Farrell et al, 1996; Pearson et al, 1998). A possible explanation is cell type specific signaling or differences in the experimental setup as well as the very rapid and transient JAK2 activation by SCF.

I. Adapter proteins Adapter proteins are proteins with several domains that specify protein-protein interactions. The multiple domains enable them to interact with several proteins simultaneously. The ability of linking proteins together through specific and many times regulated protein-protein interactions, enables signaling to be sequentially and spatially regulated (for review, see Pawson and Scott, 1997). Grb2 was originally identified as a protein interacting with the phosphorylated EGF receptor (Lowenstein et al, 1992) and found to mediate activation of the Ras/Erk pathway by RTKs. Grb2 is a ubiquitously expressed protein containing one SH2 domain and two SH3 domains. Tyrosine phosphorylated c-Kit has been shown to associate with Grb2 (see above under C “The Ras/Erk pathway”) through Y703 and Y936 of c-Kit (Thömmes et al, 1999). The adapter protein Gads (also denoted Mona, Grap2, GrpL or Grf40) is closely related to Grb2 and expressed in hematopoietic cells (for review, see Liu et al, 2001) and has been shown to interact with c-Kit in a manner similar to Grb2 (Liu and McGlade, 1998). However, it is not known whether Gads interact with the same tyrosine residues as Grb2. Another member of the same family of adapter proteins with a very similar structure, Grap, also interacts with c-Kit (Feng et al, 1996). ShcA is a ubiquitously expressed adapter protein that contains one SH2 domain and a PTB domain, that both enable ShcA to interact with phosphorylated proteins (for review, see Ravichandran, 2001). Phosphorylation of ShcA by RTKs, directly or indirectly via SFKs, leads to the creation of high affinity binding sites for Grb2, which in turn leads to activation of the Ras/Erk pathway. In vitro data suggest that ShcA interacts with the juxtamembrane domain of c-Kit (Price et al, 1997). The adapter protein Grb7 belongs to a family of closely related proteins consisting of Grb7, Grb10 and Grb14 that each exist as several alternatively spliced variants (for review, see Han et al, 2001). Grb7 contains an SH2 and a so-called GM region (for Grb and Mig) which includes a PH domain and shows sequence homology with the C. elegans protein Mig-10, that has been implicated in embryonic migration. Grb7 interacts with activated c-Kit through Y936 in the carboxyterminal tail of the receptor (Thömmes et al, 1999). However, the exact role of Grb7 in c-Kit signaling remains to be elucidated. Grb10 was identified in a yeast two-hybrid screen using the D816V constitutively active mutant of c-

H. The JAK/STAT pathway The Janus kinases (JAKs) are a family of closely related cytoplasmic tyrosine kinases that are activated through ligand stimulation of cytokine receptors or RTKs. Downstream targets of the JAKs are the signal transducers and activators of transcription (STAT) that are phosphorylated by JAKs and thereby activated. STAT proteins are a class of transcription factors with DNA binding domains, an SH2 domain and a carboxy-terminal transactivating domain. Upon tyrosine phosphorylation, STATs dimerize through interaction between the phosphorylated tyrosine residues and the SH2 domain of STAT, leading to dimerization. STATs can form both homodimers of identical STATs or heterodimers between different members of the STAT family. The dimerized STATs translocate to the nucleus, where they regulate expression of responsive genes (for review, see Kerr et al, 2003). The JAK/STAT pathway is activated following SCF stimulation. c-Kit stimulates rapid and transient tyrosine phosphorylation of JAK2 (Brizzi et al, 1994). JAK2 was found to be constitutively associated with c-Kit, with increased association after ligand stimulation of c-Kit (Weiler et al, 1996). A role of JAK2 in c-Kit mediated signaling was suggested by the finding that treatment of cells with JAK2 antisense oligonucleotides resulted in a marked decrease in SCF-induced proliferation. In addition, SCF-induced growth of fetal liver cells from mice carrying a targeted deletion of JAK2 was shown to be reduced compared to cells from wild-type mice (Radosevic et al, 2004). Furthermore, JAK2 was also required for differentiation of the Kit+ progenitor cells into mast cells. Ligand-stimulated activation of c-Kit leads to physical association with and activation of STAT1# STAT3, STAT5A and STAT5B (Brizzi et al, 1999; Deberry et al, 1997; Ning et al, 2001; Ryan et al, 1997). It has been shown that STAT3 activation is required for the

11


Lennartsson et al: c-Kit signal transduction Kit as a bait (Jahn et al, 2002). The interaction between Grb10 and c-Kit was shown to be mediated through its SH2 domain, while the PH domain mediates interaction with the serine/threonine kinase Akt. It was further demonstrated that Grb10 and c-Kit are able to activate Akt in a synergistic manner. The adaptor protein Lnk belongs, together with APS and SH2-B, to a family of closely related adapter proteins. All three proteins share a common domain structure in that they contain a conserved amino-terminal domain that includes a proline-rich stretch, a PH domain and an SH2 domain. They all contain a conserved tyrosine residue in their carboxy-termini that is presumed to be a phosphorylation site mediating interaction with Cbl (Iseki et al, 2000). Using transgenic mice lacking the expression of Lnk, it was shown that B cell precursor cells were hypersensitive to SCF stimulation (Takaki et al, 2000), leading to proportional accumulation of B cell precursors in the bone marrow and B cells in the spleen of transgenic mice. Thus, these data suggest that Lnk has a negative regulatory role in B cell production. APS was originally identified in a yeast two-hybrid screen using constitutively active c-Kit as bait (Yokouchi et al, 1997). When APS is phosphorylated in its carboxyterminal tail, it physically associates with Cbl (Wakioka et al, 1999; Yokouchi et al, 1999). Being a ubiquitin E3-ligase, Cbl is able to monoubiquitinate activated RTKs, leading to their internalization and degradation in the lysosomes (Haglund et al, 2003). The primary associations sites for APS in c-Kit have been shown to be phosphorylated Y568 and Y936 (Wollberg et al, 2003). Mutation of both Y568 and Y936 was necessary to completely block binding of APS to c-Kit. Recently it was shown that APS exists as a dimer (Hu et al, 2003), which might explain why both sites are needed for full binding of APS to c-Kit. Interestingly, in the viral form of kit, v-Kit, both Y568 and Y936 are missing (Herbst et al, 1995). Thus, it has been speculated that loss of binding of APS, or maybe SH2-B or Lnk, in v-Kit could possibly lead to reduced ubiquitination and prolonged receptor signaling, that could possibly contribute to transformation. A number of transforming mutants of RTKs have been shown to lack association sites for Cbl, leading to reduced ubiqutination and stabilization of active receptors (Peschard and Park, 2003). However, the physiology of mice with a targeted deletion of APS does not support a major role for APS in c-Kit signaling. The effects are mainly related to the immune system (Iseki et al, 2004), although mast cells derived from APS knock-out animals show a markedly augmented degranulation in response to c-Kit stimulation, as well as lower levels of F-actin (Kubo-Akashi et al, 2004). In contrast, targeted deletion of either Lnk or SH2B did not lead to any marked effect on mast cell behavior. Dok-1 is an adapter protein of 62 kDa originally identified as a tyrosine-phosphorylated protein associated with p120-RasGAP in fibroblasts transfected with v-Src (Ellis et al, 1990). It contains a phosphotyrosine binding (PTB) domain and a PH domain. Cells from Dok-1 knockout mice hyperproliferate in response to stimulation with a number of growth factors and cytokines, suggesting

a role of Dok-1 as a negative regulator of cell proliferation (Yamanashi et al, 2000). Dok-1 was found to associate with activated c-Kit in chronic myelogenous leukemia progenitor cells (Carpino et al, 1997). The Gab/Dos family of adaptor proteins are a family of scaffolding adaptors with similar overall structural organization (reviewed in Gu and Neel, 2003), containing an N-terminal PH domain, proline-rich motifs that can interact with SH3 domains and multiple tyrosine phosphorylation sites that can serve as docking sites for SH2 domain containing proteins. Both Gab-1 and Gab-2 are phosphorylated in response to SCF-stimulation (Nishida et al, 1999). Gab-2 is required for mast cell development and c-Kit signaling (Nishida et al, 2002), while Gab-1 does not seem to be essential for c-Kit signaling. Bone marrow mast cells derived from Gab-2deficient mice grew poorly in response to SCF and activation of both Erk and Akt were impaired. The Crk family of adapter proteins consists of one SH2 domain, as well as one or two SH3 domains. The family consists of four members: CrkI and CrkII (alternative splice forms of the same gene), CrkL (Feller, 2001) and the recently discovered CrkIII (Prosser et al, 2003). Ligand stimulation of c-Kit leads to phosphorylation of CrkL (Sattler et al, 1997), which indirectly associates with c-Kit through the p85 subunit of PI3-kinase. In addition, CrkL mediates interaction with Cbl, which thus likely contributes to c-Kit ubiquitination and degradation. The closely related protein CrkII was also shown to be phosphorylated in response to SCF stimulation and interact with c-Kit also indirectly via the p85 subunit of PI3-kinase (Lennartsson et al, 2003). This interaction was dependent on phosphorylation of Y900 in the second part of the kinase domain, which is not an autophosphorylation site but is phosphorylated through the action of SFKs.

J. Protein tyrosine phosphatases The two closely related protein tyrosine phosphatases (PTPs) SHP-1 and SHP-2 constitute a family of proteins consisting of two amino-terminal SH2 domains, a PTP domain and a carboxyterminal tail (Neel et al, 2003). A number of SHP binding proteins have been reported including activated cytokine receptors and RTKs, as well as scaffolding adaptors, such as the Gab proteins. Activation of SHPs occurs through binding of the SH2 domains to tyrosine phosphorylated peptides, in particular biphosphorylated. Binding of the phosphopeptide leads to opening up of the phosphatase structure. SHP-1 associates with phosphorylated Y570 in c-Kit (Kozlowski et al, 1998) and is involved in negative regulation of c-Kit signaling (see also “Negative regulation of c-Kit signaling�). In contrast, SHP-2 is, despite the fact that it is a phosphatase, a positive regulator of signaling. SHP-2 physically interacts with SCF-stimulated c-Kit and becomes phosphorylated on tyrosine residues (Tauchi et al, 1994). The site of interaction was shown to be Y568 in the juxtamembrane region of c-Kit (Kozlowski et al, 1998), which also constitutes the docking site for a number of other signal transduction molecules, such as SFKs, CHK and APS. In most RTK signaling, SHP-2 12


Cancer Therapy Vol 3, page 13 activation is required for full activation of the Ras/Erk pathway, e. g. the PDGF "-receptor (Rรถnnstrand et al, 1999). SHP-2 also plays an important role in mediating embryonic stem cell differentiation and hematopoiesis (Chan et al, 2003). The tyrosine phosphatase PTP-RO is despite its lack of SH2 domains still able to associate with the c-Kit receptor (Taniguchi et al, 1999). Furthermore, PTP-RO becomes phosphorylated on tyrosine residues after SCF stimulation. By use of antisense oligonucleotides the function of PTP-RO could be inhibited, which led to significantly inhibited proliferation of Mo7e cells (Taniguchi et al, 1999). Similar to PTP-RO, the protein tyrsoine phosphatase PTP-PEST was shown to bind to c-Kit in a liganddependent manner (Markova et al, 2003). However, the mode of interaction, the site of interaction and its role in cKit signaling remains to be elucidated.

tuning of the regulation of transcriptions factors downstream of c-Kit. Another transcription factor involved in c-Kit signaling is Slug, a member of the Snail family of zinc finger transcription factors. Mice with a targeted deletion of Slug show pigment deficiency, gonadal defects, and impairment of hematopoiesis, very much reminiscent of the phenotype of loss-of-function mutations in c-Kit (Perez-Losada et al, 2002). It was demonstrated that cells from Slug knockout animals despite the expression of cKit were defective in SCF-induced migration, suggesting a role for Slug downstream of c-Kit. It was recently shown that Slug function in c-Kit mediated radioprotection (Perez-Losada et al, 2003). Furthermore, in malignant mesothelioma, which is often resistant to conventional chemotherapy, it was established that c-Kit-dependent upregulation of Slug conferred drug resistance (Catalano et al, 2004). In contrast, down-regulation of Slug using RNAi made the cells susceptible to apoptosis induced by chemotherapeutic drugs. Thus, Slug appears to have a central position in SCF-mediated protection from apoptosis induced by chemotherapy. Therefore development of therapeutic agents that target Slug or Slug regulated genes may provide ways to sensitize tumors for conventional chemotherapy.

K. Transcription factors A number of genes are induced upon SCFstimulation of cells. One is the Mitf protein, which is a member of the MYC superfamily of transcription factors (Hodgkinson et al, 1993; Hughes et al, 1993; Boissy and Nordlund, 1997) and is closely related to three other basic Helix-Loop-Helix Leucine Zipper (bHLHZip) transcription factors, Tfe3, Tfeb and TfeC. The phenotype of Mitf mutant mice is strikingly similar to that of mice with loss-of-function mutations of c-Kit or its ligand (spotted fur color, mast cell deficiency; reviewed in Boissy and Nordlund, 1997). This suggested a functional link between the Mitf transcription factor and c-Kit and its ligand. This has been proven by a number of investigators. In vitro experiments have demonstrated that the activity of the Mitf transcription factor is regulated by signaling through the c-Kit receptor tyrosine kinase. Ultimately, this signaling results in effects on the activation potential and/or stability of the Mitf protein (Hemesath et al, 1998; Wu et al, 2000; Weilbaecher et al, 2001). SCF stimulation of c-Kit results in Erk2-dependent phosphorylation of S73 of Mitf, which regulates its transcriptional activity. Furthermore, Price et al. have shown that only the phosphorylated version of Mitf can interact with the p300 co-activator protein (Price et al, 1998). Phosphorylation of S409 by the p90/Rsk kinase, downstream of Erk, is a second phosphorylation event that has been shown to link c-Kit and Mitf. This phosphorylation has been shown to affect the protein stability such that Mitf protein phosphorylated on S409 is degraded more rapidly than a mutant S409A Mitf protein. The increased degradation was shown to correlate with increased polyubiquitination of the protein that targets it for proteosome-dependent degradation (Wu et al, 2000). Recently, a functional cross talk between Mitf, protein inhibitor of activated Stat3 (PIAS3) and Stat3 was demonstrated (Sonnenblick et al, 2004). PIAS proteins are a class of proteins that act as inhibitors of Stat activity (Wormald and Hilton, 2004). Upon SCF-stimulation, PIAS3 is translocated from Mitf to the activated Stat3 due to phosphorylation of Mitf at S409. It is sugggested that such a mechanism leads to a fine-

L. Other signal trandsduction molecules SWAP-70 is an unusual protein that was originally found as a part of a multiprotein DNA recombination complex in activated B cells (Borggrefe et al, 1998). It was later shown to also be expressed in mast cells. It carries a PH domain, that is required for membrane localization, three nuclear localization motifs and a nuclear export signal, and a Dbl domain. Recently, it was shown that mast cells derived from mice carrying a targeted deletion of SWAP-70 display impaired responses to c-Kit activation (Sivalenka and Jessberger, 2004). SCFinduced migration was deficient both in in vitro and in vivo assays. The reduced chemotaxis could be explained by aberrant polymerization of F-actin and impaired Rac activation. Furthermore, both SCF-induced calcium fluxes and Akt activation were impaired in SWAP-70-/- mast cells.

M. Negative regulation of c-Kit signaling In a number of cases, receptor tyrosine kinases have been shown to be regulated by serine/threonine kinases, such as protein kinase C (PKC), including the Met receptor (Gandino et al, 1994), the EGF receptor (Morrison et al, 1993) and the insulin receptor (Bollag et al, 1986). Blume-Jensen et al. could demonstrate that also the tyrosine kinase activity of c-Kit can be modulated through phosphorylation by PKC (Blume-Jensen et al, 1994). Down-modulation of c-Kit activity by PKC occurs through dual mechanisms. Activated PKC phosphorylates S741 and S746 in the kinase insert region of c-Kit, which leads to inhibition of kinase activity (Blume-Jensen et al, 1994; Blume-Jensen et al, 1995). Conversely, treatment of cells with the PKC inhibitor calphostin C, resulted in enhanced kinase activity of c-Kit and, furthermore, selectively increased activation of PI3-kinase (Blume13


Lennartsson et al: c-Kit signal transduction Jensen et al, 1994). Mutation of S741 and S746 to alanine residues, resulted in a gain of function and markedly increased tyrosine kinase activity of c-Kit (Blume-Jensen et al, 1998). In addition, treatment of cells with phorbol myristate acetate (PMA), an activator of PKC, results in proteolytic release of the ligand-binding domain of c-Kit, that leads to decreased responsiveness to SCF stimulation (Yee et al, 1993, 1994). Stimulation of c-Kit with soluble SCF results in phosphoinositide 3’-kinase (PI3-kinase) dependent activation of phospholipase D (Kozawa et al, 1997), leading to release of phosphatidic acid, which can be dephosphorylated to yield diacylglycerol (DAG), an activator of PKC. The protein tyrosine phosphatase SHP-1 interacts with Y570 of c-Kit and negatively regulates c-Kit signaling (Yi and Ihle, 1993; Kozlowski et al, 1998). SHP1 consists of two SH2 domains and a carboxyterminal protein tyrosine phosphatase domain. The motheaten (me) mice express a loss-of-function mutation in SHP-1 and show a hyperproliferative phenotype of their hematopoietic progenitor cells (Shultz et al, 1993). However, loss of SHP-1 function did not affect SCFinduced proliferation of bone-marrow derived mast cells, suggesting that the role of SHP-1 might to some extent be cell-type specific (Lorenz et al, 1996). The suppressors of cytokine signaling (SOCS) are a family of proteins that were originally cloned based on their ability to suppress cytokine signaling (for review, see (Wormald and Hilton, 2004)). They have a central SH2 domain flanked by an N-terminal domain of variable length and a C-terminal domain of 40 amino acids denoted the SOCS box. In a yeast two-hybrid screen using c-Kit as a bait, SOCS-1 was identified as an interactor with c-Kit (De Sepulveda et al, 1999). Its expression is induced upon stimulation of mast cells with SCF, and it associates with c-Kit via its SH2 domain. In contrast to its function in cytokine signaling, SOCS-1 selectively suppressed c-Kitstimulated mitogenesis, while not affecting survival signals. The mechanism does not involve inactivation of the tyrosine kinase activity of c-Kit, but rather binding of Grb2 to SOCS-1 via its SH3 domain. SOCS-1 in its turn binds to Vav (De Sepulveda et al, 1999). Interestingly, targeted deletion of SOCS-1 did not lead to enhanced cKit signaling in bone marrow derived mast cells, as one might have expected, but rather a reduced proliferative response to SCF-stimulation (Ilangumaran et al, 2003). Furthermore, deletion of SOCS-1 led to increased levels of proteases, leading to degradation of signal transduction molecules.

functions of this receptor/ligand pair in the hematopoietic system, fertility, pigmentation, gut movement as well as in the nervous system (Russell, 1979; Keshet et al, 1991; Lev et al, 1994). Below is a short summary of the major in vivo functions of c-Kit signaling.

A. Hematopoiesis Hematopoietic stem cells (HSC) have the ability to self-renew as well as differentiate into all hematopoietic cell lineages. HSC often divide asymmetrically, i.e. the daughter cells become a new HSC whereas the other starts to differentiate and lose the ability to self-renew. During the process of lineage commitment the ability to selfrenew decreases but the proliferative activity increases leading to an expansion of cell number. In general, c-Kit is expressed on primitive hematopoietic cells such as stemand progenitor cells and is lost during differentiation (Ogawa et al, 1991; Okada et al, 1991; Broudy, 1997; Lyman and Jacobsen, 1998). In fact, less than 0.1% of peripheral blood cells express c-Kit suggesting a minor role for c-Kit in differentiated hematopoietic cells (Ashman et al, 1991; Papayannopoulou et al, 1991; Broudy et al, 1992). Primitive hematopoietic cells depend on SCF for growth and survival, often in synergy with other growth factors and cytokines. An important exception among the hematopoietic cells are mast cells that retain a high c-Kit expression even as fully differentiated cells and depend on c-Kit signal transduction for their survival, growth and function (Oliveira and Lukacs, 2003). As a single factor SCF can induce differentiation of primitive hematopoietic cells from fetal mouse livers into mast cells (Radosevic et al, 2004). Hematopoietic defects in W and Sl mice include the development of erythrocytes, megakaryocytes and mast cells (Lev et al, 1994; Russell, 1979). W/W mice can be rescued by transplantation of wild-type HSC indicating that the lethality of these mice is due to anemia. A recent study demonstrated that transgenic expression of Epo can rescue W/W mice and thus support hematopoiesis in the absence of c-Kit (Waskow et al, 2004). This indicates that signals emanating from c-Kit that support hematopoiesis are not unique and can be replaced, at least in a mouse model, by signals from other receptor/ligand pairs. Among the lymphoid lineages, c-Kit plays a role for the maintenance of the immune system in adult animals (Waskow et al, 2002). Thus, treatment with c-Kit inhibitors for an extended time period might affect the immune system. Indeed, in older mice, treatment with the kinase inhibitor STI-571 (Gleevec, imatinib mesylate) leads to a defect in development of pro-B and pro-T-cells (Agosti et al, 2004). These results are important to consider before initiating a long-term STI-571 treatment in patients.

III. c-Kit in homeostasis There exist a large number of natural loss-of-function mutations in the W and Sl loci in mice, encoding for c-Kit and SCF respectively. The consequence of these mutations range from minor defects in catalytic activity to complete loss of c-Kit kinase activity as well as reduced to complete loss of expression of both c-Kit and SCF (Lev et al, 1994). These mutations have enabled scientists to appreciate the diverse effect c-Kit signaling has during mouse development and adult life. Detailed analysis of this extensive panel of mouse mutants has suggested critical

B. Fertility The fertility deficiency in W and Sl mice is probably linked to the loss of the normal ability of c-Kit to protect germ cells from apoptosis and to induce their migration and proliferation (Loveland and Schlatt, 1997). One important pathway for the ability of c-Kit to inhibit apoptosis is the PI3-kinase/Akt pathway (Blume-Jensen et 14


Cancer Therapy Vol 3, page 15 al, 1998). Indeed, knock-in studies using a mutant c-Kit receptor (c-Kit Y719F) unable to activate PI3-kinase resulted in sterile male mice (Blume-Jensen et al, 2000 ; Kissel et al, 2000). In the work by Kissel et al. also female mice had a reduced fertility (Kissel et al, 2000). These studies suggest that c-Kit in germ cell biology is involved in the processes of oogenesis, folliculogenesis and spermatogenesis. Furthermore, the function of c-Kit in germ cells is strictly dependent on its ability to activate PI3-kinase. Interestingly, these mice displayed no other phenotype, indicating that other cell types have redundant systems to compensate for the loss of PI3-kinase signaling downstream of c-Kit. In addition there exists a truncated form of c-Kit (tr-Kit) that is produced by an alternative intronic promotor which is active during spermatogenesis (Rossi et al, 2003). Even though tr-Kit lacks kinase activity it can activate the mouse egg if microinjected, in a SFK- and PLC-! dependent manner (Sette et al, 1997, 1998, 2002). It is likely that tr-Kit becomes phosphorylated by intracellular kinases, e.g. Src family kinases, and functions as an adaptor protein. However, the molecular function is presently not clear. The fact that trKit was recently discovered in human prostate cancer (Paronetto et al, 2004) warrants further investigation of this protein also in relation to cancer.

function of the hippocampal region of the brain (Motro et al, 1996; Katafuchi et al, 2000). Moreover, c-Kit expression has been found in neuroproliferative zones in the adult rat brain as well as in neuronal cultures (Jin et al, 2002). Furthermore, administration of SCF leads to proliferation of primitive neurons in vivo. It was recently demonstrated that c-Kit signaling is important for the migration of neuronal stem- and progenitor cells to injured areas of the brain (Sun et al, 2004). The precise role of cKit signal transduction for the development and function of the nervous system is not clear and needs further investigation.

IV. c-Kit as a therapeutic target in cancer treatment The tyrosine kinase receptor c-Kit is associated with several malignant human diseases. As mentioned above, cKit is expressed in the hematopoietic system, in certain cells in the gastrointestinal system (i.e. ICC), nervous system, germ cells, and melanocytes. Indeed, c-Kit driven tumors in these tissues have been observed. Mechanisms leading to uncontrolled activation of c-Kit involve both autocrine loops as well as mutational activation. Autocrine loops, i.e. the same cells produce both c-Kit and its ligand SCF, have been found in small cell lung cancer (SCLC), gynecological tumors and neuroblastomas (Inoue et al, 1994; Krystal et al, 1996; Vitali et al, 2003). Mutational activation of c-Kit has been found in patients with mastocytosis, gastrointestinal stromal tumors (GIST) and acute myeloid leukemia (AML). These mutations are predominantly located within the kinase domain or in the juxtamembrane region of c-Kit. The juxtamembrane mutations are believed to release an inhibitory alpha-helix and thereby result in uncontrolled receptor activation (Ma et al, 1999). In a study using molecular modelling Torrent et al. showed that kinase domain mutations in c-Kit and FLT3, a close relative to c-Kit, result in constitutive activation by destabilizing the inactive conformation, and not by directly stabilizing the active structure (Torrent et al, 2004). Unregulated c-Kit activity probably contributes to malignancies through increased proliferation and suppression of apoptosis. In all tumors in which c-Kit is the driving force for cellular transformation its kinase activity is essential. This is irrespective of the mode of kinase activation, whether it is activated by mutation(s) or by autocrine stimulation. Thus, inhibitors that specifically inhibits c-Kit enzymatic activity should be promising therapeutic agents. Several of these have been developed and perhaps the clinically approved STI-571 is the best known. This inhibitor was developed to target the kinase activity of the fusion oncoprotein Bcr-Abl, which is a result of the Philadelphia chromosome translocation and is commonly associated with chronic myelogenous leukemia. Subsequently STI-571 has been found to efficiently inhibit also c-Kit and the PDGFRs (Buchdunger et al, 2000). Recently, STI-571 was co-crystallized with the c-Kit kinase domain (Mol et al, 2004). This study showed that STI-571 binds to c-Kit in a way that is similar to its interaction with the Abl tyrosine kinase, i.e. near the active site of the inactive kinase and thereby stabilizing this conformation (Nagar et al, 2002). The crystal structure

C. Pigmentation The observation that mice with reduced c-Kit function have defective pigmentation is believed to be linked to the ability of SCF to induce proliferation and guide migration of melanocytes from the neuronal crest to the dermis (Scott et al, 1994; Mackenzie et al, 1997; Wehrle-Haller, 2003). A recent study from Bernstein and co-workers demonstrated that the two tyrosine residues 568 and 570 in the juxtamembrane region of c-Kit, which among other proteins couple the receptor to SFKs, are needed for normal pigmentation (Kimura et al, 2004). Moreover, loss-of-function mutations in c-Kit have been detected in the majority of humans with Piebaldism syndrome which is characterized by areas of hypopigmentation on the stomach, extremities and hair due to a lack of melanocytes (Spritz, 1994).

D. Gastrointestinal tract In the W and Sl mice there is a depletion of Interstitial cells of Cajal (ICC). ICC, also known as pacemaker cells, regulate the gut movement by their ability to communicate with both nerve and smooth muscle cells. Loss of ICC is connected with several human diseases involving defects in gut movement such as slow transit constipation (Lyford et al, 2002). ICC express !c-Kit and in the W and Sl mice there is a constipation phenotype suggesting an important role for c-Kit in ICC development and/or function (Huizinga et al, 1995; Ward et al, 1995; Ward et al, 1994). In addition, treatment of mice with an inhibitory c-Kit antibody leads to loss of ICC and consequently impaired gut movement. (Maeda et al, 1992)

E. Nervous system Studies using W and Sl mutant animals have established a role for c-Kit signaling in the spatial learning 15


Lennartsson et al: c-Kit signal transduction of STI-571 in complex with c-Kit also revealed that optimization of the STI-571 structure, removing unfavorable sterical interactions, could lead to a c-Kit inhibitor with higher affinity and specificity (Mol et al, 2004). Notably, STI-571 interacts with the inactive conformation of the kinase and hence will have limited efficacy on c-Kit with mutations in the kinase domain resulting in constitutive activity. Indeed, it has been shown that STI-571 does not effectively inhibit the c-KitD816V mutant (Frost et al, 2002; Ma et al, 2002). Therefore, the development of an inhibitor that targets the activating mutants of c-Kit would be beneficial for the treatment of many c-Kit driven tumors. Optimally this inhibitor will only inhibit the mutant form of c-Kit but not the wild-type protein, resulting in minimal side-effects. Effective treatment with kinase inhibitors requires exact knowledge regarding the presence or absence of specific mutations in c-Kit. Thus, development of methods to diagnose the c-Kit status in tumor tissues at the molecular level, e.g. using DNA sequencing as well as probing tumor tissues with antibodies raised against active and/or mutated forms of cKit, will be critical for successful treatment of these patients. As with most, if not all, enzymatic inhibitors used in cancer treatment the tumors eventually become tolerant or even resistant to the drug. This can occur through different mechanisms including increased expression of the oncoprotein, point mutations and/or expression of drugresistance-proteins that can for example remove the drug from the cell or interact with the drug in a way that blocks its therapeutic activity. In the case of STI-571 resistance has been observed due to increased oncoprotein expression as well as point mutations within the kinase domain (Gorre et al, 2001; Weisberg and Griffin, 2001; Shah and Sawyers, 2003). Strategies to overcome problems with resistance include combinational therapy using several drugs that simultaneously target the receptor and/or downstream signal transduction pathways. Thus, a detailed understanding of the cell biological and biochemical properties of receptors and their downstream effectors is critical for the development of novel targeted therapeutic interventions. Below follows a brief account of some of the cancers in which c-Kit has been implicated. The list is by no means complete and is constantly growing as more tumors are found to express c-Kit.

Kit kinase domain (D816V or D816Y) (Ferrao et al, 1997). Interestingly, the mutant c-Kit is often found in combination with mutations in the core-binding transcription complex (Beghini et al, 2000). However, a functional connection between c-Kit and the core-binding mutations has not been established. It has been speculated that the mutation(s) in the core binding factor result in defects in differentiation and c-Kit activation provides anti-apoptotic and proliferative signals complying with the 2-hit hypothesis of AML etiology (one hit affecting differentaition and one hit affecting proliferation) (Reilly, 2002). Notably, also the c-Kit related tyrosine kinase FLT3 has been found activated in AML (O'Farrell et al, 2003). Using the low molecular weight inhibitors SU5416 and SU6668, that target c-Kit, FLT3 and VEGFR-2, leads to reduction of c-Kit phosphorylation and increased apoptosis in human AML blasts (Smolich et al, 2001; O'Farrell et al, 2004). These data support the idea that cKit (and/or FLT3 and VEGFR-2) can drive AML blast proliferation and survival. A recent phase I trial of SU11248, an inhibitor of FLT3, c-Kit, VEGFR-2 and PDGFRs, in treatment for AML showed partial short term remission and reduced c-Kit, Stat3, Akt and VEGFR-2 phosphorylation (Fiedler et al, 2004). In two studies analyzing the effect of STI-571 on bcr-abl-negative AML patients, one showed a hematological response whereas the other did not (Cortes et al, 2003; Kindler et al, 2004). However, in the study that failed to observe a response the investigators used a lower dose (400mg/d) compared to the study where a response was seen (600mg/d). Thus more work is needed before we can fully appreciate the efficacy of STI-571 in the treatment of bcr-abl-negative AML patients. For detailed information on c-Kit and FLT3 in leukemogenesis refer to (Reilly, 2002).

B. GIST GIST are the most common cancer in the digestive tract with mesenchymal origin. However, this class of cancers only accounts for about 1% of all tumors in the gastrointestinal tract. There is evidence to suggest that GIST originate from ICC, which are known to express cKit (Wang et al, 2000; Duensing et al, 2004). Gain-offunction mutations found in GISTs are commonly located in the c-Kit intracellular juxtamembrane domain, but mutations have also been found in the extracellular domain (duplication of A501 and Y502) and in the kinase domain (K642E, N822K and N822H) (Lux et al, 2000: Rubin et al, 2001; Kinoshita et al, 2003). GISTs that contain mutated c-Kit have high cellularity and mitotic activity (Kim et al, 2004). In addition, the presence of cKit mutations indicates poor prognosis. However, all GISTs do not have mutations in c-Kit and it was recently shown that 35% of these c-Kit mutation negative patients had an activated mutation in the related PDGFR# (Heinrich et al, 2003). Moreover, activating mutations in c-Kit or PDGFR# appear to be mutually exclusive since no tumor has been found expressing both these oncoproteins. Primary GIST can be treated with surgical removal of the tumor. In the case of metastatic GIST this treatment is combined with conventional chemotherapy. Lately the

A. AML AML is exceedingly variable with relation to cell morphology and genetic changes. Expression of c-Kit has been found in about 85% of human AML cells (Heinrich et al, 2002). Since c-Kit primarily is expressed on stemand progenitor cells in the hematopoietic system this suggests that AML cells are in a primitive differentiation stage. In many AML samples c-Kit is constitutively phosphorylated. This is achieved through different mechanisms. First, SCF stimulates proliferation of AML cells in vitro and a co-expression of SCF and c-Kit has been detected in AML blasts (Ikeda et al, 1991, 1993; Kanakura et al, 1993; Pietsch, 1993). Thus, autocrine stimulation is probably involved in driving tumorigenesis. Second, activating mutations have been detected in the c16


Cancer Therapy Vol 3, page 17 tyrosine kinase inhibitor STI-571 that targets c-Kit, PDGFRs and Bcr-Abl fusion protein has been demonstrated to be therapeutically successful against GIST (Joensuu et al, 2001; Tuveson et al, 2001). Actually, STI-571 is the first effective treatment for GIST and more than 80% of patients respond to this drug. This highlights the importance of c-Kit signaling in this type of cancer. Recently, point mutations in the c-Kit kinase domain (V654A or T670I) making the GIST tumor resistant to STI-571 have been found in patients treated for a prolonged period of time with this drug (Chen et al, 2004; Tamborini et al, 2004). For detailed information on GIST biology and tumorigenesis please refer to Duensing et al, 2004 and Corless et al, 2004.

One obvious phenotype of W and Sl mice is their lack of fur pigmentation, which is due to a loss of melanocytes (Lev et al, 1994). Thus melanocytes depend on c-Kit for their function and therefore melanomas have been investigated in regard to c-Kit involvement (WehrleHaller, 2003). In general, it appears that c-Kit is downregulated during tumor progression towards metastatic cutaneous melanoma (Easty and Bennett, 2000). In addition, re-expression of c-Kit into certain melanoma cell lines leads to reduced tumor growth and SCF-induced apoptosis (Huang et al, 1996). Hence it is possible that loss of c-Kit allows the transformed melanocytes to escape from apoptosis. In contrast, c-Kit expression has been detected on choroidal and uveal melanoma cell lines (Mouriaux et al, 2003; Lefevre et al, 2004). Furthermore, a significant c-Kit phosphorylation has been found in uveal melanoma cell lines, but no activating mutations (AllEricsson et al, 2004; Lefevre et al, 2004). However, coexpression of SCF was observed thus establishing an autocrine/paracrine loop. Therefore the efficacy of STI571 was tested on these cell lines and the results are encouraging with reduced proliferation (All-Ericsson et al, 2004; Lefevre et al, 2004). Welker et al. demonstrated reduced expression of membrane-associated SCF but not soluble SCF in melanoma cell lines, particularly those that metastasize in vivo, compared to normal melanocytes (Welker et al, 2000). Thus, it is possible that the membrane-restricted form of SCF makes it difficult for melanocytes to move. Loss of this anchor allows the transformed melanocyte to metastasize to distant regions of the body. In conclusion, the involvement of c-Kit in melanoma is complex and varies between different types of melanoma, e.g. cutaneous versus choroidal and uveal melanomas. Hence, further investigation is needed to clarify the importance of c-Kit as a therapeutic target in choroidal and uveal melanomas.

C. Mastocytosis Mastocytosis is a disease characterized by accumulation of mast cells in various tissues. The disease shows large variations in clinical symptoms, with prognosis varying form non-lethal to lethal. c-Kit and its ligand SCF are critical for normal development and function of mast cells. In fact, mast cells are the only differentiated hematopoietic cell type that relies on c-Kit for its function. Activating mutations in the c-Kit kinase domain (D816V) have been identified in mast cells from patients with mastocytosis. This mutation renders c-Kit constitutively active. Notably, familial and pediatric mastocytosis may occur without mutations in the kinase domain of c-Kit indicating that the molecular reasons behind mastocytosis is variable and not exclusively dependent on a single oncoprotein. However, it is also possible that these patients have not yet identified mutations in other regions of c-Kit. Currently, there is no effective treatment of mastocytosis and consequently pharmacological interventions aim to reduce discomfort. Unfortunately, the catalytic domain mutant of c-Kit, most often found in mastocytosis patients, is not well inhibited by STI-571 rendering this drug inefficient in treatment of mastocytosis (Zermati et al, 2003). Thus, there is a need to develop therapeutic agents that target the kinase activity of the cKitD816V mutant. Recently Corbin et al. published a study demonstrating that the kinase inhibitor MLN518 could inhibit the activity of c-KitD816V (Corbin et al, 2004). Moreover, MLN518 showed low toxicity in a phase I trial for AML (DeAngelo et al, 2003). Thus, this is a promising compound for treatment of mastocytosis expressing activating mutation in c-Kit kinase domain. Furthermore, it has also been demonstrated that inhibition of NF-%B using IMD-0354 reduced proliferation of mast cells expressing c-Kit V560G and/or D816V but not normal mast cells (Tanaka et al, 2004). This suggests that interfering with NF-%B function might provide a novel therapeutic approach for mastocytosis. For detailed discussion of mastocytosis refer to Castells, 2004.

E. SCLC SCLC represents about 20% of all lung cancers, and if untreated it is a very aggressive form. The molecular biology behind SCLC is not clear, but it has been reported that about 70% of small-cell lung cancer cell lines overexpress c-Kit (Krystal et al, 1996; Sekido et al, 1991). Indeed c-Kit kinase inhibitors, such as STI-571, SU11248, SU5416 and SU6597, have been shown to reduce growth of SCLC cell lines and human SCLC xenografts in mice (Krystal et al, 2000; Wang et al, 2000: Krystal et al, 2001; Abrams et al, 2003). The in vivo activity of STI-571 on SCLC needs to be addressed in patients with tumors that are positive for c-Kit overexpression. Notably, a recent study failed to detect a correlation between the expression or mutation status of c-Kit and SCLC survival (Boldrini et al, 2004). Moreover, Haas and co-workers showed in a retrospective study of 203 patients with SCLC that lack of c-Kit expression was associated with shorter survival compared to patients with tumors that expressed c-Kit (Rohr et al, 2004). In contrast, Micke et al. found that high c-Kit expression is correlated with poor prognosis (Micke et al, 2003). Thus the involvement and importance of c-Kit

D. Melanoma The genetic changes that occur in melanoma are tremendously variable, but a common characteristic is an increased presence of tyrosine phosphorylated proteins compared to normal melanocytes (McArdle et al, 2003). 17


Lennartsson et al: c-Kit signal transduction in SCLC is not clear and further investigations are justified. Since SCLC is generally sensitive to both chemoand radiotherapy these are the standard therapies for this disease (Stupp et al, 2004). However, the overall prognosis of SCLC is poor due to its tendency to metastasize early in the disease (Jafri et al, 2003). Autocrine and paracrine loops are believed to be important for the ability of SCLC to form metastases. Thus, development of strategies that interfere with the autocrine loops could result in significantly better long-term prognosis for patients with SCLC. A recent study found that simultaneous targeting both c-Kit/SCF and IGF-1R/IGF-1 signaling using the inhibitors AG1024 and AG1296 synergistically reduced growth and increased apoptosis in SCLC cells (Camirand and Pollak, 2004).

understood. Some studies were conducted on transfected fibroblasts that express a different repertoire of signal transduction molecules than hematopoietic cells, possibly giving rise to activation of different signal transduction pathways. Also the differentiation state of hematopoietic cell lines is likely to influence the response elicited by SCF stimulation. Several splice forms of c-Kit have been demonstrated to exist, with sometimes different signaling capabilities, both quantitatively and qualitatively. Very little is known about how the expression of these different splice forms is regulated during development and differentiation. It is not unlikely that differences in signaling shown in the literature might be due to differences in expression of various splice forms of c-Kit in different cell types. Furthermore, the qualitative differences in signaling of the membrane bound versus soluble form of SCF have been demonstrated in a number of studies. The use of transgenic animals with targeted deletions of individual signal transduction molecules and the use of so-called knock-in methodology to introduce specific mutants of c-Kit in animals, have proven invaluable tools for our understanding of c-Kit signaling. In order to be able to study c-Kit signaling in hematopoietic development, more sensitive methods for the study of signaling in individual cells will be of utmost importance. Understanding of the mechanisms of synergy between SCF and various cytokines is also a challenging field for future research. Increased knowledge of molecular mechanisms of c-Kit signaling in diseases, such as cancer, is paramount to the potential development of targeted therapies. It is likely that c-Kit signal transduction, resulting in suppression of apoptosis and enhanced proliferation, at least contributes to the progression of many tumors. Hence, c-Kit is a clinically relevant target for drug development. Importantly, in cases where c-Kit activation is due to overexpression or an autocrine loop, the tumors are likely to respond to drugs that target wild-type c-Kit, e.g. STI-571. In contrast, tumors that contain c-Kit with mutations in the catalytic domain will not respond well to STI-571. Therefore we would like to emphasize the importance to develop new therapeutic agents which target the catalytic domain mutant of c-Kit that can be applied in the clinic. Moreover, it is important to identify differences in signal transduction pathways downstream of normal and oncogenic forms of c-Kit since these would be excellent targets for therapeutic intervention, with minimal effect on the function wild-type c-Kit, in order to complement drugs acting directly on the receptor. It is likely that simultaneous targeting of different signaling molecules will lead to synergistic anticancer effects and circumvent or at least delaying the emergence of drug resistance.

F. Other tumors Expression of c-Kit has been detected in several other tumors not described above including neuroblastoma, cervical, testicular, thyroid, breast, colon, bladder and renal carcinoma (Arber et al, 1998; Bellone et al, 2001; Caceres-Cortes et al, 2001; Hines et al, 1999; Lin et al, 2004; Pan et al, 2004; Tian et al, 1999; Vitali et al, 2003). In the case of breast cancer, c-Kit expression is lost during transformation from normal breast tissue to invasive breast cancer (Ulivi et al, 2004; Yared et al, 2004). Thus monitoring c-Kit expression can be of prognostic value. A recent report identified expression of the tr-Kit in a prostate cancer cell line and in 28% of primary prostate cancers (Paronetto et al, 2004). Tr-Kit has been thought only to be expressed in sperm. Therefore tr-Kit might provide an almost tumor specific target. Two recent investigations regarding the involvement of c-Kit in neuroblastoma have highlighted the complexity of this disease. In one study it was shown that c-Kit expression indicates a good prognosis (Krams et al, 2004). c-Kit expression was associated with differentiation and this is a possible explanation for c-Kit being a favorable prognostic marker. On the other hand, Beppu et al, demonstrated using neuroblastoma cell lines that STI-571 treatment leads to reduced proliferation and viability (Beppu et al, 2004). Furthermore, STI-571 treatment was associated with reduced c-Kit and PDGFR" phosphorylation as well as reduced VEGF expression. In order to target c-Kit clinically these discrepancies between in vitro and in vivo studies must be resolved.

V. Concluding remarks Since the discovery of SCF as the ligand of c-Kit almost fourteen years ago, numerous studies have contributed to our knowledge about the mechanism of action of c-Kit. A multitude of signaling pathways are activated by SCF leading to diverse biological responses, such as chemotaxis, proliferation, differentiation and survival. Using a number of different cell systems, investigators have many times found similar mechanisms of action of c-Kit, but sometimes also differences. The exact reason for these discrepancies is not fully

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Weilbaecher KN, Motyckova G, Huber WE, Takemoto CM, Hemesath TJ, Xu Y, Hershey CL, Dowland NR, Wells AG and Fisher DE (2001) Linkage of M-CSF signaling to Mitf, TFE3 and the osteoclast defect in Mitf(mi/mi) mice. Mol Cell 8, 749-758. Weiler SR, Mou S, DeBerry CS, Keller JR, Ruscetti FW, Ferris DK, Longo DL and Linnekin D (1996) JAK2 is associated with the c-kit proto-oncogene product and is phosphorylated in response to stem cell factor. Blood 87, 3688-3693. Weisberg E and Griffin JD (2001) Mechanisms of resistance imatinib (STI571) in preclinical models and in leukemia patients. Drug Resist Updat 4, 22-28. Welker P, Schadendorf D, Artuc M, Grabbe J and Henz BM (2000) Expression of SCF splice variants in human melanocytes and melanoma cell lines: potential prognostic implications. Br J Cancer 82, 1453-1458. Williams DE, Eisenman J, Baird A, Rauch C, Van Ness K, March CJ, Park LS, Martin U, Mochizuki DY, Boswell HS and et al. (1990) Identification of a ligand for the c-kit protooncogene. Cell 63, 167-174. Wisniewski D, Strife A and Clarkson B (1996) c-kit ligand stimulates tyrosine phosphorylation of the c-Cbl protein in human hematopoietic cells. Leukemia 10, 1436-1442. Vitali R, Cesi V, Nicotra MR, McDowell HP, Donfrancesco A, Mannarino O, Natali PG, Raschella G and Dominici C (2003) c-Kit is preferentially expressed in MYCN-amplified neuroblastoma and its effect on cell proliferation is inhibited in vitro by STI-571. Int J Cancer 106, 147-152. Wollberg P, Lennartsson J, Gottfridsson E, Yoshimura A and Rรถnnstrand L (2003) The adapter protein APS associates to the multifunctional docking sites Tyr568 and Tyr936 in cKit: possible role in v-Kit transformation. Biochem J 370, 1033-1038. Wormald S and Hilton DJ (2004) Inhibitors of cytokine signal transduction. J Biol Chem 279, 821-824. Vosseller K, Stella G, Yee NS and Besmer P (1997) c-kit receptor signaling through its phosphatidylinositide-3'kinase- binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion and membrane ruffling. Mol Biol Cell 8, 909-922. Voytyuk O, Lennartsson J, Mogi A, Caruana G, Courtneidge S, Ashman LK and Rรถnnstrand L (2003) Src family kinases are involved in the differential signaling from two splice forms of c-Kit. J Biol Chem 278, 9159-9166. Wu M, Hemesath TJ, Takemoto CM, Horstmann MA, Wells AG, Price ER, Fisher DZ and Fisher DE (2000) c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi. Genes Dev 14, 301-312. Yamanashi Y, Tamura T, Kanamori T, Yamane H, Nariuchi H, Yamamoto T and Baltimore D (2000) Role of the rasGAPassociated docking protein p62(dok) in negative regulation of B cell receptor-mediated signaling. Genes Dev 14, 11-16. Yarden Y, Kuang WJ, Yang-Feng T, Coussens L, Munemitsu S, Dull TJ, Chen E, Schlessinger J, Francke U and Ullrich A (1987) Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J 6, 3341-3351. Yared MA, Middleton LP, Meric F, Cristofanilli M and Sahin AA (2004) Expression of c-kit proto-oncogene product in breast tissue. Breast J 10, 323-327. Yee NS, Hsiau CW, Serve H, Vosseller K and Besmer P (1994) Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase and protein kinase C. J Biol Chem 269, 31991-31998. Yee NS, Langen H and Besmer P (1993) Mechanism of kit ligand, phorbol ester and calcium-induced down- regulation

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Cancer Therapy Vol 3, page 27 of c-kit receptors in mast cells. J Biol Chem 268, 1418914201. Yi T and Ihle JN (1993) Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand. Mol Cell Biol 13, 3350-3358. Yokouchi M, Kondo T, Sanjay A, Houghton A, Yoshimura A, Komiya S, Zhang H and Baron R (2001) Src-catalyzed phosphorylation of c-Cbl leads to the interdependent ubiquitination of both proteins. J Biol Chem 276, 3518535193. Yokouchi M, Suzuki R, Masuhara M, Komiya S, Inoue A and Yoshimura A (1997) Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation. Oncogene 15, 7-15. Yokouchi M, Wakioka T, Sakamoto H, Yasukawa H, Ohtsuka S, Sasaki A, Ohtsubo M, Valius M, Inoue A, Komiya S and Yoshimura A (1999) APS, an adaptor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis. Oncogene 18, 759-767. Zeng S, Xu Z, Lipkowitz S and Longley JB (2004) Regulation of stem cell factor receptor signaling by CBL family proteins (CBL-B/c-CBL) Blood. Zermati Y, De Sepulveda P, Feger F, Letard S, Kersual J, Casteran N, Gorochov G, Dy M, Dumas AR, Dorgham K, et al (2003) Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit

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Lars Rรถnnstrand

27

Johan Lennartsson


Lennartsson et al: c-Kit signal transduction

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Cancer Therapy Vol 3, page 29 Cancer Therapy Vol 3, 29-30, 2005

Miliary tuberculosis peritonitis mimicking advanced ovarian cancer Case Report

Malihe Hasanzadeh* and Hasan Malekoti Gynecology Oncology, Mashhad University of Medical Sciences, Mashhad, Iran.

__________________________________________________________________________________ *Correspondence: Malihe Hasanzadeh, Obstetric Gynecology Department Cheam Hospital, Mashhad, Iran.Phon/Fax: 98-511-8409612; e-mail:Malhasanzadeh@yahoo.com Key words: Ovarian cancer, Peritoneal Tuberculosis, Abdominal mass, CA-125 Received: 17 January 2005; Accepted: 28 January 2005; electronically published: February 2005

Summary Primary peritoneal tuberculosis is a rare presentation of this disease. It is usually associated with ascites, raised CA125 levels and abdominal mass. A patient with a pelvic mass, ascites, raised CA-125 underwent an exploratory laparatomy for presumed ovarian cancer. Final pathology revealed peritoneal tuberculosis without any pulmonary involvement. Acid -Fast bacilli were confirmed with polymerase chain reaction in the surgical specimen. This case demonstrated a high rate of misdiagnosis between advanced ovarian cancer and peritoneal tuberculosis. An elevated CA- 125 is not specific for ovarian malignancy. In her first visit, she had abdominal pain and increasing abdominal girth and weigh loss (20 pound) from 3 month ago. CT-Scan showed a huge heterogenic ovarian mass (solid-cystic components). Serum CA125 was elevated at 200(u/ml). The patient had any history of fever, chills, night sweats. The patient's family history was significant for a brother who treated tuberculosis 2 year ago. Laboratory data revealed a normal white blood cell count. A preoperative chest X-ray was normal. The patient underwent an exploratory laparatomy.We could not enter to abdominal cavity. There are disseminated nodules in the peritoneum of the parietal wall. The specimen send to pathologic department, frozen section was performed. Pathology revealed chronic granulomatous changes and no malignancy. Post operatively all sputum samples, blood cultures and peritoneal fluid analyses were negative for acid-fast bacilli. Polymerase chain reaction was positive for acidfast bacilli in the surgical tissue specimen. The patient was treated with Isoniazid, Rifampicin, Pyrazinamide and Etamburol. After 4 months, in her visit abdominal mass was disappeared. Treatment continued for 1 year.

I. Introduction Ovarian cancer is the leading cause of death from gynecologic malignancies. Because early ovarian cancer produces few specific symptoms, most women present with advanced stage disease where the prognosis is poor. Although there have been advanced in the evaluation and treatment of ovarian cancer, most patients continue to present with advanced disease when survival is limited (Disaia and Creasman, 2002). Annual serum CA-125 levels, pelvic examination and transvaginal ultrasound are being used for screening, has not adequate sensitivity or specificity (Karl and Platt, 1995). In advanced stages, there are many nonspecific signs and symptoms and we require surgery and pathologic evaluation for diagnosis. Tuberculosis of the peritoneum is a rare disease. There are several case reports that point to uncertainly in the preoperative differential diagnosis of peritoneal tuberculosis and advanced ovarian cancer (Lachman et al, 1985; Gurgan et al, 1993; Groutz et al, 1998). This paper presents one additional case of peritoneal tuberculosis and emphasize that we must be careful when interpreting a positive results.

III. Discussion

II. Case

This patient is a case of tuberculosis peritonitis diagnosed with tissue pathology. Peritoneal tuberculosis is one of the generalized pathologies that present with nonspecific sign and symptoms such as ascites and pelvic,

In May 2002,a 22 year old woman(primiparous)was referred to obstetric Gynecology Department in Ghaem hospital, Mashhad, Iran.

29


Hasanzadeh and Malekoti: Miliary tuberculosis peritonitis mimicking advanced ovarian cancer abdominal pain and mass, which mimic ovarian cancer (Martin and Bradsher, 1986). The pelvic tuberculosis most often in patients between the ages of 20 and 40 years. Ovarian cancer is rare before the age of 40 and peaks at age 65 to 75 (Sutherland, 1980). An elevated serum CA-125 level in a patient with a pelvic mass and ascites raises the suspicion of ovarian cancer (Gurgan et al, 1993). Many malignancies that are not ovarian, such as endometrial and gastrointestinal adenocarcinoma,have also been associated with elevated serum CA-125 levels (Kramer et al, 1993; Simsek et al, 1996). Differential diagnosis in elevated serum CA-125 is extremely difficult and needs surgical intervention. Futhermore,elevation in CA-125 serum levels are present in a number of benign ovarian tumors, including endometrioma, inflammatory disease of the ovaries and serous cyst adenoma (Barbieri et al, 1986). More than two thirds of the cases (tuberculosis peritonitis) are diagnosed at the time of laparatomy performed for some other diagnosis. This disease should be included in the differential diagnosis of this devastating malignancy, especially in developing countries where it remains endemic. Progression of tuberculosis peritonitis often takes months or even years. Chest Roentgenograms are not of help either in distinguishing patients with Tuberculosis peritonitis (Bhansali, 1977). Son graphic features of tuberculosis peritonitis illustrate adenexal mass, adhesions and septated or particulate ascites (Kramer et al, 1993). In computerized tomography examination, presence of a smooth peritoneum with minimal thickening and pronounced enhancement suggest peritoneal tuberculosis, where as nodular implants and irregular peritoneal thickening suggest peritoneal carcinomatosis (Rodriguez and Pombo, 1996). The literature reports other cases of tuberculosis peritonitis diagnosed solely by tissue pathology. Final pathology provided the crucial information and diagnosis for this patient. Frozen section can be used to confirm malignancy intraoperatively (Straughn et al, 2000). In this case frozen section would have revealed chronic granulomatous reaction and inflammation which would be consistent but not diagnostic for tuberculosis. Histology confirmation of Tuberculosis peritonitis can be difficult, but the lack of malignant cell would have made extra ovarian carcinoma less likely.

In addition standard microscopic section, the specimen can be examined by fluorescent antibody technique. Acid-fast Staining of tissue is effective in detecting the organism (Bilgin et al, 2001). Tuberculosis peritonitis should be considered in the differential diagnosis of a patient with pelvic mass and elevated CA-125.

References Barbieri RL, Niloff JM, Bast RC, Schaetzl E, Kistner RW, Knapp RC (1986) Elevated serum concentrations of CA-125 in patients with advanced endometriosis. Fertil 45, 630-40. Bhansali SK (1977) Abdominal tuberculosis. Am J Gastroenterol 67, 324-37. Bilgin T, Karabay A, Dolar E, Develioglu OH (2001) Peritoneal tuberculosis with pelvic abdominal mass, ascites and elevated CA-125 mimicking advanced ovarian carcinoma: A series of 10 cases. Int J Gynecol Cancer 11, 290-294. Disaia PJ, Creasman W (2002) Epithelial ovarian cancer, in Disaia PJ, Creasman WT editors.Clin Gynecology Oncol, Sixth ed.St.louis Mosby-year book, pp 289-351. Groutz A, Carmon E, Gat E (1998) Peritoneal tuberculosis versus advanced ovarian cancer: a diagnostic dilemma. Obstet Gynecol 91, 868. Gurgan T, Zeyneloglu H, Urman B, Develioglu O, Yarah H (1993) Pelvic peritoneal tuberculosis with elevated serum and peritoneal fluid CA-125 levels. Gynecol Obstetric Invest 53, 60-1. Karl BY, Platt LD (1995) Ovarian cancer screening. The role of ultrasound in early detection of ovarian cancer. Cancer 76, 2011-5. Kramer BS, Gohagan J, Prorok PC, Smart C (1993) A national cancer institute sponsored trial for prostatic, lung, colorectal and ovarian cancers. Cancer 71, 589-93. Lachman E, Moodley J, Pitsoe SB (1985) Peritoneal tuberculosis initiating ovarian carcinoma (special category). Acta Obstetric Gynecol Scand 64, 677-79. Martin RE, Bradsher RW (1986) Elusive diagnosis of tuberculosis peritonitis. South Med J 79, 1076-1079. Rodriguez E, Pombo E (1996) Peritoneal tuberculosis versus peritoneal carcinomatosis: distinction based on CT findings. J Comput Assist Tomogr 20, 269-72. Simsek H, Kadayifci A, Okan E (1996) Importance of serum CA-125 levels in malignant peritoneal mesothelioma. Tumor Biol 17, 1-4. Straughn JM, Robertson ME, Partridge EE (2000) Case Report: A patient presenting with a pelvic mass, Elevated CA-125 and fever. Gynecol Oncol 77, 471-472. Sutherland AM (1980) Surgical treatment of tuberculosis of the female genital tract. Br J Obstetric Gynecol 87, 610-612.

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Cancer Therapy Vol 3, page 31 Cancer Therapy Vol 3, 31-40, 2005

Quality of surgery in soft tissue sarcoma: a single centre experience with the French Sarcoma Group (FSG) surgical system Research Article 1

2

3

3

Eberhard Stoeckle *, Jean-Michel Coindre , Guy Kantor , Laurence Thomas , 1 3 4 5 Antoine Avril , Philippe Lagarde , Michèle Kind , Binh Nguyen Bui 1

Department of Surgery,

2

Department of Pathology,

3

Department of Radiotherapy,

4

Department of Radiology,

5

Department of Medicine, Institut Bergonié, Regional Cancer Centre, 33076 Bordeaux Cedex, France

__________________________________________________________________________________ *Correspondence: Dr. E. Stoeckle, Department of Surgery, Institut Bergonié, Regional Cancer Centre, 229 cours de l’Argonne, 33076 Bordeaux Cedex, France; Phone: (33) 5 56 33 33 33; Fax: (33) 5 56 33 33 87; E-mail: stoeckle@bergonie.org Key words: Soft tissue sarcoma, Local recurrence, Quality of surgery, French Sarcoma Group (FSG), FSG surgical system, Resection type, UICC, Abbreviations: co-operative group of surgical oncology, (CAO); External beam radiotherapy, (EBRT); French Federation of Cancer Centres, (FNCLCC); French Sarcoma Group, (FSG); local recurrence, (LR); patients, (pts.); soft tissue sarcoma, (STS) Received: 24 January 2005; revised: 28 January 2005 Accepted: 1 February 2005; electronically published: February 2005

Summary The UICC recommends to report results of surgery in soft tissue sarcoma (STS) according resection type (R), but does not indicate how to determine (R). For this purpose, the French Sarcoma Group (FSG) developed a surgical system, based on a collective determination of (R), evaluated here in a single institution. One hundred ten patients (pts.) with primary trunk wall and extremity STS operated consecutively at Institut Bergonié from 1996 to 1999 were prospectively allocated to resection types (R) by FSG criteria. Evaluation of the FSG system was based on repartition of resection types (R) and on local recurrence rates according to resection types (R). There were 75 (68%) resections R0, 32 (29%) resections R1 and 3 (3%) resections R2. Repartition of resection type (R) depended on tumour invasion. At a median follow-up of 70 months (range: 43 - 106 months) for surviving pts., local recurrence (LR) occurred in 10/107 pts (9%) with initial gross excisions, giving an actuarial local control rate of 91% at 5 years. LR was 4% (3/75 pts.) after resection R0 and 22% (7/32 pts.) after resection R1 (p< 0,01). By this system, patients R0 can be foreseen not to recur locally in 96% of cases, whereas patients R1 harbour a 22% risk of LR. The FSG surgical system combines simplicity and accuracy in determining quality of surgery. Highly predictive of local outcome, it may be useful in decision making concerning multidisciplinary treatment and help to improve local outcome of STS.

UICC does not give instructions, how to determine resection type (R)! Therefore, quality of surgery in STS is often determined individually by either surgeons or pathologists. Surgeons mostly transpose Enneking’s classification (Enneking et al, 1980) to the Rclassification, meaning wide margins = R0, close margins = R1 and intra-lesional excision = R2. However, surgeons

I. Introduction Reporting quality of surgery in soft tissue sarcoma (STS) should be done according resection - type (R) as recommended by the UICC (Union Internationale Contre le Cancer), meaning resection R0 = in sano, resection R1 = microscopic residual disease, resection R2 = macroscopic residual disease (Hermanek et al, 1992). However, the 31


Stoeckle et al: French Surgical System in sarcoma tend to overestimate quality of surgery. This fact has been demonstrated in an overview of practice within the German co-operative group of surgical oncology (CAO) by Junginger, et al (2001), where resection rates R0 are reported in up to 82% of assessable extremity sarcoma and in 64% of retroperitoneal sarcoma, a rate hardly achievable in this localisation (Stoeckle et al, 2001). Pathologists, on the other hand, do not see the ongoing of excision, but a post-operative specimen. Analysis of margins can be erroneous, especially after shrinkage of the specimen, giving falsely view to the tumour capsule, due to the retraction of muscles initially covering the tumour surface. Currently however, in about half of cases of re-excisions recommended for contaminated margins, no residual tumour is found in the re-excision specimen. Hence, at a period where soft tissue sarcoma are treated conservatively by a multidisciplinary approach (Rosenberg et al, 1982; Brennan et al, 1987; Sarcoma Meta-Analysis, 1997; Yang et al, 1998), an inexact appreciation of quality of surgery, either by overor underestimation, leads to inadequate treatment, concluding either to over- or undertreatment. The consequences are excessive sequels or high local recurrence rates. Moreover, the results of the different series are to be considered with caution as the initial definitions may differ. In order to clarify reporting of surgical results in soft tissue sarcoma, the French Sarcoma Group (FSG) decided to elaborate a surgical system deemed to be easily reproducible. A first step consisted in 1995 to review retrospectively ten operation forms of sarcoma patients in each of the eight participating centres in order to gather pertinent information about surgery. As a result, technique of surgery was well described, but information concerning the tumour (size, localisation, depth) was sparse and

moreover, quality of surgery mostly could not be determined correctly. A check-list of surgery reporting was therefore established (Table 1). Importance was given to descriptive items, asking the surgeon to describe excision, but not to interpret it. Essential terms for this purpose were the notions whether the tumour was seen, whether there was rupture of tumour and how theses critical margins were managed. Good acceptance of this check-list was confirmed after a multicentric survey by questionnaire. It confirmed reliability to pathological findings especially in primary operations, whereas more difficulties were seen in re-excisions (Stoeckle et al, 1997). A third step consisted to establish recommendations for pathological reporting in soft tissue sarcoma (Ghnassia et al, 1998) (Table 2). Finally, the FSG recommended to determine quality of surgery collegially according resection type R, by confronting surgical and pathological reports, at a regular common staff meeting. This procedure was aimed to gain more objectivity in defining quality of surgery by confronting purely descriptive reports from each, surgeon and pathologist, allocating surgery to resection type (R) as a result of a consensual decision. At Institut Bergonié, one of the participating centres to FSG, this procedure was introduced in 1996. The objective of the present study is to report the prospective experience with the FSG surgical system in this single centre. Endpoints considered for validation of the procedure were distribution of resection types (R) and local recurrence (LR) rates, reflecting finally optimal adequacy between result of surgery and complementary treatment. To obtain sufficient follow-up data, the four-year period from 1996 to 1999 was considered for analysis. The final follow-up date was December 2004.

Table 1. FSG check-list for surgery reporting in soft tissue sarcoma The operative form is established according to the surgeon’s own practice. For soft tissue sarcoma, the following informations should figure systematically: 1. Title of operation 2. Clinical presentation • tumour size • tumour depth/fascia 3. What has been done? • excised structures (muscles, nerves, vessels, organs). • tumour bed marked • description of tumour (if seen): pseudcapsula, focality, site 4. Conclusion: what remains? • excision complete?

yes/no

• tumour seen?

yes/no? site? re-excision?

• rupture of tumour?

yes/no? site? re-excision?

• margins?

site? quality? distance (mm)?

32


Cancer Therapy Vol 3, page 33 Table 2. FSG recommendations for pathology reporting in soft tissue sarcoma (from (Ghnassia et al, 1998), modified) 1. Presentation of operative specimen • fresh, unprepared specimen (avoid picric acid) • presentation following previous agreement with surgeon • do not change afterwards 2. Pathological report • Macroscopy – tumour size – depth/fascia • Microscopy – confirmation of malignancy – histological type – histological grade – local extension: focality, neurovascular involvement, tumour emboli • Status of margins – localisation of nearest margin – description of margins: distance (in mm), quality (fascia) – status of complementary margins 3. Conclusion (descriptive, do not interpret quality of surgery!) • Definition of tumour type and grade • Description of margins (distance in mm, quality) primary treatment failure: (1) one patient with an extensive, rapidly growing, inoperable radiation-induced sarcoma of the thoracic wall had primary excisional biopsy only and progressed under chemotherapy; (2) one patient with an advanced angiosarcoma of the forearm refused amputation and underwent incomplete, conservative surgery; (3) one patient with an advanced liposarcoma of the groin involving the femoral bifurcation underwent two successive incomplete surgeries and refused proposed amputation. Post-operative radiotherapy was indicated after resection of deep tumours (stages T1b and T2b). Twelve patients with deep tumours did not undergo irradiation because of previous radiotherapy in 4 cases, post-operative complications in 4 cases, palliative treatment in three cases, refusal in one case. Eighty seven patients (79%) underwent post-operative radiotherapy. The treatment volume included the tumour compartment when present, comprising the tendons and insertions of the muscles involved, but sparing a strip of unirradiated tissue in the extremities. External beam radiotherapy (EBRT) was delivered by high energy photons from a linear accelerator in accordance with a technique described elsewhere (Lagarde et al, 1990). Treatment recommendations at our Institut required a dose of 50 Gy after resections R0, whereas a complementary boost was indicated after resections R1, either by post-operative radiotherapy, delivering 10 Gy to a reduced volume in the tumour bed, or by afterloading brachytherapy delivering 15 Gy according to a technique described elsewhere (Thomas et al, 1994). In cases of previous brachytherapy, the dose of postoperative radiotherapy was lowered to 45 Gy. Twenty seven patients received a radiation boost, 15 in patients R1, 12 in patients R0. The total delivered radiation dose was 52 Gy (range: 44-65 Gy). Apart from the indication as first-line treatment in patients with locally advanced tumours, complementary chemotherapy was usually recommended for criteria other than quality of surgery (e.g. metastatic presentation, high tumour grade, highrisk pathology). During the study period, an anthracyclin-based chemotherapy was used, mostly by combination chemotherapy with the MAID regimen (Mesna, Doxorubicin 60 mg/m2, Ifosfamide 7.5 mg/m2, Dacarbazine 900 mg/m2; days 1-3), exceptionally with Doxorubicin alone.

II. Materials and methods A. Selection of patients From January 1996 to December 1999, all consecutive patients undergoing resection of primary STS at Institut Bergonié, a regional cancer centre in south west of France, were prospectively included in the study. For the present analysis, only primary STS of the extremities and trunk wall, operated first at the centre, or re-operated after referral, were considered. This excluded patients operated elsewhere and having subsequent, non surgical treatment at our Institut, patients with other locations of STS, or not true soft tissue sarcoma, e.g. fibromatosis, and patients with recurrent STS. One hundred and ten patients fulfilled the inclusion criteria and are analysed here.

B. Patient and tumour characteristics (Table 3) There were 54 females and 56 males. Median age was 60 years (range: 15-84 years). Median tumour size was 10 cm (range: 2-30 cm). Malignant fibrous histiocytoma, liposarcoma and leiomyosarcoma predominated among a wide variety of histopathologic subtypes. Most tumours were high grade and deep. Locally advanced disease, as defined by neurovascular or bone involvement, multifocal spread or both, was present in 37% of patients. Four patients had sarcoma located within radiation fields. Table 3 details tumour characteristics. Sixty one patients were re-operated after referral from another institute. Forty nine patients had their first operation at Institut Bergonié, preceded by biopsy in 46 cases (16 core needle biopsies, 30 surgical biopsies).

C. Realised treatment Function-sparing conservative surgery was the goal in all patients. In 21 patients with high grade, locally advanced tumours not suitable directly to conservative surgery, tumour reductive first-line chemotherapy was performed. In 18 patients with a close dissection between the tumour and a functional structure, intra-operative brachytherapy was performed in order to allow a conservative resection. With this multidisciplinary approach, satisfactory conservative surgery could be performed in 106 patients. One patient with a sarcoma of the hand underwent a finger amputation. Three patients experienced

33


Stoeckle et al: French Surgical System in sarcoma Table 3. Characteristics of tumours in 110 operated patients Tumour characteristics

N

Median tumour size (range) Location Upper extremity Shoulder girdle (shoulder, axilla) Trunk wall Pelvic girdle (buttock, groin) Lower extremity

(%) 10 cm

(2-30 cm)

(15) (12) (18) (8) (57)

(14) (11) (16) (7) (52)

Major tissue invasion (41) (multifocal: 24 pts; neurovascular/bone involvement: 23 pts; both: 6 pts)

(37)

T

(9) (22) (2) (77)

(8) (20) (2) (70)

(9)

(8)

(30) (22) (16) (9) (5) (5) (3) (3) (3) (7) (7)

(27) (20) (14) (8) (5) (5) (3) (3) (3) (6) (6)

(21) (19) (70)

(19) (17) (64)

T1a T1b T2a T2b

N1 or M1 (N1: 4 pts; M1: 7 pts; both: 2 pts) Histological subtypes Malignant fibrous histiocytoma Liposarcoma Leiomyosarcoma Synovial sarcoma Clear cell sarcoma Extraskeletal osteosarcoma Rhabdomyosarcoma MPNST Angiosarcoma Others Undifferentiated, unclassified sarcoma Grade

G1 G2 G3

Six cycles of treatment were usually delivered According to former experience with concomitant radio- and chemotherapy (Lagarde et al, 1998) showing no supplementary toxicity despite the use of anthracyclins, post-operative chemotherapy was usually associated with post-operative radiotherapy. Sixty-six patients (60%) underwent chemotherapy.

2.Standardised pathological report Pathological work-up was done in accordance with the recommendations from the pathologists of the FSG (Ghnassia et al, 1998) (Table 2). Histological subtype was established according to WHO recommendations (Weiss et al, 1994). Grading was done in accordance with the French Federation of Cancer Centres (FNCLCC) classification (Trojani et al, 1984).

D. Determination of resection quality

3. Definition of quality of surgery

1.Surgical report

As recommended by the FSG, resection type was determined collegially at a weekly group meeting. After

Surgery was prospectively encoded in accordance with the FSG checklist for surgery (Table 1).

34


Cancer Therapy Vol 3, page 35 confrontation between the surgical and pathological reports, type of resection was then expressed according to UICC R criteria.

square test. Actuarial local recurrence-free estimations were made with the Kaplan-Meier method.

E. Database, follow-up and statistics

F. Outcome of patients

Information on patientsâ&#x20AC;&#x2122; charts comprised patient identification, tumour specification, treatment and follow-up data. Final follow-up date was December 2004. The interval of follow-up was calculated for surviving patients from date of surgery to date of last follow-up. For deceased patients, the intervals between first surgery and death were calculated. Comparisons between frequencies were obtained using the Chi-

At update on December 31, 2004, seventy eight patients were alive and 32 patients were dead, twenty four of whom have died from cancer and two from treatment complications. Median interval to death was 19 months. Median follow-up of living patients was 70 months (range 43 â&#x20AC;&#x201C; 106 months). Ninety six percent of patients (75/78 patients) were followed-up over four years and seventy seven percent (60/78 patients) over five years. Actuarial overall survival at 5 years was 75% (Figure 1).

Figure 1. Actuarial overall survival in 110 patients.

Figure 2. Actuarial local control rate in 107 patients with complete tumour excision

35


Stoeckle et al: French Surgical System in sarcoma

G. Validation criteria

wall, for whom quality of surgery was determined prospectively in accordance to the surgical system of FSG. Determination of type of resection at a weekly staff meeting rapidly proved to be consensual. Decisions for reexcision or complementary treatment were made collegially. The reliability of the system showed to be good. The current series reports a resection rate R0 of 68% and an actuarial local recurrence rate at five years of 9%. At a median follow-up of 70 months, the risk of LR is 4% in patients R0, 22% in patients R1, reaching 35% in patients R1 lacking a radiation boost. The rate of resections R0 in the current monocentric series (68%) is lower than that reported by Junginger (Junginger et al, 2001) in their multicentric survey (82%), notwithstanding the fact that monocentric studies usually show superior results when compared to multicentric studies. In the current patient group, 37% of patients had locally advanced disease with vasculonervous – or bone invasion, or multifocal spread. Vasculonervous or bone invasion has shown to be an independent, negative prognostic factor for resectability of STS (Sastre et al, 1997). The difficulty to obtain satisfactory resection in this patient subgroup with locally advanced disease is underlined in the current study by a significant lower resection rate R0 in this subgroup when compared to the subgroup of patients with limited disease. In our mind, a rate of 68% of resections R0 is a rate closer to what really can be done by surgery in a continuous series of patients with soft tissue sarcoma. Without doubt, the collegial determination of resection type R gives a more objective appreciation of quality of surgery as if it was determined by the sole surgeon. Local recurrence rates beneath 10% are usually reported only in series of selected patients, like limited tumours selected for surgery alone (Rydholm et al, 1991; Baldini et al, 1999), superficial tumours (Gibbs et al, 1997) or in patients responding to neoadjuvant therapy (Eilber et al, 2001) or selected for pre-operative radiotherapy excluding patients already operated (Sadoski et al, 1993). In unselected series, local recurrence rates rank from 10 to 30% (Le Vay et al, 1993; Coindre et al, 1996; Lewis et al, 1997; Karakousis et al, 1999; Fleming et al, 1999; Pitcher et al, 2000; Trovik et al, 2000; Zagars et al, 2003: pp 2530 - 2543). We think that the better local results observed in the current series, are mostly due to the better appreciation of quality of surgery, allowing therefore to adapt best treatment strategy. That includes reexcision, which surely allows to obtain more resections R0, as shown in patients re-operated after prior surgery elsewhere with higher resection rates R0 (74% vs. 61%) than in patients operated directly at our centre who underwent mostly one single operation. The same findings have been made by others (Zagars et al, 2003: pp 25442553). Furthermore, knowing best quality of surgery, complementary treatment can be better adapted. In our patient subgroup R1, LR is only 7% when a radiation boost is added to external beam radiotherapy as recommended, but attains 35% when the boost is omitted. On the contrary, given the 96% probability of absence of local recurrence in patients defined R0 by the FSG system,

The reliability of the FSG surgical system was measured on (1) the repartition of resection types, and (2) local recurrence rates.

III. Results A. Repartition of resection types 1. All patients Resections R0 were performed in 75 patients (68%), resections R1 in 32 (29%) and resections R2 in three patients (3%).

2. Resection types according to referral In order to achieve a satisfactory resection, 52 of 61 referred patients were re-operated once, six patients two times and three patients three times. Residual tumour was found in 31 patients (51%). Final distribution of resection types in referred patients was resections R0 in 45 patients (74%), R1 in 14 patients (23%), R2 in 2 patients (3%). In 49 patients operated directly at Institut Bergonié, re-operations for insufficiency of surgery were performed in six patients. Distribution of resection type was resections R0 in 30 patients (61%), R1 in 18 patients (37%) and R2 in 1 patient (2%).

3. Resection types according to tumour growth In 69 patients with limited disease there were 61 resections R0 (88%) and 8 resections R1 (12%). In 41 patients with locally advanced disease (multifocality or neurvascular or bone involvement) there were 14 resections R0 (34%), 24 resections R1 (59%) and three resections R2 (7%). The difference in resection rates R0 between limited disease (61/69 patients) and locally advanced disease (14/41 patients) was significant (p < 0.001).

B. Local recurrence LR was considered in 107 patients with complete tumour excision (R0 + R1). Ten patients recurred locally giving a crude LR rate of 9%. Actuarial local recurrencefree rate at 5 years was 91% (Figure 2). Median interval to LR was 12 months (range: 5-70 months). Nine of ten LR’s occurred within 33 months. The incidence of LR differed significantly between patients of groups R0 and R1 with respectively 3/75 (4%) and 7/32 (22%) LR’s (p < 0.01). Therefore, in the current series, the prevalence of LR is 9% (9/107 patients), the positive predictive value (risk for patients R1 having LR) is 22% (7/32 patients) and the negative predictive value (probability of patients R0 not having LR) is 96% (72/75 patients). There was a difference in LR within 32 patients R1 whether or not a radiation boost was delivered. In 15 patients with a boost, one LR occurred (7%), whereas in 17 patient with no boost, six LR occurred (35%). This difference is significant (p < 0.05).

IV. Discussion We report a continuous series of patients operated at our centre for primary STS of the extremities and trunk 36


Cancer Therapy Vol 3, page 37 complementary radiotherapy could probably be withdrawn in a selected subgroup of these patients. Before testing this hypothesis, the results of an ongoing multicentric evaluation of the FSG system are awaited in order to confirm the multicentric reproducibility of the system. Meanwhile, results of the current study (e.g. lack of radiation boost in 17 patients R1, excessive boost in 12 patients R0) have already permitted to improve treatment strategy for soft tissue sarcoma at Institut Bergonié, helping to elaborate the current algorithm for local treatment as shown Figure 3. The cornerstone of the FSG surgical system is the collegial determination of quality of surgery. In groups with a similar, collective approach defining surgery, equal low rates of local recurrences are reported (Gerrand et al, 2003). In this Canadian series, local recurrences are 10% in 480 consecutive patients with extremity soft tissue sarcoma. Whatever the system which has been elaborated, it seems that a collective determination of quality of surgery shows to be superior, more precise, than an individual approach, either by the surgeon or the pathologist. The advantage of the FSG system is its simplicity, with easily reproducible items (tumour seen or

ruptured) and the descriptive approach, allowing rapid consensus within the participants.

V. Conclusion The surgical classification for STS presented in this study is simple to apply. It shows to reflect most accurately the real tumour extent and therefore the possibilities of resection and complementary treatment. The cornerstone of the FSG system is a consensual, multidisciplinary and not an individual determination of quality of surgery. Such a multidisciplinary approach means that STS should be treated in specialised centres, in accordance with former recommendations (Gustafson et al, 1994). This strategy ensures patients to be treated with an individually tailored, tumour extension adapted treatment, a guarantee for better outcome.

Acknowledgements Special thanks to Véronique Picot for statistical analysis and to Dorothée Quincy for assistance in preparing the manuscript.

Figure 3. Algorithm for local treatment strategy at Institut Bergonié according to resection type (R).

37


Stoeckle et al: French Surgical System in sarcoma

Lagarde P, Kantor G, Tawfiq N, Salem N, Thomas L, Stöckle E, Bui NB (1998) Chimiothérapie des sarcomes des tissus mous de l’adulte. Cancer Radiother 2, 747-751. LeVay J, O’Sullivan B, Catton C, Bell R, Fornasier V, Cummings B, Hao Y, Warr D, Quirt I (1993) Outcome and prognostic factors in soft tissue sarcoma in the adult. Int J Radiat Oncol Biol Phys 27, 1091-1099. Lewis JL, Leung D, Heslin M, Woodruff JM, Brennan MF (1997) Association of local recurrence with subsequent survival in extremity soft tissue sarcoma. J Clin Oncol 15, 646-652. Pitcher ME, Ramathan RC, Fish S, A’Hern A, Thomas JM (2000) Outcome of treatment for limb and limb girdle sarcomas at the Royal Marsden Hospital. Eur J Surg Oncol 26, 548-551. Rosenberg SA, Tepper J, Glatstein E, Costa J, Baker A, Brennan M, DeMoss EV, Seipp C, Sindelar WF, Sugarbaker P, Wesley R (1982) The treatment of soft-tissue sarcomas of the extremities. Prospective randomized evaluations of (1) limbsparing surgery plus radiation therapy compared with amputation and (2) the role of adjuvant chemotherapy. Ann Surg 196, 305-315. Rydholm A, Gustafson P, Röoser B, Willen H, Akerman M, Herrlin K ( 1991) Limb-sparing surgery without radiotherapy based on anatomic location of soft tissue sarcoma. J Clin Oncol 9, 1757-1765. Sadoski C, Suit HD, Rosenberg A, Mankin H, Efird J (1993) Preoperative radiation, surgical margins, and local control of extremity sarcomas of soft tissues. J Surg Oncol 52, 223230. Sarcoma meta-analysis collaboration (1997) Adjuvant chemotherapy for localised resectable soft-tissue sarcoma of adults: meta-analysis of individual data. Lancet 350, 16471654. Sastre-Garau X, Coindre JM, Leroyer A, Terrier P, Ollivier L, Stoeckle E, Bonichon F, Collin F, Le Doussal V, Contesso G, Vilain MO, Jacquemier J, Bui BN (1997) Predictive factors for complete removal in soft tissue sarcomas: a retrospective analysis in a series of 592 cases. J Surg Oncol 65, 175-182. Stoeckle E, Coindre JM, Bonvalot S, Kantor G, Terrier P, Bonichon F, Bui BN (2001) Prognostic factors in retroperitoneal sarcoma. A multivariate analysis of a series of 165 patients of the French Cancer Federation Sarcoma Group. Cancer 92: 359-368. Stoeckle E (1997) Nouvelles techniques chirurgicales des sarcomes des tissus mous. Cancer Radiother 1, 453-456. Thomas L, Delannes M, Stoeckle E, Martel P, Bui BN, DalySchveitzer N, Pigneux J, Chevreau C, Kantor G (1994) Intraoperative interstitial Iridium brachytherapy in the management of soft tissue sarcomas : preliminary results of a feasibility phase II study. Radiother Oncol 33, 99-105. Trojani M, Contesso G, Coindre JM, Rouëssé J, Bui NB, de Mascarel A, Goussot JF, David M, Bonichon F, Lagarde C (1984) Soft-tissue sarcoma of adults; study of pathological prognostic variables and definition of a histopathological grading system. Int J Cancer 33, 37-42. Trovik CS, Bauer HC, Alvegard TA, Anderson H, Blomqvist C, Berlin O, Gustafson P, Saeter G, Walloe A (2000) Surgical margins, local recurrence and metastasis in soft tissue sarcoma: 559 surgically-treated patients from the Scandinavian Sarcoma Group Register. Eur J Cancer 36, 710-716. Weiss SW, Sobin LH (1994) Histological typing of soft tissue tumors. WHO International Classification of Tumours. 2nd ed. Berlin: Springer Verlag.

References Baldini EH, Goldberg J, Jenner C, Manola JB, Demetri GD, Fletcher CD, Singer S (1999) Long-term outcomes after function-sparing surgery without radiotherapy for soft tissue sarcoma of the extremities and trunk. J Clin Oncol 17, 32523259. Brennan MF, Hilaris B, Shiu MH, Lane J, Magill G, Friedrich C, Hajdu SI (1987) Local recurrence in adult soft tissue sarcoma. A randomized trial of brachytherapy. Arch Surg 122, 1289-1293. Coindre JM, Terrier P, Bui NB, Bonichon F, Collin F, Le Doussal V, Mandard AM, Vilain MO, Jacquemier J, Duplay H, Sastre X, Barlier C, Henry-Amar M, Mace-Lesech J, Contesso G (1996) Prognostic factors in adult patients with locally controlled soft tissue sarcoma. A study on 546 patients from the French Federation of Cancer Centers sarcoma group. J Clin Oncol 14, 869-877. Eilber FC, Rosen G, Eckardt J, Forscher C, Nelson SD, Selch M, Dorey F, Eilber FR (2001) Treatment-induced pathologic necrosis: a predictor of local recurrence and survival in patients receiving neoadjuvant therapy for high-grade extremity soft tissue sarcomas. J Clin Oncol 19, 3203-3209. Enneking WF, Spanier SS, Godman MD (1980) A system for the surgical staging of musculoskeletal sarcoma. Clin. Orthoped. 153, 106-120. Fleming JB, Berman RS, Cheng SC, Chen NP, Hunt KK, Feig BW, Respondek PM, Yasko AW, Pollack A, Patel SR, Burgess MA, Papadopoulos NE, Plager C, Zagars G, Benjamin RS, Pollock RE, Pisters PW (1999) Long-term outcome of patients with American Joint Committee on Cancer stage IIB extremity soft tissue sarcomas. J Clin Oncol 17, 2772-2780. Gerrand CH, Bell RS, Wunder JS, Kandel RA, O’Sullivan B, Catton CN, Griffin AM, Davis AM (2003) The influence of anatomic location on outcome in patients with soft tissue sarcoma of the extremity. Cancer 97, 485-492. Ghnassia JP, Vilain MO, Coindre JM, Bertrand G, Château MC, Collin F, Depardieu C, Fiche M, Guillou L, Jacquemier J, Larsimont D, Le Doussal V, Leroux-Broussier A, Mandard AM, Marques B, Ranchère D, Sastre X, Terrier P, Trassard M (1998) Recommandations pour la prise en charge anatomo-pathologique des sarcomes des tissus mous de l’adulte. Ann Pathol 18, 505-511. Gibbs CP, Peabody TD, Mundt AJ, Montag AG, Simon MA (1997) Oncological outcomes of operative treatment of subcutaneous soft-tissue sarcomas of the extremities. J Bone Joint Surg 6, 888-897. Gustafson P, Dreinhofer KE, Rydholm A (1994) Soft tissue sarcoma should be treated at a tumor center. A comparison of quality of surgery in 375 patients. Acta Orthop Scand 65, 47-50. Hermanek P, Sobin LH (1992) TNM classification of malignant tumours. Fifth edition, second revision, Springer, Berlin. Junginger TH, Kettelhack C, Schönfelder M, Saeger HD, Rieske H, Krummenauer F, Hermanek P (2001) Therapeutische Strategien bei malignen Weichteiltumoren. Ergebnisse der Weichteiltumor-Registrierstudie der CAO. Chirurg 72, 138148. Karakousis CP, Driscoll DL (1999) Treatment and local control of primary extremity soft tissue sarcoma. J Surg Oncol 71, 155-161. Lagarde P, Kantor G, Bussières E, Stöckle E, Coindre JM, Tramond P, Avril A, Bui NB, Marée D (1990) Radiothérapie post-opératoire des sarcomes des parties molles des membres. Analyse des volumes et des doses d’irradiation sur une série de 31 cas. Bull Cancer Radiother 77, 101-109.

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Cancer Therapy Vol 3, page 39 Yang JC, Chang AE, Baker AR, Sindelar WF, Danforth DN, Topalian SL, DeLaney T, Glatstein E, Steinberg SM, Merino MJ, Rosenberg SA (1998) Randomized prospective study of the benefit of adjuvant radiation therapy in the treatment of soft tissue sarcomas of the extremity. J Clin Oncol 16, 197203. Zagars GK, Ballo MT, Pisters PW, Pollock RE, Patel SR, Benjamin RS, Evans HL (2003) Prognostic factors for patients with localized soft-tissue sarcoma treated with conservation surgery and radiation therapy: an analysis of 225 patients. Cancer 97, 2530-2543. Zagars GK, Ballo MT, Pisters PW, Pollock RE,Patel SR, Beamin RS (2003) Surgical margins and resection in the management of patients with soft tissue sarcoma using conservative surgery and radiation therapy. Cancer 97, 2544-2553.

Eberhard Stoeckle

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Cancer Therapy Vol 3, page 41 Cancer Therapy Vol 3, 41-56, 2005

Current role of erythropoietin in the management of patients with haematological and solid malignancies Review Article

Max Mano*, Priska Butzberger, Anne Reid, Alan Rodger, Richard Soutar, John Welsh Beatson Oncology Centre, Glasgow

__________________________________________________________________________________ *Correspondence: Dr Max Mano, Consultant in Medical Oncology, Beatson Oncology Centre, Western Infirmary, Dumbarton Rd, Glasgow G11 6NT; Tel: 0044 141 116299; Fax: 0044 141 2111866; Email: max.mano@northglasgow.scot.nhs.uk Key words: erythropoietin, haematological and solid malignancies, clinical activity, anaemia, Quality of Life (QOL), Iron supplementation, cognitive impairment Abbreviations: blood transfusions, (BT); chemotherapy, (CT); Epoetin, (EPO); European Cancer Anaemia Survey, (ECAS); European Organisation for Research and Treatment of Cancer`s, (EORTC); haematological response, (HR); haemoglobin, (Hb); intravenous, (IV); National Cancer Institute, (NCI); overall survival, (OS); performance status, (PS); Quality of Life, (QOL); radiotherapy, (RT); recombinant human erythropoietin, (RHuEPO) Received: 20 January 2005; Accepted: 28 January 2005; electronically published: February 2005

Summary Epoetin (EPO) proteins have been considered the treatment of choice for chemotherapy (CT)-induced anaemia (after exclusion of other causes), and there is growing evidence that this should also be the case for cancer-related anaemia. However, the impact of EPO on survival is currently unknown. Although two recent randomised trials have suggested a negative impact of EPO on outcome, in a recent meta-analysis (of trials with a lower haemoglobin (Hb) target, i.e. 12-13 g/dL) EPO was associated with a trend toward an improved survival. All currently available agents and schedules seem to have similar efficacy. Serious complications with these agents are unusual, though the incidence of thromboembolic events seems to be slightly increased in cancer patients. The use of EPO for cancer and/or CT-induced anaemia significantly reduces the need for blood transfusions (BT) and spares stocks of blood, but is likely to result in increased costs. Therefore, its cost-effectiveness should be determined on a local basis (EORTC) toxicity grading criteria (Table 1). Table 2 depicts the prevalence of anaemia in patients with different tumour types. During the survey, the frequency of anaemia at some point increased to 67% (Hb <10 g/dL in 39%). In this study, the mean Hb level that triggered initiation of treatment was 9.7 g/Dl, and the anaemia was treated in only 39% of the cases ((EPO)= 17.4%; blood transfusion (BT)= 14.9%; and iron= 6.5%). This landmark study has also confirmed what was already known from the clinical practice, i.e. that there is a strong correlation between levels of Hb and PS (Ludwig et al, 2004).

I. Introduction Prevalence of anaemia in cancer patients Anaemia is a common problem in patients with cancer. In a large survey of patients with non-myeloid malignancies included in (published) chemotherapy clinical trials (therefore, presumably with a good baseline performance status (PS)), the incidence of anaemia requiring transfusion was as high as 50-60% in patients with lymphoma, lung, gynaecological and genito-urinary cancer (Groopman and Itri, 1999). Recent results of the European Cancer Anaemia Survey (ECAS), which investigated a total of 15,367 cancer patients (approximately 80% with solid and 20% with haematological malignancies) from 748 centres in 24 countries, reported a baseline prevalence of anaemia of 39.3% (Hb <10g/dL in 10%). In this study, anaemia was defined as haemoglobin (Hb) <12 g/dL, based on the National Cancer Institute (NCI) and the European Organisation for Research and Treatment of Cancer`s

II. Anaemia and changes in Quality of Life (QOL) As in chronic renal failure, the correlation between anaemia and QOL has been well described in cancer patients (Demetri, 2001; Glaspy, 2001; Littlewood et al, 2001; Ludwig and Strasser, 2001; Fallowfield et al, 2002; Straus, 2002). However, the best data so far are the recently published results of ECAS (Ludwig et al, 2004). 41


Mano et al: EPO in haematological and solid malignancies management In this study, Hb levels significantly correlated with mean PS at enrolment (p<0.001) (Table 3), and similar trends were observed all over the survey. Performance Status scores of 3-4 were uncommon in patients with higher Hb values, but much more frequent in patients with lower Hb levels (2.5%, 5.5%, 12.3% and 24% in patients with Hb levels of >=12 g/dL, 10-11.9 g/dL, 8-9.9 g/dL and <8 g/dL, respectively).

III. Correction of improvements in QOL

anaemia

the use of EPO. Most of these studies have shown consistent improvements in QOL with increases in Hb levels (Abels, 1993; Glaspy et al, 1997; Pawlicki et al, 1997; Demetri et al, 1998; Dammacco et al, 2001; Gabrilove et al, 2001; Littlewood et al, 2001; Seidenfeld et al, 2001; Quirt et al, 2001; Wilkinson et al, 2001; Crawford et al, 2002; Fallowfield et al, 2002; Osterborg et al, 2002; Pronzato et al, 2002; Thomas et al, 2002; Vansteenkiste et al, 2002; Boogaerts et al, 2003; Hedenus et al, 2003; Janinis et al, 2003; Iconomou et al, 2003; Shasha et al, 2003; Chang et al, 2004; Charu et al, 2004; Jones et al, 2004; Savonije et al, 2004; Witzig et al, 2004) (Table 4).

and

Several studies have looked at changes in QOL with the use of interventions aiming at increasing Hb, such as

Table 1. Grading of anaemia used in ECAS and that proposed by NCI (Hb in g/dL)

MILD MODERATE SEVERE LIFE-THREATENING

ECAS 11.9-10.0 9.9-8.0 <8.0 NA

NCI 10.0-WNL 8.0-10.0 6.5-7.9 <6.5

Hb= haemoglobin; NA= not applicable; WNL= within normal limits (12-16 for women, 14-16 for men); NCI= National Cancer Institute

Table 2. Prevalence of anaemia per tumour type (Ludwig et al, 2004)

Hb<12

Breast (n*=3123)

Lung (n=2002)

GI-CRC (n=2402)

H&N (n=684)

Gyn (n=1675)

30.4%

37.6%

38.9%

24.9%

49.1%

g/dL

Lymphoma/ Myeloma (n=2260) 52.5%

Leukaemia (n=624)

U-G (n=894)

53%

29.2%

OVERALL PREVALENCE= 39.3%

n= number of patients; Hb= haemoglobin; GI-CRC= gastro-intestinal and colorectal; H&N= head & neck; Gyn= gynaecology; U-G= uro-genital * Evaluable population

Table 3. Correlation between Hb levels and PS in ECAS, at enrolment (Ludwig et al, 2004) Hb (g/dL) n Mean (WHO) PS

<8 187 1.7

8-9.9 1242 1.4

10-11.9 4214 1.0

>=12 8750 0.8

Hb= haemoglobin; WHO= World Health Organisation; n= number of patients; PS= performance status; ECAS= European Cancer Anaemia Survey

Table 4. Changes in QOL in patients receiving interventions to correct Hb Trial

n

Abels, 1993

413

Osterborg et al, 2002

349

Design

Population RANDOMISED TRIALS r-HuEPO (100 U/kg 3xw Anaemic (Hb<=10.5g/dL) for non-CT, 150 U/kg pts on CT (n=289) or not 3xw for CT pts) (n=124) vs placebo EPO-! (150 IU/kg 3xw) Transfusion-dependent pts with haematological vs malignancies and low serum placebo erythropoietin concentration

42

Results EPO corrected anaemia in all groups, and seemed to improve functional capacity in responding pts Significant improvements in QOL in the EPO-! group


Cancer Therapy Vol 3, page 43 Witzig et al, 2004

344

EPO-" (40,000) U qw vs placebo

Anaemic (Hb<11.5 g/dL and <10.5 g/dL for males and females, respectively), after receiving CT

Littlewood et al, 2001

375

EPO-" (150 to 300 IU/kg 3xw) (2:1) vs placebo

Anaemic (Hb <=10.5 g/dL, or > 10.5 g/dL but <or= 12 g/dL after a Hb #of >or= 1.5 g/dL per cycle since starting CT) pts with solid or nonmyeloid haematological malignancies receiving nonplatinum CT

Chang et al, 2004 Boogaerts et al, 2003

354

Anaemic (Hb =12 g/dL) breast cancer pts on CT Anaemic (Hb<or=11 g/dL) pts with lymphoid or solid tumour malignancies

EPO-" was effective in improving QOL Baseline to final visit changes in QOL scores were significantly greater with EPO-!

Savonije et al, 2004

315

Mildly anaemic (Hb<= 12 g/dL) pts with solid malignancies receiving platinum-based CT

EPO-"improved QOL scores at the end of the study.

Charu et al, 2004)

170

Anaemic (Hb<=11 g/dL) pts not on CT or RT

Improvements in QOL with DA in the first 12 weeks

Vansteenkiste et al, 2002

320

Anaemic (Hb<=11 g/dL) lung cancer pts receiving CT

Pts on DA had better improvements in fatigue scores

Hedenus et al, 2003

344

Anaemic lymphoma or myeloma pts receiving CT

DA improved QOL

Pronzato et al, 2002

223

Anaemic (Hb 10-12 g/dL) breast cancer pts receiving CT

Early treatment with EPO-" resulted in significant improvements in QOL scores

Thomas et al, 2002

130

Wilkinson et al, 2001

182

Iconomou et al, 2003

122

Mildly anaemic (Hb < 12.0g/dL) cancer pts undergoing CT Anaemic (Hb 10-12 g/dL) ovarian cancer pts on platinum-based CT Anaemic (Hb </=11.0 g/dl) pts on CT

Early treatment with EPO-" resulted in significant improvements in QOL scores Preliminary results from 160 pts showed significant improvements in QOL Significant improvement in QOL with EPO

Janinis et al, 2003

372

Anaemic (Hb <11 g/dL) pts on CT

Dammacco et al, 2001

145

EPO-" (40,000 U qw) vs BSC EPO-! (initial dose 150 IU kg 3xw) vs BSC EPO-" (10.000 3xw; $ 20.000 3xw as necessary) (2:1) vs BSC DA (3mcg/kg q2w);(2:1) vs observation (for 12 weeks, then DA) DA (2.25mcg/kg/qw, doubled as necessary) vs placebo DA vs placebo EPO-" (10000-20000 3xw) vs BSC EPO-" (10000 3xw) vs BSC EPO-"(10000 3xw)(2:1) vs BSC rHuEPO vs placebo EPO-"(10000 3xw) vs no EPO EPO-" (150 UI/kg 3xw) vs placebo

Positive impact on QOL which was independent of anti-tumour response Benefit seen only on univariate analysis

Fallowfield et al, 2002

262

Anaemic (Hb<11 g/dL) pts with myeloma

43

Changes in the average QOL scores from baseline to the end of the study were similar in the two study arms; a benefit was seen in the subgroup of Hb responders only. Improvement of all primary cancer- and anaemia-specific QOL domains was significantly greater for EPO-". Vast majority of pts in this trial had Hb levels <10.0 g/dL. This QOL data has been later more extensively evaluated and the significant QOL gains confirmed (Fallowfield 2002)


Mano et al: EPO in haematological and solid malignancies management

Gabrilove et al, 2001

3012

Crawford et al, 2002

4382

Shasha et al, 2003

777

Quirt et al, 2001

401

Demetri et al, 1998

2370

Glaspy et al, 1997

2342

Pawlicki et al, 1997

215

Jones et al, 2004

Seidenfeld et al, 2001

11459

851

NON-RANDOMISED TRIALS Community-based; Anaemic (Hb <or= 10.5 g/dL, multicentre, openor > 10.5 g/dL but <or= 12 label(EPO-" 40,000 U g/dL after a Hb # of >or= 1.5 3xw; $ to 60,000 U 3xw g/dL per cycle since starting CT) pts with non-myeloid depending on the Hb malignancies on CT response at 4 weeks) Retrospective review of Anaemic (Hb 8-14 g/dL) pts data from 2 open-label, undergoing chemotherapy community-based trials of EPO-" (given 3xw)

Multicentre, open-label (EPO-" 40,000 U qw; $ to 60,000 U qw if Hb $ to < or = 1 g/dL after 4 weeks) Prospective, open-label (EPO-" 150 IU/kg 3xw; $ 300 IU/kg after 4 weeks is insufficient response) Community-based (EPO-" 10,000 U 3x week; $ to 20,000 U 3xw depending on the Hb response at 4 weeks) Community-based, openlabel (EPO-" 150 U/kg 3xw; $ to 300 U/kg if insufficient response)

Anaemic (Hb <or=11 g/dL) pts with non-myeloid malignancies receiving RT concomitantly or sequentially with CT Anaemic (symptomatic or Hb<11 g/dL) pts on CT (n=218) or not (n=183) Anaemic (Hb <= 11g/dL) pts with non-myeloid malignancies on CT

Anaemic (non-specified) pts receiving CT

Open-label, multinational Cancer pts with anaemia (EPO-" 150 IU/kg 3xw) secondary to platinum- or non-platinum-based CT META-ANALYSES Meta-analysis of 23 Anaemic pts with cancer published and unpublished, randomised/ controlled and single-arm studies that included at least 20 pts per arm Meta-analysis of 22 trials Cancer pts undergoing anti(n=1927) to estimate the cancer treatment and receiving odds of BT EPO Also, evaluation of 9 trials (n=581 evaluable) that have reported QOL analysis

Significant improvements in functional status and fatigue; improvements in QOL parameters correlated significantly with increases in Hb levels Non-linear relationship and significant positive correlation between high Hb levels and high QOL scores; a benefit was seen at all levels of Hb but was the largest when Hb levels of 11-13 g/dL were achieved In 359 pts who were evaluable for QOL assessment, EPO-" significantly improved overall QOL from baseline to the time of final evaluation. Improvements in QOL scores, which correlated with increases in Hb levels; similar benefits were observed in the 2 cohorts EPO-" increased Hb levels, which correlated with an improvement in QOL scores; this was independent of tumour response EPO-" was effective in improving the functional status and QOL, as well as increasing Hb level and decreasing BT requirements; improvement in functional status was attributed to increases in Hb levels. Significant improvement in QOL and PS.

EPO-" improved QOL in pts with cancer. Results adjusted for confounding factors remained consistent. The favourable impact on QOL was significant only in studies with mean baseline Hb <10 g/dL Insufficient data to show whether initiating EPO before Hb drops to less than 10 g/dL resulted in improved QOL; these data were however considered insufficient for a meta-analysis.

QOL= quality of life; Hb= haemoglobin; EPO= epoetin; CT=chemotherapy; vs= versus; BT= blood transfusion; RT= radiotherapy; pts= patients; qw= once a week; $ = increase; #= decrease; WHO= World Health Organisation; PS= performance status; 3xw= three times a week; DA= darbepoetin; r-HuEPO= recombinant human epoetin; BSC= best standard of care

44


Cancer Therapy Vol 3, page 45 The cloning of the erythropoietin gene in the mid 1980s allowed the development and quick introduction of recombinant human erythropoietin (RHuEPO) into clinical practice. These agents were first used in the management of anaemia of chronic renal failure, but their use has been extended to other conditions such malignant diseases, HIV infection, prematurity and surgery. Four different RHuEPOs are currently available: alpha, beta, delta and omega, with some differences in pharmacokinetics and pharmacodynamics. Alpha and beta have been the most commonly used RHuEPOs in Europe. The erythropoietin analogue darbepoetin alfa is a unique molecule that carries two additional glycosylation sites. This agent stimulates erythropoiesis through the same mechanism as endogenous erythropoietin, but has a longer half-life allowing less frequent administration. In summary, there is currently substantial evidence that the use of EPO improves QOL in patients with CTinduced (non-platinum or platinum-based) or cancerrelated anaemia, as compared to conventional care (comprising BT as necessary). This is supported by a number of randomised trials and recently ratified by at least one meta-analysis (level I evidence (i.e., based on randomised controlled clinical trials and/or metaanalyses)). In this meta-analysis, EPO-" improved Cancer Linear Analog Scale (CLAS) (20-25%), Functional Assessment of Cancer Therapy (FACT)-Fatigue (17%), and FACT-Anemia (12%) scores (P = 0.05) and, whilst PS worsened for control cohorts (P = 0.05), EPO-" cohorts remained unchanged. Four of the Short-form 36 Questionnaire (SF-36) subscales, namely Physical Function, Role Physical, Vitality, and Social Function, improved with EPO-" (P = 0.05) (Jones et al, 2004). Despite this, ECAS revealed that more than 60% of the patients who were ever anaemic (at baseline or during the study) did not receive any treatment for this, and most of those who did had their treatment initiated only when Hb levels dropped below 10 g/dL. At present, the most robust evidence of a favourable impact of EPO on QOL is in patients with Hb <= 10 mg/dL, and this was actually the threshold recommended by an ASCO panel in 2002 (Rizzo et al, 2002). However, it is important to point out that losses in QOL scores can be observed with even more subtle drops in Hb levels (<12g/dL) (Ludwig et al, 2004), and there is some clinical data suggesting that the maximal gain in QOL occurs when Hb levels of 12 g/dL (range 1113 g/dL) are achieved (Crawford et al, 2002). This concept has also been supported by the results of several randomised trials that are assessing the impact of earlier intervention with EPO (Hb 10-12 g/dL) on QOL (Wilkinson et al, 2001; Pronzato et al, 2002; Thomas et al, 2002; Chang et al, 2004; Rearden et al, 2004). Of note, a European panel has recently recommended levels of 9-11 g/dL to be used as threshold for initiation of EPO treatment (Bokemeyer et al, 2004).

randomised clinical trials (Table 4) (Abels, 1993; Glaspy et al, 1997; Pawlicki et al, 1997; Demetri et al, 1998; Dammacco et al, 2001; Gabrilove et al, 2001; Littlewood et al, 2001; Seidenfeld et al, 2001; Quirt et al, 2001; Wilkinson et al, 2001; Crawford et al, 2002; Fallowfield et al, 2002; Osterborg et al, 2002; Pronzato et al, 2002; Thomas et al, 2002; Vansteenkiste et al, 2002; Boogaerts et al, 2003; Hedenus et al, 2003; Janinis et al, 2003; Iconomou et al, 2003; Shasha et al, 2003; Chang et al, 2004; Charu et al, 2004; Jones et al, 2004; Savonije et al, 2004; Witzig et al, 2004). Overall, these trials have shown HR in the range of 50% to 80%, and the mean increase in Hb is in the range of 1.8 to 2.0 g/dL. There seems to be little difference in activity among the various agents and schedules currently available. Doubling the dose in nonresponding patients has typically resulted in a further 1015% HR rate (Bokemeyer et al, 2004). In a recent metaanalysis of 14 trials, the HR rate was 48%, and there were significant reductions in requirements for BT (Table 5) (Bohlius et al, 2004). These findings were similar to those reported in a previous meta-analysis of 12 trials (Seidenfeld et al, 2001). In the largest trials, the HR rates were in the range of 60-70% (Tables 4 and 5) (Abels, 1993; Glaspy et al, 1997; Pawlicki et al, 1997; Demetri et al, 1998; Dammacco et al, 2001; Gabrilove et al, 2001; Littlewood et al, 2001; Seidenfeld et al, 2001; Quirt et al, 2001; Wilkinson et al, 2001; Crawford et al, 2002; Fallowfield et al, 2002; Osterborg et al, 2002; Pronzato et al, 2002; Thomas et al, 2002; Vansteenkiste et al, 2002; Boogaerts et al, 2003; Hedenus et al, 2003; Janinis et al, 2003; Iconomou et al, 2003; Shasha et al, 2003; Chang et al, 2004; Charu et al, 2004; Jones et al, 2004; Savonije et al, 2004; Witzig et al, 2004). Therefore, there is currently level I evidence supporting the role of EPO in raising the Hb and reducing the need for BT (of about 20%) in patients with cancer-associated and CT-induced anaemia (Bokemeyer et al, 2004). The Cohcrane meta-analysis has also provided level I evidence that prophylactic use of EPO in non-anaemic patients receiving cytotoxic CT can prevent anaemia (Bohlius et al, 2004), though the safety and clinical significance of this approach has not been fully established.

V. Impact of anaemia on cancer outcomes The presence of anaemia has been considered an adverse prognostic factor in patients with cancer (Caro et al, 2001). This has been better demonstrated in gynaecological (Girinski et al, 1989; Pedersen et al, 1995; Lentz et al, 1998; Obermair et al, 1998; Grogan et al, 1999; Obermair et al, 2001, 2003; Dunst et al, 2003; Lavey et al, 2004; Munstedt et al, 2004) and head & neck cancer (Bryne et al, 1991; van Acht et al, 1992; Fein et al, 1995; Dubray et al, 1996; Kumar et al, 1997; Glaser et al, 2001; Reichel et al, 2003; Chua et al, 2004; Haugen et al, 2004), but has also been shown in other solid tumours such as lung (Albain et al, 1991; Armour et al, 2003; Langendijk et al, 2003) and testicular cancer (Bokemeyer et al, 2002), and in several lymphoproliferative diseases (in some of which Hb levels have been incorporated into the staging system) (Caro et al, 2001).

IV. Evidence of clinical activity: haematological response (HR) rates The HR rates to EPO in anaemic cancer patients have been evaluated by numerous randomised and non-

45


Mano et al: EPO in haematological and solid malignancies management Table 5. Meta-analyses evaluating the efficacy of EPO agents (as assessed by HR rates) Trial Seidenfeld et al, 2001

n 1390

Design Meta-analysis of 12 trials to estimate the odds of BT

Population Cancer pts undergoing anti-cancer treatment and receiving EPO

Bohlius et al, 2004

2347 and 3069

Meta-analysis of 14 trials for estimation of HR (n=2347) and 25 trials for estimation of odds for BT (n=3069)

Anaemic cancer pts receiving EPO

Results EPO reduced the odds of BT (RR= 0.38 [95% CI 0.28-0.51] The number of pts needed to prevent one transfusion was 4.4. Insufficient data to show whether initiating EPO before Hb drops to less than 10 g/dL resulted in reduction of BTs HR= 48% (vs 11% in the control group) Hazard ratio for HR= 3.60 (95% CI 3.07-4.23) EPO reduced the odds of BT (0.44 [95% CI 0.36 to 0.54]); there was a reduction in the risk of BT of approximately 33% ; in the subgroup of responding pts (48%), this benefit is likely to be higher but could not be analysed

rHuEPO= recombinant human epoetin; EPO= epoetin; 3x= three times; Hb= haemoglobin; vs= versus; RR= response rate; EPO-"= epoetin; CI=confidence Intervals; alpha; HR= haematological response; qw= once a week, q2w= twice a week; q3w= three times a week

Interestingly, there is some preclinical data showing that optimal tissue oxygenation can improve the efficacy of radiotherapy (RT) and CT in cancer, and EPO is one of the agents that has been investigated in this context (Thews et al, 1998; Silver and Piver, 1999). There is also some evidence that these agents could potentially compensate for the negative impact of anaemia on outcome, though this is either based on retrospective data, or on studies in which the clinical outcome had not been the primary endpoint (which were therefore underpowered for such conclusions) (Table 6) (Littlewood et al, 2001; Bohlius et al, 2004; Glaser et al, 2001; Antonadou et al, 2001; Littlewood et al, 2003; Sloan et al, 2002; Henke et al, 2003; Leyland-Jones, 2003; MĂśbus et al, 2004). Based on these encouraging data, several prospective randomised trials looking at the specific question of the impact of EPO on survival have been initiated. Unfortunately, preliminary results of these trials were disappointing, two of those having been prematurely closed due to a potential negative impact of EPO on survival and loco-regional control (Henke et al, 2003; Leyland-Jones, 2003). In the BEST trial (Leyland-Jones, 2003), 939 nonanaemic (Hb>13g/dL) patients with metastatic breast cancer receiving first-line CT were randomised to receive EPO-" or placebo. The one-year overall survival (OS) was significantly worse in the EPO-" arm (70% vs 76%, p=0.0117). Curiously, most of the deaths occurred within the first 4 months, and were due to either disease progression, thromboembolic and/or vascular events. Of note, this trial had some design problems, comprising an imbalance of baseline prognostic factors (worse in the EPO arm). Similarly in the ENHANCE trial (Henke et al, 2003), 351 patients with head & neck cancer (T3/4 or nodepositive) and Hb level <12 g/dL (for women) or <13 g/dL (for men) were randomised to receive EPO-! plus RT

(starting 10-14 days prior to RT) or RT alone. After 208 events, loco-regional progression free survival, locoregional progression and survival were significantly worse with EPO-! (adjusted relative risk 1.62 (p=0.0008), 1.69 (p=0.007) and 1.39 (p=0.02), respectively). Interestingly, EPO-! had a particular negative impact on the outcome of patients irradiated for manifested cancer. This trial also had an imbalance of prognostic factors, i.e. more smoking patients in the EPO-! arm, and it is important to point out that more than 80% of the patients achieved very high Hb levels (more than 14 g/dL in women and 15 g/dL in men). This trial has also been criticised for unusually poor outcomes by international standards and by the large number of protocol violations (the differences in outcome between treatment and control groups were no longer significant when the analysis was limited to the â&#x20AC;&#x153;perprotocolâ&#x20AC;? population). More vascular disorders have been reported in the EPO-! arm (11% vs 5%). The authors correctly point out that EPO receptors, which may be targeted by EPO, are known to be present in tumour cells (Okuno et al, 1990; Acs et al, 2001) and seem to be functional (Arcasoy et al, 2002). Furthermore, it is possible that tumour cells can use EPO for angiogenesis and growth (Miller et al, 1992; Yasuda et al, 2003). However, in contrast with these data, EPO has already been shown to have anti-tumour activity in preclinical models (Mittelman et al, 2001). In summary, for the time being, the clinical significance of these interesting preclinical data remains largely unknown. The results of these two clinical trials are in clear contrast with those of a recent meta-analysis of 19 randomised trials (n=2865) (Bohlius et al, 2004), which showed a trend towards an improved survival in patients receiving EPO in addition to anti-cancer treatment. It is worth noting that the final mean Hb in these trials ranged from 10.01 g/dL to 13.86 g/dL, thus significantly lower than those achieved in the 2 trials above. 46


Cancer Therapy Vol 3, page 47 Table 6. Studies investigating the effect of EPO on the outcome of patients receiving anti-cancer treatment Trial

n

Glaser et al, 2001

191

Littlewood et al, 2001 Littlewood et al, 2003

375

Sloan et al, 2002

344

Antonadou et al, 2001

385

Henke et al, 2003

351

LeylandJones, 2003

939

Möbus et al, 1284 2004

Bohlius et al, 2004

2865

Design

Population Results RETROSPECTIVE Retrospective. Pts with SCC of the oral On multivariate analysis, Hb level and Pts with a low Hb before or cavity or oropharynx treated use of r-HuEPO were independent during CH-RT received rwith neoadjuvant CH-RT prognostic factors for response to CHHuEPO 10,000 IU/kg s.c. 3- and surgery RT and locoregional control (p < 0.01). 6x week until surgery Placebo-controlled, double- Anaemic (<or= 10.5 g/dL, Kaplan-Meier estimates showed a trend blind, randomised (EPO-" or > 10.5 g/dL but <or= 12 in OS favouring EPO-" (P =.13, logg/dL after a Hb # of >or= vs placebo; 2:1). rank test), and Cox regression analysis EPO-" 150 to 300 IU/kg 1.5 g/dL per cycle since showed an estimated hazards ratio of starting CT) pts with solid 1.309 (P =.052) favouring EPO-" 3xw or non-myeloid (retrospective analysis) haematological malignancies receiving nonplatinum CT Double-blind, placeboPts with advanced cancer Tumour response and survival between controlled, randomised and with anaemia (Hb<11.5 the two groups were virtually identical (EPO-" vs placebo). g/dL and 10.5 g/dL for (17% vs 20% response for EPO and males and females, placebo respectively, p=0.57; 7% vs 6% EPO-" 40,000 U qw respectively) after receiving death rate at study completion myelosuppressive CT respectively, p=0.65) PROSPECTIVE, RANDOMISED, MULTICENTRE RT ± r-HuEPO 10.000 U Pts with pelvic The addition of r-HuEPO to the daily 5x/week malignancies receiving RT treatment course of RT significantly improved 4-year DFS and local control RT ± EPO-! Anaemic (Hb <12g/dL for Significantly worse loco-regional women and <13 g/dL for control and survival in EPO-! arm; men) pts receiving radical however, the design has been criticized RT for locally-advanced head and neck cancer CT ± EPO-" Non-anaemic metastatic Significantly worse one-year OS in breast cancer pts receiving EPO-" arm; however, the design has CT been criticized CT ± EPO-" (2nd Non-anaemic early breast No impact on DFS or survival cancer pts receiving randomisation) adjuvant CT META-ANALYSES Meta-analysis of 19 Anaemic pts receiving anti- Inconclusive effect of EPO on survival, randomised clinical trials cancer treatment (in 14 but a trend for improved survival with (none designed to baseline Hb <10g/dL; in 2 the use of EPO was observed (HR investigate survival as between 10-12 g/dL,; in 3 0.81;95% CI 0.67-0.99) primary endpoint) Hb > 12 g/dL) In 9 studies the CT was platin-based, in 7 nonplatin-based

AWNH= average weekly nadir Hb; BT= blood transfusion; DFS= disease-free survival; OS= overall survival; SCC= squamous cell carcinoma; RT= radiotherapy; CH-RT= chemoradiotherapy; CT= chemotherapy r-HuEPO= recombinant human epoetin; EPO= epoetin; RR= relative risk; PFS= progression-free survival; pts= patients; 3xw= three times a week; 5xw= five times a week

Furthermore, in another prospective randomised trial in non-anaemic early breast cancer patients receiving adjuvant CT, EPO had no detrimental effect on outcome (Möbus et al, 2004). In short, in light of statistical and design problems (especially targeting non-anaemic patients or the use of high Hb targets), and also of conflicting results from other studies (Bohlius et al, 2004; Möbus et al, 2004), the

interpretation of the results of these 2 prospective trials remains difficult. It is unlikely, based on the results depicted in Table 6, that patients with lower Hb levels (in whom EPO is licensed for use) could also be at risk of increased mortality when treated with EPO. Although this question is currently being investigated by a number of clinical trials, in the meantime physicians should avoid over-increasing the Hb (to more than 13-14 g/dL) or 47


Mano et al: EPO in haematological and solid malignancies management prolonging the treatment duration (to more than 16 weeks, as in the pivotal trials). Also, patients with Hb levels higher than 12 g/dL should not receive these agents outside the context of a clinical trial. Of interest, some (retrospective) studies in the past have looked at the impact of BT on the outcome of cancer patients, with some also conflicting results in terms of its effect on outcome (Girinski et al, 1989; Lentz et al, 1998; Landers et al, 1996; Grogan et al, 1999).

VI. Optimal schedule and agent There is little evidence that either agent or schedule could make a significant difference in terms of efficacy or toxicity. However, there may be some differences in terms of convenience and costs (Ng et al, 2003; Cremieux et al, 2004). As shown in Table 7, the efficacy of different agents and schedules seemed very similar in the pivotal trials.

Table 7. Comparisons between different EPO agents and schedules Trial

n

Design RANDOMISED TRIALS Pts with lymphoproliferative disorders Randomised (EPO-! 10.000 U 3xw vs 30.000 U qw) Randomised (1:1), open-label, multicentre EPO-" 40000 U qw vs DA 200 mcg q2w; pts on CT

Cazzola et al, 2003 Waltzman et al, 2004

241

Mirtsching et al, 2002

375

Pooled analysis of 3 DA trials

Schwartzberg et al, 2004

312

Rearden et al, 2004

204

Thatcher et al, 1999

130

Pooled analysis of 3 identical multicentre, prospective, randomised 1:1 (DA 200 mcg q2w or EPO-" 40000 U qw Randomised, prospective Early (immediate treatment) vs late intervention (when Hb dropped below 10 g/dL) Agent: DA 300 mcg q3w Multicentre, randomised (no additional treatment [n = 44], EPO-" 150 IU kg 3xw [n = 42] or 300 IU kg 3xw [n = 44]) Pts on CT

Hesketh et al, 2004 Granetto et al, 2003

242

McKenzie et al, 2002 Schwartzberg et al, 2003

Patton et al, 2004 Thames et al, 2004

Gabrilove et al, 2001

First 123

Results Comparable efficacy Preliminary results suggested that EPO-" may be slightly more active rHuEPO and DA were equality effective, but DA seemed to result in less BT requirements No differences in efficacy and safety Early intervention resulted in reductions in BTs and improved QOL Significantly fewer (P < 0.05) EPO-" pts experienced anaemia (Hb <10 g/dL) during the course of CT (300 IU kg= 39%; 150 IU kg= 48%; untreated= 66%); Comparable efficacy

Randomised phase II (fixed or weight-based dose after a front-loading schedule) 546 Multicentre, open-label, randomised (EPO-" fixed Similar efficacy vs weight-based dose) RETROSPECTIVE AND PROSPECTIVE/NON-RANDOMISED STUDIES 1238 Retrospective, community-based analysis of pts Comparable efficacy receiving EPO-" 40000 U qw(50%), q2w(32%), or q3w or less frequently(17%) 1391 Multicentre, retrospective cohort study (practice Both agents seemed equally patterns of the use of DA-" and EPO-" for CTeffective. Darbepoetin-" (q2w) induced anaemia) has reduced frequency of dosing as compared to EPO-" (qw) 408 Retrospective, observational (practice patterns of the Comparable efficacy use of DA-" and EPO-") 330 Retrospective, multicentre chart review DA-" is effective in treating CTinduced anaemia in both EPO-"na誰ve pts and those switched from EPO-" 3012 Community-based; multicentre, open-label, nonComparable efficacy (with that randomised. of a historical control arm) EPO-" 40,000 U 3xw; $ to 60,000 U 3x week depending on the Hb response at 4 weeks

q2w= every 2 weeks; q3w= every 3 weeks; qw= once a week; EPO= epoetin; CT= chemotherapy; n= number of patients; HR= haematological response; 3xw= three times a week; DA= darbepoetin; Hb= haemoglobin; rHuEPO= recombinant human erythropoietin; pts= patients; BT= blood transfusion

48


Cancer Therapy Vol 3, page 49 More recently, prospective randomised trials have compared different schedules and agents, with no major surprises. As expected, surveys have shown that patients prefer less frequent visits to clinics to receive EPO injections (Schwartzberg et al, 2004; Tauer et al, 2004). Considering the currently available agents, the possible schedules for use in daily practice are: EPO-" (10.000 U thrice a week or 40.000 U once a week); EPO-! (10.000 U thrice a week or 30.000 U once a week) and Darbepoetin (100 mcg once a week or 200 mcg once every 2 weeks). Other schedules may also be feasible, but have been less extensively investigated and should not be recommended routinely. The optimal duration of treatment seems to be 12-16 weeks, as in most clinical trials. The optimal target Hb is currently unknown, but 12-13g/dL has been accepted as a reasonable target. The treatment should be probably interrupted if the Hb rises above 13-14 g/dL.

transferrin saturation <20% and baseline serum ferritin >100ng/mL, the typical picture of anaemia of chronic disease. In short, the role of IV iron supplementation as an adjunct treatment to EPO in patients with cancerassociated anaemia deserves further investigation. Preliminary evidence suggests that IV, but not oral iron, may increase HR rates in subgroups of anaemic cancer patients receiving EPO agents. It remains to be determined whether this increased activity could result in cost savings (by further reducing the need for BT and/or allowing the use of lower doses of EPO).

VIII. Prediction of response EPO is a costly treatment, and is not exempt from toxicity. Considering that up to 30-50% of patients will not respond to this treatment, it would be important to identify those anaemic cancer patients who are most likely to respond. As discussed in the previous section, patients with functional or absolute iron deficiency may be less likely to respond to treatment with EPO, but the HR rate may be significantly improved by the administration of IV iron supplementation. In a small study, the presence of <5% of hypochromic erythrocytes at baseline and an increase in reticulocytes of >= 50% after 2 weeks of treatment were strongly predictive of response to EPO, whilst the presence of >5% hypochromic erythrocytes at baseline indicated functional iron deficiency, requiring the use of IV iron (Katodritou et al, 2004). There is also good evidence that low levels of endogenous erythropoietin may be predictive of response to EPO (Cazzola et al, 1995; Osterborg et al, 2002; Boogaerts et al, 2003; Bamias et al, 2003; Hedenus et al, 2003). Since the majority of the patients included in these studies had haematological malignancies, the evidence is currently considered less robust for solid malignancies (Bokemeyer et al, 2004). Furthermore, not all studies have confirmed the predictive value of low levels of endogenous erythropoietin (Littlewood et al, 2003; Chang et al, 2004). There is also general agreement that HR in the first 4 weeks is predictive of response to EPO (Gonzalez-Baron et al, 2002), though it is also known that a further 10-15% of patients may still respond to EPO after the starting dose is doubled. There is also some data suggesting that baseline Hb levels (>=90 g/dL) (Bokemeyer et al, 2004; Chang et al, 2004) and age (<60 years) (Bokemeyer et al, 2004) may also be predictive factors of response to EPO in patients with CT-induced anaemia. In short, apart perhaps from erythropoietin endogenous concentration in patients with haematological malignancies and the presence of functional or absolute iron deficiency, for the time being the variables above cannot be routinely used for prediction of response to EPO in daily practice.

VII. Iron supplementation Despite these encouraging results, approximately 30% to 50% of cancer patients with CT-related anaemia fail to achieve a meaningful response to EPO. As already demonstrated in patients with anaemia of chronic renal failure (Fishbane et al, 1995; Macdougall et al, 1996; Sepandj et al, 1996), intravenous (IV) iron seems to increase HR rates in patients with CT-related anaemia. The rationale for this is the fact that, during treatment with EPO, large amounts of iron are required to sustain the demands of accelerated erythropoiesis. In patients with chronic renal failure, oral iron supplementation has not resulted in a meaningful benefit (Fishbane et al, 1995; Macdougall et al, 1996). Of note, the risk of anaphylaxis, a feared complication of earlier IV iron preparations, seems to be a rare occurrence (<1%), at least in patients with chronic renal failure (Sepandj et al, 1996). In a recent randomised clinical trial (Auerbach et al, 2004), 157 patients with CT-induced anaemia ((Hb <or= 105 g/L, serum ferritin <or= 450 pmol/L or <or= 675 pmol/L with transferrin saturation <or= 19%) were randomised to one of 4 arms: (1) EPO alone, (2) EPO plus iron 325 twice daily orally, (3) EPO plus iron Dextran 100 mg IV or (4) EPO plus iron Dextran total dose IV. The best HR rates were seen in both IV iron arms, though these results will need confirmation in larger randomised trials. Serious side effects were uncommon in this trial. As a note of caution, the HR rates to EPO in the “no iron supplementation” and “oral iron” arms seemed significantly lower than those reported in the pivotal trials, though this may be because of the selected population of patients with functional or absolute iron deficiency included in this trial, who may well be poor responders to EPO. Similar results were observed in another trial (Henry et al, 2004), in which 187 anaemic (Hb <11 g/dL, with ferritin >100 ng/mL and/ or transferrin saturation >15%) cancer patients receiving CT and EPO were randomised to no iron, iron 325 mg tid orally or IV ferric gluconate. Haematological response rates were better with IV iron than with no iron supplementation or oral iron (73% vs 41%(p=.0029) vs 46%(p=.0099), respectively). The benefit seemed to be the highest among patients with

IX. Effect on cognitive impairment Several studies have linked the administration of CT to a degree of cognitive impairment (van Dam et al, 1998; Schagen et al, 1999; Brezden et al, 2000; Wefel et al, 49


Mano et al: EPO in haematological and solid malignancies management 2004). Interestingly, EPO receptors are known to be expressed throughout the central nervous system (Marti et al, 1997; Juul et al, 1999; Nagai et al, 2001), and at least one small randomised trial has suggested that EPO may have a “neuroprotectant” effect in patients receiving cytotoxic CT for early breast cancer (O'Shaughnessy et al, 2002). Although this potential “neuroprotectant” effect of EPO in patients with CT-induced cognitive dysfunction (and other neurological diseases/brain injury) has been an area of intense research, for the time being this should not be considered an indication for the use of EPO in daily practice.

probably need to be assessed taking into account local realities, so that results of studies are not automatically applicable in different countries or services. In a retrospective study of patients receiving CT for breast cancer, EPO-" given prophylactically was significantly more costly than BTs, though one confusing factor was the absence of guidelines for the indication of BT (Meadowcroft et al, 1998). In another American retrospective study performed in 1997, conventional treatment (with BT as necessary) resulted in cost savings of more than US$ 8,000 per patient, compared to EPO (Sheffield et al, 2003). In a more recent study of 55 metastatic breast cancer patients receiving non-platinum-based CT, a high probability of favourable cost-utility outcomes with EPO" was suggested by the authors (Martin et al, 2003). Stage IV breast cancer therapy costs were collected by surveying UK oncologists, and utilities for associated health states were from published sources. In short, though such estimations are usually difficult, the use of EPO in anaemic patients with cancer is likely to result in increased costs. This should be weighed against the benefits, i.e. improved QOL and saving of blood stocks/risks associated with BT and, in countries like the UK, the risk of variant Creutzfeldt-Jacob disease transmission. Interestingly, in health services that rely totally on BT for the treatment of cancer-associated anaemia, an interesting dilemma has been observed: despite compelling evidence that QOL is improved by the correction of anaemia, there is considerable reluctance to offer early BT to patients, mainly because of potentially unknown risks.

X. Safety profile Epoetin proteins have been generally considered safe agents. There is a larger experience in patients with chronic renal failure, and data is starting to accumulate in cancer patients. The most feared toxicities with these compounds are thromboembolic events and hypertension, both having been shown to be slightly increased in patients with CT induced anaemia receiving EPO (level I evidence) (Bokemeyer et al, 2004). In the BEST study (Leyland-Jones, 2003), a prospective randomised trial closed early because of an increased mortality associated with the use of EPO, some of the early deaths were attributed to thromboembolic events, though it is important to point out that this trial was investigating the role of EPO in patients with normal baseline Hb levels. Another prospective randomised trial investigating the role of EPO in patients with breast cancer has also been prematurely closed due to an increased incidence of thromboembolism (Rosenzweig et al, 2004). In the ENHANCE trial, a prospective randomised trial investigating the role of EPO in patients with head and neck cancer, an increased incidence of vascular events was noted in the EPO arm (11% vs 5%) (Henke et al, 2003). As in the BEST trial, very high levels of Hb were achieved in this study, suggesting that this may be a risk factor for thromboembolic events in this population. In the Cochrane meta-analysis (Bohlius et al, 2004), which included 1738 patients from 12 clinical trials, thromboembolic events occurred in 43/1019 and 14/719 patients in the EPO and control groups respectively (absolute risk difference of 0.02 (95% CI 0.00 to 0.04)). In this review, neither thromboembolism nor hypertension could be definitively linked to the administration of EPO, but under-reporting was a concern with the former. In this same meta-analysis, there was no definite evidence that EPO increased the risk of other adverse events such as rash, irritation, pruritus, haemorrhage and thrombocytopenia. So far, pure red-cell aplasia, a rare but grave complication of treatment with EPO, has been seen only in patients with chronic renal failure (Casadevall et al, 2002; Locatelli and Del Vecchio, 2003), possibly because of the deficient immunity typically present in cancer patients.

XII. Conclusion Although EPO has been considered the preferred treatment for CT-induced anaemia (after exclusion of other causes), there is a surprising variation in its use in different countries. In addition, there is emerging evidence that EPO should also be considered the treatment of choice for cancer-related anaemia. However, the impact of EPO on survival is currently unknown. The disappointing results of some recent prospective randomised trials, which showed a possible negative impact of EPO on outcome, must be balanced against evidence from other trials showing just minor or no increased incidence of thromboembolism and no negative impact on outcome, particularly when a lower Hb target is used (such as 12-13 g/dL). All currently available agents and schedules seem to have similar efficacy. Serious complications are unusual with these agents, the most feared in cancer patients being the risk of thromboembolic events. The use of EPO for cancer and/or CT-induced anaemia significantly reduces the need for BT and spares stocks of blood, but is likely to result in increased costs. Therefore, its cost-effectiveness should be determined on a local basis, and in light of the resources available. It is still too early to routinely use predictive factors of response to EPO in the clinical practice, though serum baseline erythropoietin levels in patients with haematological malignancies and the presence of functional or absolute iron deficiency may be occasionally useful. The role of IV iron supplementation

XI. Cost-utility analysis There is very limited data on the cost-effectiveness of EPO in anaemic cancer patients. Furthermore, costs 50


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as an adjunct to EPO in subgroups of patients with cancerassociated anaemia should be further investigated.

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Cancer Therapy Vol 3, page 55 regulates tumour growth Carcinogenesis 24, 1021-29

Study of Epoetin " Versus Placebo in Anemic Patients With Cancer Undergoing Chemotherapy. J Clin Oncol, in press Yasuda Y, Fujita Y, Matsuo T, Koinuma S, Hara S, Tazaki A, Onozaki M, Hashimoto M, Musha T, Ogawa K, Fujita H, Nakamura Y, Shiozaki H, Utsumi H (2003) Erythropoietin

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Cancer Therapy Vol 3, page 57 Cancer Therapy Vol 3, 57-64, 2005

Targeting of cytostatic bile acid derivatives toward tumours of the enterohepatic circuit Review Article

Jose J.G. Marin*, Marta R. Romero, Marta Vallejo, Maria J. Monte Laboratory of Experimental Hepatology and Drug Targeting, University of Salamanca, Salamanca, Spain

__________________________________________________________________________________ *Correspondence: Dr. Jose J.G. Marin, Department of Physiology and Pharmacology, Campus Miguel de Unamuno, E.D. S-09, 37007Salamanca, Spain; Phone: 34 923 294674; Fax: 34 923 294669; E-mail: jjgmarin@usal.es Key words: Cancer, Chemotherapy, Drug targeting, Intestine, Liver Abbreviations: bile salt export pump, (BSEP); cis-diammine-bisursodeoxycholate-platinum(II), (Bamet-UD2); cis-diamminechlorocholylglycinate-platinum(II), (Bamet-R2); ileal bile acid transporter, (IBAT); Na+-taurocholate cotransporting polypeptide (NTCP); organic anion-transporting polypeptides, (OATP) Received: 8 February 2005; Accepted: 11 February 2005; electronically published: February 2005

Summary Two important limitations to the chemotherapy available for the treatment of tumours of the enterohepatic circuit are low patient response to cytostatic agents, together with the appearance of undesirable side effects. One interesting strategy to overcome these drawbacks consists of using drugs with enhanced tumour cell selectivity. In this sense, based upon the existence in the plasma membrane of hepatocytes, cholangiocytes and cells of the intestinal mucosa of proteins able to carry out efficient uptake of bile acids and their derivatives, several targeted agents have been synthesized by coupling cytostatic moieties to these natural acid steroids. The best results in preclinical screenings have been obtained for cisplatin derivatives such as cis-diamminechloro-cholylglycinateplatinum(II) (Bamet-R2) and, in particular, cis-diammine-bisursodeoxycholate-platinum(II) (Bamet-UD2). These compounds are efficiently incorporated into hepatic and intestinal tumour cells and they are also taken up and efficiently excreted into bile by normal hepatocytes. The potential clinical usefulness of Bamets as anti-tumour agents is based on their strong ability to form adducts with DNA, which results in pro-apoptotic and cytostatic activity and in vivo anti-tumour effects, together with enhanced liver and intestinal organotropism, which probably accounts for minor non-specific toxicity and a marked overall increase in the life span of nude mice bearing liver implants of wild-type or cisplatin-resistant liver or colon tumours. This beneficial effect is enhanced by the encapsulation of Bamets into liposomes. In conclusion, novel cytostatic agents with enhanced targeting towards enterohepatic tumours are now available for further investigations in the preclinical stage and, eventually, in clinical trials. resistance to the cytostatic drug (Canon et al, 1990). Thus, in order to treat tumours of the enterohepatic circuit, more efficient anticancer drugs are needed. One way to increase the beneficial characteristics of novel drugs is to enhance their cell selectivity. The objective of drug targeting, especially for cancer chemotherapy, is to enhance the effectiveness of the drug by concentrating it at the target site and minimizing its effects at non-target sites. This is one of the main aims of modern research in the field of gut and liver cancer chemotherapy. To achieve this goal, many different strategies have been devised and several targeting systems, usually based on the biological properties of several molecules coupled to cytostatic agents, have been proposed.

I. Introduction A large proportion of diagnosed cancers are found in tissues of the enterohepatic circuit, which includes the liver, the biliary tree, the gallbladder and the intestine, and the incidence of some of them, such as hepatocellular carcinoma, is rapidly increasing (Cahill et al, 2004). In addition, their prognosis is usually very poor and some of them are quite difficult to treat (Jarnagin et al, 2003; O'Connell et al, 2003; Si et al, 2003). Current therapy for these types of tumour includes surgery, when feasible, and this is often combined with radiotherapy and/or chemotherapy (Cahill et al, 2004). However, the latter alternative very often faces two major drawbacks that seriously limit its clinical usefulness. These are the appearance of noxious side effects and the development of

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Marin et al: Targeting of cytostatic agents toward enterohepatic tumours Examples of research into intestinal drug targeting include studies on the usefulness of cisplatin-encapsulated transferrin-polyethylene glycol liposomes to treat the peritoneal dissemination of gastric cancer (Iinuma et al, 2002). Moreover, several targeted drug delivery systems for the delivery of anticancer drugs to the colon have been reviewed recently (Minko, 2004). One of these possibilities is the targeting/drug delivery of celecoxib using guar gum as a carrier (Krishnaiah et al, 2002). Regarding liver targeting, owing to the existence in parenchymal hepatic cells of specific plasma membrane receptors for asialoglycoproteins there exists the posibility of coupling antitumour drugs to moieties containing branched galactose residues (Yu et al, 2000; Ohya et al, 2001; Wu et al, 2002). Other interesting alternatives include albumin-conjugates (Stehle et al, 1997), combinations with biotinyl saccharides (Ouchi et al, 2004), the gelatin powder (Gelfoam) used in regional chemotherapy (Pohlen et al, 2001) and nanoparticles (Yamada et al, 2003). Intracellular targets have been also explored, and include the use of anti-sense oligonucleotides and ribozymes (Huesker et al, 2002). Furthermore, plasma membrane proteins expressed in both hepatic and intestinal cells and involved in the efficient uptake of the so-called cholephilic organic anions, such as bile acids (Meier et al, 2002), have been investigated by our group, and others, for use as molecular targets for directing anticancer drugs towards tumours of the enterohepatic circuit (Marin et al, 2001).

in bile acid secretion into bile (Meier et al, 2002). Most bile acid molecules secreted into bile are re-absorbed at the intestine, mainly in the ileum, by very efficient uptake mechanisms, including the ileal bile acid transporter (IBAT, gene symbol SLC10A2). Thus, a minor daily faecal loss (approximately 5% of the total bile acid pool) occurs, which is replaced by de novo synthesis. The sodiumdependent transporter NTCP (gene symbol SLC10A1), and several members of the organic anion-transporting polypeptides (OATP) family, in particular OATP-C or according to the new nomenclature (Hagenbuch et al, 2004) OATP1B1 (gene symbol SLCO1B1), and OATP8 or OATP1B3 (gene symbol SLCO1B3) are responsible for the efficient uptake of bile acids from sinusoidal blood (Meier et al, 2002). As a result, only a small proportion of the bile acid pool escapes from the enterohepatic circuit to the systemic circulation, where bile acid concentrations are usually lower than 10 ÂľM. The marked enterohepatic organotropism of these compounds has prompted several investigators to propose the possible usefulness of bile acids or their analogues as shuttles for drugs towards tissues located in the enterohepatic circuit (Ho, 1987; Betebenner et al, 1991; Stephan et al, 1992; Kramer et al, 1996). The most evident advantage of this strategy in cancer chemotherapy is that selective drug uptake will be enhanced if the resulting molecule is recognized as a substrate by plasma membrane carrier proteins expressed in tumours of the enterohepatic circuit. Therefore, a key aspect in designing these new drugs is to know at exactly which point of the bile acid molecule the active agent should be bound. In principle, the possibilities for conjugating a drug to a bile acid include the hydroxyl groups, in particular the one located at the 3!-position, and the carboxyl group on the side chain (Figure 1).

II. Enterohepatic organotropism of bile acid derivatives Bile acids are synthesized from cholesterol in hepatocytes. Proteins of the superfamily of ATP binding cassette proteins or ABC proteins, such as the bile salt export pump (BSEP, gene symbol ABCB11) are involved

Figure 1. Schematic representation of a typical bile acid (cholic acid) showing potential positions for coupling to pharmacologically active moieties. (A) Chemical structure (3!, 7!, 12!-trihydroxy-5"-cholanoic acid). (B) Spatial representation of the amphipathic characteristics endowing bile acid derivatives the ability to be efficiently encapsulated into liposomes.

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Cancer Therapy Vol 3, page 59 Some bile acid transporters, such as NTCP in hepatocytes and IBAT in cells of the intestinal epithelium and cholangiocytes, which carry out efficient co-transport of bile acids with sodium (Figure 2A) seem to interact with the side chain of the bile acid molecule (Kramer et al, 2001), and therefore this part of the molecule must not be

used to bind the active agent if it is desired that the resulting drug be taken up by these transporters. In contrast, bile acid derivatives obtained by coupling an active agent to the bile acid side chain are efficiently taken up by several members of the human OATP family when expressed in Xenopus laevis oocytes (Briz et al, 2002).

Figure 2. Schematic representation of the plasma membrane carriers involved in normal uptake/secretion of bile acids (BA) by normal hepatocytes (A) and targeting of cytostatic bile acid derivatives to liver tumour cells (B). NTCP, Na+-taurocholate-cotransporting polypeptide; OATP, organic anion-transporting polypeptide; A-, exchanged anion; BSEP, bile salt export pump; MRP2, multidrug resistance-associated protein-2.

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Marin et al: Targeting of cytostatic agents toward enterohepatic tumours The whole of the strategy described above is based on the hypothesis that proteins able to take up bile acids are expressed in enterohepatic tumours. But is this in fact true? Although markedly reduced, the expression of carrier proteins and the ability to take up cholephilic organic anions that are typically substrates of OATPs and NTCP are still present in several, but not all, tumour cell lines of enterohepatic origin (Kroker et al, 1978; Von Dippe et al, 1990; Marchegiano et al, 1992; Kullak-Ublick et al, 1996). Moreover the specific expression of several of these transporters has been found in human hepatocellular carcinomas (Kullak-Ublick et al, 1997). Using real-time quantitative RT-PCR we have also recently detected the expression of these transporters in biopsies collected from intestinal adenomas and carcinomas (unpublished results). Moreover, the ability of liver tumour cells to take up bile acids has been confirmed in laboratory animals. Thus, cells isolated from the liver of rats undergoing a protocol of hepatocarcinogenesis induction were able to take up bile acid derivatives in a selective and sodium-independent way (Monte et al, 1999). This was consistent with the major role of members of the OATP family in this process. Although the efficiency of tumour cells to take up bile acids could be expected to be lower than that of normal epithelial cells, it should be noted that tumour cells are not polarized, and hence drug uptake might not be accompanied by efflux to a similar extent (Figure 2B). Indeed, when overall drug accumulation was analyzed after administering bile acid derivatives to nude mice that had previously been ortothopically implanted with murine liver tumour cells, the amount of drug in the tumours was higher than that found in healthy tissue of the same liver (Briz et al, 2003). Moreover, as expected, there is a relationship between tumour cell load and sensitivity to the cytostatic effect of these compounds (Larena et al, 2002). Although originally the main reasons for synthesizing cytostatic bile acid derivatives were to use them against tumours located in tissues of the enterohepatic circuit (Marin et al, 2001) or to enhance their water miscibility (Maeda et al, 1990), an additional interest coming from their organotropic characteristics is to extend their use to regional treatment of tumours located outside to the enterohepatic circuit. In these cases, the pharmacological advantage of these compounds would be due to the ability of the liver to efficiently take up and eliminate into bile the drug that, escaping from the tumour, might reach the general circulation during regional therapy (Macias et al, 1998; Larena et al, 2001).

the characteristic shared by all members of this family of compounds is that they contain a bile acid-like moiety and an atom of a DNA-reactive transition metal. The reason for choosing this tandem is the small size of the resulting molecule, which would increase the probability of maintaining both the substrate properties as regards bile acid transporters and the reactivity versus DNA, and hence the antiproliferative effect of these metals, in particular platinum(II) such as in cisplatin - cisdiamminedichloro platinum(II) - (Muggia, 1991). Bamets containing transition metal atoms other than platinum, such as gold (Carrasco et al, 2001) are less efficient cytostatic agents than those containing Pt(II) in the reactive moiety. Regarding the organic moiety of the molecule, two types of variable have been assayed. These are the bile acid moiety and the nature of the linker placed between this and the transition metal atom alone or as part of cisplatin-derivatives (Marin et al, 2001). More recently, other groups have expanded the list of variations in the Bamet family by synthesizing several carboplatin-bile acid derivatives (Paschke et al, 2003). Two of the best studied and most promising compounds of the Bamet family are cis-diamminechloro-cholylglycinate platinum(II) (BametR2) (Criado et al, 1997) and cis-diamminebisursodeoxycholate platinum(II) (Bamet-UD2) (Criado et al, 2000).

IV. Mechanism of the cytostatic effect The mechanism of DNA-Bamet interactions is expected to be similar to that of the moiety endowing them with cytostatic ability; namely, cisplatin in the case of Bamet-R2 and Bamet-UD2. Cisplatin forms DNA adducts mainly at the guanine-N7 position (Horacek et al, 1971; Pinto et al, 1985). By different approaches, indirect evidence for the existence of Bamet-induced changes in DNA structure have also been found (Marin et al, 1998a; Martinez-Diez et al, 2000). Thus, in vitro experiments have shown that Bamets are able to change the eletrophoretic mobility of supercoiled covalently-closed and open forms of the circular plasmid pUC18 from Escherichia coli. Moreover, incubation of double-strand DNA with Bamets resulted in an increase in the DNA melting temperature, suggesting the existence of stronger interactions between both strands. This would presumably be due to the formation of inter-strand Bamet-DNA adducts. Scatchard analysis of the results obtained from ethidium bromide displacement assays reveal a marked reduction in the number of DNA sites for ethidium bromide intercalation after in vitro incubation of DNA with Bamets. The Bamet-DNA interaction probably plays an important role in the ability of these compounds to induce the inhibition of DNA synthesis by proliferating cells, as has been determined by measurement of the incorporation of radiolabeled thymidine into DNA using rat hepatocytes in primary culture and several tumour cell lines (Marin et al, 1998a; Martinez-Diez et al, 2000). The sensitivity to the cytostatic effect of Bamets is not similar for all compounds and all cell lines assayed. However, in general,

III. Coupling bile acids to transition metals to generate cytostatic agents Although the possibilities of targeting cytostatic agents to the enterohepatic circuit are very broad, and in this respect the coupling of bile acids to organic antitumour drugs, such as chlorambucil (Kramer et al, 1992) as well as other organic moieties (Eunok et al, 2001) has been achieved and investigated, we shall focus the present review on the family of compounds for which this targeting strategy has been most deeply explored so far, i.e., Bamets. This name is an acronym for BA (bile acids) and MET (metal), since 60


Cancer Therapy Vol 3, page 61 in the case of Bamet-R2 and Bamet-UD2 this ability is comparable to that of cisplatin. In addition to blocking DNA replication, the cytostatic effect of Bamets is believed to be due to both a mild pro-necrotic effect and strong pro-apoptotic activity. This is a relevant issue, since cytostatic activity due to the induction of necrosis may lead to the appearance in vivo of noxious inflammation-associated side effects. Apoptosis has been suggested to be an important mechanism in cell killing by cisplatin (Ormerod et al, 1994). In studies on enterohepatic tumour cells, the release of lactate dehydrogenase to the culture medium and caspase 3 activity in treated cells have been measured to evaluate pro-necrotic versus pro-apoptotic effects, respectively. Apoptosis was confirmed by DNA fragmentation as studied by DNA-ladder formation, TUNEL, single-cell electrophoresis or â&#x20AC;&#x153;cometâ&#x20AC;? assays, Hoechst-33258 staining, and flow cytometry. Bamet-UD2, Bamet-R2, and cisplatin induce a similarly low degree of necrosis. However, pro-apoptotic ability in tumour cells is particularly high for Bamet-UD2. When administered orally, this compound is also able to induce an increase in the rate of apoptosis observed in normal intestinal mucosa of mouse small intestine, although this effect is mild enough as to not affect the structure and function of this organ (Marin et al, 2004).

When after multiple drug administration to rats for 40 days, the platinum contents in different tissues were measured by flameless atomic absorption spectrometry, the amounts of platinum found in kidney, nerve, brain, bone marrow, lung, heart and muscle were significantly lower in rats treated with Bamet-R2 and Bamet-UD2 than in those receiving cisplatin. By contrast, platinum liver contents at short-term (3 h after administration of a single dose) were higher in Bamet-treated rats than in animals receiving cisplatin. Nevertheless, the level of platinum in liver tissue was more efficiently reduced later on (14 days after administration of a single dose) in animals receiving Bamets. This particular time course in the liver handling of these drugs is probably due to the following three features: i) The ability of hepatocytes to take up Bamets (Macias et al, 1998); ii) Bamets are efficiently secreted into bile, with no major biotransformation (Macias et al, 1998; Larena et al, 2001); iii) Although Bamets are absorbed in the intestine, such absorption is significantly lower than the biliary excretion rate (Marin et al, 1996). Thus, Bamets differ from the parent drug cisplatin in their cholephilic characteristics, which are responsible for an important shift in the predominance of faecal versus renal pathways of elimination from the body (Marin et al, 1998b; Macias et al, 1998; Palomero et al, 1998; Macias et al, 1999; Larena et al, 2001).

V. Efficient fecal elimination and low systemic toxicity

VI. Circumvention of resistance to chemotherapy

As mentioned above, two important drawbacks, also limit the clinical usefulness of chemotherapy based on cisplatin-related cytostatic drugs (Muggia, 1991). These are the development of resistance by tumour cells (Canon et al, 1990), and the appearance of undesirable side effects, which mainly include nephrotoxicity, but also myelotoxicity, and neurotoxicity (Von Hoff et al, 1979; Zhang et al, 1994). By coupling cisplatin to bile acids these drawbacks can be markedly reduced. One of the most interesting properties of certain members of the Bamet family is their low overall toxicity. However, this characteristic is not shared by all Bamets. For instance, Bamet-D3, which proved to be highly effective as a cytostatic agent was found to induce renal toxicity in rats (Larena et al, 2001). The side effects induced by Bamet-R2 are significantly lower that those induced by cisplatin in rats (Dominguez et al, 2001). Moreover, and more importantly, despite the marked antitumour activity of Bamet-UD2, no toxicity to the liver, kidney, bone marrow or nervous system has been detected for this drug in in vivo studies (Dominguez et al, 2001). The reason for the low toxicity of Bamets, in particular Bamet-UD2, as compared to cisplatin is most probably related to the organotropic properties of these compounds, which are assumed to be due to the presence of the bile acid moiety in the molecule. In addition, in the case of Bamet-UD2 the bile acid moiety is ursodeoxycholic acid, a bile acid with well-known cytoprotective properties (Botla et al, 1995; Saksena et al, 1997) that cooperate to reduce the noxious effects of the cisplatin moiety on normal cells exposed to the drug.

Another interesting aspect of cytostatic bile acid derivatives is their potential ability to circumvent resistance of tumour cells to chemotherapy. To evaluate this possibility, monoclonal cell lines of enterohepatic origin with several-fold enhanced resistance to cisplatin have been obtained. In all cases, resistance was characterized as mainly being due to an up-regulation of the export pumps of the ABC superfamily of proteins; namely multidrug resistance-associated protein-2 (MRP2), which is believed to play a key role in cisplatin resistance (Cui et al, 1999; Leonard et al, 2003). As free drugs, Bamet-R2 and Bamet-UD2 are not able to overcome cisplatin resistance in cells of nonenterohepatic origin, such as resistant COR-L23/R nonsmall-cell lung cancer cells. In contrast, both drugs were equally effective as cytostatic agents in wild-type and cisplatin-resistant cells derived from intestinal and liver tumours. This can probably be explained in terms of the ability of these cells to strongly reduce the intracellular content of cisplatin, whereas they are not able to prevent the efficient uptake of Bamet-R2 and Bamet-UD2 (Briz et al, 2000). In addition to their interaction with carrier proteins located in the plasma membrane of normal and tumour cells, which is responsible for the specific drug targeting of cytostatic bile acid derivatives, they have another physicochemical characteristic of great interest in the fight to overcome resistance to chemotherapy: as they are amphypathic compounds, that is, they have a hydrophilic region while another part of the molecule is hydrophobic, they can be efficiently encapsulated into liposomes. 61


Marin et al: Targeting of cytostatic agents toward enterohepatic tumours Liposomes can be used both to encapsulate hydrophilic compounds in the aqueous core and to load lipophilic compounds in the hydrophobic layers, while amphipathic molecules can be loaded in both regions of the liposomes. This is particularly interesting in the case of multilamellar liposomes, in which the encapsulation of Bamets is several fold more efficient than that obtained for cisplatin (Briz et al, 2003). When cisplatin-resistant tumour cells were treated with Bamet-R2 or Bamet-UD2 encapsulated in different types of liposomes the cytostatic effect was enhanced to values similar or even higher than those found for cisplatin in wild-type cells (Briz et al, 2003).

BFI2003-03208) and the Junta de Castilla y Leon (Grant SA013/04), Spain. The group is member of the Spanish Network for Cooperative Research on Hepatitis. Instituto de Salud Carlos III, Spain (Grant G03/015). Secretarial help by M.I. Hernandez, technical help by E. Flores and English revision of the manuscript by N. Skinner are gratefully acknowledged.

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VII. In vivo antitumoral activity Although cisplatin-related drugs are among the most effective cytostatic agents against certain types of tumours (Boulikas et al, 2003), so far regimens including these drugs have had only moderate success in the treatment of hepatic and intestinal tumours (Lee et al, 2003; Mancini et al, 2003; O'Dwyer et al, 2003). Enhanced targeting of cisplatin using Bamets may change this situation, and such expectations are indeed supported by preclinical investigation in laboratory animals. Subcutaneous implantation of human colon carcinoma cells or mouse hepatoma cells into the backs of nude mice resulted in the formation of single tumours. When these animals were treated by intratumour injection of saline, cisplatin, Bamet-R2 or Bamet-UD2, cisplatin, as well as the Bamets assayed, significantly reduced tumour growth. The order of antitumour activity at all doses used was cisplatin>Bamet-UD2>Bamet-R2. However, at the most effective anti-tumour dose, cisplatin caused 33% mortality, versus no deaths recorded in any of the groups of animals during treatment with Bamets (Dominguez et al, 2001). Similar results have been obtained in a model closer to the clinical situation. After subcutaneous implantation and growth in the back of a donor nude mouse wild-type or resistant enterohepatic tumours were harvested and re-implanted in the livers of several nude mice. These animals were then treated by intraperitoneal injection of saline, cisplatin or Bamet-UD2. Both cisplatin and Bamet-UD2 were able to inhibit tumour growth. However, although some mice treated with cisplatin survived longer than those receiving only saline, no significant differences in mean survival times were found between these two groups. By contrast, treatment with Bamet-UD2 significantly prolonged the survival of mice bearing a tumour in their livers. This difference in antitumour effectiveness was particularly evident when cisplatin-resistant tumours were implanted. The life span of animals treated with Bamet-UD2 was markedly increased when the mice were treated with Bamet-UD2 encapsulated in anionic liposomes, and even more so when this drug was loaded into cationic liposomes (Briz et al, 2003).

Acknowledgements This study was supported in part by the Ministerio de Ciencia y Tecnologia, Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (Grant

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Cancer Therapy Vol 3, page 65 Cancer Therapy Vol 3, 65-76, 2005

Thalidomide and its use in renal and prostate cancer Review Article

Simon Pridgeon1, Marcus Drake2,* 1

Department of Urology, Freeman Hospital, High Heaton Road, Newcastle Upon Tyne, NE7 3DN, UK School of Surgical Sciences, The Medical School, University of Newcastle, NE2 4HH, UK

2

__________________________________________________________________________________ *Correspondence: Marcus Drake, School of Surgical Sciences, 3 rd Floor Wm Leech building, Medical School, University of Newcastle, NE2 4HH, UK; Tel: +44 191 222 7157; Fax: +44 191 222 8514; E-mail: M.J.Drake@ncl.ac.uk Key words: Prostate cancer, Pharmacology, Adverse drug effects, Tumour biology and angiogenesis, Renal cell carcinoma, Clinical trials Abbreviations: androgen independent prostate cancer, (AIPC); cyclooxygenase-2, (COX-2); fibroblast growth factor, (FGF); Genedirected Enzyme Prodrug Therapy, (GDEPT); I!B kinase, (I!K); interferon-"2a, (IFN-"2a); interferon–"2b, (IFN-"2b); Prostatic Specific Antigen, (PSA); Renal cell carcinoma, (RCC); System for Thalidomide Education and Prescribing Safety, (S.T.E.P.S); vascular endothelial growth factor, (VEGF) Received: 9 February 2005; Accepted: 11 February 2005; electronically published: February 2005

Summary Thalidomide was marketed as a sedative and antiemetic in the 1950’s, but it was withdrawn due to teratogenicity. It has since shown clinical benefit in certain benign diseases, and recently interest has focussed on its potential use in cancer management. It has anti-angiogenic properties, which are mediated by a variety of mechanisms including growth factor inhibition. First line management of advanced prostate cancer generally uses androgen ablation therapy. Progression and metastasis of prostate cancer following androgen ablation arises from growth factormediated mechanisms, in which angiogenesis is an important component. Clinical trials with thalidomide have shown partial response in a proportion of patients with androgen-independent prostate cancer. These trials provide some insight into possible mechanisms of cancer progression, and support future research into the role of thalidomide as an adjunctive therapeutic agent. Progression of renal cell cancer is poorly understood. Management of metastatic disease is unsatisfactory, since the tumours show little response to chemotherapy or radiotherapy, while systemic immunotherapy is effective in a minority of patients. Several clinical trials in renal cell cancer have employed thalidomide, but results have generally not suggested substantial benefit. related cachexia. In addition, substantial interest surrounds the immunomodulatory, anti-inflammatory and antiangiogenic properties of thalidomide in the treatment of malignant diseases. A number of studies have demonstrated it has significant activity in the treatment of multiple myeloma (Singhal et al, 1999; Rajkumar et al, 2002) as well as other haematological malignancies. This article reviews the mechanism of action of thalidomide and its potential use as an anti-cancer agent in the treatment of urological malignancies.

I. Introduction Thalidomide was introduced in the 1950’s as an over-the-counter sedative and subsequently an anti-emetic for the treatment of pregnancy-induced morning sickness. Reported association between thalidomide use and severe congenital abnormalities prompted its withdrawal in 1961. By this time about 12,000 children had been affected with birth defects, including dysmelia (stunted limb growth) and deformities of the heart, kidneys, eyes and spinal cord. Thalidomide has re-emerged as a drug of scientific interest following recognition of its activity in reactive lepromatous leprosy (Sheskin, 1965). Thalidomide was approved by the U.S. Food and Drug Administration in 1998 as a treatment for the acute cutaneous manifestations of erythema nodosum leprosum and as maintenance therapy to prevent its recurrence. Thalidomide has since been shown to be effective in the treatment of other nonmalignant diseases including mycobacterium tuberculosis infection, graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosis, Crohn’s disease and HIV-

II. Pharmacology Thalidomide –"- (N-phthalimido )glutarimide– contains a phthalimide ring and a glutarimide ring (Figure 1). The glutarimide ring has a chiral centre with dextrorotatory (R) and levorotatory (S) forms. The enantiomers have different biological properties; sedative effects are associated with the R-enantiomer, whereas immunological effects are more closely associated with the S-enantiomer. They readily interconvert at physiologic 65


Pridgeon and Drake: Thalidomide and its use in urological malignancies an axonal neuropathy. By nine months all three patients who continued the trial developed a neuropathy. Further reported side effects of thalidomide include somnolence, nausea, dry mouth and skin, constipation, increased appetite, headache, hypertension, bradycardia, dizziness and orthostatic hypotension, altered temperature sensitivity, irregularities in menstrual cycles, hypothyroidism and peripheral oedema (Stirling, 2000). Constipation can be controlled with an aggressive laxative regimen. Somnolence, nausea, and skin rashes are dosedependent and generally resolve with discontinuation of therapy. Thalidomide-induced sedation can be antagonized with the central nervous system stimulants methylphenidate and methylamphetamine (Somers, 1960) but this has not been evaluated in controlled trials. The impact of somnolence can be minimized by administration of the drug at bedtime. The immunomodulatory action of thalidomide does not appear to be associated with an increased incidence of infections; evidence suggests that thalidomide does not directly suppress lymphocyte proliferation (Geitz et al, 1996) although inhibition of proliferation of already stimulated lymphocytes has been shown (Keenan et al, 1991). Thalidomide is teratogenic when taken in early pregnancy (Nowack, 1965). The drug was found to be teratogenic only during days 34-50 after the last menstruation. To safeguard patients and prevent foetal exposure, the U.S. manufacturer of thalidomide developed a restricted prescribing and dispensing program called S.T.E.P.S. (System for Thalidomide Education and Prescribing Safety) (Warren, 2001). This program requires registration of doctors and pharmacists involved in the dispensing process as well as the provision of educational material. Women of child bearing age are instructed to use two forms of contraception and undergo pregnancy testing before and during a course of treatment. Males are required to abstain from sexual intercourse or use a condom during therapy and up to one month thereafter. The optimal dosing strategy for thalidomide is uncertain, and is influenced by the biology of the disease being treated, the idiosyncratic nature of the adverse effects profile and the unpredictable gastrointestinal absorption. Most clinical trials for cancer have used doses from 100-1200 mg per day either as a single dose at night or in two divided doses. Intolerance of the side effect profile is an important factor in patient withdrawal from trials.

Figure 1: The chemical structure of thalidomide

pH making total separation of their clinical effects unfeasible. Thalidomide has poor solubility in both water and ethanol. Its poor aqueous solubility means that no intravenous preparation is available and enteral absorption of the drug is slow. The time to peak plasma levels following oral administration increases with increasing dose, ranging from 3 to 6 hours (Warren, 2001). Thalidomide undergoes rapid spontaneous non-enzymatic hydrolysis which is affected by both pH and temperature, generating at least 12 metabolites in humans. In vitro studies suggest thalidomide induces cytochrome P-450 isoenzymes in rats. However, in humans thalidomide does not inhibit or induce its own metabolism over a 21-day period, and thus very little metabolism of thalidomide is thought to occur via the hepatic cytochrome P-450 system (Tsambaos et al, 1994; Scheffler et al, 1999). The mean elimination half life in healthy individuals is 4.7 hours with an apparent mean clearance of 10 and 21 L/h for the R- and S- enantomers respectively. Both single and multiple dosing of thalidomide in older prostate cancer patients revealed a significantly longer half-life at a higher dose (1200 mg daily) than at a lower dose (200 mg daily) â&#x20AC;&#x201C; 18.52h and 6.52h respectively (Figg et al, 1999). Metabolites of thalidomide are rapidly eliminated in urine, but little unchanged thalidomide is excreted this way.

III. Adverse drug effects Thalidomide-associated peripheral neuropathy is generally characterized by painful symmetric paraesthesiae in the toes and feet; electrophysiologic findings suggest axonal degeneration without demyelination (Fullerton and O'Sullivan, 1968; Aronson et al, 1984; Clemmensen et al, 1984). Related symptoms include muscle cramps or weakness, signs of pyramidal tract involvement, and carpal tunnel syndrome. Improvement is usually observed with discontinuation of therapy; however, cases of irreversible or longstanding sensory loss have been reported. The incidence of neuropathy is increased in older patients and after administration of high cumulative doses. Molloy et al, (2001) investigated the incidence of thalidomide-induced neuropathy in a group of patients treated for metastatic prostate cancer. Seventy five percent of the patients who remained on treatment for 6 months or longer developed

IV. Mechanisms of action The anti-inflammatory, anti-angiogenic and immunomodulatory properties of thalidomide have been studied both in vitro and in vivo and are likely to be due to its ability to affect cytokine production and cell function. The understanding of the in vivo modes of action of thalidomide have been difficult to elucidate due to the spontaneous inter-conversion between S- and Renantiomers, making total separation of their effects impossible. Thalidomide appears to undergo an as yet uncharacterised metabolic activation since its in vitro

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Cancer Therapy Vol 3, page 67 effects are modest when compared to its observed potency in vivo (Bauer et al, 1998; Stirling, 2000). Initial clues as to thalidomide’s role in angiogenesis came from observations by D’Amato et al, who found that it inhibited neovascularisation induced by basic fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) in the rabbit cornea micropocket assay (D'Amato et al, 1994). Because this inhibition occurred only after oral administration of thalidomide it has been suggested that an active metabolite is formed in vivo (D'Amato et al, 1994; Bauer et al, 1998). Animal studies support the antiangiogenic effect of thalidomide since treatment has been shown to decrease vascular density in granulation tissue (Adeoti et al, 1998). The antiangiogenic effects are thought to be clinically beneficial in diseases dependant on new vessel formation Thalidomide decreases production of tumour necrosis factor-· (TNF-") in monocytes and macrophages by accelerating degradation of TNF-" mRNA (Sampaio et al, 1991; Moreira et al, 1993). An alternative action is via binding to "1-acid glycoprotein, which is known to have anti- TNF-" activity (Keifer et al, 2001). In addition thalidomide decreases the binding activity of the transcription factor nuclear factor-!B (NF-!B), an antiapoptotic gene product involved in immune responses and cellular growth, which in turn controls the activation of the TNF-" gene (Turk et al, 1996). Anti TNF-" activity may

play a role in thalidomide treatment of multiple myeloma. Increased pre-treatment TNF-" levels appear to predict progression- free survival after thalidomide. In addition DNA polymorphisms involving the TNF-" gene have been correlated with response to thalidomide treatment in myeloma (Thompson et al, 2003). A role for the enzyme CYP2C19 has recently been reported for thalidomide metabolism. It is suggested that thalidomide undergoes hydroxylation under the influence of this enzyme (Ando et al, 2002). A mechanism has been proposed whereby CYP2C19 converts thalidomide to a bioactive form that interferes with NF!B activation. NF!B is usually bound to the inhibitory protein I!B" in the cytoplasm and dissociates from this complex following activation of the enzyme I!B kinase (I!K). NF!B is then free to translocate into the nucleus to activate genes which promote cellular proliferation, angiogenesis and inhibition of apoptosis (Figure 2). The hydroxylated metabolites of thalidomide reduce I!K activity through interleukin-1‚ (IL-1#) and TNF-" thereby inhibiting the release of free NF!B (Ando et al, 2002). Pro-angiogenic factors other than TNF-" are altered by thalidomide. Thalidomide inhibits interleukin-6, a potent growth factor for malignant cells, and interleukin12 production by monocytes (D'Amato et al, 1994).

Figure 2 : Mechanisms of action of thalidomide. Metabolites formed from the oxidation of thalidomide by CYP2C19 inhibits TNF-" and/or IL-1# activation of I!K. This in turn prevents the dissociation of of I!K from the I!K-NF!B complex. NF !B usually enters the nucleus to induce genes that promote angiogenesis, cellular proliferation and inhibition of apoptosis as well as promoting further TNF-" transcription; this function of NF!B is thus inhibited by thalidomide and its metabolites. Thalidomide also promotes the degredation of TNF-" mRNAand interacts with "-1 acid glycoprotein to inhibit the actions of TNF-" function.

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Pridgeon and Drake: Thalidomide and its use in urological malignancies Further effects of thalidomide may be relevant to clinical management of malignancy. Inhibition of polymorphonuclear leukocyte chemotaxis has been reported in an in vitro model (Faure et al, 1980). Thalidomide also inhibits neutrophil chemotaxis (Dunzendorfer et al, 1997). Leukocyte migration to sites of inflammation may be affected by downregulation of cellular adhesion molecules (Settles et al, 2001). Thalidomide has been reported to decrease the ratio of circulating T-helper cells to suppressor T cells in healthy subjects as the result of a reduction in the production of helper cells and increase in production of suppressor cells. Another possible mode of action for thalidomide involves modulation of cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins (Fujita et al, 2001). COX-2 is highly expressed in various human cancers including prostate cancer, and has been shown to be required for angiogenesis in a rat corneal model (Yamada et al, 1999; Daniel et al, 1999). The results are in keeping with the anti-angiogenic response of human prostate cancer cells treated both in vitro and in vivo with a COX-2 Inhibitor (Kirschenbaum et al, 2001). Thalidomide has been demonstrated to inhibit COX-2 and subsequent prostaglandin-E2 biosynthesis in a dose dependant manner (Fujita et al, 2001).

stage. Mean microvessel counts are significantly greater in the primary tumours of patients with metastatic disease than in those without metastases, and a correlation between microvessel density and increasing pathological grade has been observed. Increased microvascularity also correlates with a poorer prognosis (Lissbrant et al, 1997). Rogatsch et al (1997) investigated whether the quantification of tumour microvessels can be reliably applied to prostatic core biopsies, as a means of detecting likely local tumour extension prior to radical prostatectomy. They observed a high degree of correlation between microvessel density in biopsies and the subsequent prostatectomy samples. The median microvessel density in core biopsies predicted the local tumour stage. Malignant prostatic epithelial cells show increased expression of numerous angiogenic substances, including; basic FGF, (Mansson et al, 1989; Gleave et al, 1992; Nakamoto et al, 1992; Warren, 2001) transforming growth factor (TGF) " and -#, (Ikeda et al, 1987; Hofer et al, 1991; Harper et al, 1993; Truong et al, 1993; Cohen et al, 1994; Eastham et al, 1995; Steiner et al, 1994) epidermal growth factor (Ching et al, 1993; Ware, 1993) and platelet derived growth factor (Fudge et al, 1994; Sitaras et al, 1988). Elevated serum levels of TNF-" and basic FGF can be seen in patients with prostate cancer (Meyer et al, 1995; Nakashima et al, 1995).

V. Prostate cancer Prostate cancer is the most commonly diagnosed malignancy affecting men beyond middle age in developed countries. Although detection of early prostate cancer is increasing, many patients still present with metastatic disease. Hormone treatment in the form of androgen deprivation is an established therapeutic option for metastatic prostate cancer. In patients who respond to hormone manipulation, subsequent clonal expansion of hormone insensitive cancer cells results in loss of response with a mean time to tumour progression of less than 18 months. Prostatic Specific Antigen (PSA) is used as a clinical marker of tumour load and a serial increase in PSA values after initial successful androgen ablation usually indicates clinical progression to androgen independent prostate cancer (AIPC). Androgenindependence can be defined as a rising PSA value of at least 20ng ml-1 on two consecutive occasions after the nadir of response to androgen ablation therapy, or a rise of at least 5ng ml-1 if the absolute PSA value was less than 20 ng ml-1 (Bubley et al, 1999). Various agents have been tested in an attempt to achieve second-line response in AIPC, including; corticosteroids, diethylstilboestrol, tamoxifen, aminoglutethamide, ketoconazole, suramin, estramustine, taxanes, mitozantrone and herceptin. Benefits in terms of disease suppression and survival are uncertain. The prognosis for patients with hormone refractory disease is poor; median overall survival is 12 18 months.

B. Clinical trials With the emergence of evidence implicating the involvement of angiogenesis in prostate cancer, the use of thalidomide as a potential therapeutic agent has been investigated. Figg et al, (2001b) have compared low-dose (200mg/day) and high dose (up to 1200 mg/day) thalidomide in patients with AIPC. The high dose regime commenced at 200mg/day with incremental rises of 200mg /day every two weeks up to a maximum of 1200mg where tolerated. Fifty patients were randomised to the low dose arm. In the high dose arm, thirty percent of patients were unable to tolerate doses of above 200mg/day due to the side effect profile. As a result of the high incidence of adverse effects, and since no patient showed a substantial decline in PSA, the high dose arm was discontinued after 13 patients were enrolled. The median time on study was 2.1 months for the low dose arm and 2.0 months for the high dose arm (range 35-247 days). Response to treatment was evaluated in terms of PSA reduction, changes in metastatic lesions on bone scan and measured tumour load as detected on serial CT scans. The assessment of circulating growth factors (basic FGF, VEGF, TNF-" and TGF-#) was also carried out. Reductions in PSA were observed in 58% of patients in the low dose arm and 68% of the high dose arm. Nine out of the 63 patients (14%) showed declines in PSA of $50%, all of whom were in the low dose arm of the study. The failure of PSA response in the high dose arm may result from the up-regulation of PSA secretion which is seen in prostate cancer cell lines exposed to thalidomide (Dixon et al, 1999). Of the patients who had measurable disease on CT scanning, 14% had some reduction in tumour load. Two patients had radiographic improvement of bone scan lesions and both

A. Tumour biology and angiogenesis The role of angiogenesis in the growth and metastasis of prostate cancer has been demonstrated by Weidner et al, (1993) who showed that increased microvessel density in prostate cancer specimens can predict advanced tumour 68


Cancer Therapy Vol 3, page 69 had sustained reductions in serum PSA. Declines in serum TGF-# were seen in the eight patients with PSA reductions at four months. There were also reductions in basic FGF in six of these patients. Figg and colleagues concluded from this trial that thalidomide may have modest anti-tumour activity through its anti-angiogenic properties in patients with hormone refractory prostate cancer. Drake et al, (2003) examined the benefits of low dose thalidomide in patients with rising PSA after initial response to hormone manipulation therapy. Response to treatment was evaluated in terms of serum PSA reduction and growth factor levels. Twenty patients were enrolled in this study and given 100mg thalidomide once daily at bedtime. The mean time on study was 109 days (range 4–184 days) with 16 patients continuing thalidomide treatment for more than two months. Three men had PSA falls of $50% with five other patients showing some decline. Of the 16 men who tolerated treatment for more than two months, six showed a fall in their serum PSA by a mean of 48%. In this study serum growth factors were analysed in 11 patients at three months. There was an overall mean rise in both basic FGF and VEGF. However, when analysed according to PSA response, five out of the six men who manifested a decline in PSA showed a decline in mean values of both basic FGF and VEGF; at the time of loss of PSA response, there was a coincident rise in growth factor levels. Patients who demonstrated increasing PSA levels had associated rises in growth factors. Three patients discontinued low dose thalidomide due to adverse effects. The most commonly reported adverse reaction was constipation (n=9) followed by sedation hangover (n=3) and dizziness (n=2). Clinical trials of single agent docetaxel in AIPC have demonstrated response rates of 20-60% (Logothetis, 2002). Accordingly, thalidomide has also been studied in combination with docetaxal in a phase II trial enrolling 75 patients with AIPC (Figg et al, 2001a; Dahut et al, 2004). Patients were treated with intravenous docetaxel, with or without a single night-time dose of thalidomide 200mg. 35% of those receiving docetaxel alone showed decrease in PSA of at least 50%, while 53% of patients in the combination arm demonstrated an equivalent response. At 18 months, overall survival in the docetaxel group was 42.9%. Compared with 68.2% in the combined arm. Of the first 43 patients treated with docetaxel and thalidomide, nine patients developed deep venous thrombosis and a further three suffered a cerebrovascular incident. There were no thromboembolic complications in the docetaxel group. Subsequent patients enrolled into the combined group were given low molecular weight heparin, preventing further thrombotic events. Thalidomide combination therapy with the cytotoxic agents paclitaxel and estramustine has been evaluated (Daliani, 2003). Thirty patients with AIPC were given weekly paclitaxel for 2 of 3 weeks together with oral estramustine and escalating doses of thalidomide, titrating from 200 mg a day to 600 mg a day. Seventy-two percent of the 25 evaluable patients achieved a sustained decline in PSA levels of 50% or more. Twelve percent showed a drop of 80% or more. The men who experienced a drop in

PSA also reported less bone pain. Four of the 29 men evaluable for toxicity developed Grade 3-4 deep venous thrombosis, despite warfarin prophylaxis, requiring treatment discontinuation in two patients. There were no cases of grade 2-4 neuropathy. The U.S. National Cancer Institute is undertaking a phase II trial of Estramustine, Docetaxel and Thalidomide in AIPC. A phase II trial using a combination of thalidomide (100-200mg) with granulocyte macrophage colony stimulating factor in 17 AIPC patients with soft tissue or bone disease has been carried out (Dreicer et al, 2002). Partial response was seen in five of 13 evaluable patients. Treatment was generally well tolerated

VI. Renal cell carcinoma Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies. Incidence of RCC has risen over the last 30 years, with greater use of ultrasonography and CT scanning increasingly detecting incidental tumours. The identification of early tumours has seemingly led to improved survival. However, the overall mortality rates for RCC have increased over this time, suggesting a possible change in tumour biology, perhaps due to environmental factors (Chow et al, 1999). One third of patients with RCC present with metastatic disease and 40% of the rest eventually develop distant metastases. The prognosis for these patients is poor, as RCC is resistant to most chemotherapeutic agents. Patients who have nonbulky metastatic disease and who are of good performance status may be suitable for immunotherapy. Nonetheless, only 10-15% of patients experience an objective response to immunotherapy with either interleukin-2 (IL-2) or "interferon (·-IFN)(Negrier et al, 1998).

A. Tumour biology and angiogenesis RCC is one of the few tumours in which spontaneous regression is recognised, with an estimated incidence of 0.3% (Vogelzang et al, 1992). Most cases have been in patients with pulmonary metastases and have occurred after nephrectomy, but regression of primary RCC has also been documented even in the absence of any treatment (Vogelzang et al, 1992). RCC’s are highly vascular tumours, as demonstrated by the distinctive neovascular pattern on renal angiography. The up-regulation of a variety of angiogenic factors and the renin angiotensin system has been reported (Nicol et al, 1997; Hii et al, 1998; Horie et al, 1999). Increased levels of mRNA for VEGF have been found in the majority of hypervascular renal cell carcinomas, while hypovascular tumours have exhibited low levels of this transcript (Brown et al, 1993; Takahashi et al, 1994). Increased levels of VEGF have also been found in the serum of patients with RCC and a correlation with stage and grade has been noted (Kato et al, 2000). Elevated serum levels of basic FGF have also been demonstrated in RCC and other possible angiogenic growth factors contributing include placental growth factor, TGF-#1, angiogenin, interleukin-8 and hepatocyte growth factor (Campbell, 1997). Sporadic and familial RCC’s commonly demonstrate mutations or deletions in the Von Hippel Lindau gene, and this genetic change may lead to 69


Pridgeon and Drake: Thalidomide and its use in urological malignancies increased expression of VEGF (Stebbins et al, 1999). The angiogenic dependence of RCCâ&#x20AC;&#x2122;s and the involvement of such genes and growth factors have led to the investigation of a variety of antiangiogenic treatments including thalidomide, endostatin, interleukin-2, squalamine and neutralising antibodies to VEGF.

metastatic melanoma, renal cell carcinoma and ovarian and breast cancer. Three of 18 patients with RCC showed a partial response to treatment (100mg daily). A further three patients had stable disease for at least three months. A further phase II trial by the same group evaluated the use of high dose oral thalidomide (600mg daily) (Stebbing et al, 2001). 25 patients were enrolled in this study with advanced metastatic RCC. Nearly all had undergone prior surgery and systemic immunotherapy or immunochemotherapy. Progressive disease was identified in all patients prior to study entry. Three patients were removed from the trial at an early stage due to toxicity

B. Clinical trials Numerous trials employing thalidomide in RCC have been undertaken. These are summarised in Table 1. Eisen et al, (2000) carried out a study to assess the efficacy and toxicity of thalidomide in the treatment of patients with

Table 1. Summary of trials of thalidomide in renal cell carcinoma Trial

Eisen, (1999)

No. of Additional Thalidomide Prior patients drug dose nephrectomy

No

% Stebbing No (2001) % Novik No (2001) % Motzer No (2002) % Daliani No (2002) % Li (2001) No

Srinivas (2002)

% No

18

none

100mg

Efficacy: Complete response

Partial response

n/s

0

3

Stable disease /minor response 13

17 2

Progres Median Median sion free survival time to survival progression 17% >3 months

n/s

n/s

72 12

n/s

9 months

n/s

n/s

n/s

n/s

32% > 6 months

n/s

4 months

n/s

18.3 months

4.7 months

32% >5 months

n/s

n/s

n/s

9.3 months

n/s

24% >6 10 months months

n/s

25

none

100-600mg

24

0 0

27

none

100-1000mg

96 21

0 0

9 0

54.5 7

0 0

0 0

26 16

26

none

200-800mg

78 15

20

none

200-1200mg

58 19

0 0

0 2

64 9

0 0

10.5 2

47 9

36

none

200-1200mg

95 31

14

none

200-1200mg

86 11

0 0

6 0

31 6

400-1200mg

79 32

0 0

0 0

46 9

400-1200mg

80 19

0 1

6 2

27 3

n/s

3.5 months

2.3 months

100-400mg

79 n/s

0 0

4 0

12.5 0

n/s

n/s

n/s

100-1000mg

17

0 0

0 2

0 8

n/s

68 weeks

n/s

200-1000mg

57 190

0 0

6.7 5

26.7 50

n/s

10.8 months

n/s

100-200mg

74 29

0 1

3.1 1

31.3 0

n/s

n/s

n/s

200-600mg

93 15

3 1

3 5

0 2

n/s

n/s

n/s

100

10

50

20

% Escudier No 40 none (2002) % Minor No 29 none (2002) % Nathan No 13 IFN-"2a (2002) % IFN-" Clarke No 30 (2004) % Gordon No 175 in IFN-"2b (2004) thalidom ide arm % Olenki No 31 IL-2 (2003) % Amato No 15 IL-2 (2002) %

70


Cancer Therapy Vol 3, page 71 and five other patients required dose reduction due to peripheral neuropathy. Of the 22 assessable patients, two (9%) achieved a partial response and 12 had stable disease for 12 months, with dose reduction due to toxicity. In many of these patients there was a significant reduction in serum TNF-". Minor et al, (2002) investigated the activity of thalidomide in patients with advanced RCC in a phase two study involving 29 patients. All patients had advanced progressive metastatic RCC disease and many were poor performance status. 72% had received prior therapy in the form of interleukin-2, "-interferon and cytotoxic chemotherapy. Patients were started on a daily dose of thalidomide of 400mg rising in increments of 200 mg to a maximum dose of 1200 mg. Tumour responses were evaluated using South Western Oncology Group criteria and in some patients serum levels of VEGF 165 was monitored. Of the 24 patients assessable for response, only one demonstrated a partial response. Three patients had stable disease for over six months. The median time to progression for all 29 patients was 2.3 months and median survival was 3.5 months. VEGF 165 was measured in 8 patients and no obvious change in plasma levels was demonstrated. The largest trial to date involved 40 patients treated with thalidomide doses starting at 400mg daily rising to 800mg and then 1200mg (Escudier et al, 2002). At 12, 18 and 24 weeks 31, 25 and 17 patients remained on treatment respectively. At nine months, only eight patients were still on study therapy, with all patients stopping treatment after one year due to severe neuropathy. After 6 months, two patients (5%) achieved partial response, with 9 patients (22%) having stable disease. The one year survival was 38%. In this study, neuropathy and thromboembolism were significant side effects. A further trial examining the use of escalating doses of thalidomide was carried out by Daliani et al, (2002). Twenty patients with metastatic RCC were enrolled and treated with doses starting at 200mg, increasing in weekly increments up to a target maximum dose of 1200mg per day. Nineteen patients were evaluable for response, of whom two patients (10.5%) had a partial response and nine patients had stable disease. Median time to progression was 4.7 months (range 0.7-31.3 months). Thirteen patients remained on the study at three months and 12 of these tolerated a maximum dose of 1200mg. Delayed dose reductions were required for three patients because of peripheral sensory neuropathy. A further three patients developed thomboembolic events believed to be related to thalidomide. Motzer used doses of thalidomide up to 800mg in advanced RCC (Motzer et al, 2002). Twenty five patients were assessable for response. No patient achieved a partial or complete response; stable disease was observed in 16 patients (64%), which was maintained for a mean duration of 6 months. The median time to disease progression was 4 months. The one-year survival was 57%. A further small study recruited 14 patients (Srinivas and Guardino, 2002) comparing low dose thalidomide (200mg/day) to higher escalating doses (800–1200mg/day). Six patients (46%) achieved stable disease, mainly in the low dose arm, with

no objective responses. Stable disease was the best response obtained by Novik et al, (2001) in a phase II trial involving 27 patients with metastatic RCC, including lung, brain and liver metastases. Li et al, (2001) treated 36 patients with thalidomide (200-1200mg) All of these patients had failed to respond to prior interleukin-2 therapy. Seventeen of the patients managed to tolerate the maximum dose. Partial responses were seen in 2 of the 29 evaluable patients with stable disease in 9 further patients. Thalidomide has also been investigated in combination with immunotherapeutic agents for the treatment of RCC. In a phase II trial to examine the efficacy of interferon "-2a (IFN"-2a) with thalidomide (Nathan et al, 2002), no responses to treatment were seen. Five patients experienced serious adverse side effects, which were attributed to the membrane destabilising effects of both of these agents. The authors concluded that caution is needed in combining these two therapies. A further phase II trial (Clarke et al, 2004) looking at thalidomide in combination with IFN-" in 30 patients reported an objective response rate of 7% with no complete responses, 2 partial responses and 8 patients achieving stable disease. One third of patients experienced toxicity that required discontinuation of thalidomide. In a phase III study of the Eastern Co-operative Oncology Group (Gordon et al, 2004), interferon–"2b (IFN-"2b) in combination with thalidomide (200-1000mg daily) was compared with IFN–"2b alone. There was no difference in objective response, but there was a higher percentage of patients with stable disease in the combination therapy arm (31.3% vs 18.5%). This is consistent with thalidomide’s known cytostatic activity, but there was no discernable difference in the progression free survival curves. Furthermore, no clinically significant difference in quality of life was achieved between the arms at baseline or through treatment. As single therapy for metastatic RCC, IL-2 can elicit clinical responses, but only in a minority of patients. A phase I trial of combination IL-2 and oral thalidomide involving 31 patients (Olencki et al, 2003) demonstrated moderate to severe toxicity with one complete response and one partial response. More promising responses have been reported by (Amato et al, 2002), administering combination therapy for 9 months, followed by 6 months of thalidomide monotherapy. An initial phase I trial, involving only a small number of patients (n=15), showed 5 partial responses and 1 complete response. Later data from the same trial reported further responses, which were sustained for at least 12-18 months. A phase II trial recruited 37 patients, of whom 33 were assessable for response. This produced an objective response rate of 39% with 2 complete responses, 11 partial responses and 10 patients with stable disease.

VII. Conclusions Thalidomide has numerous biological actions; suppression of growth factor levels gives rise to antiangiogenic and tumour cytostatic effects, but also substantial adverse drug reactions. The biological actions have led to the application of thalidomide in various clinical scenarios on an empirical basis, particularly where 71


Pridgeon and Drake: Thalidomide and its use in urological malignancies human prostate tissue and cell lines. Mol Cell Biochem 126, 151-8.

current management options are limited. The current treatment options for AIPC and advanced RCC are primarily palliative. The events leading to progression in prostate cancer include angiogenesis. Clinical trials of thalidomide in AIPC indicate a response in a subgroup of patients, which correlates with alterations in growth factor levels. This has not been translated into a clinical management option as yet, but does provide some insight into the biology of androgen-independence and the potential for pharmacological manipulation of this complex process. The use of thalidomide in combination with other chemotherapeutic drugs appears more promising. RCCs are chemotherapy-resistant; objective responses to thalidomide also appear to be infrequent (Table 1). The greater clinical benefit with low dose thalidomide in certain clinical trials in AIPC and RCC indicates that the immunomodulatory effects of thalidomide can outweigh the cytokine responses.

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Dr. Simon Pridgeon

Dr. Marcus Drake

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Cancer Therapy Vol 3, page 77 Cancer Therapy Vol 3, 77-84, 2005

Genetic predisposition in preinvasive lesions of the breast Review Article

George C. Zografos1,*, Flora Zagouri1, Costantinos Fotiadis2, John Bramis1 1

1st Department of surgery, School of Medicine, Athens Univrsity, Greece, 3rd Department of surgery, School of Medicine, Athens University, Greece,

2

__________________________________________________________________________________ *Correspondence: George C. Zografos, Professor of Surgery, University of Athens, Vas. Sofias 101, 11521 Ampelokipi, Athens Greece; Tel: +302106426390; e-mail: gzografo@med.uoa.gr Key words: Genetic predisposition, precancerous lesions, breast cancer, histological models, invasine cancer, preinvasive cancer, ALH/LCIS, UDH/ADH, DCIS Abbreviations: atypical ductal hyperplasia, (ADH); atypical lobular hyperplasia, (ALH); ductal carcinoma in situ, (DCIS); lobular carcinoma in situ, (LCIS); Relative risk, (r.r); Van Nuys Prognostic Index (VNPI) Received: 25 January 2005; Accepted: 16 February 2005; electronically published: February 2005

Summary Preinvasive breast lesions, which include: atypical ductal hyperplasia, ductal carcinoma in situ, atypical lobular hyperplasia, and lobular carcinoma in situ, represent a spectrum of diseases some of which are more aggressive. Uncertainities in the prognosis have caused a controversial discussion about adequate treatment, with a wide range of treatment approaches from observation to mastectomy, resulting in diverse and confusing clinical recommendations. The aim of this review article is to help the clinician, in the common clinical practice, to estimate the risk for each individual patient of developing invasive cancer, by evaluating her genetic predisposition. Significant genetic changes are present in all preinvasive breast lesions. Knowledge of genetic alterations specific for certain phenotypes of premelignant disease, and for the subsequent transition to invasive carcinomas, should provide clues for the development of mechanism-based treatments. The aim of this review article is to identify genetic changes in preinvasive breast lesions and to correlate the relative risk of these lesions for the subsequent transition to invasive carcinoma, in order to give certain answers, and to improve treatment decision in the common clinical practice. According to our knowledge there is only one review article in the English literature focused on genetic changes in preinvasive lesions and their role in the clinical and therapeutical decision.

I. Introduction Breast cancer, is thought to involve through a sequential progression from normal to proliferate epithelium, to in situ, and finally to invasive carcinoma (Dupont and Page, 1985; Buerger et al, 1999a). Preinvasive lesions of the breast, which include: atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS), have an increased relative risk for the development of infiltrating carcinoma (Bradley et al, 1990; Rosen and Obermann, 1993; Fitzgibbons et al, 1998). It has become apparent, that preinvasive breast lesions represent a spectrum of neoplasmatic disease, some of which are more aggressive, while others display a more protracted course. Uncertainties in the prognosis have caused a controversial discussion about adequate treatment, with a wide rage of treatment approaches from observation to mastectomy, resulting in diverse and confusing clinical recommendations, distressing to both patients and clinicians (Buerger et al, 1999a; Schnitt, 2003).

II. Methods We searched for published manuscripts, of molecular markers with a positive prognostic value in all precancerous lesions of the breast, that means in UDH, ADH, DCIS, ALH, and in LCIS. We systematically searched MEDLINE from January 1982 to June 2004 using the Medical Subject Heading terms or key words: precancerous lesions of the breast, preinvasive breast lesions, ADH, DCIS, ALH, LCIS combined with genetic changes, genes, genetic predisposition, c-erbB2, p53, Ecathedrine, bcl-2, ki67, p27, apoptosis, apoptotic index, proliferation, proliferative index, necrosis, oestrogen 77


Zografos et al: Genetic predisposition in preinvasive lesions of the breast receptor. We manually searched bibliographies of original and review articles identified in MEDLINE.

true precursor of invasive carcinoma, and belongs to preinvasive lesions of the breast (Reis-Filho and Lakhani, 2003). ALH is “a small amount of LCIS”, with a relative risk of breast cancer 3. Differences in risk between ALH and LCIS were the main reason to distinguish these lesions (Page et al, 2003; Van de Vijver and Peterse, 2003). Over the past twenty years or so, a histological model of human breast cancer evolution was predominant. Stem cells in normal TDLUs give rise to atypical hyperplasias (ADH, ALH), which progress to in situ carcinoma (DCIS, LCIS), which eventually develop into invasive and metastatic disease (Allred et al, 2001) (Table 3). Although, recently new models have been proposed. Farabegoli et al, (2002) proved, that DCIS is a possible but not an obligate precursor of invasive breast cancer and suggest that, pure DCIS and DCIS associated with IDC may be genetically distinct (Boecker et al, 2001). The evolution from DCIS to IDC may follow multiple pathways and not a linear model. Leong et al, (2001) suggested that in most cases low-grade DCIS is associated with low-grade invasive carcinoma and high-grade DCIS with high-grade invasive carcinoma, ie, a horizontal progression. They supported that intermediate-grade DCIS is heterogenous, so it is possible that this group represents some cases of DCIS that have progressed from low-grade DCIS and cases that may progress to high-grade DCIS, so progression may not be entirely horizontal in intermediategrade DCIS (Table 4). It has become apparent though, that preinvasive lesions of the breast biologically and clinically represent a spectrum of neoplastic diseases, some of which are more aggressive. It is important to evaluate the individual relative risk of developing invasive breast carcinoma rather than the relative risk of each preinvasive lesion. Accounting for this fact, various morphological classification schemes of preinvasive lesions have been proposed (Schulze-Garg et al, 2000).

III. Preinvasive lesions of the breasthistological models of their progression to invasine cancer Preinvasive lesions of the breast, as noted above, include four diseases: ADH, DCIS, ALH, and LCIS that have an elevated risk for developing invasive carcinoma (Table 1). The most compelling evidence that all these lesions may be procusors comes from studies showing identical genetic abnormalities with synchronous ipsilateral IBC (O'Connell et al, 1998) (Table 2). ADH represents a proliferative lesion that fulfils some but, not all the criteria for a diagnosis of low-grade, non-comedo type DCIS and represents usually an early stage in the formation of low-grade DCIS. ADH, identified in only 4% of bening breast biopsies, is associated with a relative risk for subsequent cancer 4.4. When ADH is combined with a positive family history the relative risk for invasive cancer arises to 9.7 (Dupont et al, 1993; Oyama et al, 1999; Van de Vijver and Peterse, 2003; Locke et al, 2004). DCIS of the mammary gland, that represents an early, pre-invasive stage in the development of invasive breast cancer, is increasingly diagnosed since the introduction of high-quality mammography screening (Schulze-Garg et al, 2000). Presently constitutes 10-20% of screen detected malignancies of the breast (Schnitt et al, 1988; Zografos et al, 2004), and it is known that a diagnosis of this form confers an 8-10-fold elevated risk for the development of infiltrating ductal breast cancer (Page et al, 1982; Lagios, 1995). LCIS, an uncommon lesion with a distinctive appearance, is a “marker of increased risk” rather than a Table 1. Precancerous lesions of the breast

Relative risk (r.r) • Atypical Ductal Hyperplasia (ADH) • Ductal Carcinoma In Situ (DCIS) • Atypical Lobular Hyperplasia (ALH) • Lobular Carcinoma In Situ (LCIS)

4,4 8–10 3 7

Table 2. Genetic predisposition in precancerous breast lesions

ADH: ALH: DCIS: LCIS:

Genetic predisposition Losses 1q, 2p, 6q, 9p, 11p, 11q, 13q, 14q, 16q, 17p, 17q, Xq 11q, 16p, 16q, 17p, 22q 1p, 7q, 2p, 2q, 3p, 3q, 4p, 6p, 6q, 7p, 7q, 8p, 8q, 9p, 11p, 11q, 12p, 13q, 14q, 15q, 16p, 16q, 17p, 17q, 18q, 21q 11q, 13q, 16p, 16q, 17p, 17q, 22q

78

Gains Unknown 6q 1q, 3q, 6q, 6p, 8q, 17q, 20q, Xq 6q


Cancer Therapy Vol 3, page 79 Table 3. Fischerâ&#x20AC;&#x2122; s model

Table 4. Horizontal progression

Nevertheless, these approaches still leave a host of open questions with respect to practical and conceptual matters, as mirrored by our difficulties in diagnosis and treatment.

IV. Classifications breast disease

of

V. Genetic predisposition preinvasive breast lesions

in

It has become apparent, that preinvasive lesions of the breast are a heterogenous group and a uniform approach to treatment is therefore not appropriate. Some lesions require no treatment other than excisional biopsy; others benefit from complete excision plus radiation therapy, and some will require mastectomy. The challenge is to use available clinical and pathologic data to select the most appropriate therapy for each individual patient. Significant genetic changes are present in all preinvasive breast lesions. Knowledge of genetic alterations specific for certain phenotypes of premelignant disease, and for the subsequent transition to invasive carcinomas, should provide clues for the development of mechanism-based treatments (Schulze-Garg et al, 2000) (Table 2).

preinvasive

Currently the decision for treatment of preinvasive lesions is solely based on morphological criteria. In fact, there is no specific classification for ADH, ALH, LCIS while for DCIS the Van Nuys classification is the most widely accepted method for risk estimation and has replaced the Holland, the Bellamy, the Leal and the Lagios classifications (Leong et al, 2001). The Van Nuys Prognostic Index (VNPI) combines three significant predictors of local reccurance: tumor size, margin width, and pathologic classification (nuclear grade, comedo-type necrosis). Scores of 1 (best) to 3 (worst) were assigned for each of the three predictors and then totaled to give an overall VNPI score ranging from 3 to 9, by which DCIS is classified into three groups with low, intermediate, or high risk of local reccurence after breast conserving therapy (Bellamy et al, 1993; Silverstein et al, 1996) (Table 5). It is obvious that, genetic predisposition associated with specific forms of preinvasive lesions and their progression to invasive carcinoma should provide improved and, more reliable tools for the diagnosis and prognosis.

A. ALH/LCIS Loss of material from 16p, 16q, 17p, and 22q and gain of material from 6q have been found at similar high frequencies in both LCIS and atypical lobular hyperplasia (ALH) (Lu et al, 1998; Moinfar et al, 2000; Reis-Filho and Lakhani, 2003). Comparison of the CGH results from LCIS/ALH with those from DCIS and invasive carcinoma showed some similarities at the chromosomal level, but it also showed significant differencies, including gain of 1q and 8q and evidence for genomic amplification, which were not found in LCIS/ALH (Lu et al, 1998).

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Zografos et al: Genetic predisposition in preinvasive lesions of the breast

Table 5. Van Nuys prognostic index scoring system

Van Nuys prognostic index scoring system Size (mm) Score I: less than 15 mm Score II: 15-40 mm Score III: more than 41mm Margin width (mm) Score I: 10mm or more Score II: 1 – 9mm Score III: < 1mm Pathologic classification (nuclear grade, necrosis) Score I: absence of high nuclear grade, absence of comedo-type necrosis Score II: absence of high nuclear grade, presence of comedo-type necrosis Score III: presence of high nuclear grade, presence of comedo-type necrosis Total VN score 3 – 4: well differentiated DCIS 5 – 6 – 7: intermed differentiated DCIS 8 – 9: poorly differentieted DCIS

other hand, cytoceratin expression can be influenced by nutritional and hormonal factors as has been shown for vitamin A and oestrogens (Moll et al, 1982; Raju et al, 1990; Aldaz et al, 1995; Clover et al, 1997). This study provides evidence that cells undergoing malignant transformation tend to be fairly advanced in the glandular lineage of differentiation (Boecker et al, 2002).

B. UDH/ADH Lakhani et al, (1995) demonstrated that LOH identified at loci on 16q and 17p in DCIS is also present in ADH with a similar frequency. Their findings also demonstrated that ADH is a monoclonal, and hence, neoplastic proliferation rather than a hyperplastic (polyclonal) condition as its name suggests (Noguchi et al, 1994; Walker, 2001) Similar results were reported by Amari et al, (1999). As expected, owing to the morphological overlap with low grade DCIS, losses of 16q and 17p were the most frequent changes found in ADH (Reis-Filho and Lakhani, 2003). Gong et al, (2001) and Gong et al, (2001) saw in their study frequent sharing of genetic changes between ADH and UDH in the same biopsy. These data support the likelihood that UDH is a precursor of ADH, at least in some cases representing neoplastic growth. The frequencies of 16q and 17p losses suggest that alterations of candidate genes located in these chromosomal regions may play a role early in breast carcinogenesis. Candidate genes that might be responsible for some of these losses include E-cathedrin on 16q and p53 on 17p, also17q12 is the locus of erbB2/HER2-neu and 11q13 the locus of cyclin D1 (Buerger et al, 1999b) On the other hand, Boecker et al, (2002) and Otterbach et al, (2000) supported the idea that UDH of the breast is a committed stem (progenitor) cell lesion distinct from ADH and DCIS, as UDH appears to be a Ck5-positive committed stem (progenitor) cell lesion with the same differentiation potential as seen in the normal breast. This is in sharp contrast to ADH/DCIS, which display the differentiated glandular immunophenotype (Ck8/18/19positive, but Ck5-negative) (Bocker et al, 1992; Moinfar et al, 1999; Boecker et al, 2001) There are two possible explanations for the lack of cytokeratin 5 in neoplastic breast epithelia. On the one hand, neoplastic cells of ADH, DCIS, and infiltratig carcinomas may develop from Ck 5negative glandular precursor cell, since cytoceratin polypeptide patterns are largely preserved during malignant transformation (Kasami et al, 1997). On the

C. DCIS It is known, that significant qualitive and quantitive genetic changes are assossiated with clinical stage and morphological phenotype. According to Aldaz et al, (1995) in DCIS there is a tendency for association between high nuclear grade and higher frequency of allelic losses and imbalances. This is in agreement with previous histopathological studies that indicated that high nuclear grade appears to identify subsets of DCIS with worse prognosis (Radford et al, 1993) The broad spectrum of phenotypically heterogenous DCIS with significantly different aberrations raises the question of multiple pathways for invasive breast cancer with DCIS as a precursor lesion. Considering the wide rage of genetic alterations overall and, particularly, within the subgroups of DCIS, it is obvious that DCIS is far more heterogenous as a group than its morphology suggests. The loss of genetic material of 16q22-24 is almost exclusive restricted to DCIS, well and intermediately differentiated (James et al, 1997; Buerger et al, 1999a). Studying allelic imbalances, a correlation of loss of heterozygosity of 16q22-24 with the grading of the tumours was discussed in cotroversial form (Tsuda et al, 1994, 1995; Radford et al, 1995; Fujii et al, 1996) Losses of 16q material were seen almost exclusively in well- and intermidiately differentiated DCIS. These two groups differed in the average number of genetic imbalances, 2.5 and 5.5 respectively. Additionally, a higher frequency of gains of 1q and losses of 11q material was seen in intermediatellydifferentiated in contrast to well differentiated DCIS. The

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Cancer Therapy Vol 3, page 81 high overall frequency of LOH of 16q in poorlydifferentiated DCIS might reflect a gross genetic instability of this chromosomal locus. Losses of putative tumour suppressor genes such as BBC1 and E-cathedrin therefore seem to mark an important stage during the onset of a low-grade neoplastic disease (Cleton-Jansen et al, 1995; Gupta et al, 1997) DCIS poorly differentiated reveals a decreased rate of gains of 1q and losses of 16q compared with DCIS, intermediately differentiated. Poorly-differentiated DCIS displayed a higher frequency of amplifications (17q12, 11q13) and a higher average rate of genetic inbalances (7.1) (Buerger et al, 1999a, b; Moore et al, 1999) Findings supported by several studies showing amplifications of ERBB2 and immunohistogically detectable overexpression of the erbb2 protein (De Potter et al, 1993; Tsuda et al, 1993; Zafrani et al, 1994; Coene et al, 1997). Although the biological impact of these findings is not yet known, it is likely that DCIS differs biologically depending on the type and number of such alterations.

VII. Summary In conclusion, it can be said that even if the characterization of preinvasive breast lesions by immunohistochemistry, fluorescent in situ hybridisation, analysis of LOH, CGH, and, more recently, cDNA microarrays and proteomics analysis will one day allow a better genetic grading with prognostic implications (Buerger et al, 1999a; Jeffrey and Pollack, 2003; ReisFilho and Lakhani, 200), today the clinician, in the common clinical practice, can estimate the risk for each individual patient with preinvasive lesion of the breast of developing invasive cancer, by evaluating morphological classification, prognostic markers and genetic predisposition. Evaluating these data the clinician can select the preinvasive lesions of the breast which are at high risk of developing to malignacies, and chose the most appropriate therapy for each individual patient.

VI. Therapeutical decision

References

Therapeutical decision should be taken after concisuous and persistant study of the above mentioned genetic changes and molecular markers (c-erbB2, p53, bcl2, ki-67, p27, AAMP, gene 14-3-3!, 67LR, estrogen receptor, TGF-") (Petersen et al, 1987; Hockenbery et al, 1991; Williams et al, 1991; Pike et al, 1993; Bobrow et al, 1994; Basolo et al, 1996; Bodis et al, 1996; Bose et al, 1996; Karayiannakis et al, 1996; Siziopikou et al, 1996; Clarke et al, 1997; Fontanini et al, 1997; Kapucuoglu et al, 1997; Rudas et al, 1997; Viacava et al, 1997; El-Deiry, 1998; Henderson et al, 1998; Khan et al, 1998; Potten et al, 1998; Gobbi et al, 1999; Shoker et al, 1999; Viacava et al, 1999; Roger et al, 2001; Umbricht et al, 2001; Vogelstein et al, 2001; Wykoff et al, 2001; Adeyinka et al, 2002; Gasco et al, 2002; Man et al, 2003). The clinician should be aware on the recent progress of the molecular biology, individualising every patient based on her risk factors, her genetic profile, her tumour markers, her complete histological evaluation, her psychological and social status, etc. All these parameters should be carefully evaluated before the final decision be undertaken. In Table 6 is shown the genetic changes with a bad prognostic value for developing IBC.

Adeyinka A, Emberley E, Niu Y, Snell L, Murphy LC, Sowter H, Wykoff CC, Harris AL, Watson PH (2002) Analysis of gene expression in ductal carcinoma in situ of the breast. Clin Cancer Res 8, 3788-3795. Aldaz M, Chen T, Sahin A, Cunningham J, Bondy M (1995) Comperative allelotype of in situ and invasive human breast cancer, High frequency of microsatellite instability in lobular breast carcinomas. Cancer Res 55, 3976-3981. Allred DC, Mohsin SK, Fuqua SA (2001 Histological and biological evolution of human premalignant breast disease. Endocr Relat Cancer 8 47-61 Amari M, Suzuki A, Moriya T, Yoshinaga K, Amano G, Sasano H, Ohuchi N, Satomi S, Horii A (1999) LOH analyses of premalignant and malignant lesions of human breast: frequent LOH in 8p, 16q, and 17q in atypical ductal hyperplasia. Onc Rep 6, 1277-1280. Basolo F, Pollina L, Pacini F, Fontanini G, Menard S, Castronovo V, Bevilacqua G (1996) Expression of the Mr 67, 000 laminin receptor is an adverse prognostic indicator in human thyroid cancer: an immunohistochemical study. Clin Cancer Res 2, 1777-1780. Bellamy CO, McDonald C, Salter DM, Chetty U, Anderson TJ (1993) Noninvasive ductal carcinoma of the breast: the relevance of histologic categorization. Hum Pathol 24, 1623. Bobrow LG, Happerfield LC, Gregory WM, Springall RD, Millis RR (1994) The classification of ductal carcinoma in situ and its association with biological markers. Semin Diagn Pathol 11, 199-207. Bocker W, Bier B, Freytag G, Brommelkamp B, Jarasch ED, Edel G, Dockhorn-Dworniczak B, Schmid KW (1992) An immunohistochemical study of the breast using antibodies to basal and luminal keratins, #-smooth muscle actin, vimentin, collagen IV and laminin. Part II: Epitheliosis and ductal carcinoma in situ. Virchows Arch A Pathol Anat Histopathol 421, 323-330. Bodis S, Siziopikou KP, Schnitt SJ, Harris JR, Fisher DE (1996) Extensive apoptosis in ductal carcinoma in situ of the breast. Cancer 77, 1831-1835 Boecker W, Buerger H, Schmitz K, Ellis IA, van Diest PJ, Sinn HP, Geradts J, Diallo R, Poremba C, Herbst H (2001) Ductal epithelial proliferations of the breast: a biological continuum? Comparative genomic hybridization and high-

Table 6. Molecular markers of preinvasive breast lesions – – – – – – – – – – – –

c – erbB2 (Her – 2neu) p – 53 E – cathedrine TGF – " AAMP Ki – 67 p 27 bcl – 2 67 LR Gene 14-3-3 ! (!) Estrogen receptors Progesteron receptors

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George C. Zografos

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Cancer Therapy Vol 3, page 85 Cancer Therapy Vol 3, 85-94, 2005

Head and neck cancer in elderly patients Review Article

Daniele Bernardi1,2,*, Domenico Errante1, Luigi Barzan5, Giovanni Franchin3, Luigi Salvagno1, Antonio Bianco1, Luca Balestreri4, Umberto Tirelli2 and Emanuela Vaccher2 1

Division of Medical Oncology, Ospedale Civile, Vittorio Veneto (TV), Italy Division of Medical Oncology A, National Cancer Institute, Aviano (PN), Italy 3 Division of Radiotherapy, National Cancer Institute, Aviano (PN), Italy 4 Department of Radiology, National Cancer Institute, Aviano (PN), Italy 5 Division of Otolaryngology, Ospedale S. Maria degli Angeli, Pordenone, Italy 2

__________________________________________________________________________________ *Correspondence: Dr. Daniele Bernardi, U.O. Oncologia Medica, Ospedale Civile, Via Forlanini 71, 31021 Vittorio Veneto (TV), Italy; Tel. +39 0438 665371; Fax. +39 0438 665432; e-mail: daniele.bernardi@ulss7.it Key words: Head and neck, cancer, elderly, review Abbreviations: 5-fluorouracil, (5-FU); American Society of Anesthesiology, (ASA); complete remission, (CR); Comprehensive Geriatric Evaluation, (CGA); erythropoietin, (rhEpo); Geriatric Radiation Oncology Group, (GROG); Head and Neck, (H-N); local control, (LC); loco-regional control, (LRC); no evidence of disease, (NED); Performance status, (PS); quality of life, (QOL); Radiation Therapy and Oncology Group, (RTOG); Surveillance, Epidemiology and End Results data base, (SEER) Received: 21 February 2005; Accepted: 23 February 2005; electronically published: March 2005

Summary Head and Neck cancers occur mostly in the fifth and sixth decade; their onset in patients older than 60 years is not a rare event, though. In almost all case series from the literature, radical treatments have a lower prevalence among elderly as compared to younger patients, in particular surgery and combined treatment. The advances in anesthesiology techniques, in peri-operative monitoring and in post-operative support allow now to deal with lower risks surgical procedures also in older patients. Elderly patients with N0 disease but at high risk of relapse or distant metastases should be offered appropriate surgical treatment and chronological age should not be considered a limit for neck dissection. Radiotherapy is a feasible treatment in elderly patients, and, in the era of organ preservation, the combination of chemotherapy and radiotherapy has a paramount importance, even if very few data exist on combined treatment in the elderly patients. Elderly patients who are functionally independent and do not show severe comorbidities must be treated in the same manner as younger patients, but during anti-cancer treatment, special attention should be paid to supportive treatment as well. Therapeutical planning must be based not only on tumor characteristics, but also on the physiological, rather than the chronological age of the patient. Therefore, in patients aged 70 or older, a selection of patients to be administered anticancer treatment has to be performed. A Complete Geriatric Assessment and a multidisciplinary approach are the crucial points. Cefaro 1997). Elderly patients aged 70 to 75 years represent 6 to 32% of all patients with H-N cancers in mono-institutional case series. The most frequent histologic type is squamous cell carcinoma and the most common sites of disease are larynx and oral cavity and, less frequently, oropharynx and hypopharynx. The distribution of stages does not differ from that of the younger patients, with the exception of some case series where a prevalence of stage N0 is present in elderly patients (Table 1). A peculiar characteristic of almost all case series from the literature is the lower prevalence of radical treatments among elderly patients as compared to younger patients (30-74% vs 67-91%, p<0.001), in

I. Introduction Approximately 60% of all tumors arise in patients older than 65 years and 70% of all deaths due to cancer occur in this age (Fentiman et al, 1990; Kennedy, 2000; Balducci and Beghe, 2001; Repetto et al, 2001). Although the majority of Head and Neck (H-N) cancers are seen between the fifth and sixth decade, their occurrence in elderly patients is not rare. In a retrospective survey conducted by the Italian Geriatric Radiation Oncology Group (GROG), H-N cancers were present in 12% of patients older than 70 years with different tumors, referred to 37 radiation therapy centers in Italy (Olmi and Ausili-

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Bernardi et al: Head and neck cancer in elderly patients

Table 1. Clinical characteristics of H-N cancers in elderly patients in the main case series from literature

Tot Age, years Year of diagnosis Site of disease: Oral cavity Oropharynx Larynx Hypopharynx TNM Stage - T1-T2 T3-T4 - N0 N1 N2-N3 - M1 UICCb Stage I-II III-IV a

Olmi (1997) % 365/1114 (32%) >70 1960-92

Hirano (1998) % 751/2508 (30%) >70 1971-95

Sarini (2001) % 273/4610 (6%) >75 1974-83

SEER (2001) % 9386 >65 1985-93

Vaccher (2002) % 181/2143 (8%) >75 1975-98

32 28 40 --

12 17 22 25

40 29 25 9

39 20 42 --

23 17 49 10

1

61 39 72 13 14 2

62 38 81a

40 60 60 --1

19 --

31 69

52 48

N0+N1; UICC = Union Internationale Contre le Cancer.

b

particular surgery and combined treatment of surgery plus radiation therapy or chemotherapy and radiation therapy. Overall, survival is significantly lower in elderly patients, with an actuarial rate at 5 years of 17-31% vs 30-44% (p<0.001) in younger patients in the same case series (Olmi et al, 1997; Hirano and Mori 1998; Barzan et al, 1999; Sarini et al, 2001; Reid et al, 2001; Vaccher et al 2002). In the analysis of the case-control study from the Surveillance, Epidemiology and End Results data base (SEER), on 2508 case of carcinoma of the larynx, tongue and tonsil in patients older than 50 years, cancer specific survival of patients older than 70 years has been shown to be similar to that of patients of 50-69 years, with the exception of stage I and IV glottic carcinoma and stage III tonsil carcinoma, whose cancer-specific prognosis has been demonstrated to be worse and better in elderly patients, respectively (Bhattacharyya et al, 2003). Both groups were homogeneous according to sex, year of diagnosis, tumor characteristics and type of treatment. According to the same study, the overall medical morbidity and mortality rates were 5.65% and 2.98%, respectively. Postoperative pneumonia was the most common medical complication (3.26%) and was associated with a mortality rate of 10.94% (odd ratio for mortality, 4.4). Acute myocardial infarction and stroke were rare and were not statistically associated with increased mortality. Procedures that involved the esophagus carried the highest mortality rate (8.38%). Nevertheless, in the analysis of the prognostic factors for overall survival performed on the whole case-series in the SEER elderly patients, constituted by 9386 patients older than 65 years with the same type of H-N tumor but not

selected by stage and/or therapy, the main prognostic factor has been shown to be comorbidity according to the Charlson score. The presence of one comorbidity whatsoever is prognostically more important in patients with an age between 65 and 74 years as compared to those older than 85 years, probably due to the lower life expectancy of the latter group (HR 1.53, 95% CI 1.38-1.69 vs 1.32, 95% CI 1.09-1.84) (Reid et al, 2001). Ageing is always associated with a multiorgan functional decline, an increase in comorbidity and a decline of cognitive functions (Kennedy 2000; Balducci and Beghe 2001; Repetto et al, 2001). The presence of these failures is very heterogeneous in the population of elderly patients and anagraphic age by itself cannot be the only criterion for the therapeutic planning. In a quality of life (QOL) analysis carried out by a Dutch group, treatment does not affect QOL differently in older (!70 years) and younger (45-60 years) patients affected by H-N cancer (Derks et al 2004).

II. Surgery In general, solid tumors, including H-N cancer, are still most frequently treated with surgery. Elderly patients, though, have a higher potential operative risk of morbidity and mortality due to the presence of comorbidity and physiologic reduction of functional reserve connected to ageing. Elderly patients are more sensitive than younger patients to the volume depletions that are often associated to wide resections and/or longer surgical procedures typical of surgical oncology, and less resistant to postoperative infections due to the progressive impairment of the immune system (Kemeny et al 2000; Kennedy 2000; Balducci and Beghe 2001; Repetto et al, 2001). The first 86


Cancer Therapy Vol 3, page 87 studies on demolitive surgery in H-N cancers in elderly patients date back to the 1970s and 1980s and show a significant increase in mortality in patients older than 6570 years with a rate ranging from 3.5-7.4% vs 0.8-1.4% in younger patients (Morgan et al, 1982; McGuirt et al, 1997). The first study of the 1990s was published by Barzan and co-workers and focused on the impact of demolitive surgery on a group of 107 patients older than 70 years, compared with 135 patients aged 60-69 years and 196 patients younger than 59 years. As predictable, systemic contraindications to surgery and/or refusal of surgery were more frequent in elderly patients as compared to other patients. The number of patients undergoing “en-bloc” surgery was higher in the group of younger patients, but post-operative local or systemic complications were similar in all age groups (Table 2). Moreover, no difference was shown in loco-regional control (LRC) and in cancer specific survival among the age groups. Performance status (PS) and stage of disease, but not age, were the main prognostic factors for survival (Barzan et al, 1990). In a group of 43 patients older than 80 years, compared with 79 patients younger than 65 years, Clayman and co-workers demonstrated the feasibility of demolitive surgery even in very old patients. Although 93% of elderly patients fit in the high anesthesiologic risk category (Group 3-4 according the American Society of Anesthesiology [ASA]) classification) vs 63% of other patients (p<0.001), the complications were similar in the two groups, with a rate of major complication of 23% in elderly patients vs 20% in younger patients and of minor complications respectively in 28% and 23% of cases. The type of complications was different among the groups, with a higher prevalence of systemic complications, in particular cardiovascular and pulmonary, in the older patients and a higher prevalence of local complications in the younger patients. Post-operative mortality was 2% in the elderly and absent in the younger patients. LRC at 2 and 5 years in patients stratified by stage of disease was similar in the two groups, whereas overall survival was lower in elderly patients as compared to the control group (at 5-years 33% vs 63%, p<0.001), but similar to that of the population of the same age group (Clayman et al, 1998). Patients older than 75 years with locally advanced stage of disease have a higher operative morbidity and

mortality risk as compared to the other age groups (McGuirt and Davis 1995). Conservation surgery, such as supraglottic laryngectomy, reconstructive subtotal laryngectomy, conservation surgery of base of tongue and of hypopharynx, showed a moderate mortality rate in elderly patients (0-7%). The low compliance to rehabilitation in elderly patient, due to refusal and/or the lack of an adequate familial and social support, seriously affects the functional outcome of surgery and is frequently associated with an increased risk of aspiration pneumonia (Barzan et al 1999). Supracricoid partial laryngectomy, one of the surgical treatments with a higher risk of inhalation of food in the airways, is feasible in cooperative elderly patients. In fact, in a series of 69 patients older than 65 years (median age 71 years), with a carcinoma of the glottic and supraglottic larynx (stage I-II 61%), mortality was shown to be absent and the rates of surgical complications (13%) and medical complications (10%) during the operative procedure and in the immediate post-operative period were similar to that reported in younger patients. Twenty-two percent of patients showed an inhalation pneumonia in the first 6 months of follow-up and 1% died after 3 years, due to pulmonary complications. Therefore, nutritional rehabilitation after this surgical procedure must be continued for a long period of time (Lacourreye et al, 1998). Age does not affect the outcome of reconstructive surgery with free flaps, where engraftment occurs overall in 95-100% of elderly patients (Shestak et al, 1992, Bridger et al, 1994, Malata et al 1996, Shaari et al, 1998, Pompei et al, 1999, Blackwell et al, 2002). Nevertheless, patients older than 70 years, with important comorbidities, show a rate of local complications, such as ischemic necrosis, significantly higher as compared to younger patients without comorbidities (12% vs 8% in the case series of Pompei). Patients older than 80 years, 92 % with high ASA anesthesiologic risk, have a rate of medical intra- and post-surgical complications higher than that of younger patients (62% vs 15%, p=0.02), after reconstructive surgery with free flaps. Considering the same ASA class, the incidence of complications is still higher in patients older than 80 years and correlates with the duration of the surgical procedure (Blackwell et al, 2002).

Table 2. Surgical treatment and post-operative complications in a case series of 438 patients with H-N cancers, stratified by age.

Surgery Resection “En bloc” Complications Local Systemic

AGE (years) <59 % 48 a 47 a

60-69 % 33a 35a

>70 % 19 a 20 a

25 25

25 25

40 27

a

p<0.001 From Barzan et al, 1990, modified.

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Bernardi et al: Head and neck cancer in elderly patients The advances in anesthesiology techniques, in perioperative monitoring and in post-operative support allow now to face with lower risks surgical procedures in older patients as well. In general, in tumors of the oral cavity, surgical procedures including wide reconstructions (skinbone-mucosa) and revascularized flaps are more difficult to perform. In the carcinoma of the oropharynx, wide resections of base of tongue or of the lateral wall more easily can lead to chronic inhalation and therefore should not be performed. In the carcinoma of the larynx and hypopharynx, conservation surgical procedures must be weighted in relation to the entity of the predictable resection, the patientâ&#x20AC;&#x2122;s respiratory function and his/her possibility to cooperate in a post-operative rehabilitation program. Chronological age should not be considered a limit for neck dissection. Appropriate surgery treatment should be offered to elderly patients with N0 disease but at high risk of relapse or distant metastases. The deterioration of the general conditions and the diagnostic delay following the impossibility of an adequate follow-up, can often render non-feasible the salvage surgery in elderly patients (Barzan et al, 1999) In this setting, despite advances in conservative laryngeal surgery and radiotherapy, total laryngectomy remains a valuable and reliable treatment for advanced pharyngo-laryngeal cancers in elderly patients The classification of the operative risk according to the ASA score does not seem to have a predictive value in elderly patients. Transoral laser surgery, most commonly with CO2 laser, has achieved a key position in minimally invasive treatment concepts in the ears, nose and throat area, especially for the treatment of malignancies of the upper aerodigestive tract. In the hands of experienced surgeons it remains a valuable option for elderly patients since it is a minimally invasive, functional and rapidly performed treatment (Werner et al, 2002).

of Florence, Italy, published the biggest case series on 446 cases of carcinoma of the larynx, oropharynx and oral cavity in patients older than 70 years, treated exclusively with RT with curative intent, whose outcome was compared to that of patients <70 years with the same type and stage of tumor. In this case series, laryngeal cancers were mostly at early stage (T1-T2), while in both groups the other neoplasias were mostly in advanced locoregional stage. No differences in 5-year actuarial local control (LC) or survival with no evidence of disease (NED) were seen between the two age groups for laryngeal and oropharyngeal cancer. For patients with cancer of the oral cavity, LC was better in the younger patients than in those aged 70 years and older (50% vs 28%, p=0.04). There was no statistically significant difference in the NED survival between the two groups. Acute or late reactions from RT in older patients were not different from those observed in younger patients (Olmi et al, 1997). The Gustave Roussy Institute reported the experience on 331 elderly patients with an age >70 years affected by carcinoma of the larynx (28%), oropharynx (27%) and oral cavity (16%) treated with radical RT (65-70 Gy) in 84% of cases and with palliative RT (30 Gy) in the remaining 16% in poor general conditions. Overall, the treatment was well tolerated with a grade 3-4 toxicity according to the Radiation Therapy and Oncology Group (RTOG) score as follows: cutaneous 1%, mucositis only in 17%, but nasoenteral feeding was required in 54% of cases. A reduced psychological tolerance due to depression, confusion or inability to cooperate, affected the feasibility of RT in 6% of patients, with a heterogeneous distribution in the age groups (5% in patients 70-75 years old, 9% and 21%, respectively, in patients 75-80 years and 80-85 years). Overall, the LC at 3-years was 71% for patients treated with radical dose and 19% for those treated with a palliative dose. The analysis of the LC by stage of disease showed similar data to those of historical control groups with an age lower than 70 years (Table 3). Five-year survival rates of 30%, 27%, 21% and 0% were observed for the 70-75, 75-79, 80-85 and over 84 age groups, respectively. In patients treated with palliative dose, the survival rate at 5-years was only 5% (Lusinchi et al, 1990).

III. Radiotherapy A. Conventional fractionation The most widely used treatment in H-N tumors in elderly patients is represented by external beam radiotherapy with conventional fractionation (180-200 cGy/day for 5 days/week) (standard-RT). The University

Table 3. Loco-regional control (LRC) in 331 patients >70 years with H-N tumorsa treated with radiotherapy and stratified by stage (TNM) STAGE

a

LRC at 3 years %

Primary tumor T1-T2 T3-T4 Nodal disease N0 N1-N2 N3

89-66 47-41 88 71 46

oropharynx 30%, larynx 28% From Lusinchi et al, 1990, modified.

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Cancer Therapy Vol 3, page 89 Thompson and co-workers reported 2 case series in patients older than 75 years, 68 of whom had laryngeal carcinoma, treated with radical RT in 59% of cases and 33 patients with carcinoma of the hypopharynx, treated with curative intent in 52% of cases. The 3-year actuarial survival was 57% in the group of patients with laryngeal carcinoma and 22% in the group of patients with carcinoma of the hypopharynx (Thompson et al, 1996). The GROG evaluated prospectively the feasibility of radical radiotherapy in 91 elderly patients (age 70-88 years) with laryngeal carcinoma, mainly (56%) in stage III. Overall, the treatment was well tolerated, with a mild cutaneous and mucosal toxicity, respectively in 11% and 38%, and severe (G3-G4) in 1% and 5% (Olmi et al, 1997). The impact of age on the development of an acute or chronic toxicity was evaluated by Pignon and coworkers on 589 patient with H-N carcinoma treated with radical RT in 5 protocols of the EORTC, activated between 1980 and 1995. The acute normal tissue reactions (mucositis and weight loss) in elderly patients (>70 years) was not different from that of younger patients, but, considering the same objective damage, the severe subjective intolerance, defined as G3-4 functional acute toxicity, was significantly more frequent in elderly patients (Table 4). No difference was shown in the analysis of the late toxicity. In these studies, where usually patients in very good general conditions and without important comorbidities were enrolled, LRC and cancerspecific survival were similar in all age groups (Pignon et al, 1996). Data on the use of RT in very old patients (>80-90 years) are limited to few case series and have mostly been reported together with other tumors. In the case-series of Zachariah, on 203 patients older than 80 years, 50 patients (25%) had H-N cancer in different sites and stage of disease. Thirty-five of them (70%) were treated with radical RT and 15 (30%) with palliative RT. In the group treated with higher dose, 51% of patients developed a mild mucositis (G1-G2 according to RTOG), 29% a moderatesevere mucositis (G3) and only 3% a severe hemorrhagic mucositis (G4). With supportive therapy, mucositis disappeared in 4-6 weeks. In the group treated with palliative RT, G1-G2 mucositis was demonstrated in only 13% of patients. The objective response rate was 86%,

with 66% complete remission (CR) in the radically treated group, while a palliation of the symptoms of the disease was obtained in 67% of patients treated with low dose RT. Overall, patients achieving a CR presented a longer median survival of 25 months (Zachariah et al, 1997). Mitsuhashi reported on 32 patients older than 90 years, 14 of whom (44%) affected by H-N tumors, 11 (79%) treated with radical RT (median dose 61.2 Gy) and 3 (21%) with palliative RT (40 Gy). The treatment had to be discontinued for 2-3 weeks in 4 (36%) patients of the first group due to G2-G3 mucositis. The median survival in the radically treated patients was 8 months (range 3-55) while that of patients treated with palliative intent was 6 months (Mitsuhashi et al, 1999).

B. Unconventional fractionation A promising method to improve the treatment outcome in patients with H-N carcinoma is constituted by accelerated RT (fraction size of daily dose >200 cGy) and hyperfractionated RT (more than one fraction per day), often used in combination. Nonetheless, in general, elderly patients are excluded from protocols with unconventional fractionated RT, due to the fear of an increased toxicity, sometimes relevant also in younger patients. A Swiss group recently published the first study with an unconventional RT regimen (accelerated concomitant boost RT schedule), in a group of 39 patients older than 70 years with carcinoma of the hypopharynx-larynx (49%) and of the oral cavity-oropharynx (46%), compared with 81 patients < 70 years. Elderly patients were in 79% of cases in good general conditions (PS 0-1) and, in comparison with younger patients, had a more advanced T stage (T3-T4 54% vs 30%, p=0.01) but a less advanced N stage (N0 46% vs 72%, p=0.01). The primary tumor area and both sides of the neck down to the clavicles received a dose of 50.4 Gy over 5.5 weeks given daily fractions of 1.8 Gy, 5 times a week. The boost to the initial involved sites comprised 13 fractions of 1.5 Gy (total 19.5 Gy) given as a second daily fraction beginning the last day of the second week. Withdrawal of treatment due to toxicity occurred in only 8% of elderly patients and in none of the younger patients. The median dose administered and the

Table 4. EORTC Radiation Trials in H-N cancers (1589 patients): evaluation ad the impact of age on acute toxicity AGE ACUTE TOXICITY Objectivec G0 G1-G2 G3 Functionald G0 G1-G2 G3-G4

pa

<70 years %

>70 years %

1 48 51

2 41 58

NS

2 49 49

0 34 67

<0.001

a

X2 test; c1307 evaluable patients; d868 evaluable patients. From Pignon et al, 1996, modified.

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Bernardi et al: Head and neck cancer in elderly patients median treatment time were similar in the two groups. Acute and late toxicities were similar in the two age groups, as well as LRC and overall survival (Allal et al, 2000).

drugs and reduce the capacity of healthy tissues to recuperate. Moreover, polypharmacy, typical of the older age, can be responsible for pharmacokinetic and pharmacodynamic interactions between the different types of drugs. Table 5 shows data on potential toxicity of cisplatin and 5-FU in elderly patients in solid tumors (Kennedy 2000, Balducci and Beghe 2001, Repetto et al, 2001, Balducci and Corcoran 2000, Zagonel et al, 1998). Cisplatin is associated with an increase in peripheral neuropathy, anemia, and nephropathy. Generally, sensorymotor peripheral neuropathy initially arises with paresthesia, loss of deep tendinous reflex and tactile sensitivity and then with muscular weakness that sometimes severely affects patient’s autonomy (Rudd et al, 1995; Zagonel et al, 1998; Balducci and Corcoran, 2000). In vitro studies have clearly demonstrated that elderly patients have a reduced capacity to repair cisplatininduced DNA damages. Treatment with 5-FU, mostly administered in continuous infusion at high dose, determines in elderly patients a potential increase in cardiotoxicity, mucositis and leukopenia. Cardiotoxicity has its main cause in the frequent co-existence of a cardiomyopathy and/or alterations in electrolytes that occur during treatment. Mucositis is in general more severe than in younger patients and requires significantly longer time to recuperate. Leukopenia is mostly determined by a reduction in the bone marrow functional reserve and its severity is strictly related to the age of the patient (Stein et al, 1995; Zagonel et al, 1998; Balducci and Corcoran, 2000). An interesting study was performed on 71 patients aged 70 or older treated with cisplatin and 5-FU, with an age-adjusted dose regimen. Patients aged 70-79 years were treated with standard-dosage of cisplatin 100 mg/m2 day 1 and 5-FU 1000 mg/ m 2/day continuous infusion for 5 days, while those aged 80-84 years with a reduction of the dosage by 20% and those older than 85 years with a reduction of the dosage by 30%. The objective response rate was 79% (CR 52%) among the 54 patients aged 70-79 years and only 31% (CR 6%) among the 17 patients aged 80 or older. In the group of patient older than 80 years, patients responsive to chemotherapy were in better general conditions as compared to the non-responsive patients.

C. Conclusions In conclusion, RT is a feasible treatment in elderly patients, also in very advanced age groups and even with innovative schedules with unconventional fractionation. When radical doses are employed, the LRC is almost superimposable to that obtained in younger patients with the same type of neoplasia. Acute and chronic toxicities are similar to those showed in younger patients, but subjective tolerance and sometimes compliance are significantly lower as compared to the other age groups. Therefore, this data show the need to increase supportive medical and psychological therapy always during and after treatment. Frail patients seem to tolerate well palliative radiation treatment, but the data from the literature are at the moment too unclear to provide treatment guidelines in this subset of patients. Finally, the fact that in certain stages or sites of disease, in patients treated with radical therapy, the outcome in elderly patients is more unfavorable as compared to the younger ones prompts the activation of studies aimed at evaluating the impact of age on the tumor biology.

IV. Chemotherapy A background exists for an increased toxicity from chemotherapy in elderly patients, but clinical studies, aimed at evaluating the relationship between toxicity from chemotherapy and age, are very few (Balducci and Corcoran, 2000; Argiris et al, 2004). Nonetheless, elderly patients are often excluded from chemotherapy clinical trials (Fentiman et al, 1990). Standard chemotherapy for H-N carcinomas is the Al-Sarraf regimen, a sequential combination of cisplatin and infusional 5-fluorouracil (5FU) that, in the treatment of locoregional recurrences and/or distant metastases achieves a response rate of 4050% (CR 5-10%) and in neoadjuvant setting (CT-RT) for organ preservation of 70-88% (CR 40-60%) (Posner et al, 2000). The reduced functional reserve of elderly patients can potentially alter the pharmacokinetics of cytotoxic

Table 5. Possible causes of increased acute toxicity from Cisplatin and 5-Fluorouracil in elderly patients TOXICITY CISPLATIN Peripheral neuropathy Anemia Nefrotoxicity 5-FLUOROURACIL Cardiotoxicity Mucositis

CAUSE • • • •

Reduced capacity of DNA-damage reparation. Pharmacokinetic alterations. Multiorgan functional reduction. Reduced glomerular filtration rate.

• • • •

Cardiomiopathy. Pharmacokinetic alterations. Reduced intracellular concentration of dihydropirimidine carboxilase. Reduced bone marrow reserve.

Leukopenia

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Cancer Therapy Vol 3, page 91 Myocardial ischemia, the only form of cardiotoxicity that was examined in this study, was very low and similar in the two age groups, with a rate of 2% in the first group and 3% in the second (Schneider et al, 1994). Chemotherapy seems to be feasible also in patients aged 80 years or older, but a reduction in the dosage dependent only on the chronological age can seriously affect the efficacy of the treatment. The Eastern Cooperative Oncology Group (ECOG) has recently analyzed data from two randomized studies employing intensive cisplatin-based regimen for the treatment of patients with recurrent/metastatic H-N carcinoma, to evaluate the outcome of elderly patients. Fifty-three patients aged 70-80 years had comparable response rates (28% vs 33%) and survival outcomes (1year survival 26% vs 33%) compared with 346 younger patients. However, severe nephrotoxicity, thrombocytopenia and diarrhea were more common in the elderly than in the younger patients, occurring in 8% vs 2% (p=0.04), 26% vs 12% (p=0.009) and 17% vs 3% (p=0.0002), respectively (Argiris et al, 2004). Strategies to ameliorate toxicity should be pursued in the elderly. In the era of organ preservation, chemotherapy combined with RT has a paramount importance in the treatment of H-N tumors (Posner et al, 2000). Elderly patients, an emerging problem for public health in the industrialized countries, cannot be excluded a priori from organ preservation programs. Older patients who are functionally independent and do not show severe comorbidities must be treated in the same exact manner as younger patients, but during the treatment, supportive treatment must be increased. In particular, the administration of bone marrow growth factors, such as GCSF and erythropoietin (rhEpo) must be always evaluated. Data concerning the use of rhEpo in the prevention of chemotherapy-related anemia in early or advanced H-N cancer are not extensive (Tsukuda et al, 1993; Dunphy et al, 1997; Oettle et al, 2001). The role of recombinant rhEpo in preventing or correcting chemotherapy-related anemia in elderly patients with H-N cancer has been recently described (Gebbia et al, 2003). Acoording to this study, recombinant rhEpo is able to prevent anemia, to reduce transfusion requirements and to improve quality of life parameters in patients treated with carboplatin and 5FU as compared to untreated controls. The use of amifostine in the prevention of mucositis from CT is still controversial and should be eventually considered only when RT is administered (Schuchter et al, 2002). On the other hand, topical use of GM-CSF, administered as oral gargles, might accelerate the resolution of mucositis, even if an improvement of the quality of life has never been clearly demonstrated. In all patients particular attention should be paid to maintaining an adequate nutritional status, since malnutrition can affect both efficacy of chemotherapy and patients survival (Zagonel et al, 1998; Balducci and Corcoran 2000; Kennedy 2000; Balducci and Beghe 2001; Repetto et al, 2001). In fact, nutrition is often deficient in elderly patients in general, due to several reasons, such as depression, poor dentition, functional impairment, cognitive impairment, lack of appetite due to chronic

comorbid disease, and lack of caregiver. Elderly patients with cancer may also face additional problems brought on by chemotherapy, such as nausea, vomiting, diarrhea, and painful oral ulcerations. Correcting malnutrition and establishing a suitable dietary plan are simple measures that can substantially improve the patient’s clinical outcome and quality of life. The main concern with respect to emotional conditions in these patients is depression, which is common in both geriatric and oncology populations, and is therefore especially common in elderly patients with cancer. Depression and cognitive disorders can be mistaken for each other and either type of condition could adversely affect the patient’s functional status and the outcome of cancer treatment. Patients older than 80 years, patients not functionally independent and/or with severe associated comorbidities, must be treated in the setting of new treatment protocols, in which the choice of the regimen employed and the dose of the drugs must be adjusted according to a Comprehensive Geriatric Evaluation (CGA). CGA is an instrument aimed at evaluating the overall status of the patient and its efficacy has been documented by several randomized studies (Monfardini et al, 1996; Zagonel et al, 1998; Balducci and Corcoran 2000; Kennedy 2000; Balducci and Beghe 2001; Repetto et al, 2001). The preliminary results have been published of an ongoing trial using CGA to tailor the treatment of patients affected by aggressive non-Hodgkin’s lymphoma; to date, 23 patients have been treated with reasonable efficacy and toxicity (Bernardi et al, 2003). Noteworthy is the fact that in none of the studies concerning treatment in H-N cancers that have been published so far in the literature, a CGA has been used in the evaluation of the clinical status of the elderly patient.

V. Combined treatment A number of important factors should be considered in deciding the best therapy for the patient when chemoradiotherapy is used in a combined modality plan for the curative treatment of locally advanced H-N cancer (Vokes et al, 2000). No data exists in the literature on combined chemo-radiotherapy in the elderly, and there are very few experiences on retrospective subgroup analysis. It is essential to identify appropriate patients for combination therapy. Patients with underlying severe comorbidities, age-related frailty, or underlying severe psychosocial problems are not good candidates for highly intensive treatment plans. These patients may benefit less complicated or less potentially toxic treatment plans. The biology of the patient’s disease also must be considered in selecting or planning a combined modality approach. Patients with rapidly growing tumors or with advanced nodal presentation are less likely to be cured with surgery or radiation therapy alone and are most likely to benefit from the addition of chemotherapy. The location of the primary tumor is also an important factor in selecting therapy. Small lesions in the larynx, base of tongue and hypopharynx may benefit from an organ preservation approach, while similarly sized lesions in the anterior oral cavity might be better treated with direct surgical and 91


Bernardi et al: Head and neck cancer in elderly patients and neck cancer treated with cisplatin-based chemotherapy. J Clin Oncol 22, 262-268. Balducci L, Beghe C (2001) Cancer and age in the USA. Crit Rev Oncol Hematol 37, 137-45. Balducci L, Corcoran MB (2000) Antineoplstic chemotherapy of the older cancer patient. Hematol Oncol Clin North Am 14, 193-12. Barzan L, Olmi P, Franchin G, Vaccher E, Politi D, Loreggian L, Grando G, Tirelli U (1999) Carcinomi del distretto ORL. Argomenti di Oncologia 20,149-54. Barzan L, Veronesi A, Caruso G, Serraino D, Magri D, Zagonel V, Tirelli U, Comoretto R, Monfardini S (1990) Head and neck cancer and ageing: a retrospective study in 438 patients. J Laryngol Otol 104, 634-40. Bernardi D, Milan I, Balzarotti M, Spina M, Santoro A, Tirelli U (2003) Comprehensive Geriatic Evaluation in elderly patients with lymphoma: feasibility of a patient-tailored treatment plan. J Clin Oncol 21,754. Bhattacharyya N (2003) A matched survival analysis for squamous cell carcinoma of the head and neck in the elderly. Laryngoscope 113, 368-72. Blackwell KE, Azizzadeh B, Ayala C, Rawnsley JD (2002) Octogenarian free flap reconstruction: complications and cost of therapy. Otolaryngol Head Neck Surg 126, 301-06. Bridger AG, O’Brien CJ, Lee KK (1994) Advanced patient age should not preclude the use of free-flap reconstruction for head and neck cancer. Am J Surg 168, 425-28. Clayman G, Eicher SA, Sicard MW, Razmpa E, Goepfert H (1998) Surgical outcomes in head and neck cancer patients 80 years of age and older. Head Neck 20, 216-23. Derks W, de Leeuw RJ, Hordijk GJ, Winnubst JA (2004) Quality of life in elderly patients with head and neck cancer one year after diagnosis. Head Neck 26, 1045-1052. Dunphy FR, Dunleavy TL, Harrison BR, Boyd JH, Varvares MA, Dunphy CH, Rodriguez JJ, McDonough EM, Minster JR, McGrady MD (1997) Erythropoietin reduces anemia and transfusions after chemotherapy with paclitaxel and carboplatin. Cancer 79, 1623-1628. Fentiman IS, Tirelli U, Monfardini S, Schneider M, Festen J, Cognetti F, Aapro MS (1990) Cancer in the elderly: why so badly treated? Lancet 335, 1020-1022. Gebbia V, Di Marco P, Citarella P (2003) Systemic chemotherapy in elderly patients with locally advanced and/or inoperable squamous cell carcinoma of the head and neck: impact of anemia and role of recombinant human erythropoietin. Crit Rev Oncol Hematol 48 (suppl.), S4955. Hirano M, Mori K (1998) Management of cancer in the elderly: therapeutic dilemmas. Otolaryngol Head Neck Surg 118, 110-14. Kemeny MM, Busch-Deverauz E, Merriam LT, O’Hea BJ (2000) Cancer surgery in the elderly. Hematol Oncol Clin North Am 14, 169-92. Kennedy BJ. Aging and cancer. Oncology (Huntingt) 2000, 14, 1731-33. Laccourreye O, Brasnu D, Périé S, Muscatello L, Ménard M, Weinstein G (1998) Supracricoid partial laryngectomies in the elderly: mortality, complications, and functional outcome. Laryngoscope 108, 237-42. Lusinchi A, Bourhis J, Wibault P, Le Ridant AM, Eschwege F (1990) Radiation therapy for head and neck cancers in the elderly. Int J Radiation Oncology Biol Phys 18, 819-23. Malata CM, Cooter RD, Batchelor AG, Simpson KH, Browning FS, Kay SP (1996) Microvascular free-tissue transfers in elderly patients: the Leeds experience. Plast Reconstr Surg 98, 1234-41.

radiotherapy approaches. The goals of the addition of chemotherapy in a treatment plan must be considered in determining the best therapy: appropriate goals in the curative treatment of locally advanced H-N cancer include organ preservation, improved survival, optimization of quality of life and reduction in metastases (Posner et al, 2000). A recent study (Airoldi et al, 2004) assessed treatment toxicity, patient compliance, and clinical results in 40 patients >70 years who were treated with concomitant adjuvant chemoradiotherapy. The results of this study confirm previously established beliefs that adjuvant chemioradiotherapy can be successfully applied in older patients who are fit to receive such treatment. The role of the combination therapy in the postoperative setting can only be validated by phase III trials. A comparison of the results of the study by Airoldi with those of the group 70 years or older treated with radiotherapy alone suggests that superior results can be obtained with chemoradiotherapy compared with radiotherapy alone in this age group.

VI. Closing remarks The physiological, rather than the chronological age of the patient, together with tumor characteristics, should be considered when planning the treatment of H-N cancers in older patients. Elderly patients who are functionally independent and do not show severe comorbidities must be treated in the same manner as younger patients, but during anti-cancer treatment, special attention should also be paid to supportive treatment. Patients with underlying severe comorbidities, age-related frailty, or severe psychosocial problems are not good candidates for highly intensive treatment plans. The key issue is, therefore, the selection of patients to be administered anticancer treatment. In patients aged 70 or older, CGA and a multidisciplinary approach are the crucial points for an adequate therapeutical planning. A determinant factor in the prognosis of the patient with H-N tumors of any age is the multidisciplinary management of the disease. Surgeons, radiation-therapy specialists, medical oncologists and geriatricians must actively cooperate in a multidisciplinary setting.

Acknowledgements The skillful and patient assistance of Mrs. Paola Favetta in the preparation of the manuscript is gratefully acknowledged.

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Cancer Therapy Vol 3, page 95 Cancer Therapy Vol 3, 95-100, 2005

Radiotherapy and surgery in the management of non-small cell lung cancer in the elderly patients, a review of the recent literature Review Article

H. Cuneyt Ulutin* and Gorkem Aksu Gulhane Military Faculty of Medicine, Radiation Oncology Department

__________________________________________________________________________________ *Correspondence: Dr. Cuneyt Ulutin; Kizilcik sok. Kizilcikapt. 10/10 Anittepe Ankara Turkey; Telephone: 90-312-3044684; Fax: 90312-3044150; e-mail: culutin@yahoo.com Key words: Radiotherapy, surgery, non-small cell lung cancer, elderly patients Abbreviations: biologic effective dose, (BED); cancer-directed surgery, (CDS); continuous hyperfractionated accelerated radiotherapy, (CHART); gray, (Gy); gross tumor volumes, (GTV); Non-small-cell lung cancer, (NSCLC); three-dimensional conformal radiotherapy, (3D-CRT); wedge resection, (WR) Received: 27 December 2004; Accepted: 8 February 2005; electronically published: March 2005

Summary More than 50% of all patients with NSCLC are older than 65 years and about one-third of all patients are >70 years old at the time of diagnosis. However, there is no standard chronological age to consider a person as “ elderly”. In most of the historical series this was defined by the age of 65 or older, but it is not an exact definition since health status differs and there is a great heterogeneity of patients in this group according to functional organ capacity. Therefore, biological age should be defined individually and before making a treatment decision a careful evaluation of functional status, coexisting diseases, nutritional status, psychological functioning and social support should be performed. Surgery is the treatment of choice for patients in early stage (I/II) NSCLC. . Resection for stage IIIA NSCLC patients is acceptable if the affected N1 lymph nodes can be resected, or only micrometastatic disease is present in N2 lymph nodes, or restaging after neoadjuvant therapy shows no residual cancer in N2 lymph nodes. Radical radiation therapy is also used for curative intent in elderly patients who are not candidates to surgery because of poor performance status, old age or refusal of surgery in early stage NSCLC or who can not tolerate chemoradiotherapy in locally advanced disease, however, the survival rates are lower than those reported after surgery. In this review, we evaluated the role of surgery and radical radiotherapy in the management of NSCLC in the elderly patients. status, coexisting diseases, nutritional status, psychological functioning, and social support should be performed.

I. Introduction Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers. The population of elderly people increases and lung cancer is also the most common cause of cancer deaths aong this population. More than 50% of all patients with NSCLC are older than 65 years and about one-third of all patients are >70 years old at the time of diagnosis (Mountain, 1997). However, there is no standard chronological age to consider a person as “ elderly”. In most of the historical series this was defined by the age of 65 or older, but it is not an exact definition since health status differs and there is a great heterogeneity of patients in this group according to functional organ capacity. Therefore, biological age should be defined individually and before making a treatment decision a careful evaluation of functional

II. Surgery in elderly patients with early stage NSCLC The five-year survival rate for patients with Stage I NSCLC is better than 60% and surgery is the treatment of choice for patients in early stage (I/II) NSCLC. Resection for Stage IIIA patients is acceptable if the affected N1 lymph nodes can be resected, or only micrometastatic disease is present in N2 lymph nodes, or restaging after neoadjuvant therapy shows no residual cancer in N2 lymph nodes. (Mountain, 1997). Although, lung cancer resection in elderly patients is justified and has decreasing morbidity and mortality rates, age is sometimes used as an 95


Ulutin and Aksu: Radiotherapy and surgery in the management of NSCLC in the elderly patients excuse not to resect lung cancer. In a recent analyze by Dexter et al. it was determined that of the 33% of elderly patients who had stage I or II disease, only 6% underwent surgical resection (Dexter et al, 2004). Oâ&#x20AC;&#x2122;Connell et al, (2004) recently reviewed elderly patients for cancer-directed surgery for localized adenocarcinoma of the breast, esophagus, stomach, pancreas, colon, rectum, non-small-cell lung carcinoma and sarcoma and found out that rates of cancer-directed surgery (CDS) declined steadily with increasing age for all these tumors beginning at 60 years (O'Connell et al, 2004). Despite the fact that curative resection should be performed in older patients, the type of the resection is discussed in many studies since the extent of surgery directly influences the overall morbidity and mortality. In most of the studies (Bates, 1970; Whittle et al, 1991; Damhuis and Schutte, 1996; Janssen-Heijnen et al, 2004), pneumonectomy is accompanied by an increase in the morbidity and mortality in elderly patients thus especially right pneumonectomy should be performed only in highly selected patients (Teeter et al, 1987; Au et al, 1994). Lobectomy or sub-lobar resection is preferred as a curative resection in many studies for older adults. The postoperative complications are lower than the patients who undergo pneumonectomy. Oliaro et al. analyzed patients who were 70 years and older undergoing curative resection. Postoperative complication rates were 78.5% for patients receiving pneumonectomy and 58% for patients undergoing lobectomy or wedge resection. All cases of postoperative death occurred in patients who were treated with pneumonectomy. Prognostic factors were poor performance status (WHO 2 or more), chronic obstructive pulmonary disease, and elevated levels of blood urea nitrogen (Oliaro et al, 1999). In a different study by Whittle et al. most of the patients were treated with standard lobectomy. The major complication rate was 11%, and per operative death was seen in 3.7% of patients. The survival rates for patients with Stage I disease were 86% at one year, 62% at three years, and 43% at five years. The authors concluded that postoperative mortality rises with increasing age (Whittle et al, 1991). Lobectomy is now the most frequently performed procedure in the elderly NSCLC patients, though pneumonectomy can be performed in selected cases. However lobectomy, and pneumonectomy can hardly be compared directly some recent studies report similar results for pneumonectomy and lobectomy (Au et al, 1994; Sioris et al, 1999) while most of the studies report higher operative mortalities (17%-30%) with pneumonectomy (Bates, 1970; Kirsh et al, 1976; Harviel et al, 1978; Yellin et al, 1985). Since almost all deaths are caused by cardiopulmonary adverse effects (Harviel et al, 1978; Sherman and Guidot, 1987; Mane et al, 1994), it is necessary to carefully evaluate patients preoperatively and select appropriate surgical procedures (Ishida et al, 1990; Roxburgh et al, 1991). Using modern surgical and imaging techniques and advances in anesthesiology and per operative care can also reduce postoperative mortality and morbidity (Tsuchiya et al, 1981; Ginsberg et al, 1983;

Ishida et al, 1990; Thomas et al, 1993; Morandi et al, 1997). The effect of increasing age on the incidence of major complications or postoperative death is controversial. While some studies (Ginsberg et al, 1983; Sherman and Guidot, 1987; Deneffe et al, 1988; Whittle et al, 1991; Romano and Mark, 1992; Thomas et al, 1993; Cangemi et al, 1996; Damhuis and Schutte, 1996) demonstrated older age as an adverse prognostic factor, there are also studies that found no significant relationship between age and postoperative complications (Ishida et al, 1990; Breyer et al, 1981; Nagasaki et al, 1982; Kadri and Dussek, 1991). Yamamoto, et al, 2003 analyzed the surgical results of 797 patients with Stage I NSCLC and found out that patients aged 70 and older had similar 5- and 10-year survival rates compared with younger patients (Yamamoto et al, 2003). In a different study, older age (defined as 65 years or more), anemia, and higher stage were found as prognostic factors for patients who underwent surgical resection for Stages I and II NSCLC. Patients older than 65 had a shorter event-free survival time (34 vs. 55 months, p= 0.002) and overall survival (39 versus 58 months, p= 0.002) compared with younger patients (Jazieh et al, 2000). Van Rens et al. analyzed 2,361 patients who were treated with curative resection for Stages I, II, and IIIA NSCLC. The overall five-year survival rate for patients younger than 65 was 44% compared with 38% for older patients (p= 0.001); however, the authors noted that survival rates were similar for as long as four years after surgery, and thus the five-year survival rate difference may be secondary to comorbid disease (Van Rens et al, 2000). Kamiyoshihara et al. analyzed 160 patients with nonsmall cell lung cancer underwent lobectomy or pneumonectomy with mediastinal lymph node dissection. Of these, 37 (23%) were 70 years of age or older. The outcome of this group was compared with 123 non-elderly patients. Five- and 10-year survivals in the elderly patients were 35.1%, and 24.3%, respectively. In outcome more than 5 years from operation, elderly patients had a significantly poorer prognosis than non-elderly patients by any causes of death, but a similar prognosis by primary death (p=0.04). Deaths from non-tumoral reasons were more in the elderly group than non-elderly patients with no significant difference (p=0.6) (Kamiyoshihara et al, 2000). Mane et al. reviewed 1433 patients with lung cancer concerning tobacco use, stage of disease, treatment and survival rate of patients treated surgically. A comparison was made between patients aged 65 or less with those over 65. The stage of disease at the time of diagnosis was similar in both groups but the distribution by histological type showed significant differences (p < 0.05) with a higher percentage of squamous carcinoma in the younger group (54% versus 44%). Surgery was performed in 30% of the patients aged 65 years or less but only in 19% of the older cases (p < 0.05). There was no difference in the survival of younger and older patients (Mane et al, 1994).

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Cancer Therapy Vol 3, page 97 Froeschle et al. analyzed 70 patients with 70 years of age or older who underwent surgery for lung cancer. Standard lung resections were performed in 42 cases and 17 patients underwent enlarged resections. In the postoperative period, complications occurred in 32 cases (47%). The overall operative mortality rate of 13% was mainly due to concomitant cardiovascular diseases and enlarged lung resections (Froeschle et al, 1996). Pagni et al. analyzed 1506 patients with NSCLC treated by lung resection for perioperative morbidity and mortality. 385 (25.6%) patients aged 70 years and older were operated. Operations included 293 (77%) lobectomies, 24 pneumonectomies (6%), 16 bilobectomies (4%) and 52 wedge or segmental resections (13%). The pathology was bronchogenic carcinoma in 89% and metastasis or other tumours in 11% of patients. The mortality for all resections in elderly group was 4.2% (16/385) and was 1.6% for the control group. Mortality in the octogenarian group was 2.8%. Female gender correlated with a decreased risk of death, with only two of 16 deaths in females (P < 0.005). Overall morbidity was higher in the study than in control patients (34% vs. 25%), although major morbidity was similar in both groups (13.2% vs. 13%). Abnormal pulmonary-function testing and positive cardiac history did not correlate with increase overall or specific risk. Pneumonectomy carried a higher risk for death, with three of 24 deceased (12.5%; P < 0.05). The authors concluded that, age alone no longer appears to be a risk factor for mortality and pneumonectomy should undertaken cautiously in this age group. Based on this data, functional elderly patients should not be denied curative lung resection based on age alone (Pagni et al, 1998). Although Romano and Mark, (1992) identified advanced age (>79 years) as the strongest risk factor for death, Harvey et al, (1995) and Higgins and Beebe, (1967) reported that survival after surgery was negatively correlated with increasing age. In a recent study by Port et al, (2004) 61 elderly patients were evaluated. The surgical techniques were 46 lobectomies, 6 segmentectomies, 5 wedge resections, and 4 pneumonectomies. There was one perioperative death (1.6%) and the overall complication rate was 38% with a major complication rate of 13%. Overall 5-year survival was 82% for stage IA patients. Patients with more advanced disease had a significantly worse survival. The authors agree that appropriately selected elderly patients with early stage disease should be offered anatomic surgical resection for cure and should not be denied an operation on the basis of age alone (Port et al, 2004). Videothoracoscopic lobectomy can be used in the treatment of stage I and II lung cancer. Roviaro et al, (2004) analyzed 257 patients with stage I lung cancer, 193 patients underwent VATS lobectomy. There was no intraoperative mortality and no recurrence. The survival rates at 3 years and 5 years were 77.7% and 63.64%, respectively. T1N0 patients had a better survival curve at 3 years and 5 years (83.50% and 70.21%, respectively) compared to T2N0 patients (71.13% and 56.12%). Patients < 70 years of age had better 3-year and 5-year survival rates (82.37% and 73.32%, respectively) than those > 70

years of age (57.49% and 37.09%). The results showed that VATS approach match the "best" results reported in literature following conventional surgery and this minimally invasive surgery seems to imply reduced tissue damage and decreased impairment of immunologic function especially in elderly patients (Roviaro et al, 2004). The results of these studies show that surgery is the main treatment for early stage NSCLC in both young and elderly patients. The risk of postoperative complications can be minimized with the selection of the surgical procedure, careful preoperative evaluation and attentive postoperative care.

III. Radical radiotherapy in early stage NSCLC Radical radiation therapy is used for curative intent in elderly patients who are not candidates to surgery because of poor performance status, old age or refusal of surgery in early stage NSCLC or who can not tolerate chemoradiotherapy in locally advanced disease, however, the survival rates are lower than those reported after surgery. Gauden et al retrospectively analyzed 347 patients with T1 and T2N0M0 tumours who were treated with radical radiotherapy. The median age was 70 years and the minimum radiation dose was 50 Gray. 5-year survivals were 22% in the patients aged < 70 years and 34% in patients > or=70 years. Median survivals were 22 months and 26 months respectively. 5-year disease free survivals were 18% and 30% with no statistical significance. The 75-79-year group showed better survival than other age groups with the 5-year overall survival for this group being 53%, while the 5-year recurrence free survival was 45%. The toxicity in both groups was minimal. The authors concluded that radical radiation therapy with curative intent may be a viable alternative to surgery in those elderly patients who either refuse surgery or are judged to be unfit for operation (Gauden et al, 2001). Pignon et al, 1998 analyzed 1208 patients receiving chest irradiation in six EORTC randomized trials. Patients were split into six age ranges from 50 to 70 years and over. Data regarding age and acute toxicity were available for 1208 patients who experienced 640 grade > or =1 toxicities. The difference in distribution over age was not significant for acute nausea, dyspnea, oesophagitis, weakness and WHO performance status alteration. Weight loss was significantly different with regards to age with a trend toward increased weight loss in older age groups (P=0.002). 1082 grade > or =1 late toxicities were recorded in 935 patients. The mean time to complication was 13 months and was similar in all age groups. Forty percent of patients were free of complication at 4 years showing no significant difference between age groups (P=0.57). For grade >2 side-effects, late dyspnea and late weakness, there was also no significant difference, including grade >2 late oesophagitis (P = 0.1). The results of this study showed that age alone is not a sufficient reason to exclude patients in good general condition with thoracic tumor from curative radiotherapy when medically indicated (Pignon et al, 1998). 97


Ulutin and Aksu: Radiotherapy and surgery in the management of NSCLC in the elderly patients The proportion of patients who receive radiation decreases with increasing age. Among patients who receive treatment, the likelihood of receiving radiation is higher than any other therapy (p=0.0008)(41). Hillner et al. evaluated 1706 NSCLC patients of whom 1212 were age > or=65 years ("elderly"). Radiation was used more often in elderly patients compared with younger patients with local disease (30.5% vs. 14.0%) but less often in patients with distant disease (76.2% vs. 54.9%). In comparison with elderly patients, younger patients more often were treated with surgery for local disease (80.2% vs. 54.8%) and surgery alone or in combination with radiation for regional disease (51.9% vs. 32.0%) (Hillner et al, 1998). Different fractionation schedules and new applications of radiotherapy are currently available due to improvements in technology. In a recent study by Ghosh et al, 2003 lobectomy, wedge resection (WR) or continuous hyperfractionated accelerated radiotherapy (CHART) were compared in 215 patients with T1N0 NSCLC aged >70 years. Survivals at 1 and 5 years for patients undergoing WR, lobectomy and CHART were 98% and 74% vs. 97% and 68% vs. 80% and 39%, respectively (P=0.0484). The frequency of local/regional recurrence in the WR group (19.1%) was not significantly higher than in the lobectomy group (18.4%, P=0.38) when compared to the CHART group (27%, P=0.07). The results of this study showed that loco-regional recurrence and survival after WR, and lobectomy in elderly patients with stage I NSCLC were comparable and CHART was a reasonable treatment option for those who are not suitable candidates for surgery (Ghosh et al, 2003). Lester et al. used CT-planned accelerated hypofractionated radiotherapy in the radical treatment of non-small cell lung cancer. 135 patients with stage I-IIIB NSCLC were treated with CT-planned accelerated hypofractionated radical radiotherapy to a dose of 5055Gy in 15-20 fractions over 3-4 weeks. The 2-year overall and cause-specific survival for all patients was 44.4% and 47.8% respectively. Overall median survival was 21 months. There were no reports of severe acute or late treatment-related toxicities (Lester et al, 2004). Stereotactic hypofractionated high-dose irradiation for stage I nonsmall cell lung carcinoma was evaluated by Onishi et al, 2004 245 patients with a median age of 76 years (T1N0M0, n=155; T2N0M0, n=90) were treated with stereotactic hypofractionated high-dose radiotherapy. A total dose of 18-75 gray (Gy) at the isocenter was administered in 1-22 fractions. The median calculated biologic effective dose (BED) was 108 Gy (range, 57-180 Gy). Grade > 2 toxicities were observed in only 6 patients (2.4%). Local progression occurred in 33 patients (14.5%), and the local recurrence rate was 8.1% for BED > or=100 Gy compared with 26.4% for < 100 Gy (P < 0.05). The 3year overall survival rate of medically operable patients was 88.4% for BED > or=100 Gy compared with 69.4% for < 100 Gy (P < 0.05). The authors concluded that hypofractionated high-dose STI with BED < 150 Gy was feasible and beneficial for curative treatment of patients with Stage I NSCLC (Onishi et al, 2004).

Wulf et al used stereotactic radiotherapy for primary lung cancer and pulmonary metastasis in 61 medically inoperable patients.The patients were treated with stereotactic radiotherapy at 3 x 10 Gy (n=19), 3 x 12-12.5 Gy to the planning target volume enclosing 100%-isodose, with normalization to 150% at the isocenter; n=26) or 1 x 26 Gy to the planning target volume enclosing 80%isodose (n=26). The actuarial local control rate was 92% for lung cancer patients and 80% for metastasis patients > or =1 year after treatment and was significantly improved by increasing the dose from 3 x 10 Gy to 3 x 12-12.5 Gy or 1 x 26 Gy (p=0.038). The overall survival rate after 1 and 2 years was 52% and 32%, respectively, for lung cancer patients and 85% and 33%, respectively, for patients with metastasis, was impaired because of systemic disease progression. No severe acute or late toxicity was observed, and only 2 patients (3%) developed symptomatic Grade 2 pneumonitis, which was successfully treated with oral steroids (Wulf et al, 2004). The results show that stereotactic radiotherapy is a very effective treatment option for lung cancers without significant complications in medically impaired patients who are not amenable to surgery. Respiratory gated radiation therapy is developed for minimizing respiratory-induced anatomic motion and found to be practical during both simulation and treatment. With proper patient selection and training, this system is currently being used in some radiotherapy departments. Regarding the results of these studies doses ! 65 Gy with continuous fractionation are recommended and the target volume should enclose the location of tumor volume with the ipsilateral hilum and the adjacent mediastinum (Dosoretz et al, 1993).

IV. Radiotherapy alone in locally advanced NSCLC Radical radiotherapy is also a good management for locally advanced NSCLC in the elderly patients who canâ&#x20AC;&#x2122;t tolerate chemoradiotherapy. Zachariah et al. evaluated elderly patients who were treated with 59.40â&#x20AC;&#x201C;66 Gy with standard fractionation. The response rate was 43% and only 24% of patients had progressive disease The authors concluded that aggressive radiotherapy should not be withheld from older patients because of chronological age alone since older patients with good functional status tolerate radiotherapy as well as younger patients and have comparable tumor response and survival rates (Zachariah et al, 1997). Lonardi et al. evaluated the outcome of 48 patients, aged 75 years and over, treated with radiation therapy for advanced (stage IIIA-B), inoperable, symptomatic NSCLC. A median dose of 50 Gy was delivered to the primary site and mediastinum with standard fractionation. 21 patients had partial remission, 17 stable disease, and nine had progressive disease. Toxicity was negligible and mainly consisted of grade I-II esophagitis. Overall median survival was 5 months but dose-related survival was much better in patients given at least 50 Gy than in those treated with lower doses: 52% versus 35% at 6 months, and 28% versus 4% at 13 months. The results confirmed that

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Cancer Therapy Vol 3, page 99 radiation therapy could be safely delivered to very aged patients with advanced NSCLC (Lonardi et al, 2000). In a retrospective trial by Nakano et al. in locally advanced NSCLC patients, median survival was 11.5 months in the younger group and 6.3 months in the elderly group (p=0.0043). A good performance status, age of <75 years, and good response to treatment were found as significant prognostic factors (Nakano et al, 1999). Tombolini et al. analyzed 41 medically inoperable IIIA and IIIB elderly patients (aged > or=70 years), treated with radiotherapy alone. The 2-year overall survival and disease free survival were respectively 27% and 14.6%. Patients presenting with weight loss > 10% experienced 14% overall survival at 2 years compared to 58% for those without weight loss (p=0.0027). Patients with tumor size less than 4 cm had a overall survival of 64% at 2 years but patients with tumor size > 4 cm had only a 2 year survival of 7% (p=0.0009). They concluded that radiotherapy is a good management for locally advanced NSCLC in elderly patients assuring good quality of life, high rates of relief of symptoms and overall and disease free survival similar to those obtained with chemotherapy and chemotherapy plus radiotherapy (Tombolini et al, 2000). Rengan et al, 2004 investigated 72 patients with Stage III NSCLC and gross tumor volumes (GTV) of greater than 100 cc which were treated with threedimensional conformal radiotherapy (3D-CRT) to understand whether high-dose radiation improved local control. Patients were divided into two groups: those treated to less than 64 Gy (37 patients) and those treated to 64 Gy or higher (35 patients). The 1-year and 2-year local failure rates were 27% and 47%, respectively, for Stage III patients treated to 64 Gy or higher, and 61% and 76%, respectively, for those treated to less than 64 Gy (p=0.024). The median survival time for patients treated to 64 Gy or higher was 20 months vs. 15 months for those treated to less than 64 Gy (p=0.068). A 10 Gy increase in dose resulted in a 36.4% decreased risk of local failure. These data suggested that administration of higher doses using 3D-CRT improves local control in Stage III NSCLC patients with large GTVs (Rengan et al, 2004). Radiation is frequently used for palliation of lung cancer-related symptoms. Radiotherapy can palliate thoracic pain and hemoptysis in 60% to 80% of cases and control other local symptoms in approximately 50% to 70% of cases. The median duration of benefit is 7 to 14 weeks. The main toxicity is self-limiting esophagitis (Higgins and Beebe, 1967).

advanced disease but the survival rates are lower than those reported after surgery.

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V. Conclusion Appropriate treatment for elderly patients with early stage non-small cell lung cancer should be surgical resection for cure and these patients should not be denied for an operation on the basis of age. Patients who have good performance status and no other medical problems should be offered standard resection. Extended resections should be avoided when possible. Radical radiation therapy is used for curative intent in elderly patients who are not candidates to surgery because of poor performance status, old age or refusal of surgery in early stage NSCLC or who can not tolerate chemoradiotherapy in locally 99


Ulutin and Aksu: Radiotherapy and surgery in the management of NSCLC in the elderly patients Kamiyoshihara M, Kawashima O, Ishikawa S et al (2000) Longterm results after pulmonary resection in elderly patients with non-small cell lung cancer. J Cardiovasc Surg 41, 483-486. Kirsh MM, Rotman H, Bove E, et al (1976) Major pulmonary resection for bronchogenic carcinoma in the elderly. Ann Thorac Surg 22, 369-373. Lester JF, Macbeth FR, Brewster AE et al (2004) CT-planned accelerated hypofractionated radiotherapy in the radical treatment of non-small cell lung cancer. Lung Cancer 45, 237-42. Lonardi F, Coeli M, Pavanato G et al (2000) Radiotherapy for nonsmall cell lung cancer in patients aged 75 and over, safety, effectiveness and possible impact on survival. Lung Cancer 28, 43-50. Mane JM, Estape J, Sanchez-Lloret J et al (1994) Age and clinical characteristics of 1433 patients with lung cancer. Age Ageing 23, 28-31. Morandi U, Stefani A, Golinelli M et al (1997) Results of surgical resection in patients over the age of 70 years with non small-cell lung cancer. Eur J Cardiothorac Surg 11, 432-439. Mountain CF (1997) Revisions in the International System for Staging Lung Cancer. Chest 111, 1710-7. Nagasaki F, Flehinger BJ, Martini N et al (1982) Complications of surgery in the treatment of carcinoma of the lung. Chest 82, 25-29. Nakano K, Hiramoto T, Kanehara M et al (1999) Radiotherapy alone for elderly patients with stage III non-small cell lung cancer. Nihon Kokyki Gakkai Zasshi 37, 276-281. O'Connell JB, Maggard MA, Ko CY (2004) Cancer-directed surgery for localized disease, decreased use in the elderly. Ann Surg Oncol 11, 962-9. Oliaro A, Leo F, Filosso PL, et al (1999) Resection for bronchogenic carcinoma in the elderly. J Cardiovasc Surg 40, 715-719. Onishi H, Araki T, Shirato H et al (2004) Stereotactic hypofractionated high-dose irradiation for stage I nonsmall cell lung carcinoma, clinical outcomes in 245 subjects in a Japanese multiinstitutional study. Cancer 101, 1623-31. Pagni S, McKelvey A, Riordan C et al (1998) Pulmonary resection for malignancy in the elderly, is age still a risk factor? Eur J Cardiothorac Surg 14, 40-44; discussion 4445. Pignon T, Gregor A, Schaake Koning C et al (1998) Age has no impact on acute and late toxicity of curative thoracic radiotherapy. Radiother Oncol 46, 239-48. Port JL, Kent M, Korst RJ et al (2004) Surgical resection for lung cancer in the octogenarian. Chest 126(3, 733-8. Rengan R, Rosenzweig KE, Venkatraman E et al (2004) Improved local control with higher doses of radiation in large-volume stage III non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 60, 741-7. Romano PS, Mark DH (1992) Patient and hospital characteristics related to in-hospital mortality after lung cancer resection. Chest 101, 1332-1337. Roviaro G, Varoli F, Vergani C et al (2004) Long-term survival after videothoracoscopic lobectomy for stage I lung cancer. Chest 126, 725-32. Roxburgh JC, Thompson J, Goldstraw P (1991) Hospital mortality and long-term survival after pulmonary resection in the elderly. Ann Thorac Surg 51, 800-803.

Sherman S, Guidot CE (1987) The feasibility of thoracotomy for lung cancer in the elderly. JAMA 258, 927-930. Sioris T, Salo J, Perhoniemi V et al (1999) Surgery for lung cancer in elderly. Scand Cardivasc J 33, 222-227. Smith TJ, Penberthy L, Desch CE et al (1995) Differences in initial treatment patterns and outcomes of lung cancer in the elderly. Lung Cancer 13, 235-52. Teeter SM, Holmes FF, McFarlane MJ et al (1987) Lung carcinoma in the elderly population. Influence of histology on the inverse relationship of stage to age. Cancer 60, 13311336. Thomas P, Sielezneff I, Ragni J et al (1993) Is lung cancer resection justified in patients aged over 70 years? Eur J Cardiothorac Surg 7, 246-250; discussion 250-251. Tombolini V, Bonanni A, Donato V, et al (2000) Radiotherapy alone in elderly patients with medically inoperable stage IIIA and IIIB non-small cell lung cancer. Anticancer Res 20, 4829-4833. Tsuchiya R, Miyazawa N, Naruke T et al (1981) Lung resection in patients over age of 70 years]. Kyobu Geka 34, 416-420. Van Rens MT, De La Riviere AB, Elbers HR et al (2000) Prognostic assessment of 2,361 patients who underwent pulmonary resection for non-small cell lung cancer, stage I, II, and IIIA. Chest 117, 374-9. Whittle J, Steinberg EP, Anderson GF et al (1991) Use of Medicare claims data to evaluate outcomes in elderly patients undergoing lung resection for lung cancer. Chest 100, 729734. Wulf J, Haedinger U, Oppitz U et al (2004) Stereotactic radiotherapy for primary lung cancer and pulmonary metastases, a noninvasive treatment approach in medically inoperable patients. Int J Radiat Oncol Biol Phys 60, 18696. Yamamoto K, Padilla Alarcon J, Calvo Medina V et al (2003) Surgical results of stage I non-small cell lung cancer, comparison between elderly and younger patients. Eur J Cardiothorac Surg 23, 21-5. Yellin A, Hill LR, Lieberman Y (1985) Pulmonary resections in patients over 70 years of age. Isr J Med Sci 21, 833-840. Zachariah B, Balducci L, Venkattaramanabalaji GV et al (1997) Radiotherapy for cancer patients aged 80 and older, a study of effectiveness and side effects. Int J Radiat Oncol Biol Phys 39, 1125-1129.

Drs Cuneyt Ulutin and Gorkem Aksu

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Cancer Therapy Vol 3, page 101 Cancer Therapy Vol 3, 101-104, 2005

P-glyco protein (multi drug resistance protein product) does not predict the response of laryngeal and hypopharyhgeal cancer to chemotherapy Research Article

Avi Khafif1, Elizabeth Gillis2, Jesus E. Medina3, * 1

Department of Otorhinolaryngology the Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel, Department of Pathology and 3 Department of Otorhinolaryngology, The University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma 2

__________________________________________________________________________________ *Correspondence: Avi Khafif, Department of Otorhinolaryngology, Tel Aviv Sourasky Medical Center, 6 Wiezman street, Tel Aviv; Tel: (9723) 540-0526; E-mail: khafif@tasmc.health.gov.il Key words: G-glyco protein, hypopharyhgeal cancer, chemotherapy, Immunohistochemistry, Clinical study Abbreviations: 5-fluorouracil, (5-FU); diaminobenzidenetertrahydrochloride, (DAB); multidrug resistance gene, (MDRG); proliferating cell nuclear antigen, (PCNA); P-glycoprotein, (P-GP); squamous cell carcinoma, (SCC) Received: 26 December 2004; Revised: 28 February 2005 Accepted: 28 February 2005; electronically published: March 2005

Summary Larynx preservation protocols using combinations of chemotherapy and radiation are effective in some patients with advanced resectable head and neck cancer. The purpose of the current study was to investigate whether Pglycoprotein (P-GP), the product of the multi-drug resistance gene, correlates with tumor response to chemotherapy, survival and the rate of laryngeal preservation using a larynx preservation protocol for advanced squamous cell carcinomas of the larynx and hypopharynx. Twenty-four patients with advanced cancer of the larynx and hypopharynx, were prospectively assigned to receive two cycles of cis-platinum and 5-FU followed by radiation. Immunohistochemistry for P-GP was performed and staining correlated with response to chemotherapy, survival, and the rate of laryngeal preservation. Six patients (25%) had tumors that exhibited positive staining for P-GP. Four of these patients (66%) responded to chemotherapy and three (50%) are alive at the time of last follow-up. Of the 18 patients with tumors that exhibited negative staining for P-GP, 15 (83%) responded to chemotherapy, and 9 (50%) are alive at the time of the last follow-up. The response to chemotherapy, survival and rate of laryngeal preservation was similar in the two groups of patients. Positive staining for P-GP did not correlate with a response to cis-platinum and 5-FU in patients with SCC of the larynx and hypopharynx, nor did it predict a decreased survival or a lower rate of laryngeal preservation in these small cohort of patients. significantly correlated with the rate of laryngeal preservation. However, only tumor stage correlated with response to chemotherapy and none of these factors correlated with survival. Glutathione and Glutathione STransferase play a major role in cellular protection against free radicals and oxidative stress but their levels did not predict tumor response to platinum or 5-FU in patients with SCC of the Head and Neck (Bier et al, 1996). Etienne et al, (1995) studied the effect of Dihydropyrimidine dehydrogenase, the enzyme responsible for the degradation of 5-fluorouracil (5-FU), on response of 62 patients with head and neck cancer to 5-FU. They concluded that 5-FU catabolism in target cells is probably a determinant factor for 5-FU responsiveness in cancer

I. Introduction Larynx preservation regimens using chemotherapy and radiation are being used extensively as part of experimental larynx preservation protocols in the treatment of patients with advanced squamous cell carcinoma (SCC) of the larynx and hypopharynx. The resistance of tumor cells to chemotherapy, however, is a major problem, and recent studies have focused on understanding the mechanisms of this resistance. Bradford et al (Bradford et al, 1999) investigated factors affecting the response in patients treated as part of the Veterans Affairs Laryngeal Cancer Cooperative Study. They found that a high T-stage, positive staining for p-53 and high levels of proliferating cell nuclear antigen (PCNA)

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Khafif et al: G-glyco protein as a response predictor of laryngeal and hypopharyhgeal cancer patients. This enhanced catabolism may be mediated by high expression of the multidrug resistance gene (MDRG). The purpose of this study was to determine whether response to chemotherapy could be predicted by an immunohistochemistry staining for P-Glycoprotein (PGP), the protein product of the MDRG.

Twelve patients are alive with no evidence of disease and 12 are dead of disease. The larynx was preserved in 9 patients and only three patients were salvaged with a total laryngectomy and were alive with no evidence of disease at the time of last follow-up. Altogether, six patients had tumors that exhibited a positive staining for P-GP while 18 patients had tumors that had negative staining. Four of the patients with positive staining for P-GP (66.6%) and 15 of the patients with negative staining (83%) responded (PR or CR) to chemotherapy (p>0.05, table 1). Three of the six patients with positive staining (50%) died of their disease, comparable to 9 of the 18 patients (50%) with negative staining for P-GP (p>0.05). Two of the six patients (33%) that had a positive staining for P-GP are alive with their larynx preserved compared to 7 of the 18 (38%) that had negative staining (p > 0.05).

II. Materials and Methods A. Clinical study Patients with advanced, resectable SCC of the larynx and hypopharynx were enrolled onto the study and were treated with two cycles of chemotherapy including cis-platinum (100mg/M2) and 5-FU (1gr/M2/24h for 5 days). Patients with cardiac comorbidity received carboplatin and 5-FU. If the tumor decreased to less than 50% of its original size, a third cycle of chemotherapy was usually added and was followed by a full course of radiation. If less than 50% response was observed, salvage surgery (i.e. total laryngectomy) was performed. End points for analysis were death or recurrent disease and laryngeal preservation. Follow-up data was collected for a minimal period of two years or until death. The study was approved by the institutional review board (IRB).

IV. Discussion The MDRG P-GP had been suggested as a reliable predictor of response of tumor cells to chemotherapy drugs. The results of the present study suggest that immunohistochemical staining of SCCa of the larynx and hypopharynx does not correlate with the response of the tumor to chemotherapy with cis-platinum and 5-FU patient survival or rate of larynx preservation. These results are interesting because the tumors studied represent a relatively homogeneous group of advanced tumors of only two sites within the head and neck, which were treated with a single chemotherapeutic regimen and were systematically evaluated for response to therapy. These results may shed some light on the controversy existing in the literature about the role of P-GP expression as a predictor of tumor response to chemotherapy. Recently, Lo-Muzio et al, (2000) studied tumors from 30 patients with squamous cell carcinomas of the oral cavity and normal tissues from 6 patients by immunohistochemistry staining to P-GP. They found that 66% of their normal and 80% of their tumoral tissues showed a positive staining. Interestingly, in 4 patients the tumor showed strong staining while the surrounding normal tissues were negative, and in 6 patients a strong positive staining was observed in areas of higher degree of differentiation. The authors suggest that activation of the MDRG may occur early in the carcinogenesis process and may be involved in tumor response to chemotherapy. This is supported by a recent study (Warnakulasuriya et al, 2000) reporting a significant correlation of P-GP expression and response to chemotherapy and radiation in 111 patients with advanced head and neck cancer. Cordon-Cardo et al, (1990) performed immunohistochemistry staining for P-GP in various tissues and concluded that although P-GP expression can be detected in a variety of tumors, further studies are needed to establish the significance of such findings. Filiptis et al, (1997) investigated the association between expression of the MDRG and survival or response to chemotherapy in patients with acute leukemia. Positive immunocytochemistry staining for MDRG had no impact on survival or response to induction chemotherapy in these

B. Immunohistochemistry The histopathology biopsy specimens of the 24 patients included on the study were retrieved. Representative tumor tissue slide preparations made from the formalin fixed paraffin embedded tissue. These were immunostained using antibody, NCL- PGLYm (Novocastra Labs) with specificity for Human Pglycoprotein,C-terminal cytoplasmic domain. Immunohistochemistry was performed by the avidin-biotin complex method of Hsu et al*. Following deparaffinization, inhibitor solution was optimized to remove endogenous peroxidase activity for 4 minutes at 37째C.The optimized antibody is applied and incubated for 32 mins. at 37째C. This was followed by a biotinylated horse anti-mouse immunoglobulin with 8 mins incubation at 37째C and Avidin-HRPO for another 8 mins (Ventana, Tuczon, AR). Chromogen solution consisted of 0.05% diaminobenzidenetertrahydrochloride (DAB) with 0.07% H202. Sections were counterstained with hematoxylin and carefully examined. Membrane immunoreactivity in tumor was scored as follows: positive, trace (< 10% of tumor cells staining), and negative. Liver was used as the recommended positive control. Negative controls lacking primary antibody were also evaluated in each case for background staining. Diffuse weak and diffuse strong staining were considered positive staining while focal traces or no staining were reported as a negative staining. Degree of staining was correlated with survival, response to chemotherapy and the rate of laryngeal preservation. A twotailed t-test was used to evaluate the statistical significance of this correlation and a p value equal or smaller than 0.05 was considered a significant difference.

III. Results Twenty-four patients (18 men and 6 women) were enrolled in the study between the years 1993-1995. All had locally advanced (T3-T4) primary tumors located in the larynx (n=19) and hypopharynx (n=5). Nineteen patients had a complete or partial response and received all three cycles of chemotherapy followed by radiation. Five patients failed to show more than 50% reduction and had a salvage laryngectomy. Thirteen patients (54%) had a salvage laryngectomy either for persistent or recurrent disease. All patients had a minimal follow-up of 2 years. 102


Cancer Therapy Vol 3, page 103 Bradford CR, Wolf GT, Carey TE et al (1999) Predictive markers for response to chemotherapy, organ preservation, and survival in patients with advanced laryngeal carcinoma. Otolaryngol Head and Neck Surg 121, 534-8. Cordon-Cordo C, Oâ&#x20AC;&#x2122;Brien JP, Boccia J, Casals D, Bertino JR, Melamed MR (1990) Expression of the Multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Hitochem Cytochem 38, 1277-87. Darnton SJ, Jenner K, Steyn RS, Ferry DR, Matthews HR (1997) Lack of correlation of P-glycoprotein expression with response to MIC chemotherapy in esophageal cancer. J Clin Pathol 48, 1064-6. Etienne MC, Cheradame S, Fischel JL et al (1995) Response of fluorouracil therapy in cancer patients, the role of tumoral dihydropyrimidine dehydrogenase activity. J Clin Oncol 13, 1663-70. Filipits M, Suchomel RW, Zochbauer S, Brunner R, Lechner K, Pirker R. (1997) Multidrug resistance-associated protein in acute myeloid leukemia, No impact on treatment outcome. Clin Cancer Res 3, 1419-25. Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques, a comparison between ABC and unlabelled (PAP) procedures. J Histochem Cytochem 29, 577 Lo-Muzio L, Staibano S, Pannone G, et al (2000) The human multidrug resistance gene (NDRG-1), immunohistochemistry detection of its expression in oral SCC. Anticancer Res 20, 2891-7. Rabkin D, Chhieng DC, Miller MB, Jennings T, Feustel P, Steiniger J, Parnes SM (1995) P-glycoprotein expression in the squamous cell carcinoma of the tongue base. Laryngoscope 105, 1294-9. Warnakulasuriya S, Jia C, Johnson N, Houghton J (2000) p53 and P-glycoprotein expression are significant prognostic markers in advanced head and neck cancer treated with chemo/radiotherapy. J Pathol 191, 33-8.

patients. Darnton et al. investigated the response of 27 patients with esophageal cancer to MIC chemotherapy (mitomycin, ifosfamide, cisplatin) and found that 20/27 (70%) of patients with squamous cell carcinoma responded to chemotherapy and only one expressed P-GP, whereas 3/10 (30%) of the patients with adenocarcinoma responded to chemotherapy and of these 7 showed over expression of the P-GP. These concluded that the presence of P-GP might explain the lower response of adenocarcinoma to MIC chemotherapy; however, it cannot explain the variable response rates in patients with SCC since all these tumors showed low expression of P-GP. Rabkin et al, (1995) on the other hand, reported over expression of P-GP in all squamous cell carcinomas of the base of the tongue. Analysis of these two later studies reveals that the rate of expression of P-GP was not different in most patients with squamous cell carcinomas and the different rates of expression may reflect various sensitivity for detecting P-GP. The use of P-GP as a predictor of response in these patients is thus questioned, a fact that is supported in the current study; P-GP did not predict the outcome after chemotherapy in patients with advanced Laryngeal and Hypopharyngeal squamous cell carcinomas. The reliability of P-GP expression as a predictor of response to chemotherapy in patients with laryngeal and hypopharyngeal Squamous cell carcinomas is questioned.

References Bier H, Hoffmann T, Eickelmann P, Hafner D (1996) Chemosensitivity of head and neck squamous carcinoma cell lines is not primarily correlated with glutathione levels but is modified by glutathione depletion. J Cancer Res Clin Oncol 122, 653-8.

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Cancer Therapy Vol 3, page 105 Cancer Therapy Vol 3, 105-130, 2005

Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? Review Article

Daila S. Gridley1,2, Jerry R. Williams1 and James M. Slater1 1

Department of Radiation Medicine and

2

Department of Biochemistry & Microbiology, Loma Linda University and Medical Center, Loma Linda, CA 92354, USA

__________________________________________________________________________________ *Correspondence: Daila S. Gridley, Ph.D., Chan Shun Pavilion, Room A-1010, 11175 Campus Street, Loma Linda, CA 92354; Telephone: (909) 558-8361; Fax: (909) 558-0825; E-mail: dgridley@dominion.llumc.edu Key words: radiosensitivity, radioresistance, radio-adaptation, dose-rate effect, inverse dose-rate effect Abbreviations: atomic bomb, (A-bomb); Central nervous system, (CNS); chronic myelogenous leukemia, (CML); computed tomography, (CT); different poly(ADP-ribose) polymerase-1, (PARP-1); epidermal growth factor receptors, (EGFR); Epstein-Barr virus, (EBV); Food and Drug Administration, (FDA); gastrointestinal tract, (GI); human lung fibroblasts, (HFL-1); hypoxanthine-guanine phosphoribosyl transferase, (HPRT); Lewis lung carcinoma, (LLC); intensity-modulated radiation therapy, (IMRT); linear energy transfer, (LET); lipopolysaccharide, (LPS); magnetic resonance imaging, (MRI); major histocompatibility complex, (MHC); monoclonal antibodies, (Mab); Natural killer, (NK); positron emission tomography, (PET); retinoblastoma, (Rb); subcutaneous, (s.c.); T cytotoxic, (Tc); T helper, (Th); thymidine kinase, (tk); total-body irradiation, (TBI); vascular endothelial growth factor, (VEGF) Received: 28 January 2005; Accepted: 4 February 2005; electronically published: March 2005

Summary Ionizing radiation has been a major cancer treatment modality for more than five decades. Its use has resulted in increased cure rates and prolonged survival time. The treatment paradigms that are currently applied in the clinic are limited in terms of total dose and the temporal patterns used. Part of the reason for this is due to the historical effectiveness of smaller fractions, i.e., ~2 Gy, delivered at dose rates of approximately 50 Gy/hr in near daily fractions to a total dose of approximately 60 Gy. Larger fractions have sometimes led to unacceptable normal tissue toxicity. Lower dose-rates have not been widely used due to two reasons: 1) limited studies generally indicate that reduced dose-rates delivered to the same total dose are less effective in producing tumor regression and 2) the mechanisms by which lower dose rates induce cell death and other relevant endpoints are not well understood. However, many reports now suggest that lower dose rates may be effective in cancer therapy, albeit not always when used in the same way that higher dose rates are used in conventional therapy. In addition, increasing advances in radiotherapy technologies with improved spatial dose-distribution have rejuvenated interest in optimizing radiotherapy time-dose regimens. Especially intriguing from the radiation oncology point of view are reports that 1) low-dose/low-dose-rate irradiation can be more effective in killing tumor cells than high dose-rate irradiation under certain conditions, 2) lower dose irradiation delivered over an extended period of time may preferentially sensitize neoplastic cells to a subsequent high-dose-rate exposure and 3) low-dose/low-dose-rate radiation can induce radioresistance in some normal cells. The phenomenon of radiation-induced protection against acute exposure, originally known as ‘hormesis’ and today referred to as radio-adaptation, has been especially well studied in lymphocytes. Indeed, it has been proposed that radio-adaptation in lymphocytes and perhaps other immune system cells, together with the “danger” signal provided by damaged neoplastic cells, may contribute to tumor control. Collectively, these observations suggest that alternative but effective radiotherapy may produce an improved therapeutic ratio by combining low- and high-dose-rate radiotherapy. These observations also indicate a need to better understand the dose-rate effect as it modulates tumor and normal tissue responses. This review will consider the effect of dose-rate as a variable in radiotherapy and the mechanistic processes that may underlie doserate effects. We accept that the consequences of low-dose/low-dose-rate radiation on both tumor and normal cell responses have not yet been firmly established. Published reports are sometimes contradictory, indicating that priming with protracted low dose-rate exposures may increase, decrease, or have no effect on radiosensitivity. The apparent discrepancies can be at least partly explained by the dependence of low-dose/low-dose-rate irradiation effects upon many variables, including specific cell type, dose rate, total dose delivered, radiation quality, time of testing, endpoint measured and in vitro versus in vivo exposure. Historically, the differences in biological 105


Gridley et al: Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? consequences observed between low- and high-dose-rate irradiation have been largely attributed to variations in the length of time available for DNA repair and accumulation of cells in G2 (considered to be the most radiosensitive phase of the cell cycle) during intermittent low-dose or continuous low dose-rate exposure. However, it appears now that the underlying mechanisms are more complex than originally assumed. Here we seek to accomplish three goals: 1) suggest an analytical grouping of dose-rates based on the time frame of irradiation and the time frame of relevant biological processes, 2) propose an analytical framework for analyzing lower dose-rate cell survival responses and 3) review the reported effects of low-dose/low-dose-rate radiation on the responses of both tumor and normal cells. Potential for increased tumor control with radiotherapy protocols that combine low dose-rate and high dose-rate irradiation is also discussed. It is important for this review to use terminology that is internally consistent and to conform, as far as possible, with conventions in the literature. For this review, we use six dose-rate groups and abbreviations to describe doserate ranges: ultra-high dose rate, UHDR (~105 Gy/hr); high-dose rate, HDR (10-100 Gy/hr); intermediate-dose rate, IDR (1.0-10 Gy/hr); low-dose rate, LDR (0.1-1.0 Gy/hr); very-low dose rate, VLDR (0.01-0.1 Gy/hr); and ultra-low dose rate, ULDR (<0.01 Gy/hr). These groups, together with comments are summarized in Table 1. It is also important to point out that while we use these groupings in this review, the dose-rate is not an absolute constant for any radiation because the microdosimetry that describes the relative number of ionizations produced within a microvolume varies even with so called minimally-ionizing radiation such as x-rays or !-rays. The time pattern for delivering radiotherapy has always been recognized as an important variable in outcome. Use of five daily fractions per week (9-10 Gy total/week) has emerged as the standard time-dose regimen for treating many solid tumors. Hyperfractionation and now also hypofractionation, has been evaluated in limited studies in which the fraction size, time between radiation fractions and the total dose are either reduced or increased. However, these protocols still use HDR during radiation delivery. While we believe such experiments may involve some of the same mechanisms that are involved in doserate effects, we will not emphasize these types of experiments in this review. Cellular studies of those dose rates that are used most often in the clinic, ranging from ~0.1 Gy/hr to several Gy/min, have been largely derived from experiments with cultured cells (Hall and Brenner, 1991). The higher dose rates are within the range that we refer to as the HDR range, whereas the lower rates used in brachytherapy (i.e. implantating a radioactive source) extend into the IDR and LDR ranges. Thus, we suggest that the mechanisms that influence cell killing over these different ranges may differ and need to be considered in a more detailed, segmented analysis. Overall, the regimens employed for radiotherapy have been based on empirical observation of outcome, equipment limitations and patient and staff convenience.

I. Introduction A. Overview In spite of numerous improvements in cancer management, there is an urgent need to implement new treatment approaches. Estimates made by the American Cancer Society indicate that there were 1,368,030 newly diagnosed cases and 563,700 cancer-related deaths in 2004 (Jemal et al, 2004). With current therapies almost 50% of patients will eventually die because of local recurrence, metastatic disease, or a combination of both (Lichter 2000). Furthermore, the rapid expansion of elderly individuals in the United States, as well as in many other countries, will undoubtedly lead to a substantial increase in the prevalence of cancer. Since the discovery of x-rays by Wilhelm Roentgen more than 110 years ago, radiation has become an important and often indispensable modality for cancer treatment. The development of improved radiotherapy protocols has been closely allied with understanding of factors that determine treatment outcome. One of these has been improved definition of tumors in three-dimensions with the development of magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET) and molecular imaging. Another improvement has been in the understanding of the physical and chemical processes associated with predicting dose and microdose in tumors and normal tissues. Finally, there has been a better understanding of the radiobiology associated with the response of tumor and normal tissues to different temporal patterns of dose delivery. These areas of improvement could be combined into highly sophisticated radiation therapy protocols using more advanced planning systems. For example, planning systems originally proposed by Slater and colleagues in 1974 have provided guidance in newer treatment modalities such as the use of charged heavy particle and intensity-modulated radiation therapy (IMRT) (Slater et al, 1988, 1992; Purdy, 1996; Mackie and Smith, 1999; Bastasch et al, 2002; Baumert et al, 2003; Teh et al, 2003). All of these advances have contributed to more accurate tumor targeting and thus also to increased sparing of normal tissues. However, understanding is still lacking as to how this improved dose distribution can be used to optimize the therapeutic ratio (tumor cure/normal tissue toxicity). Better understanding of dose-rate effects may aid in such optimization and thus also decrease the need to sometimes underdose the tumor in order to prevent severe normal tissue complications.

B. Proton therapy as a model of a mixed dose-rate radiation Proton radiotherapy has several characteristics that relate to dose and dose-rate considerations. These characteristics are: 1) protons are administered as a rapid sequence of â&#x20AC;&#x153;spillsâ&#x20AC;? or small packets of protons and thus

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Cancer Therapy Vol 3, page 107 Table 1. Assignment of dose-rates into groups based on temporal chemical and biological processes Dose-rate group

Dose-rate 5

Comments

Ultra-high dose-rate, UHDR

~10 Gy/hr

While some studies have been performed with “flash x-rays,” this range of dose is not well studied. We define this dose-rate as generating multiple ionizations within a time frame that is short compared to the mean lifetime of induced chemical species. Although these data are not reviewed here, this category is included since it may represent effective dose-rate in some irradiated microvolumes when densely ionizing particles traverse a microvolume.

High dose-rate, HDR

10 to 100 Gy/hr

This class of dose-rates is representative of most dose-rates used in standard multi-fraction radiotherapy. We suggest the data show that small changes, up to a factor of 2, in HDR does not induce significant modulation of radiation response.

Intermediate dose-rate, IDR

1.0 to 10 Gy/hr

This class of dose-rates has been studied in a limited number of experiments. Steele has suggested that the lower limit of this dose-rate range is the rate at which there is no longer a perceivable sparing of cell killing when cells are subjected to higher dose-rates (Steele, 1996). This encompasses a range of dose-rates over which the time needed to deliver clinically relevant dose fractions (1.5 to 5.0 Gy) varies from 1.5 to 5 hours. This is a time frame at the lower end of the duration over which survival can be manipulated by splitting the dose or maintaining cells under sub-optimum growth conditions.

Low dose-rate, LDR

0.1 to 1.0 Gy/hr

This is a range of dose-rates over which the time needed to deliver clinically relevant fractions (1.5 to 5.0 Gy) varies over times in which many biological processes that are considered relevant to cell survival are changing the response status of the cell. These include: induction of altered gene expression, sub-lethal damage repair, potentially-lethal damage repair, induction of perturbation in cell cycle redistribution and susceptibility to apoptosis. Most reports of dose-rate effects have used dose-rates in this range.

Very low dose-rate, VLDR

0.01 to 0.1 Gy/hr

This range of dose-rates has not been extensively studied due to lack of observed effects. We propose that radiosensitivity of tumor cells can be altered within this dose-rate range when challenged by larger, acutely-delivered fractions.

Ultra low dose-rate, ULDR

<0.01 Gy/hr

This is a hypothetical range and we suggest that at some doserate less than 0.01 Gy/hr it will not be possible with present tools to identify effects relevant to radiotherapy.

the dose delivered is not continual but discontinuous over a very small time scale: While the “average dose-rate” at which protons are administered are comparable to photon high dose-rate (circa 50 Gy/hr) irradiation, instantaneous dose-rates during a “spill” can reach 600 Gy/hr; 2) protons produce a higher proportion of ionization clusters so that for some cell/tissue volumes, there is an increase in effective dose-rate; and 3) improved spatial dosedistribution of deposited radiation with protons permits using larger fractions with lower risk for toxicity compared to photons, a characteristic that we suggest can be used to exploit cell sensitization by low-dose-rate irradiation. Proton therapy is maturing as a first-line radiotherapy for a large number of patients. The clearest example has occurred at the proton treatment facility at the Loma Linda University Medical Center (Slater et al, 1988,

1990; Archambeau et al, 1992). Proton beams, consisting of charged particles that deposit the bulk of their energy as they approach their stopping edge (Bragg peak), are modulated such that the Bragg peak is distributed throughout the intended treatment volume. This then distributes microregions of higher dose-rate through the tumor mass. Because of the lower integral dose delivered during proton radiotherapy, the risk for normal tissue damage is minimized, as evidenced by data presented in rapidly accumulating publications (Matsuzaki et al, 1994; Gridley et al, 1996, 1998, 2004; McAllister et al, 1997; Yonemoto et al, 1997; Slater et al, 1998, 2004; Bush et al, 1999; Rossi 1999; Lin et al, 2000; Schulte et al, 2000; Bonnet et al, 2001; Hug et al, 2002; Kirsch and Tarbell, 2004).

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Gridley et al: Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? assumed that accumulation in G2 was the major mechanism by which protracted LDR radiation sensitizes tumor cells to HDR radiation. Many reports utilizing a variety of neoplastic cell types have supported this possibility (Terasima and Tolmach, 1961; Bedford and Hall, 1963; Kal et al, 1975; Mitchell et al, 1979; van Oostrum et al, 1990; Knox et al, 1993; Skladowski et al, 1993; Fowler, 2003). The increased sensitivity in G2 may be the result of insufficient DNA repair before cells reach this stage (Parshad et al, 1984) and/or incomplete repair in G2 (Walters et al, 1974; Bases et al, 1980). However, other studies have found no association between G2 arrest and radiosensitivity (Cao et al, 1983; DeWeese et al, 1998; Mitchell et al, 2002). Most recently, it has been demonstrated that exposure to LDR radiation reduces activation of the DNA damage sensor ATM and its downstream target H2AX, resulting in decreased clonogenic survival compared to HDR radiation (Collis et al, 2004). Most data on dose-rate effects are presented as surviving fraction versus total dose. However the duration of exposure can vary from a few minutes to days. It is important, we suggest, to consider the variation of the rate of cell kill per unit dose as a function of duration of exposure. We illustrate this by replotting data previously reported by Marin, Dillehay and colleagues (Marin et al, 1991) utilizing human U251 glioblastoma cells. These authors observed little dose-rate effects from 0.086 Gy/hr to 49.0 Gy/hr, with survival clustering around the survival pattern for acute exposure. However when these data are plotted as the rate of cell kill per unit time, a different pattern is observed as shown in Figure 1.

C. Mechanisms involved in dose-rate effects 1. Fractionation over times ranging from 1 to 14 hours Early studies by Elkind and Sutton utilized split-dose radiation (i.e. fractionation) to simulate low-dose rates (Elkind and Sutton, 1959). Their findings led to the theoretical prediction that biological response to LDR radiation would be decreased compared to the same total dose delivered acutely and that the difference in the effect depended upon the repair efficiency of the resulting damage (Lajtha and Oliver, 1961). The existence of this dose rate sparing was subsequently supported by studies of HeLa cells that were also shown to accumulate in the G2 phase of the cell cycle during LDR exposure (Hall and Bedford, 1964; Bedford and Mitchell, 1973; Mitchell and Bedford, 1977). While the molecular mechanisms that underlie split dose recovery after acute irradiation are unknown, it is important to consider whether this phenomenon relates directly to dose-rate effects.

2. Inverse dose-rate effect Later studies, however, demonstrated greater HeLa cell death when radiation was delivered at an LDR rate of 0.37 Gy/hr instead of at 1.54 Gy/hr (Mitchell et al, 1979). This phenomenon became known as the inverse dose-rate effect. The explanation put forth for the increased sensitivity was that cells irradiated at LDR over an extended period of time do not repair all damage; with increasing accumulation in G2, the most radiosensitive phase of the cell cycle, enhanced radiosensitivity is manifested (Mitchell et al, 1979). Indeed, it was long

Figure 1. Survival curves for human U251 glioblastoma cells exposed to 57Cs at variable dose rates. Rate of cell killing expressed as log surviving fraction per Gy as a function of exposure time for dose rates in the very low dose-rate range (0.086 Gy/hr) and in the low dose-rate range (0.123, 0.25, and 0.49 Gy/hr) compared to the rate of cell kill for high dose-rate irradiation (~50 Gy/hr). Data were extracted from the work of Marin et al, 1991 and calculated by measuring the change in surviving fraction in logs for the preceding radiation interval and dividing it by the absorbed dose over the same time interval. The horizontal dotted lines are the rates of cell kill by high dose-rate radiation (~50 Gy/hr) measured as the slope of the survival curve at ~2 Gy (alpha) and for doses higher than 4 Gy (omega). U251 is a very radioresistant cell line derived from a human glioblastoma.

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Cancer Therapy Vol 3, page 109 In this figure we compare the rate of cell kill for these several low dose-rates to the rate of cell kill for radiation delivered acutely expressed as the slope at doses circa 2 Gy and at doses higher than 4 Gy. Alpha is the rate of cell kill induced per unit dose by high dose-rate (~50 Gy/hr) at approximately 2 Gy. Alpha is identical to the coefficient alpha as determined by linear-quadratic analysis of the acute survival curve. Omega is related to the inverse of the Do and is calculated by reiterative bestfit analysis of the terminal slope (> 4 Gy) of the acute survival curve. Thus omega represents the maximum rate of cell kill per Gy for acute irradiation. These data when plotted in a time-based format illustrate three important points. First, the rate of cell kill changes dramatically over time for different dose-rates. Second, for this cell line, the rate of cell killing increases as dose rate increases from 0.086 cGy/hr in the VLDR range and reaches a maximum at approximately 0.25 Gy/hr. Increasing the dose-rate to 0.49 cGy/hr does not increase further the rate of cell killing. The third point is most important for considering dose rates other than the HDR used in the clinic, i.e. the data in Figure 1 show that for some time points, the rate of cell kill is equally effective or more effective when delivered at 0.086 to 0.49 cGy/hr, than at rates usually associated with conventional cancer radiotherapy. A fourth point must be made to place these observations into a clinical context. There is a dramatic difference in cellspecific variation between human tumor cell lines observed by Joiner and his colleagues (Marples et al, 1997; Short et al, 1999; Joiner et al, 2001) and by Williams and his colleagues (unpublished data). In some of these studies, the rate of cell kill can be more effective for dose-rates as low as 2 cGy/hr than for acute irradiation but only in some cell lines. In other cell lines this dose-rate is completely ineffective. Joiner and his colleagues suggest this response to low dose-rate correlates with cell susceptibility to induced hypersensitivity by low acute doses (< .05 Gy). Thus, there seems to us to be a strong imperative for further study of dose-rate as a possible important clinical tool, defining the molecular and cellular characteristics that will predict the response of tumor cells to lower dose-rates. Selecting an appropriate dose rate and dose per fraction would, of course, be important. It has been proposed that a minimum dose-rate effect is most likely to occur within the range of 0.06 to 0.6 Gy/hr (Vilencheck and Knudson, 2000). Table 2 presents examples of studies in which an inverse dose-rate effect has been observed with protracted exposure to low-linear energy transfer (LET) radiations such as x-rays and !-rays.

3. Radio-adaptation augmentation

and

lymphoma (Ishii et al, 1996b) and increase the number of cells needed to produce a progressively growing tumor (Sakamoto et al, 1987). The low-dose radiation-initiated immune augmentation appears to be related to enhanced secretion of cytokines needed for immune cell activation (Sherman et al, 1991; Ishii et al, 1996a; Hosoi et al, 2001), increased expression of adhesion molecules that facilitate leukocyte trafficking (Hallahan, 1996; Hallahan et al, 1996, 1997) and preferential destruction of cells with immunosuppressive properties (Tilkin et al, 1981; North, 1986). The rationale for the latter mechanism is based partly on observations that infiltrating tumor-specific T cells are converted from a radiosensitive to a more radioresistant state due to activation by tumor antigens (Dunn and North 1991). Activated lymphocytes have long been known to be more resistant to radiation than their resting (non-activated) counterparts (Anderson and Warner, 1976). Further investigation in this area may contribute to greater efficacy of both localized and more general application of low-dose LDR radiotherapy. It has even been proposed that a radiation regimen could be implemented to enhance response to tumor antigens in individuals immunized with cancer vaccines (Friedman, 2002).

D. Dose-rate in clinical studies Interstitial and intracavitary implantation of radioactive sources (i.e. brachytherapy) was first practiced in the early 1900s and, with the exception of gynecologic malignancies, abandoned owing to poor results. However, recent improvements in placement and imaging have generated renewed interest in this form of radiotherapy. Brachytherapy is an extremely important model to consider the effectiveness of LDR irradiation. Brachytherapy delivers a mixture of dose-rates from HDR to LDR to VLDR. It can deliver very high total doses that are supra-lethal to tumor volumes immediately contiguous to implanted sources and failure of brachytherapy is usually attributed to tumor volumes on the periphery of the irradiated volume that escape lethal exposure. Although peripheral volumes receive essentially lower dose-rate irradiations (Lichter, 2000), there is little or no evidence to indicate that susceptibility of the peripheral cells to acute external-beam radiation treatment is altered. Radioimmunotherapy, involving infusion of radiolabelled monoclonal antibodies (Mab), is experiencing renewed interest. Many Mab conjugated to radionuclides such as 111In and 99mTc have already been approved by the Food and Drug Administration (FDA) for tumor imaging and conjugation to high-energy therapeutic radionuclides such as 131I or 90Y has resulted in some success. Thus, it appears that radiolabelled Mab could be exploited to concentrate low doses of LDR radiation to tumors (Knox et al, 1990, 1993; Kroger et al, 2001; Eriksson et al, 2003; Hernandez and Knox 2003) and to induce low dose-rate effects that would increase the efficacy of focal external beam therapy. It is clear that a better understanding of the key mechanisms responsible for both tumor and normal cell responses to low-dose protracted irradiation is needed before widespread clinical application can be seriously contemplated. This most

immune

Studies of low-dose total-body irradiation (TBI) with external beam sources suggest that additional benefit may be gained through up-regulation of immunological mechanisms that contribute to the overall anti-tumor effect. Pre-clinical reports indicate that low doses of TBI can significantly delay tumor growth (Anderson et al, 1982), decrease incidence of lung metastases (Hosoi and Sakamoto 1993), decrease incidence of spontaneous 109


Gridley et al: Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? Table 2. Studies in which an inverse dose-rate effect has been reported after low-LET irradiation Cells L5178Y (also known as LY-R) cells WI-L2-NS cells V79 cells (and sublines including V79-S85) TK6 cells

HeLa cells T98G cells A7 cells PC-3 cells DU145 cells RKO cells R1 cells

PC-3 cells PPC-1 cells TSU-Pr1 cells NHK 3025 cells U251 cells U87 cells Bp8 cells

38C13 tumor Lewis lung carcinoma (LLC)

Description mouse lymphoma/ leukemia cell line human lymphoblastoid cell line Chinese hamster lung fibroblast cell line human lymphoblastoid cell line human cervical carcinoma cell line human glioblastoma human glioblastoma human prostate cancer cell lines human prostate human colon cancer cell lines rat rhabdomyosarcoma cell line

human prostate cancer cell lines human cervical carcinoma cell line human glioblastoma cell lines mouse ascites sarcoma cell line

mouse B cell lymphoma mouse lung carcinoma

Endpoints HPRT mutations

Dose rate 0.006 Gy/hr

HPRT mutations

0.1998 Gy/hr

mutations that induce 6-thioguanine resistance HPRT mutations

0.0045-0.029 Gy/hr

0.027 Gy/hr

Amundson & Chen 1996

survival of single cells

0.37 Gy/hr

clonogenic survival

<1 Gy/hr

Mitchell JB et al, 1979 Mitchell CR et al, 2002

clonogenic survival

0.094 Gy/hr

Collis et al, 2004

lipid peroxidation, DNA damage, cell morphology (micronuclei, apoptosis, necrosis) clonogenic survival

4.26 Gy/hr

Przybyszewski et al, 2002

0.25 Gy/hr

DeWeese et al, 1998

clonogenic survival

<0.86 Gy/hr

Furre et al, 1999

clonogenic survival

<0.49 Gy/hr

Marin et al, 1991

flow cytometry: mitotic index, total tumor cell no., cell cycle analysis tumor growth

1.11 Gy/hr

Cao et al, 1983

0.183 Gy/hr

micronuclei (apoptosis) of LLC cells in vitro after tumor irradiation

20.4 Gy/hr

Knox et al, 1990, 1993 Widel & Przybyszewski 1998

certainly also holds true for the immune-related mechanisms that are triggered by radiation exposure.

References Furuno-Fukushi et al, 1988 Colussi & Lohman 1997 Crompton et al, 1990

reported variability and sometimes contradictory results make generalizations difficult. Selected relevant publications are summarized on the basis of tumor type in the following sections.

II. Dose-rate effects on malignant cells As noted above, evaluation of dose-rate effects on the survival of tumor cells began more than 40 years ago. Some of the early findings are described in detail in publications from the 1980s and 1990s (Kelland and Steel, 1986; Steel et al, 1987; Steel, 1991, 1996). Dose rate dependence has been noted for some, but not all, of the malignant cell populations that have been tested. The

A. Brain tumor In 2004, 18,400 new cases of brain and other nervous system cancers and 12,690 deaths were estimated to have occurred in the United States (Jemal et al, 2004). Although not among the most common neoplasms, high-grade malignant tumors of the brain are among the most deadly 110


Cancer Therapy Vol 3, page 111 and also among the most radioresistant type of cancer. The radioresistance has been well documented in reports of local recurrence within the targeted volume even after relatively high radiation doses (Wallner et al, 1989). Proposed mechanisms that may account for the resistance include radiation-induced up-regulation of antioxidant enzymes within the tumor cells (Lee et al, 2004), overexpression of epidermal growth factor receptors (EGFR) (Barker et al, 2001) and low expression of bax, a pro-apoptotic protein (Shu et al, 1998; Streffer et al, 2002). In addition, the possibility of tumor escape from immune surveillance by the production of transforming growth factor-"2 (TGF-"2), a highly immunosuppressive cytokine that is secreted by the majority of glioblastomas, is under investigation (Kingsley-Kallesen et al, 2001; Strege et al, 2004). Although TGF-"2 was not tested for, the pattern of cytokine mRNA expression in five human glioblastoma cell lines has been shown to be dependent upon radiation dose rate (Ross et al, 1997). Dose rates of 0.0035 Gy/min and 0.01 Gy/min generally reduced the mRNA levels for IL-1" and IL-6 compared to unirradiated control cells. On the other hand, exposures at 0.041 Gy/min and 2 Gy/min increased mRNA for both cytokines. This study also showed that the 0.041 Gy/min intermediate dose rate was less toxic than the lower and higher dose rates. Although cytokine gene expression did not have a clear effect on glioblastoma cell survival in this in vitro study, the investigators speculated that tumorderived cytokines may be important to radiation response in vivo by affecting immune cells, tumor stroma, vasculature, or surrounding tissues. The response of five human glioblastoma cells lines (T98G, A7, U87MG, U138 and HGL21) and one grade III astrocytoma cell line (U373) to low-dose radiation was investigated in culture (Short et al, 1999). All of these cell lines had been previously shown to be relatively radioresistant. Clonogenic survival was the measured end point after single doses of X-rays (0.05 to 5 Gy) were applied to the cells at 0.2 - 0.4 Gy/min. Low-dose hypersensitivity (i.e. less clonogenic survival than is predicted by a linear-quadratic fit to higher doses) was observed in five of the six cell lines below a total dose of 1 Gy, with the most dramatic effect occurring with the A7, U138 and T98G cells; no hypersensitivity was noted for the U373 astrocytoma cells. Lack of an inverse dose-rate effect on U373 cell survival has also been reported in other studies (Mitchell et al, 2002). Overall, this work supports the premise that low-dose hypersensitivity is a common, although not universal, feature of radioresistant human glioma cell lines. It also suggests that repair mechanisms induced by high, but not low, radiation doses may be an important component of tumor radioresistance. Large, acute doses may generate damage above a certain threshold that is needed to activate repair processes that are more efficient than those that function in constitutive DNA maintenance. However, recent studies by Marples and colleagues, in which a flow cytometry-based clonogenic survival assay was utilized to assess responses of hamster V79 and human T98G and U373 cells, have demonstrated that low-dose hyper-radiosensitivity exists for asynchronous and G2-phase enriched cell populations

(Marples et al, 2003). These findings indicate that the underlying mechanism for increased radiosensitivity at low doses may be a consequence of radiation-damaged G2-phase cells entering mitosis prematurely, rather than induction of DNA repair. These and similar data suggest the possibility that delivering a series of small doses per day (â&#x20AC;&#x2DC;ultrafractionationâ&#x20AC;&#x2122;) could result in increased destruction of radioresistant tumors compared to the same total dose given in conventional 2 Gy fractions (Marin et al, 1991; Short et al, 2001). Results for most human tumor cell lines indicate that increased sensitivity per unit dose occurs when radiation is delivered acutely within the range of 0.05 Gy to 0.5 Gy/fraction (Joiner et al, 2001). In a follow-up publication, the effect of ultrafractionated radiation on A7 glioma growing subcutaneously (s.c.) in athymic nude mice was determined (Krause et al, 2003). The A7 cells were among those that had previously shown a striking increase in radiosensitivity with ultrafractionated irradiation in vitro. Tumors were irradiated either with 126 fractions at 0.4 Gy/fraction or with 30 fractions at 1.68 Gy/fraction over a period of 6 weeks. A total dose of 50.4 Gy was delivered to both groups of animals. No increase in radiosensitivity was evident with ultrafractionation. In fact, a significant decrease in tumor growth delay occurred with ultrafractionation compared to the more conventional 1.68 Gy/fraction. The investigators noted that simplistic extrapolation from data obtained in vitro is not sufficient to predict the outcome in vivo and that comprehensive evaluation of new treatment options in animal models is essential before contemplating translation to the clinic. Other investigators have also noted lack of correlation between human glioma cell lines when exposed to fractionated radiation schemes in vitro and when treated as xenografted tumors in athymic rodents (Baumann et al, 1992), as well as increasing glioma cell survival as the radiation dose rate is reduced (Yang et al, 1990). In one of these studies, Yang and colleagues evaluated 16 clones of an early-passage human glioma cell line (IN859) based on DNA content, modal chromosome number, morphology and radiosensitivity (Yang et al, 1992). The radiation dose that gave a survival fraction of 0.01 varied by a factor of ~1.5. Comparison of the most sensitive and most resistant clones at a fixed total dose surprisingly demonstrated that the sensitive clone exhibited greater survival after split-dose irradiation. Finally, with T98G glioblastoma cells, a sparing effect was noted when the dose rate was reduced from 60 cGy/hr to 30 cGy/hr (Mitchell et al, 2002). The above findings, although somewhat disappointing, certainly do not disprove the existence of low-dose hyper-radiosensitivity for tumors originating in the CNS. In a very recent study by Chalmers and colleagues, the effects of four different poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors on low-dose (0.5 â&#x20AC;&#x201C; 0.3 Gy) radiosensitivity were evaluated using T98G and U373-MG human glioma cell lines (Chalmers et al, 2004). Hamster fibroblasts (V79-379A and CHO-K1) and mouse embryo fibroblasts (3T3) were also included in this study. Inhibition of PARP-1 resulted in sensitization of the glioma and most of the other cell lines to the low-dose

111


Gridley et al: Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? radiation; the only exception was the mouse 3T3 fibroblast line (i.e. PARP-1 knockout cells). The investigators suggested that PARP-1 inhibitors may be therapeutically valuable in radiotherapy regimens that consist of multiple small doses or continuous LDR radiation because of the enhanced inhibition of PARP-1 on rapidly dividing tumor cells. PARP-1 is an enzyme that binds rapidly to singleand double-stand DNA breaks and modulates the activity of many nuclear proteins (Dâ&#x20AC;&#x2122;Amours et al, 1999; Herceg and Wang, 2001). Deficiency in PARP-1 results in severely impaired base excision repair and genomic instability in response to low-dose radiation (Shall and de Murcia, 2000). An encouraging in vivo study by Williams and colleagues combined ULDR radiation together with HDR radiation using athymic mice that were s.c. implanted with U251 human malignant glioma cells (Williams et al, 1998). The ULDR radiation was delivered locally as intratumorally implanted 125I seeds (0.05 Gy/hr) or from an external 137Cs source (0.037 Gy/hr) that provided wholebody exposure. The external beam HDR radiation treatments were initiated 3 days later and consisted of 2 Gy x 8 daily fractions and 5 Gy x 2 daily fractions. These time-dose regimens were selected because they are clinically feasible and because previous experiments predicted equal growth delay of the tumor. The ULDR radiation alone had little or no effect on tumor progression and the two HDR external beam schedules resulted in a 20-25 day growth delay. However, the continuous administration of ULDR radiation with either 125I seeds or whole-body exposure to 137Cs significantly increased efficacy of the HDR treatments. A delay of 33-35 days in growth of the glioma was obtained with the combination treatments. 125 I seed implants together with fractionated HDR external beam radiation have been explored in treating patients with malignant brain tumors. In one study, a comparison was made between temporary 125I implants with a dose rate of 0.4 Gy/hr (total dose was 60 Gy) and permanent 125I seeds with a dose rate of 0.04 - 0.07 Gy/hr (total dose was 100-120 Gy); the two protracted regimens were delivered concurrently with external beam radiotherapy to a total of 50 Gy (Zamorano et al, 1992). The investigators concluded that use of either of these implants together with external radiation seemed to offer the best chance for long-term survival without deterioration of the clinical condition. In another report, 48 patients (after brain tumor resection) received permanent low-activity 125I implants together with conventional external beam therapy; 38 of these subjects were implanted 1-2 weeks before external beam treatment was initiated and 10 received external radiation before the implant (Fernandez et al, 1995). At the time of publication, median survival was greater than 31 months for patients with anaplastic astrocytoma and greater than 23 months in cases of glioblastoma. These are promising results, since median survival after sequential conventional radiotherapy and HDR temporary 125I implantation is only ~22 months (Scharfen et al, 1992).

B. Prostate tumor Adenocarcinoma of the prostate is the leading cancer diagnosed and the second most common cause of cancerrelated mortality in males residing in the United States. According to estimates of the American Cancer Society, there were approximately 230,110 newly diagnosed cases of prostate cancer and 29,900 deaths due to the disease in 2004 (Jemal et al, 2004). Most patients presenting with prostate cancer have localized disease and are candidates for radiation therapy. Exposure to fractionated HDR radiation is a standard treatment for localized prostate cancer. With conventional radiation time-dose regimens, historical local recurrence rates range from 25% to 62%, leaving considerable room for improvement. Protocols that increase local control are likely to be very important in mitigating the subsequent development of distant metastases and prolonging survival (Fuks et al, 1991; Kaplan et al, 1994). The still relatively high incidence of side effects (e.g. proctitis, rectal bleeding, increased urinary frequency, urethral stricture and gastrointestinal complications) is also a major concern. Studies describing tumor cell responses to LDR/HDR radiation combinations present the possibility that radiotherapy of prostate cancer could be substantially improved. Most prostate cancer cell lines require relatively large doses of HDR radiation to produce significant cell death (Wollin et al, 1989; Smalley et al, 1991; Kaver et al, 1991; Leith et al, 1993; Leith, 1994). Prostate cancer cells subjected to a single acute dose of 2 Gy (a commonly used dose/fraction in prostate cancer radiotherapy) generally have a survival fraction of approximately 0.56 (DeWeese et al, 1998), whereas the survival fraction of the more radiosensitive lymphomas is often <0.35 under similar conditions (Jones et al, 1973; Malaise et al, 1986). A study by DeWeese and colleagues showed that the LNCaP prostate cancer cell line with wild type p53 is relatively sensitive to killing by LDR radiation (0.25 Gy/hr), but that protracted exposure does not result in increased killing compared to fractionated HDR exposure (1 Gy/min) (DeWeese et al, 1998). On the other hand, PC3 cells, a human prostate cancer cell line with mutant p53, were relatively resistant to LDR radiation, but a higher fraction were killed with protracted LDR exposure than with fractionated radiation delivered at a high-dose rate. It was also found, using flow cytometry analysis, that LDR irradiation of LNCaP cells resulted in their accumulation at both G1/S and G2/M cell cycle transition points, whereas PC-3 cells were arrested only at G2/M. The investigators concluded that, rather unexpectedly, the radiation-induced cell cycle distribution pattern (G1/S and G2/M versus G2/M alone) seemed to have little effect on survival after irradiation at low-dose rates. TP53 gene status also appeared to play no significant role. In another study, these same investigators found that two related sublines exhibited different cell cycle phase distributions, but similar degrees of increased radiosensitivity after LDR irradiation (DeWeese et al, 1997). Our pilot experiments comparing the efficacy of photons (60Co !-rays) and protons have demonstrated that human LNCaP prostate carcinoma cells have a significantly lower surviving cell fraction when irradiated with proton radiation (modulated 112


Cancer Therapy Vol 3, page 113 Bragg peak of 250 MeV) than photon radiation at the same physical dose between the range of 2 â&#x20AC;&#x201C; 4 Gy (p = 0.02); by 16 hours DNA synthesis was lower for LNCaP cells irradiated with protons compared to photons (p = 0.01) (Baer et al, 2000). Based on several characteristics, the LNCaP cells resemble prostate tumor cells in patients with localized disease more closely than most other human prostate tumor cell lines in that they: a) are hormone responsive (Newmark et al, 1992); b) possess wild-type TP53 and the level of p53 protein increases upon exposure to DNA-damaging agents (Nelson et al, 1996; Newmark et al, 1992); and c) produce prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) (Gau et al, 1997; Young et al, 1991). Collectively, these observations together with the greater accuracy of proton beams in tumor targeting, support dose escalation with protons in combination with LDR protocols. In a clinical trial at the Mayo Clinic and William Beaumont Hospital, 57 patients with newly diagnosed bulky prostatic carcinoma (9% with stage B2 and 91% with stage C) were treated preoperatively with 5 Gy in one fraction, underwent pelvic lymphadenectomy and received 30-35 Gy from interstitially implantated iridium-192 ( 192Ir) seeds and received 30.6 Gy external beam irradiation in 17 fractions (Stromberg et al, 1994). The radiation from the 192 Ir seeds had an LDR of 0.7-0.8 Gy/hour and was delivered over a period of approximately 3 weeks between the two external beam treatments. The 5-year actuarial survival rate of 85% compared favorably with 5-year survival rates of 58-62% for stage C prostate cancer reported with external beam radiotherapy alone (Hanks et al, 1987; Bagshaw et al, 1990). The 5-year disease-free survival of 63% was similar to that observed after only external beam therapy (Arcangeli et al, 1991; Perez et al, 1988), indicating that distant spread is an important factor in the final outcome. The investigators suggested that the relatively high 5-year actuarial local control rates of 94% and 79.5% (clinical and pathological, respectively) may reflect a higher radiobiologically effective dose delivered to the prostate gland by the 192Ir implants. By modifying the 192Ir implantation technique, the initially high rate of rectal ulceration (24%) was reduced to 13% and only 4.5% of patients required surgical diversion.

Asynchronous and synchronized cervical carcinoma cells have been exposed to 60Co !-rays at 0.33 and 0.86 Gy/hr (Furre et al, 1999). Clonogenic survival data indicated the same degree of radiation sensitivity for exposure periods of less than 20 hr. However, with exposures of >20 hr, a 2-fold greater radiosensitivity was noted and was found to occur when 80% of the cells had accumulated in G2. Thus, in this study of cervical carcinoma cells, an inverse dose-rate effect was demonstrated (i.e. more efficient cell inactivation at lower compared to higher dose rates) when total radiation doses exceeded 7 Gy. Other investigators have also demonstrated that the total dose delivered over an extended time period can influence radiosensitivity (Lamerton and Lord, 1964; Hall et al, 1966). Dose-rate effects on human adenocarcinomas of the uterine cervix (NHIK-3025, HeLa, HeLa S3) and human squamous cell carcinomas (Me180 from the cervix and A431 from the vulva) have been investigated in xenotransplanted athymic mice (van Oostrum et al, 1990). Intratumorally implanted cesium-157 (157Cs; 117 MBq) needles 15 mm in length were used to deliver radiation at 0.5 Gy/hr. Tumor biopsies were taken from 21-160 hr after radiation initiation. One biopsy was taken from tissue that received a 10 Gy dose, whereas other biopsies were taken toward the tumor periphery that had received total doses of 2-9 Gy. Tumors were also exposed to a much higher dose rate of 3-4 Gy/min to total doses ranging from 3-10 Gy using a linear accelerator. Biopsies of these tumors were taken at times corresponding to biopsy times after the 0.5 Gy/hr irradiation. Changes in tumor cell cycle distribution were determined by flow cytometry analysis. All five tumor cell lines were arrested in G2 after the 3-4 Gy/min exposure, whereas only four were arrested in G2 after 0.5 Gy/hr irradiation. Me180 was the exception that accumulated primarily in the S phase. Maximum G2 accumulation was related to cell cycle time and not the dose. Both irradiation conditions resulted in similar changes in cell cycle progression, but a greater maximum G2 accumulation was observed with the higher dose-rate exposure. In a separate experiment, the investigators showed that the enhancement of cells in G2 correlated with radiosensitivity as determined by measurements of delay in tumor regrowth. In another study, the radiation response of three cervical carcinoma cell lines (HX155c, HX156c and HX160c) was compared in vitro and in vivo as xenografted tumors (Tonkin et al, 1989). 60Co !-rays were delivered at continuous rates of 0.03 â&#x20AC;&#x201C; 0.05 Gy/min and 0.7 â&#x20AC;&#x201C; 1.0 Gy/min. Two of the three cell lines showed significant low-dose sparing in vitro and there was a tendency for the in vivo tumors to reflect a similar pattern. However, there was less tumor growth delay in vivo than that predicted from the in vitro data. The effects of protracted radiation delivered by Mabs conjugated to radionuclides are being explored in combination with HDR external beam radiation in preclinical studies of gynecological tumors. This technique is also known as radioimmunotherapy. Success with this combination treatment was demonstrated in a recent study by Eriksson and colleagues utilizing athymic mice with

C. Cervical carcinoma In the United States, cervical carcinoma and other cancers of the female genital system accounted for about 82,550 new cases and 28,720 deaths in 2004 (Jemal et al, 2004). High-grade, advanced and/or recurrent gynecological malignancies tend to have a poor prognosis. They are difficult to cure with conventional external irradiation or any other therapeutic modality. Thus, patients with gynecological cancers are often selected for HDR, pulsed-dose-rate and other variations of brachytherapy (Jensen et al, 1998; Lessard et al, 2002; Rose, 2003). A regimen that includes low-dose protracted exposure has reportedly been beneficial in at least some of these cases. Increasing knowledge from pre-clinical studies, some of which are described below, should eventually help refine these protocols in patients.

113


Gridley et al: Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? s.c. implanted human HeLa Hep2 cervical carcinoma cells (Eriksson et al, 2003). Treatments with external beam radiation at 3 x 5 Gy and/or 100 Âľg 131I-labelled Mab against placental alkaline phosphatase or 131I-Mab against cytokeratin were administered separately or in combination; specific activity of the Mab was 120-200 Mbq/mg antibody. Although tumor growth retardation was observed with both external beam and the radiolabelled Mab, combination treatment enhanced the therapeutic effects further, resulting in a significant reduction in tumor volume. The long-lasting tumor growth inhibition was related to increases in tumor necrosis and apoptosis. The investigators suggest that this combination approach could increase therapeutic efficiency for epithelial cell-derived tumors in general.

dose rate-independent group are known to be involved in cell cycle regulation, whereas the majority of those in the dose rate-dependent cluster have roles in apoptosis. The findings were consistent with the significant decline in the percentage of apoptotic cells with decreasing dose rate that was observed with fluorescence microscopy. In spite of this, the investigators stated that for the majority of genes responding to low doses of radiation, a protective doserate effect does seem to apply in the case of this human myeloid leukemia cell line. A slight, but statistically significant, increase in survival has also been reported for LX830 mouse leukemia cells with dose rates decreasing from 30 mGy/hr to 6.2 mGy/hr (Furuno-Fukushi and Matsudaira, 1989). In this latter study, no differences in mutation frequency were associated with dose rate. Other investigators have found little or no dose-rate effects on survival of repair-deficient mutants such as mouse lymphoma LYS-s, xrs5, xrs6 and irs20 cells (Evans et al, 1985; Nagasawa et al, 1989; Stackhouse and Bedford, 1993).

D. Leukemias and lymphomas Approximately 110,960 individuals were newly diagnosed and 55,100 deaths occurred in the United States in 2004 owing to hematological malignancies (Jemal et al, 2004). Collectively, leukemia, lymphoma and multiple myeloma represent the fourth most common form of cancer. Current radiation and multiple-agent chemotherapy regimens can be curative in a substantial percentage of patients with certain forms of these diseases. Nonetheless, more children die of leukemia than any other disease and the death rates for non-Hodgkinâ&#x20AC;&#x2122;s lymphoma and multiple myeloma are increasing in the U.S.A. It seems possible that implementation of LDR radiation may be beneficial in at least some of these cases. In some studies of hematological malignancies, the apoptotic effect of protracted radiation has been demonstrated to be independent of p53 status. In one of these investigations, the Raji model that mimics therapyresistant human lymphomas with mutant p53 and increased bcl-2 expression was used (Kroger et al, 2001). Athymic nude mice bearing Raji lymphoma xenografts were treated with 67Cu-2IT-BAT-Lym-1 antibody (335500 ÂľCi) and observed for toxicity and tumor response for 84 days. Subsets of animals were euthanized at 3, 6 and 24 hr after therapy so that tumors could be examined for evidence of apoptosis and for p53, bcl-2, p21, GADD45, TGF-"1 and c-myc gene expression and protein level. Apoptosis was greatly increased in the treated xenografts, whereas bcl-2 gene and protein expression were substantially decreased. These changes occurred despite only modest cumulated radiation doses of approximately 0.56 Gy at 3 hr. Apoptosis preceded tumor regression by 4-6 days and 29% of the tumors were cured by cumulated tumor doses of ~18 Gy. Amundson and colleagues have studied gene expression patterns following LDR and acute !-irradiation of the human ML-1 myeloid leukemia cell line (Amundson et al, 2003). ML-1 cells have wild type p53 and are relatively responsive to ionizing radiation in that gene expression changes have been induced with as little as 0.1 Gy. The study evaluated a wide range of genes by cDNA microarray after irradiating the ML-1 cells at dose rates of 0.0028 Gy/min to 2.9 Gy/min, with total doses ranging from 0.02 Gy to 0.5 Gy. Two major clusters of genes were radiation-induced. The majority of genes in the

E. Other tumor types In a study by Williams and co-workers, four human tumor cell lines were evaluated in colonial survival assays after exposure to protracted irradiation: colon (GEO and LS174T), squamous cell (SQ-20B) and hepatocellular (HepG2) carcinoma (Williams et al, 1992). Analysis of cell survival data was performed using a 7-parameter simulation model (Dillehay, 1990). Tritiated water added to cell culture media was used for ULDR irradiation according to the formula 0.08 mCi/ml = 0.01 Gy/hr, whereas HDR radiation at 1.1 Gy/min was administered using a !-ray irradiator. Data for three of the cell lines (GEO, LS174T and SQ-20B) showed diverging rates of cell kill per unit dose of HDR radiation. With protracted irradiation, inactivation rates diverged at low doses, but tended to converge at higher doses. The HepG2 cells, tested only with LDR radiation at 0.03 Gy/hr, exhibited no redistribution in the cell cycle after 24, 48, or 72 hours of exposure. Neuroblastoma is a common malignancy of early childhood, most often arising from the adrenal medulla. A study of two human neuroblastoma cell lines (HX138 and HX12) demonstrated no dose-rate effects above 0.02 Gy/min (Holmes et al, 1990). In addition, when the dose rate was reduced below 0.02 Gy/min, both cell lines exhibited increased survival. Clonogenic survival and double-strand DNA breaks were examined in the RT112 human bladder carcinoma cell line using radiation dose rates ranging from 0.01 Gy/min to 1.28 Gy/min (Ruiz de Almodovar et al, 1994). Immediately after irradiation, cell survival increased with decreasing dose rate, as did the number of double-strand breaks measured by pulsed-field gel electrophoresis. However, when a 4-hour repair period was allowed after exposure, all dose rates resulted in approximately the same amount of damage. The investigators concluded that the level of un-rejoined double-strand DNA breaks did not correlate with survival at different dose rates, when detected by this particular gel electrophoresis technique.

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Cancer Therapy Vol 3, page 115 Tumor cells vary in terms of their radiosensitivity dependence on cell cycle distribution, as well as expression of pro-apoptotic genes (Grdina, 1980; Keng et al, 1984; Brock et al, 1987). In the case of some tumor cell types, the degree of radiosensitivity has been associated with levels of pro-apoptotic proteins such as p53 and bax. Ohnishi and collaborators found that cultured human squamous carcinoma cells primed with !-radiation at 0.001 Gy/min to a total dose of 1.5 Gy and then challenged with !-rays at 1 Gy/min exhibited decreased apoptosis and depressed accumulation of p53 and bax (Ohnishi et al, 2001). A recent study by Furre and co-workers investigated the radiosensitivity of T-47D human breast cancer cells (Furre et al, 2003). 60Co !-rays were delivered at dose rates of 0.37 and 0.94 Gy/hr; cell survival was quantified by the colony formation assay. No increase in radiation sensitivity was noted when the T-47D cells were accumulated in G2 (i.e. no inverse dose-rat effect). Irradiation at both dose rates resulted in nearly the same degree of radiosensitivity. Two-parametric flow cytometry analysis for presence of the retinoblastoma (Rb) protein and DNA content revealed that ~15% of the cells in G2 had the Rb gene product bound in the nucleus. The investigators proposed that the Rb protein could play a role in protecting the G2-arrested cells against radiationinduced death. In a study of TP53 gene mutational status and expression levels of related genes (p21, GADD45 and bcl-2), HBT 3477 human breast carcinoma cells were xenotransplanted into immunodeficient mice (Winthrop et al, 1997). The animals were injected with 260 ÂľCi of yttrium-90 (90Yt)-DOTA-peptide-ChL6 (Y-90-ChL6), before and after tumor cell implantation and then euthanized at 3, 24 and 48 hr later. Tumor growth rate was also determined. The investigators found that tumors regressed 4-7 days after treatment with Y-90-ChL6, resulting in a 79% tumor response that was due to p53independent apoptosis. In addition, down-regulation of bcl-2 appeared to be the key to the apoptotic response induced by the relatively low dose-rate radioimmunotherapy. Induction of micronuclei was used as an indicator of apoptosis in Lewis lung carcinoma (LLC) subjected to !radiation dose rates of 0.34 Gy/min and 1 Gy/min while growing in mice (Widel and Przybyszewski, 1998). Tumors were excised, cultured in vitro and scored for micronuclei at 24-hour intervals. In the range of 0 to 6 Gy, the frequency of cells containing micronuclei was linearly dependent on dose. However, an inverse dose-rate effect was evident, with the lower dose rate resulting in a higher frequency of micronuclei per cell than the higher dose rate. Since the difference in exposure times between the two dose rates was not great, the investigators speculated that a differential radiation effect on cell kinetics during tumor irradiation could not explain the results. Rather, it appeared possible that the differential effect was dependent upon division delay and redistribution of cells in the phases of the cell cycle during in vitro incubation. In other words, they hypothesized that more cells in the higher dose-rate group died in culture because of interphase death than in the lower dose-rate group and

hence the cells were no longer available for the micronucleus assay.

III. Dose-rate effects on normal cells Normal cells respond to ionizing radiation with a delay in progression through the cell cycle so that DNA damage can be repaired before DNA replication and mitosis take place. The delay decreases the chance that genomic instability or mutant phenotypes will appear among the cell progeny. However, if the damage is too extensive, the cell will be eliminated because of postmitotic or apoptotic death. Surviving cells reenter the cell cycle after various lengths of time, depending upon the radiation dose and the specific cell type involved. The delay occurs primarily in the G2 phase, although radiation can also delay cells in G1 and S. Expression of the TP53 tumor suppressor gene is especially important in these events (Grdina, 1980; Keng et al, 1984; Rauth, 1992; Bristow et al, 1996). If TP53 is lacking or is functionally deficient, survival of cells with DNA damage and thus also risk for subsequent malignancy, is increased (Kato et al, 2002). However, the degree of radiosensitivity varies substantially among normal cell populations, especially under conditions of protracted low-dose exposure.

A. Radiation-induced normal toxicities during radiotherapy

tissue

1. Acute versus late effects With conventional radiotherapy protocols, both acute and late toxicities are possible (Lichter, 2000; Yamada et al, 2000; Gopal et al, 2001). Acute effects, seen during and up to 3 months following the end of treatment, occur primarily in rapidly proliferating tissues such as the skin, bone marrow and mucosa of the oropharynx, gastrointestinal tract, rectum and bladder. Erythema, esophagitis, pneumonitis, diarrhea, dysuria and leukopenia are among the most common manifestations. Late effects occur 3 months or longer after the end of radiotherapy and can involve virtually any organ or tissue. Most late reactions include progressively increasing fibrosis, blockage of blood vessels resulting in tissue anoxia and/or ulceration. Late effects are generally more serious and more difficult to manage than the acute toxicities. Eventually, death may occur due to organ failure or other complications. Development of secondary tumors within the irradiated volume is another consequence of radiotherapy that is likely to become more prevalent with increasing survival of cancer patients (Schneider et al, 2000; Strojan et al, 2000; Kranzinger et al, 2001).

2. Total-body irradiation (TBI) Injury to normal lung tissue and the gastrointestinal tract (GI) remains a major problem after totalâ&#x20AC;&#x201C;body irradiation (TBI) in bone marrow recipients with refractory hematological malignancies and serious immunodeficiencies (e.g. severe combined immunodeficiency disease). It was proposed several decades ago that radiation-induced lung toxicity may be ameliorated by the use of continuous protracted or fractionated radiation, since repair of sublethal damage is greater for the lung and 115


Gridley et al: Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? GI tract than for hematopoietic tissue and probably also leukemic cells (Dutreix et al, 1981). Many preclinical studies have employed localized lung irradiation, rather than TBI. In a study with mice, radiation was delivered to the upper body at dose rates ranging from 0.02 to 1.0 Gy/min (Down et al, 1986). A continuous increase in normal tissue tolerance was observed for early radiation-induced pneumonitis with decreasing dose rate, but a less pronounced sparing effect was noted for late complications. To evaluate the effects of adriamycin on radiation-induced pulmonary and upper GI tract toxicity, the thoracic region of mice was irradiated at dose rates of 0.05, 0.15 and 0.70 Gy/min (Sherman et al, 1982). The beneficial effect of the lower dose rates was markedly diminished, although still evident when the drug was administered pre-irradiation. In other words, this study showed that adriamycin can significantly increase the oral esophageal and pulmonary toxicity of radiation and can almost abrogate the sparing effect of dose rate. In a mouse model of bone marrow transplantation, Safwat and colleagues compared TBI delivered at a rate of 0.08Gy/min with TBI administered at a rate of 0.71 Gy/min, both with and without cyclophosphamide (Safwat et al, 1996). The drug was administered 24 hr before irradiation and the transplant was performed 4-6 hr after the last treatment. Lung damage, assessed using ventilation rate and mouse lethality, was decreased using the lower dose-rate regimen. However, administration of the drug markedly facilitated the TBI-induced damage. Somewhat unexpectedly, the combination of protracted radiation plus drug was found to be more toxic than acute radiation plus drug. In another study, mice given cyclophosphamide before thoracic irradiation at dose rates ranging from 0.05 Gy/min to 1.0 Gy/min developed pneumonitis at 4-9 weeks post-exposure (Lockhart et al, 1986). Without the drug, pneumonitis appeared at 14-16 weeks, regardless of radiation dose rate. In a rat model of TBI, fractionation had little effect on bone marrow ablation, but resulted in increased gastrointestinal and renal tolerance (Moulder and Fish, 1989). Results from studies in patients receiving various TBI regimens in association with bone marrow or stem cell transplantation have generally supported use of LDR or increased fractionation as a means to minimize normal tissue toxicities, increase the total dose of radiation that can be safely delivered and improve tumor control (Evans, 1983; Regnier, 1992; Corvo et al, 1999; Gopal et al, 2001; Song et al, 2003). Radiation therapy at low doses is now commonly employed in cases of chronic lymphocytic leukemia and low-grade non-Hodgkinâ&#x20AC;&#x2122;s lymphoma. The radiation is administered to the entire body at a very low dose per fraction (0.1 to 0.25 Gy) several times a week until a total dose of approximately 1.5 to 2 Gy is reached. Interestingly, the anti-tumor effects have been reported to be more pronounced than what would be expected from direct tumor cell destruction by radiation. It appears that immune enhancement is among the mechanisms by which TBI results in long-term remissions in the majority of patients. Pre-clinical studies have demonstrated that TBI up-regulates immune responsiveness in a number of ways: a) increases IFN-! and IL-2 production; b) increases

expression of IL-2 receptors on T lymphocytes; c) enhances signal transduction in T cells; d) augments T cell proliferation in response to mitogens; e) lowers serum corticosterone and increases catecholamine in spleen; and f) eliminates T cells with immunosuppressive activity (Anderson et al, 1982, 1988; Safwat, 2000a, 2000b). Low-dose TBI has been recently tested in combination with IL-2. In one study, mice were inoculated i.v. with B167F1 malignant melanoma cells on day 0 (Safwat et al, 2003a). A single whole-body dose of 0.75 Gy was administered on day 7; IL-2 was initiated on day 8 and given twice daily for 5 consecutive days. Pulmonary tumors were quantified and examined for tumorinfiltrating cells on day 14. In groups treated with either modality alone, tumor burden was similar to that in nontreated controls. However, the combination of low-dose TBI and IL-2 resulted in a synergistic anti-tumor effect. Natural killer (NK) cells and macrophages were identified as the most likely participants in the highly significant outcome of combination treatment. In a subsequent study of malignant melanoma, these same investigators used two doses of TBI and IL-2 (Safwat et al, 2004). Combining TBI with high-dose IL-2 led to a further significant reduction in tumor and less severe vascular leakage syndrome compared to high-dose IL-2 alone. In this case, therapeutic efficacy was associated with the number of tumor-infiltrating NK cells. The investigators concluded that the combination treatment was not only more effective, but also less toxic than IL-2 alone. Although very few human studies have been conducted in this area, data suggest that low-dose radiation-induced immune defense mechanisms may be operating (Safwat 2003b). Based on these types of data, some have suggested that administration of low-dose TBI may be beneficial for patients with AIDS (Shen et al, 1989; Shen et al, 1997). In contrast, TBI has also been associated with enhanced tumor progression (Duhrsen and Metcalf, 1988; Gridley et al, 1997). The discrepancies in reported data may be related to tumor type and load, as well as the dose rates and total doses of radiation utilized.

3. Central nervous system (CNS) Radiotherapy for neoplasms of the brain inevitably results in some damage to neurons. The damage can trigger a series of events, leading to destruction and malfunction of a relatively large volume of tissue due to secondary degeneration (Ikonomidou and Turski, 1996; Yoles and Schwartz, 1998). Side effects ranging from headaches, dizziness, nausea and cognitive defects to development of secondary CNS neoplasms following treatment remain problematic and are likely to become more obvious with increased survival times (Leibel SA and Sheline 1987; Schultheiss et al, 1995; Strojan et al, 2000; Kranzinger et al, 2001). The success of antiinflammatory drugs such as corticosteroids in controlling edema and increased intracranial pressure suggests that cells of the immune system play a role in the development of at least some of the observed toxicities. Corticosteroids have a number of non-specific anti-inflammatory and immunosuppressive properties including: a) they inhibit

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Cancer Therapy Vol 3, page 117 neutrophil migration to sites of tissue damage and thus minimize the level of neutrophil-derived vasoactive amines that increase vascular permeability and produce edema; b) they suppress IL-1 production by cells of the monocyte/macrophage lineage, a cytokine that is needed for T cell activation: and c) they impair synthesis and secretion of IL-2 by T lymphocytes, which in turn reduces the ability of the T cells to proliferate and produce proinflammatory cytokines. For many decades, the CNS was considered to be an immunologically privileged site, with little or no interaction occurring between the immune and central nervous systems; if immune activity did occur, it was thought to be detrimental (Streilein, 1993; Popovich et al, 1996). However, a series of recent studies have demonstrated that recruitment of systemic T lymphocytes to the injured site helps the innate branch of the immune system to ward off toxicity (Moalem et al, 1999; Schwartz and Cohen, 2000; Fisher et al, 2001; Kipnis et al, 2001, 2002). In contrast to healthy tissue, the injured CNS is more accessible to circulating lymphocytes because of blood-brain barrier breakdown and expression of adhesion molecules, chemokines and major histocompatibility complex (MHC) class II molecules. The in-migrating T cells belong to the Th1 subset that secretes cytokines such as IL-2 and IFN-!. In addition, they are autoreactive and respond to specific â&#x20AC;&#x2DC;selfâ&#x20AC;&#x2122; antigens (e.g. anti-myelin protein) that are presented to them by activated microglia. Indeed, it has been demonstrated that rats and mice that are either deficient in mature T cells or that lack autoreactive T cells lose their ability to withstand injury to the CNS (Kipnis et al, 2000; Schori et al, 2001). The existence of a protective role for the immune system has been strengthened by the demonstration that passive transfer of a subpopulation of T suppressor cells (i.e. the naturally occurring regulatory CD4+/CD25+ T cells) can eradicate irradiation-induced Th1 cell-derived neuroprotection (Kipnis et al, 2002). Most recently, a study was done to determine whether !-radiation benefits neuronal survival by alterating immune system status (Kipnis et al, 2004). Mice and rats were subjected to optic nerve crush or contusive injury to the spinal cord. Total-body or totallymphoidal irradiation (3.5 Gy) resulted in significant increases in neuronal survival (in some cases more than 3fold) and more rapid recovery from injury. The beneficial effects were noted when radiation was administered up to 3 days post-injury.

with HDR exposure. Since the majority (>60%) of cells irradiated with HDR were in G1 and those irradiated with LDR went through the S-phase and on into G2, the investigators proposed that the difference in mutation size may be related to the stage of the cell cycle. Other studies have demonstrated that decreasing the dose rate results in an inverse dose-rate effect as manifested by increased mutagenesis (Furuno-Fukushi et al, 1988; Crompton et al, 1990; Amundson and Chen, 1996). In contrast, some investigators have reported that protracted radiation exposures protect against mutation induction both in vitro (Evans et al, 1990) and in vivo (Lorenz et al, 1994) or have no significant effect compared to acute irradiation (Furuno-Fukushi et al, 1996). In some cases, discrepancies have been observed between in vitro and in vivo data (Lorenz et al, 1993). Reports by Furuno-Fukushi and colleagues using non-transformed, near-diploid m5S mouse cells demonstrated that cell proliferation during protracted irradiation has a strong influence on mutagenesis (Furuno-Fukushi et al, 1993). In plateauphase cultures, lowering the dose rate from 30 Gy/hr to 13 mGy/hr resulted in an increase in cell survival and a marked decrease in mutation frequency. However, when the cells were in log-phase culture, the magnitude of the dose-rate effect was not nearly as marked, especially when the total dose was below 5 Gy. An interesting study of mutagenesis was recently performed in which chronic low-dose rate !-irradiation was used to mimic the environment experienced by residents during the Chernobyl accident (Wickliffe et al, 2003). Big Blue# mice were exposed to !-rays for 90 days, resulting in a cumulative dose of 3 Gy (0.0014 Gy/hr). No significant increase in mutation frequency was observed in the irradiated mice based on DNA analysis of liver tissue (Big Blue# Transgenic Rodent Mutagenesis Assay System). The above-mentioned studies, as well as others, indicate that variations in experimental conditions may at least partly account for the apparent contradictions that have been noted in radiation-induced mutagenesis. Overall, the preponderance of evidence indicates that the risk for radiation-induced mutagenesis in somatic and germ line cells decreases with decreasing dose rate (Vilencheck and Knudsen, 2000).

B. Radio-adaptive response The consensus that radiation is harmful to normal cells has prevailed for many decades. Among the most serious consequences are immunodeficiency, mutations and development of malignancies such as leukemia and multiple myeloma (reviewed by Dainiak 2002). Numerous studies of radiation-induced chromosomal and DNA aberrations have been performed on lymphocytes and lymphoid cells (Hofer et al, 1994; Kronenberg, 1994; Moiseenko et al, 1997; Wu et al, 1997, 1999; Blakely and Kronenberg, 1998; Durante et al, 1998, 1999, 2000, 2002; Yamada et al, 2000; Gauny et al, 2001; Grosovsky et al, 2001; Holl et al, 2001; Wiese et al, 2001; Schulz-Ertner et al, 2002). These studies have revealed important radiationinduced genomic abnormalities, as well as variations due to differences in radiation quality. Although lymphoid organs have long been known to be highly radiosensitive,

4. Mutations and carcinogenesis Numerous studies of dose-rate effects on mutagenesis have been performed using human and rodent lymphoid cells (Lorenz et al, 1993; Lorenz et al, 1994; Amundson and Chen, 1996; Furuno-Fukushi et al, 1996). The hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (tk) loci have arguably received the greatest attention. In a study of mutation size at the HPRT locus in human lymphoblastoid WI-L2-NS cells, an inverse dose-rate effect was found after a total dose of 4 Gy (Colussi and Lohman, 1997). Significantly larger deletions were produced by LDR exposure than

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Gridley et al: Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? the susceptibility of different lymphocyte populations varies greatly, possibly owing to differences in repair mechanisms, maturation stage, activation state and/or innate rate of proliferation (Stefani and Schrek 1964; Miller and Cole, 1967; Anderson and Warner 1976; Anderson et al, 1977, 1988; Prasad 1995). An additional important observation is that irradiation can up-regulate many genes, including some that encode cytokines that can modify cellular responses to radiation (Hallahan et al, 1993; Hallahan, 1996; Barcellos-Hoff, 1998;). It is clear that radiation, when delivered at a slow continuous rate or by fractionation may have strikingly different effects compared to the same dose delivered acutely. Early studies demonstrated that monkeys could tolerate whole-body exposures up to 19 Gy with minimal suppression of the hematopoietic system when the radiation was delivered at a rate of 1 Gy/year (Spalding et al, 1972). The 19 Gy is approximately three times the lethal dose when applied acutely. Soon thereafter, it was reported that protracted radiation exposure may induce beneficial effects (i.e. radio-adaptation, previously known as â&#x20AC;&#x2DC;hormesisâ&#x20AC;&#x2122;) (Anderson and Warner, 1976; Anderson et al, 1977). The radio-adaptive response has gained much attention in recent years because of its obvious importance in setting accurate guidelines for radiation-associated human health risks, in basic radiobiology research and now also in radiation therapy. The phenomenon has been extensively discussed in a number of publications (Bhattacharjee and Ito, 2001; Feinendegen 2003; Pollycove and Feinendegen, 2003). In this review, only a few highlights of special interest are mentioned. Various endpoints have been used to detect and quantify radio-adaptation, including chromosome aberrations (Shadley et al, 1987; Wolff et al, 1988; Sankaranarayanan et al, 1989; Farooqi and Resavan, 1993), mutations and DNA strand breaks (Shadley and Wolff, 1987; Kelsey et al, 1991; Rigaud et al, 1993), micronucleus formation (Azzam et al, 1994) and cell survival (Olivieri et al, 1984; Yoshida et al, 1993; Sasaki, 1995). In many of these studies, low-dose irradiation of lymphocytes has been found to minimize the harmful effects of a subsequently delivered high dose of radiation. For example, although lymphocytes from individuals occupationally exposed to chronic doses of radiation have higher frequencies of spontaneous micronuclei than nonexposed individuals, after 1 and 2 Gy irradiation of the lymphocytes in vitro this frequency is lower for the radiation workers (Gourabi and Mozdarani, 1998). It has also been reported that low-dose irradiation can enhance the production of IL-1 by lipopolysaccharide (LPS)stimulated splenocytes, responsiveness to T cell mitogens (Ishii and Watanabe 1996) and expression of IL-2 receptors on the surface of peripheral blood lymphocytes (Xu et al, 1996). Many of these types of reports indicate that a significant radio-adaptive response is measurable within a few hours after low-dose priming (Farooqi and Resavan, 1993; Ishii and Watanabe, 1996; Park et al, 2000; Venkat et al, 2001). With intact mammalian models, radio-adaptation in immune cells has been shown to protect mice injected with the Friend leukemia virus, a member of the retrovirus

family that includes HIV-1 (Shen et al, 1989; Wolff et al, 1989; Fujiki and Suganuma, 1994). It has also been noted that the onset of thymic lymphoma is significantly delayed in Swiss mice pre-conditioned with 0.01 Gy and then acutely irradiated with 2 Gy (Bhattacharjee, 1996). Similar results have been obtained for acute myeloid leukemia (Mitchell et al, 1999) and some transplanted solid tumors in mice (Bhattacharjee and Sarma, 1999). In one very recent report, mice were subjected to single low-level exposures of 0.1 Gy or 0.2 Gy X-rays and injected i.v. 2 hr later with syngeneic L1 sarcoma cells (Cheda et al, 2004). Pre-treatment with either dose of radiation significantly reduced the number of tumor colonies in the lungs compared to the non-irradiated counterparts. In addition, significantly enhanced NK cell cytotoxic activity was observed in spleens from the irradiated animals and elimination of the NK cells by injection of anti-asialo GM 1 antibody totally abrogated the radiation-induced tumorinhibitory effect. Studies by other investigators support these findings. Suppression of pulmonary nodule development with single doses of X-rays (0.05 Gy to 0.15 Gy) 24 hr before i.v. injection of B16 melanoma and Lewis lung cancer cells has been reported in mice (Cai, 1999). Hashimoto and co-workers have shown that lung and lymph node metastases are decreased and that metastatic foci are infiltrated by lymphocytes in rats exposed to 0.2 Gy !-rays 14 days after s.c. injection of hepatoma cells (Hashimoto et al, 1999). In this latter study, in vitro irradiation of the tumor cells or localized irradiation of tumor in vivo at the same dose did not affect either primary tumor growth or degree of spontaneous metastases. Irradiation of mice with X-ray doses of <0.2 Gy has also been reported to suppress local tumor growth when malignant cells are injected after the radiation exposure (Anderson et al, 1982; Cai, 1999). Some investigators have suggested that the enhanced anti-tumor effect may be related to secretion of TNF-$ and other cytokines by cells of the monocyte-macrophage series that become activated by debris due to radiation-induced damage (Hallahan et al, 1989; Sherman et al, 1991; Weichselbaum et al, 1991). TNF-$ may add to the lethality of radiation because it has both direct and indirect anti-tumor effects. Although radio-adaptation was not specifically addressed, our previous studies have demonstrated that s.c. growth of Lewis lung carcinoma in C57BL/6 mice is consistently slower when the animals are exposed to 3 Gy !-rays 2 hr prior to tumor cell implantation (i.e. representing a small neoplastic focus that appears after the irradiation event has taken place) and that the anti-tumor effect was related to immune enhancement during repair of normal tissue damage (Miller GM et al, 2002, 2003a, 2003b). The data also showed that a dramatic expansion of NK cells occurred in the blood and spleen of tumorbearing animals 10 days after irradiation (28.0 x 105 NK cells/spleen in irradiated mice compared to 8.9 x 105 NK cells/spleen in non-irradiated controls). Enhanced production of IL-12 and IL-18 by spleen cells was consistent with augmentation of the NK cell response. Significant reductions in TGF-"1 and vascular endothelial growth factor (VEGF), both of which are associated with 118


Cancer Therapy Vol 3, page 119 immune suppression, were also noted. Enhanced NK cell activity after total-body irradiation with 0.075 to 0.5 Gy X- or !-rays has been reported by other investigators (Kojima et al, 2002, 2004; Liu et al, 1994). The important role of NK cells in nonspecific immune surveillance against aberrant cells and metastatic growth has been well established (Talmadge et al, 1980; Hanna, 1985; Wiltrout et al, 1985; Moretta et al, 1994; Reyburn et al, 1997; Barao and Ascensao, 1998). These findings are consistent with the radiation-induced â&#x20AC;&#x153;dangerâ&#x20AC;? signal originally proposed by Matzinger in 1994 and recently reviewed by Friedman (2002). Based on studies such as those above, it has been suggested that low-dose whole-body irradiation may be useful in treating some cancer patients (Pollycove and Feinendegen, 2000). However, it should be mentioned that the incidence of radiation-induced carcinogenesis increases in humans when doses exceed approximately 0.3 Gy (Pollycove and Feinendegen, 2003). Radio-adaptation has also been reported in normal cells other than those of the immune system. Broome and colleagues demonstrated that exposure of cultured AG1522 human fibroblasts to total doses ranging from 0.0001 Gy to 0.5 Gy at dose rates ranging from 0.001 to 0.003 Gy/min prior to a challenge dose of 4 Gy !-rays exhibited reduced frequency of micronuclei (Broome et al, 2002). There was no significant difference in the degree of protection induced by the two extremes of total doses used and a similar degree of protection was induced with protracted exposure to !-rays and 3H beta particles. These latter findings demonstrated that doses as low as one track per cell (0.0001 Gy) produced the same maximum adaptive response as did doses that resulted in many tracks per cell (0.5 Gy) and that the two types of radiations were similar in this respect. In addition, induction of radioadaptation in the fibroblasts occurred even when the cells were incubated at 0o C during delivery of the priming dose, a temperature at which DNA repair and other metabolic processes are inactive. In a study of human erythrocytes subjected to !radiation, identical total doses delivered in a single fraction or split into two identical fractions with a 3.5 hr interval between exposures, resulted in a 2.4-fold reduction in hemolysis when the split doses were administered (Koziczak et al, 2003). The results also suggested that the reduced damage to the red blood cells occurred because of a decrease in the level of damage to membrane lipids. An interesting study of genetic damage in erythrocytes in the bone marrow of SHK mice has been recently performed (Zaichkina et al, 2003). The effect of low-dose !-radiation (0.1 and 0.2 Gy total doses at 0.125 Gy/min) on high-dose (1.5 Gy total dose at 1 Gy/min) radiation-induced and spontaneous levels of cytogenetic damage was evaluated over the entire lifetime of the animals. The amount of micronucleated polychromatic erythrocytes in primed, primed and challenged and control groups was assessed at various time points. A single lowdose exposure induced cytogenetic radio-adaptation at 1, 3, 6, 9 and 12 months after priming, regardless of mouse age at the time of priming irradiation. In addition, the lowdose priming exposure resulted in decreases in cytogenetic damage to a level below the spontaneous rate at the end of

the lifetime (20 months) of the animals. These data demonstrated that the mechanisms underlying radioadaptation in erythrocytes protect against chromosome damage induced by high-dose irradiation and appear to also minimize spontaneous mutagenesis during aging. Although the mechanisms underlying radio-adaptive responses remain unclear, a possible scenario has been proposed by Ikushima and colleagues (1996.) and reviewed by Bhattacharjee and Ito (2001). In this model, low dose pre-irradiation damages a small amount of nuclear DNA, ultimately leading to enhanced capacity to repair serious DNA damage induced by a subsequent high dose irradiation event. In support of this possibility are recent studies demonstrating that continuous exposure to X-rays at a low-dose rate leads to no detectable misrejoining of double-strand DNA breaks in cultured mammalian cells (Kuhne et al, 2002). This finding suggests that the probability of inappropriate repair decreases dramatically when the breaks are separated in time and space. Using cell lines deficient in nonhomologous end-joining, the investigators concluded that this is an efficient pathway for correct rejoining of separated broken ends, but that it generates genomic rearrangements if the breaks are close in time and space. In this same study there was no significant decrease in double-strand DNA misrejoining after exposure of the cells to fractionated doses of alpha particles, indicating that radiation quality may be a significant factor in determining whether or not protracted low-dose radiation results in protection. However, the overall evidence for more efficient DNA repair in radio-adaptation is largely indirect and efforts continue to identify other mechanisms. For example, Takahashi et al, reported that splenocytes from C57BL/6N mice primed with whole-body irradiation to a total dose of 1.5 Gy !-rays delivered at a dose rate of 0.001 Gy/min over 25 hr, exhibited significant suppression in p53, bax and apoptosis after a 3 Gy challenge dose of Xrays delivered at a high-dose rate (1 Gy/min) (Takahashi et al, 2001). In subsequent studies, these same investigators demonstrated that the apoptosis induced by acute 3 Gy irradiation was significantly suppressed in the splenic white pulp of wild-type, but not SCID (severe combined immunodeficiency), mice and that DNA-dependent protein kinase activity may play a major role in the radioadaptive response following pre-irradiation at low doses (0.15 - 0.6 Gy) (Takahashi et al, 2002, 2003). Others have reported that protein kinase C-mediated signaling may be a key step for transducing the low dose-induced protective signal (Rigaud and Moustacchi, 1996). Using chromosomal aberrations as the indicator of damage in human lymphocytes, it has been demonstrated that the anti-mutagenic action observed with low-dose irradiation is similar to that seen with interferon pre-treatment (Tskhovrebova et al, 1995; Makedonov et al, 2000). These results suggest that priming with low-dose radiation and the action of IFN-! may induce common pathways of protection. Some reports indicate that reactive oxygen species play an important role in inducing radioprotection. In the body, oxygen radicals are generated not only by the direct effects of ionizing radiation, but also by

119


Gridley et al: Low-dose/low-dose-rate radiation: a feasible strategy to improve cancer radiotherapy? inflammatory cells that migrate to the site of tissue damage (Gridley et al, 2005). Feinendegen has recently proposed that the site, type and size of oxygen radical bursts, as well as the time interval between them, may be an integral component of the mechanisms leading to cellular radio-adaptation (Feinendegen, 2002). Reactive oxygen species at low concentrations appear to induce cellular protection, whereas the reverse is true at high concentrations. In a study of bystander effects, Iyer and Lehnert transferred supernatants from human lung fibroblasts (HFL-1) irradiated with a 0.01 Gy dose of !rays to non-irradiated HFL-1 cells (Iyer and Lehnert, 2002). When the unirradiated cells were subjected to 2 and 4 Gy doses of acute !-radiation, clonogenic survival was enhanced. The radio-adaptive bystander effect was preceded by an increase in intracellular oxygen radicals, an increase in the redox and DNA repair protein APendonuclease and a decrease in p53 protein. Studies with plants suggest that exposure to very low-dose-rate !-rays (66 mSv/hr or approximately 66 mGy/hr) leads to an efficient induction of superoxide dismutase and other enzymes that protect against reactive oxygen species (Zaka et al, 2002).

et al, 1998), frequency of T cells capable of secreting type 1 cytokines (Kusunoki et al, 2001), T cell reactivity to mitogens (Akiyama et al, 1983) and T cell response to allogeneic cells (Akiyama et al, 1989). Signs of T cell impairment, including increased reactivation of the potentially oncogenic Epstein-Barr virus (EBV), have been reported in these individuals (Kanamitsu et al, 1966; Kato et al, 1980; Akiyama et al, 1989, 1993). Yamoaka and associates recently demonstrated that A-bomb exposure induced long-lasting deficits in both naĂŻve T helper and T cytotoxic cell populations, along with increased proportions of memory T cells belonging to these subsets (Yamoaka et al, 2004). These findings indicate poor maintenance of T cells that are newly generated post-exposure. Additional data in support of defective T cell activities come from studies of persons exposed to fallout from the nuclear power plant accident at Chernobyl (Lukjanova et al, 1995; Stepanova et al, 1995; Chernyshov et al, 1997). In one of these investigations it was found that long-lasting depletion in the Th1 subset exists concomitantly with excessive proliferation of the Th2 subset and that an activating stimulus may be necessary to reveal radiation-induced immune disturbances (Dainiak, 2002). Studies of Belarussians residing near Chernobyl report differences in gene expression patterns for subjects exposed to >10 mSv compared to those receiving <10 mSv (Karkanitsa et al, 2000). In these investigations, the upregulated genes were primarily those encoding proteins associated with mitotic and apoptotic death in T cells and monocytes. Emphasis has also been placed on radiation-induced mutations that lead to carcinogenesis. In vitro exposure of human peripheral blood lymphocytes to low doses of Xrays (0.25 to 1.5 Gy) at a dose-rate of 1 Gy/min resulted in increased expression of several proto-oncogenes at 5 hr and 17 hr post-exposure (Miller AC et al, 2002). The upregulation was especially striking for c-Haras. The investigators suggested that c-Haras may be useful as an early biomarker indicative of radiation exposure. In Abomb survivors, increased incidence of leukemias, especially chronic myelogenous leukemia (CML) and other myeloproliferative diseases has been reported (Ichimaru et al, 1991). The threshold for CML in Hiroshima is thought to be between 0.09 Gy to 0.5 Gy, whereas for acute leukemia the threshold appears to be approximately 1 Gy. Some epidemiological studies of low-level exposures have concluded that cancer incidence and mortality among residents living in high versus low background areas is not significantly different (Jagger, 1998; Luckey, 1999). The effects of long-term, low-level !-irradiation among individuals exposed in radiocontaminated buildings has also been studied. Increased frequencies of various chromosomal aberrations, especially breakpoints, inversions and translocations on chromosomes 7 and 14, have been reported (Hsieh et al, 2002). This is of interest, since the genes that code for the T cell antigen receptor and the heavy chains of antibodies are found on these two chromosomes. Thus, mutations in these chromosomes could lead to significant immunodeficiency.

C. General population Studies of human populations previously exposed to various radiation doses and dose rates may provide information that is useful in radiotherapy, especially with respect to effects on cells that constitute the immune system. The functions of these cells include continuous surveillance for aberrantly (including neoplastically) transformed cells, as well as destruction of tumor cell targets, and hence they may contribute to the final outcome in radiotherapy patients. In addition, preservation of the bone marrow (hematopoietic system) is of great importance in mitigating the risk for infection, anemia, and other complications in cancer patients. Studies of survivors after accidental irradiation events, such as those that took place at Three Mile Island and Chernobyl (Lenschow et al, 1996; Thompson, 1994), may help elucidate dose and dose rate effects on bone marrow and other normal cells and tissues. Furthermore, in recent years the possibility of radiation exposure due to a nuclear (radiological) terrorism attack has come to the forefront of public and government attention. There is now great urgency to identify the best radioprotectants already available, as well as develop and validate new and better compounds. It is very likely that one or more of these radioprotectants will become incorporated into radiotherapy regimens for normal tissue protection before, during, and perhaps even after treatment to minimize damage due to oxygen radicals released by inflammatory cells. Much attention has been paid to the lymphocytes, especially the T cells, because they regulate the bodyâ&#x20AC;&#x2122;s most sophisticated immune defenses. Loss or malfunction of these cells can lead to dire consequences as exemplified by AIDS patients and organ transplant recipients. Studies of atomic bomb (A-bomb) survivors have revealed significant decreases in mature T lymphocytes, T helper (Th) cells (but not T cytotoxic, B, or NK cells) (Kusunoki 120


Cancer Therapy Vol 3, page 121 Some additional findings indicate that doses as low as 0.05 Gy to 1 Gy increased the incidence of cardiovascular disease in both A-bomb survivors and victims of the Chernobyl accident (reviewed by Trivedi and Hannan, 2004). Furthermore, wounds heal more slowly under conditions of radiation exposure, with the delay in wound healing being related directly to the dose of radiation (Ran et al, 2003; Shi et al, 2003). During wartime, large numbers of military personnel may experience prolonged exposure to radiation that occurs simultaneously with wounding. Astronauts are inevitably exposed to chronic irradiation and may also become injured while on board a spacecraft. Combined injury also occurs when surgery and radiation are used for cancer therapy. The mechanisms by which radiation delays wound healing are not clear, but may include reduction in the number of platelets and inflammatory and other tissuerepairing cells that accumulate at the injured site (Qu et al, 2003).

the significant anti-tumor effect following whole-body irradiation that exceeds what is seen when the same dose is delivered only to the tumor are phenomena that also warrant further investigation. From the information currently available, it appears likely that hyperradiosensitivity and adaptation leading to increased radioresistance are distinct examples of â&#x20AC;&#x2DC;activeâ&#x20AC;&#x2122; responses to low-dose radiation exposure that are associated, at least partly, with the rate of DNA damage and the extent of DNA repair (Joiner et al, 1999; Marples et al. 1997). With the rapid progress currently on-going in elucidating radiation-induced chromosomal events, genes that control DNA repair and signal transduction pathways, incorporation of protracted low-dose radiation in cancer therapy may eventually become common in the clinic. We suggest that the data, when taken in toto, indicate that future application of reduced dose-rates in the clinic may have several important roles: 1) direct tumor cell cytotoxicity that is greater than what is observed with acute irradiation; 2) induced changes that result in greater tumor control by HDR external-beam radiation; 3) radioadaptation in normal cell populations that decrease risk for complications; and 4) up-regulation of anti-tumor immune responses. Increased knowledge of mechanistic effects of reduced dose-rate irradiation on both tumor and normal tissues and the subsequent impact on the therapeutic ratio is needed for future clinical utility. Finally, we accept that the database that we have reviewed is complex and currently it is difficult to proceed to clinical trials with confidence in the application of low dose-rate therapy. However, we do suggest that there are laboratory and clinical research projects that should be undertaken with some priority. First, we need increased knowledge of mechanistic effects of reduced dose-rate irradiation on tumor cells, especially defining the molecular processes that are shared by induced hypersensitivity, adaptive response, and their contribution to cell killing by mitotic and apoptotic processes. Further, it is important that these observations be extended to model tumor systems such as xenografts growing in the nude mouse to determine whether cell-based observations in vitro can be extended to in vivo response of model tumors comprised of cells that do or do not exhibit low dose, low dose-rate radiosensitivity. We think that certain clinical studies should be considered. First, the combination of low dose-rate irradiation delivered by brachytherapy and radiolabelled antibody therapy with external beam high dose-rate radiotherapy is immediately feasible. Second, as soon as variation of response to low dose-rate irradiation and to combined LDR and HDR of brain tumors can be predicted for such tumors with some confidence, then such therapy should be given priority. Also, radioresistant tumors such as certain sarcomas, when located in sites accessible to direct injection of carriers of radionuclides could be considered for LDR/HDR therapy. Last, but not least, a better understanding of the response of normal tissues and the subsequent impact on the therapeutic ratio is needed for future clinical utility.

IV. Conclusions It is often stated that the major goal of cancer radiotherapy is to select a treatment regimen that has the highest probability for uncomplicated cure. A large amount of data suggests that differences in radiosensitivity between tumor and normal cell populations could be exploited to achieve a substantial increase in the therapeutic ratio. The overall implication of the discussed studies is that increased control of at least some types of cancers may be achieved by combining protracted irradiation with standard external beam therapy. However, at this point in time, it is difficult to predict with a high degree of certainty the tumor types for which this treatment approach may be beneficial and those for which the strategy may be a poor choice. Induction of hyper-radiosensitivity in tumor cells by protracted radiation exposure is clearly not a universal phenomenon. In addition, there appears to be lack of a consistent trend in the extent that tumor cells repair sublethal radiation-induced damage compared to normal cells, although a correlation between the degree of sublethal damage repair and radioresistance does appear to exist. Using a series of assumptions and mathematical modeling, Hahnfeldt and Hlatky have demonstrated that there is a tendency toward an inverse dose-rate effect even when heterogenous and cycling cell populations are exposed to low-LET radiations (e.g., photons and protons) in a protracted manner (Hahnfeldt and Hlatky, 1998). However, the mechanisms by which protracted irradiation increases tumor cell radiosensitivity in relation to acute iurradiation remain elusive. A recent publication by Marples and colleagues elegantly summarizes the most pertinent literature in this regard (Marples et al, 2004). The paper also proposes a three-component model consisting of damage recognition, signal transduction and damage repair. Undoubtedly, many questions still remain unanswered. Especially intriguing is why some tumor cells fail to exhibit hyper-radiosensitivity and why there is inconsistency between accumulation in the radiosensitive G2 phase of the cell cycle and hyper-radiosensitivity. The mechanisms underlying the radio-adaptive response and 121


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Acknowledgements We thank Larry E. Dillehay, Ph.D., Research Associate and Director of Experimental Radiators in the Department of Radiation Oncology & Molecular Biology at Johns Hopkins School of Medicine for use of data depicted in the figure and William Preston, Ed.D. for expert editorial assistance.

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Cancer Therapy Vol 3, page 131 Cancer Therapy Vol 3, 131-138, 2005

Micro and nano drug delivery systems in cancer therapy Review Article

Gorka Orive, Rosa María Hernández, Alicia R. Gascón, José Luis Pedraz* Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, Vitoria–Gasteiz, Spain

__________________________________________________________________________________ *Correspondence: Dr. José Luis Pedraz, Professor of the Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, Vitoria -Gasteiz. Spain; Phone: +34 945-013091; Fax: +34 945-013040; E-mail: knppemuj@vc.ehu.es Key words: Micro and nano drug delivery systems, Micro and nanotechnologies, cancer therapy, Macromolecular conjugation Abbreviations: blood-brain barrier, (BBB); Food and Drug Administration, (FDA); leuteinizing-hormone-releasing hormone, (LHRH); permeability and retention effect, (EPR); polyethylene glycol, (PEG); poly(lactic-co-glycolic), (PLGA); recombinant human growth hormone, (rhGH); tissue inhibitor of metalloproteinase, (TIMP) Received: 23 February 2005; Accepted: 1 March 2005; electronically published: March 2005

Summary The delivery of any drug at the right time and in the target where it is needed and at the level that is required is essential to realize the full potential of therapeutic molecules. These requirements are already more important in the case of cancer chemotherapies due to their high toxicity which could lead to serious side effects. In the last few years, a great number of new drug delivery technologies have been optimised including the micro and nano-systems as well as polymer conjugation. Together, these drug delivery systems would not only improve drug administration and the efficiency and safety of conventional chemotherapies, but also revolutionize the pharmaceutical and biomedical industries in cancer therapy. Currently, a variety of drug delivery approaches are FDA-approved or are in clinical development as anticancer treatments, including polymer microcapsules and microspheres, liposomes, polymer conjugates and nanoparticles (Figure 1). Others such as chemotherapy wafers, microchips and osmotic pumps are also in testing stage to treat human cancers but will not be the scope of this work since they have been reviewed in detail elsewhere (Moses et al, 2003b). This article focuses on the potential of micro and nanotechnology as well as polymer conjugation as a platform for developing drug delivery systems in cancer treatment.

I. Introduction The development of drug delivery systems has improved the therapeutic and toxicological properties of existing chemotherapies and facilitated the implementation of new ones. By including the drug in technologically optimized drug delivery systems or conjugating the drugs with different polymers, it is possible to modify the pharmacokinetics and biodistribution of the drugs, improving the efficacy and security of the therapy (Moses et al, 2003a). Some of the strongest arguments for the use of drug delivery systems are that they avoid or at least reduce some potential disadvantages of cancer chemotherapy including toxicity, pain management, short in vivo halflives and repeated administrations (Allen and Cullis, 2004). The possibility of designing different drug delivery systems for a controlled and continuous release of the therapeutic molecule has impact broadly the clinical application of the chemotherapies and improve the lifequality of the patients.

II. Current chemotherapies

limitations

of

One of the main limitations of chemotherapies is their high toxicity which could lead to serious side effects, reducing the administrable and the therapeutic effect. To address this issue, it is essential to transport the therapeutically active molecule mainly to the target where

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Figure 1. Examples of different drug delivery approaches that are FDA-approved or are in clinical development as anticancer treatments. Reproduced from Moses et al, 2003 with kind permission from Cancer Cell.

it is needed and at the required time and level (Orive et al, 2003a). This could be achieved by embedding the drugs into nontoxic and biodegradable polymers from which the drug will be released in a sustained manner (Duncan, 2003). For example, the use of doxorubicin in the treatment of metastatic breast cancer is associated with cardiotoxicity. But when the drug is included in a liposome, it is possible to obtain a controlled release of doxorubicin, reducing the peak levels of the drug and consequently its toxicity (Theodoulou and Clifford, 2004). Another strategy to concentrate cancer drugs only in their target tissue is through a mechanism known as enhanced permeability and retention effect (EPR) which happens in some pathological conditions such as solid tumors (Maeda et al, 2000; Hashizume et al, 2001). In fact, the network of blood vessels in many solid tumors has been shown to differ considerably from normal vasculature and to contain gaps in which tumor cells lack close contact with perfusing vessels, which ultimately leads to increased permeability (Jain, 2001; Bergers and Benjamin, 2003). In this situation, drug delivery systems which are usually excluded from entering into tissues can extravasate into tumors and increase drug concentration 10-fold or more than administration of the same dose of free drug (Northfelt et al, 1996). In the last few years, many peptides and proteins have shown biological activity which makes them potential candidates as anticancer agents (Torchilin and Lukyanov, 2003). However, their use as therapeutic drugs is often

hampered by the uptake by the reticulo endothelial system and their short half-lives in vivo which makes difficult their administration. The inclusion of a drug into a micro or nanoparticle or the coupling of a polymer decreases renal or hepatic drug clearance and immune recognition, altering the pharmacokinetics and biodistribution of the free drug. In fact, when a drug is associated with a carrier the volume of distribution and the clearance decreases, the area under time-versus-concentration increases and the half-life of the drug increases administrations (Allen and Cullis, 2004). This knowledge has provided the basis for different commercially available injectable delivery systems such as Decapeptyl速 and Zoladex速 (http://hcp.zoladex.net/Article/501611.aspx). Drug lifetime can also be prolonged by conjugating the drug molecules with water-soluble molecules such as polyethylene glycol (PEG) (Harris and Chess, 2003). The complex created is designed to increase protein solubility, stability and to reduce protein immunogenicity. In fact, by preventing rapid renal clearance and protein uptake of cells by reticuloendothelial system, conjugates with PEG are used to prolong plasma half-time (Gabizon et al, 2003; Shorr et al, 2004). The use of drug delivery systems have revolutionised the areas of cancer prevention and pain management related to classical cancer chemotherapy. Furthermore, the encapsulation of the drug in a microreservoir can improve drug solubility and stability as well as reduce drug resistance in some human carcinomas. For instance,

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Cancer Therapy Vol 3, page 133 recently it has been demonstrated that liposomally encapsulated tamoxifen not only induces pharmacological effects but reduces anti-estrogen resistance in several breast tumour models (Reiner et al, 2004). Finally, the entrapment of labile therapeutic agents can protect the latter from premature degradation by hydrolysis or by enzymes present in the plasma.

particle differs also considerably and the scale-up could be a challenge for some of these devices. An important issue to be considered when fabricating these systems is the drug load that the reservoir can carry. This drug load depends on the size and the structure of the device, ranging from some few molecules of the drug to a few tens. Therefore, selection of drugs with potent pharmaceutical activity is necessary in order to have therapeutic effects in the released dose. Furthermore, it is essential that the drug will not be altered during the fabrication process and the storage. Finally, interactions between drug and the reservoir must be optimized to facilitate drug release only in the target where it is needed and at the desired kinetic-release. Natural and synthetic polymers including albumin, fibrinogen, alginate, chitosan and collagen have been used for the fabrication of micro and nanoparticles . However, among all of them, lactic-glycolic acid copolymers are the most frequently employed materials due to their biocompatibility and biodegradability. Following a multiple emulsion process, a drug can be entrapped into a poly(lactic-co-glycolic) (PLGA) microsphere and released

III. Micro and nanotechnologies Tremendous opportunities exist for using micro and nanoparticles as controlled drug delivery systems for cancer treatment (Panyam and Labhasetwar, 2003; Birnbaum and Brannon-Peppas, 2004). The term “microparticle” refers to a particle with a diameter of 11000 µm, while “nanopaticle” is used when the particle is <1 µm in size. However, under this term it is possible to distinguish several reservoirs including micro/nanocapsules, micro/nano-spheres, liposomes, etc. All these devices differ not only in the structure (Figure 2) but also in their biopharmaceutical properties and therapeutic uses (Orive et al, 2003b). The fabrication protocol of each

Figure 2. Schematics of different nanotechnology based drug delivery systems for cancer therapy. Reproduced from Sahoo and Labhasetwar, 2003 with kind permission from Drug Discovery Today.

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Orive et al: Micro and nano drug delivery systems in cancer therapy at a zero-order kinetic by diffusion of the drug through the polymer reservoir and the slow degradation of the polymer matrix. These advantages resulted in the first two PLGAmicroparticle extended-release formulations that were approved by the US Food and Drug Administration (FDA). One of them released the recombinant human growth hormone (rhGH) (Cleland, 1997) whereas the other microparticle-based drug delivery system released the leuteinizing-hormone-releasing hormone (LHRH) agonist leuprorelin acetate (Okada, 1997). The latter is currently on the market under the name of LupronÂŽ Depot and it is approved in the United States for the palliative treatment of advanced prostate cancer. However, there are still few microencapsulated formulations on clinical trials addressing cancer treatments. In fact, a recent review of National Centre Institute revealed that from the 1200 open clinical trials in the United States only one to be testing a microparticulate system for controlled drug delivery (Birnbaum and Brannon-Peppas, 2004). Experts, however, predict that within the next 5-10 years some of the formulations currently under study might progress to the clinical evaluation and perhaps become marketed therapy not so far (http://www.zycos.com/press/release15.html; Hedley et al, 1998). Nanotechnology is a multidisciplinary field which encompasses research and development at the atomic, molecular or macromolecular level (Emerich and Thanos, 2003). Due to their extremely small size, nanoscale structures have unique properties for the controlled and targeted release of therapeutic products (Sahoo and Labhasetwar, 2003). In the last few years, investment in nanotechnology worldwide has increased considerably and recently the National Cancer Institute has announced a major commitment to nanotechnology for cancer research in the form of $144.3 million, five-year initiative.

Although the total drug-load is reduced considerably and the manufacture process is more complex, the nanoscale devices present some advantages over the micro-systems. In fact, submicron systems show higher intracellular uptake than microsized particles, thereby allowing drug-release in different cellular compartments such as cytoplasm and nucleus. Nanoparticles can be also easily conjugated with a ligand to favour a targeted therapeutic approach and as it has been reported, some nanoparticles can cross the blood-brain barrier (BBB). For example, doxorubicin bound to polysorbate-coated nanoparticles can cross the intact BBB, reaching therapeutic concentrations in the brain. When these particles were administered in glioblastoma-bearing rats, a very aggressive human cancer with short survival times, significantly higher survival times were observed in the treated animal group compared with all other groups (Steiniger et al, 2004). Depending on the elaboration method and the materials employed different nanosystems can be distinguished including micelles, nanocapsules, dendrimers, nanospheres, solid lipid nanoparticles and ceramic nanoparticles. The principal characteristics and some of the recent research using each nano-systems is reviewed in Table 1. Liposomes are one of the most well-known drug delivery carriers employed in the treatment of cancer. Due to their advantages, liposomal formulations provide a substantial increase in antitumor efficacy comparing with the free drug or standard chemotherapy regimens (Drummond et al, 2004). Liposomes are composed of a double lipid bilayer which encloses an aqueous space that can be employed to transport anticancer drugs.

Table 1. Examples of different nanoparticles and their applications as cancer treatments Nanoparticle

Description

Nanocapsules

Vesicular systems in which the drug is surrounded by a polymeric membrane

Nanospheres

Matrix systems in which the drug is physically and uniformly dispersed

Micelles

Amphiphilic block copolymers that can self-associate in aqueous solution Nanoparticles fabricated using inorganic compounds including silica, titaniaâ&#x20AC;Ś Artificial spherical vesicles produced from natural phospholipids and cholesterol Macromolecular compound that comprise a series of branches around an inner core Nanoparticles made from solid lipids

Ceramic nanoparticles Liposomes Dendrimers SLN particles

Recent applications

Reference

Stability of the cisplatin nanocapsules has been optimized by varying the lipid composition of the bilayer coat Bovine serum albumin nanospheres containing 5-fluorouracil show higher tumour inhibition than the free drug Micelle delivery of doxorubicin increases cytotoxicity to prostate carcinoma cells Ultra fine silica based nanoparticles releasing water insoluble anticancer drug Radiation-guided drug delivery of liposomal cisplatin to tumor blood vessels results in improved tumour growth delay Targeted delivery within dendrimers improved the cytotoxic response of the cells to methotrexate 100-fold over free drug SLN powder formulation of all-trans retinoic acid may have potential in cancer chemoprevention and therapeutics.

Velinova, 2004

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Santhi, 2002 McNaealy , 2004 Roy, 2003 Geng, 2004 Quintana, 2002 SooJeong, 2004


Cancer Therapy Vol 3, page 135 Some factors must be taken into account when preparing the liposomal formulations including the size, the surface charge (Senior, 1987) and the membrane fluidity (Gregoriadis and Senior, 1980). All these formulation issues have implications on the pharmacokinetics, biodistribution and bioavailability of the entrapped therapeutic product. There are many liposomes for the treatment of malignancies that are already approved, awaiting approval or in clinical trials. The most frequently studied drug family is the anthracyclines since they present activity against a wide range of tumours (Young et al, 1981). For example, doxorubicin liposomes have shown significant activity against AIDS-related Kaposi´s sarcoma, breast and ovarian cancers in different clinical trials (Amantea et al, 1997; Muggia, 1997; Ranson et al, 1997). Liposomes containing daunorubicin have been also successfully employed in the treatment of Kaposi´s sarcoma and are currently currently being evaluated with some effectiveness for the treatment of central nervous system tumors (Zucchetti et al, 1999). Another promising liposomal product is the vincristine liposome. In fact, in a preliminary phase II trial for the treatment of nonHodgin´s lymphomas, liposomal-vincristine showed efficacy against the transformed or aggressive nonHodgin´s lymphomas and presented less neurotoxicity than the free-drug (Sarris et al, 2000). Another interesting approach is the immobilization of therapeutic product-secreting cells within microcapsules (Orive et al, 2003c, 2004). Enclosing the biologically active material within a polymeric matrix surrounded by a semipermeable membrane it is possible to circumvent immune rejection while enabling the controlled and continuous release of the active substance. Several attemps of applying this technology to treatment of different malignancies have been described and reviewed in the literature (Orive et al, 2003d). For instance, using CYP2B1-transfected cells which activate the prodrug ifosfamide at the site of tumor, the median survival of mice transplanted with a human pancreatic carcinoma was the double than the control group (Lörh et al, 2002). The safety of this protocol was evaluated in a phase I/II trial in 14 patients with pancreatic cancer. Results showed that the tumours of four patients regressed after treatment and those of other ten individuals who followed the study remain stable (Lörh et al, 2001). However, the inhibition of tumour angiogenesis has been so far the most frequently targeted phenomenon by microencapsulated cells. Angiogenesis is the process by which new blood vessels are formed to promote tumour growth and metastasis (Folkman, 1971). The available evidence suggest that optimal anti-angiogenic therapy requires prolonged exposure to low drug concentrations (Cristofanalli et al, 2002; Kerbel and Folkman, 2002; Sweeney et al, 2003). Furthermore, the discovery of endogenous antiangiogenic factors (O´Really et al, 1994, 1997) has opened the door to the genetic manipulation of cells and consequently their encapsulation in polymer matrices (Boüard et al, 2003; Cirone et al, 2003). Using this strategy, genetically engineered kidney epithelial cells expressing the anti-angiogenic agent endostatin were

assayed for the treatment of glioblastomas (Joki et al, 2001). Mice inoculated with human glioma U87MG cells were divided in three groups. The first group received a single injection of microcapsules containing endostatin secreting cells (BHK-endo). The second received an injection of neomycin-resistant gene-transfected cells that did not secrete endostatin (BHK-neo) and the third group did not receive any treatment (control). Results showed that in comparison with controls, the growth of glioma tumours was suppressed by 62% by 21 days post administration of the encapsulated BHK-neo cells (Figure 3A). Furthermore, the BHK-endo group showed a 72% reduction in tumour weight when compared to the other two groups (Figure 3B) (Joki et al, 2001). A similar approach done by other research group resulted in analogous results with a tumor growth reduction of >70% (Read et al, 2001). In addition, encapsulated cells secreting angiostatin have been combined with immunotherapy against a mouse melanoma model showing improved survival with 30% of the animals surviving tumour free (Cirone et al, 2004).

IV. Macromolecular conjugation The pharmacokinetics and biodistribution of the cancer drugs can be improved by conjugating the latter with water soluble polymers, peptides and proteins. PEG is the main representative of the polymers used to conjugate with the therapeutic drug in order to improve the pharmacokinetics of the latter. In fact, since the first PEGylated protein (PEG-adenosine deaminase) entered the market in 1990 (Levy et al, 1988), a large number PEGylated pharmaceuticals have followed. For instance, PEGinterferon !-2b is under clinical evaluation in a phase II randomized study in patients with metastatic or unresectable carcinoid tumours whereas PEGylated-Lasparaginase (Oncospar ®) is used in the treatment of acute leukaemia. The latter can be administered every 2 weeks instead of the 2-3 times per week needed for the native enzyme. In addition, a Phase II clinical trial is undergoing to evaluate PEGylated-L-asparaginase in multiple myeloma (http://www.clevelandclinic.org/myeloma /pegasp.htm. This strategy can be also used to improve targeting and EPR effect of polymer particles. In fact, by conjugating specific vector molecules to micro and nanoparticles it is possible to improve the affinity towards different tissues or tumours. Targeted therapy enables a higher bioavailability of the therapeutic molecules whereas reduces considerably possible side-effects. Vector molecules capable of recognizing tumors include antibodies, lectins, peptides, hormones, folate and vitamins. For example, monoclonal nuclear antibodies can be attached to the drug carriers to promote drug release only to tumour cells and not normal cells (Iakoubov et al, 1995). Interestingly, the high affinity of folic acid for folate receptors provides a unique opportunity to use folic acid as a targeting ligand to deliver chemotherapeutic agents to cancer cells. In vitro experiments using folatetethered liposomes containing calcein or doxorubicin showed a selective drug release in both human cervical cancer HeLa-IU1 cells and human colon cancer Caco-2 cells overexpressing folate receptors (Zhang et al, 2004) 135


Orive et al: Micro and nano drug delivery systems in cancer therapy Figure 3. The effect of encapsulated endostating secreting cells (BHKendo), encapsulated cells that did not secrete endostatin (BHK-neo) and no treatment (control) on tumor growth in vivo. A) Tumour size and B) tumour weight of subcutaneous U87MG human glioma cell xenograft. Reproduced from Joki et al, 2001 with kind permission from Nature Biotechnololy.

In another approach, Hasokawa et al. used a human monoclonal antibody formulated as PEG-modified immunoliposomal doxorubicin to treat selectively human stomach cancer. In vivo experiments showed greater antitumor activity than the unmodified liposome or the free drug (Hosokawa et al, 2003). Anti-tumor antibodyconjugated polymeric micelles called immunomicelles have shown successful in vitro and in vivo delivery of taxol into various cancer cells (Torchilin et al, 2003). In

fact, when inhibition of murine Lewis lung carcinoma tumour growth in mice was evaluated with different taxol preparations, the average weight of excised tumours in the group treated with taxol incorporated in immunomicelles was 0.67±0.35 g compared with 1.58±0.48 g and 1.37±0.36 g in groups treated with free taxol or taxol in plain PEG-PE micelles, respectively (P < 0.05 in both cases) (Figure 4). Figure 4. Inhibition of murine Lewis lung carcinoma tumour growth in mice with different taxol preparations including free taxol, plain micelles and taxol releasing 2C5-immunomicelles. Reproduced from Torchilin et al, 2003 with kind permission from Proc. Natl. Acad. Sci. U.S.A.

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Cancer Therapy Vol 3, page 137 delivery systems in cancer therapy, Ed. Brown DM, Humana Press, 191-213. Duncan R (2003) The dawning era of polymer therapeutics. Nat Rev Drug Discov 2, 347- 360. Emerich DF, Thanos CG (2003) Nanotechnology and medicine. Expert Opin Biol Ther 3, 655-663. Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285, 1182-1186. Gabizon A, Shmeeda H, Barenholz Y (2003) Pharmacokinetics of pegylated liposomal doxorubicin - Review of animal and human studies. Clinical Pharmacokinetics 42, 419-436. Geng L, Osusky K, Konjeti S, Fu A, Hallahan D (2004) Radiation-guided drug delivery to tumor blood vessels results in improved tumor growth. J Control Rel 99, 369381. Gregoriadis G, Senior J (1980) The phospholipid component of small unilamellar liposomes controls the rate of clearance of entrapped solutes from the circulation. FEBS Lett 119, 4346. Harris JM, Chess RB (2003) Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2, 214-221. Hashizume H, Baluk P, Morikawa S, McLean JW, Thurston G, Roberge S, Jain RK, McDonald DM (2000) Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol 156, 1363-1380. Hedley ML, Curley JM, Langer RS (1998) Microparticles for delivery of nucleic acid, USA Patent 5783567, July 21; assigned to Pangaea Pharmaceuticals. Hosokawa S, Tagawa T, Niki H, Hirakawa Y, Nohga K, Nagaike K (2003) Efficacy of immunoliposomes on cancer models in a cell-surface-antigen-density-dependent manner. Br J Cancer 89, 1545-1551. Iakoubov L, Rokhlin O, Torchilin V (1995) Antinuclear autoantibodies of the aged reactive against the surface of tumor but not normal cells. Immunol Lett 47, 147-149. Jain, RK (2001) Normalizing tumor vasculature with antiangiogenic therapy: a new paradigm for combination therapy. Nat Med 7, 987-989. Joki T, Machluf M, Atala A, Zhu J, Seyfried NT, Dunn IF, Abe T, Carroll RS, Black P.McL (2001) Continuous release of endostatin from microencapsulated engineered cells for tumor therapy. Nat Biotechnol 19, 35-39. Kerbel R, Folkman J (2002) Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2, 727-739. Levy Y, Hershfield MS, Fernandez C, Polmar SH, Scudiery D, Berger M, Sorensen, RU (1988) Adenosine deaminase deficiency with late onset or recurrent infections: response to treatment with polyethylene glycol modified adenosine deaminase. J Pediatr 113, 312-317. Lim SJ, Lee MK, Kim CK (2004) Altered chemical and biological activities of all-trans retinoic acid incorporated in solid lipid nanoparticle powders. J Control Rel 100, 53-61. Lörh M, Hoffmeyer A, Kröger JC, Freund M, Hain J, Holle A, Karle P, Knöfel WT, Liebe S, Müller P, Nizze H, Renner M, Saller RM, Wagner T, Hauenstein K, Günzburg WH, Salmons B (2001) Microencapsulated cell-mediated treatment of inoperable pancreatic carcinoma. Lancet 357, 1591-1592. Lörh M, Hummel F, Faulmann G, Ringel J, Saller R, Hain J, Günzburg WH, Salmons B (2002) Microencapsulated, CYP2B1-transfected cells activating ifosfamide at the site of the tumor: the magic bullets of the 21st century. Cancer Chemother. Pharmacol. 49, 21-24. Maeda H, Wu J, Sawa T, Matsumura K, Hori K (2000) Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review J Control Rel 65, 271-284.

Finally, modified dendrimers have been also fully investigated for targeted drug delivery. Dendrimers associated with an anionic oligomer were elaborated for delivering angiostatin and tissue inhibitor of metalloproteinase (TIMP)-2. In vivo results showed that angiostatin release inhibited tumour growth by 71% while (TIMP)-2 by 84%. Moreover, combined therapy resulted in 96% inhibition of tumour growth (Vincent et al, 2003).

V. Conclusions The present paper reviews the use of micro and nanotechnology as well as macromolecular conjugation as strategies to deliver existing chemotherapies and novel therapeutic molecules in a controlled manner to malignancies. These technologies come along with other exciting drug delivery approaches such as patches, microchips and osmotic pumps. In general, the technologies described here improve significantly the pharmacokinetics and biodistribution of the free drugs and reduce considerably their side-effects. Furthermore, some of them have been approved by FDA and are currently in the market. In the future, some challenges need to be addressed including the adaptation of each drug delivery system to the particular needs of each malignancy, improvement of interactions between the drug and some of the components of the carrier and development of multifunctional systems able to deliver several drugs at the same or different time with different kinetic releases.

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Transvaginal color Doppler in the assessment of cervical carcinoma Review Article

Juan Luis Alcázar* Department of Obstetrics and Gynecology, Clínica Univeristaria de Navarra, School of Medicine, University of Navarra, Pamplona, Spain

__________________________________________________________________________________ *Correspondence: Juan Luis Alcázar, Department of Obstetrics and Gynecology, Clínica Univeristaria de Navarra, School of Medicine, University of Navarra, Pamplona, Spain, Avenida Pio XII, 36 31008 Pamplona, Spain; e-mail: jlalcazar@unav.es Key words: Transvaginal color Doppler, cervical carcinoma Abbreviations: flow velocity waveform, (FVW); pulsatily index, (PI); peak systolic velocity, (PSV); resistance index, (RI); vascular index, (VI) Received: 13 January 2005; revised: 28 January 2005 Accepted: 2 March 2005; electronically published: March 2005

Summary Transvaginal color Doppler is a non-invasive ultrasound-based technique that allows an in vivo assessment of tumor vascularization. Several papers in the last decade have evaluated the role of this technique in assessing carcinoma of the cervix. In this paper we will review the role of transvaginal color Doppler sonography in assessing cervical carcinoma vascularization and its correlation with tumor characteristics as well as for predicting therapeutic response to treatment such as radiotherapy and chemotherapy. waveform (FVW). And calculating velocity of blood flow throughout the cardiac cycle, including peak systolic velocity (PSV, cm/sec), mean velocity and end-diastolic velocity, and resistance to flow by calculating some velocimetric indexes such as resistance index (RI) or pulsatility index (PI). The higher the velocity and the lower the RI or PI would means the higher blood flow (Figure 1). 2. Color Doppler. It is based in the same principle than pulsed Doppler (Doppler frequency shift) and allows the visualization of vessels by color-coding. This method is useful to determine the presence of vessels and provides information about the amount of vessels (subjective), their distribution and arrangement. It is usually used in combination with pulsed Doppler (Figure 2). 3. Power Doppler. This method is based in the amplitude shift of Doppler signal but not in frequency shift. It has some advantages over color Doppler, which make it more suitable for assessing vascularity (Guerriero et al, 1998) (Figure 3). As a matter of fact, recent studies have shown that color and power Doppler sonography can be used to depict flow within arterioles and venules (> 100 µm) (Fleischer et al, 2000). Furthermore, the recent development of sonographic microbubble contrast agents and the ability to use harmonics and pulse inversion techniques have

I. Introduction Angiogenesis is the production of new vessels in a specific area (Folkman et al, 1989). It has been demonstrated that neoangiongenesis is an essential event for tumor growth and progression (Folkman et al, 1989). The process of angiogenesis initiates with the degradation of the capillary vessel basement membrane, through which migrating endothelial cells form a sprout and proliferate to create new lumen and further vessel maturation (Abulafia et al, 2000) Specifically, in cancer of the cervix, angiogenesis has been shown to be an independent prognostic factor (Schelenger et al, 1995; Tjalma et al, 1995; Kaku et al, 1998; Obermair et al, 1998) and to predict recurrence (Wiggins et al, 1995; Dinh et al, 1996) Tumoral angiogenesis is usually assessed by inmunohistochemical staining of factor VIII (Schelenger et al, 1995; Tjalma et al, 1995; Kaku et al, 1998; Obermair et al, 1998). This means that a histological piece is needed to perform the assessment. Transvaginal Doppler ultrasound allows an in-vivo non-invasive assessment of tumor angiogenesis (Cosgrove et al, 2003). Doppler ultrasound may be used in several modes (Maulik et al, 1997): 1. Pulsed Doppler. Allows the assessment of blood flow velocity and resistance in a given vessel at a given moment by analyzing the so-called flow velocity 139


Alcรกzar et al: Transvaginal color Doppler in the assessment of cervical carcinoma

Figure 1. Flow velocity waveform from two cases of cervical cancer. In one (a) resistance and pulsatility indexes are high (RI = 0.57, PI = 0.89), whereas in the other case (b) are low (RI = 0.30, PI = 0.34).

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Figure 2. Transvaginal color Doppler from a cervical carcinoma. A highly vascularized area is clearly seen within the cervix. Vessels are seen on red or blue depending on blood flow direction. Yellow vessels indicate that blood flow velocity is too high to ascertain direction of flow.

Figure 3. Transvaginal power Doppler from a cervical cancer. All vessels are depicted on the same color (no information of blood flow direction is provided by this method). However, vessels are more clearly defined.

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Alcázar et al: Transvaginal color Doppler in the assessment of cervical carcinoma enabled depiction of microvasculature (< 7-10 µm) (Foster, 2000). The objective of this paper is to review the application of transvaginal color and power Doppler in the assessment of angiogenesis in cervical cancer.

reported that this method was highly reproducible and they found that the higher VI was, the higher tumoral stage, the deeper stromal invasion, the higher lymphovascular space invasion rate and the higher pelvic lymph node metastases rate was. More interestingly, this VI had a good correlation with intratumoral microvessel density as assessed inmunohistochemically (r=0.586). Although this paper was the first to demonstrate the correlation between inmunohistochemically assessed angiogenesis and power Doppler assessed angiogenesis, the main problem that the method is a two-dimensional assessment of tumor vascularity in a given scanning section of the tumor. But the tumor is a three-dimensional structure. Furthermore, other authors have not reproduced this method. The same group reported a further series but using color Doppler in 60 women diagnosed as having a stage Ib-IIa cervical cancer (Cheng et al, 1999b). They found color signals in 58% of the cases. The presence of color signals was associated with a higher probability of lymph node metastases (sensitivity 80%, specificity 48%) and parametrial involvement (sensitivity 91%, specificity 57%). In our experience color signals may be identified in 100% of cervical cancers (Alcázar et al, 2003). We found that tumor blood flow as assessed by transvaginal color Doppler was correlated with some tumor characteristics. Tumor vascularization was higher in squamous cell cancers, moderately or poorly differentiated lesions and advanced – stage tumors. However, in all these studies assessment of tumor blood flow color mapping has the inherent bias of subjectivity and this may represent a problem for interand intra-observer reproducibility when applying this technique in clinical settings. In the last years three-dimensional power-Doppler has been introduced as a new method for assessing tumor vascularization (Figure 4). This technique allows a global assessment of vascularization in a giver area including the whole region of interest, thus representing a more accurate and actual estimation of tumor vascularity (Suren et al, 1998). Furthermore, this technique has been demonstrated to be highly reproducible (Raine-Fenning et al, 2003). To date reported results on the use of 3 D Power Doppler sonography for assessing tumor vascularization in cervical cancer are controversial. Testa et al, (2004) did not find any correlation between 3D-derived vascular indices and clinicpathological characteristics in a series of 74 cervical cancer. However, they found that 2D spectral and color Doppler parameters were correlated with tumor stage and diameter. The higher vascularization was the larger tumor and advanced stage were these results were similar to ours (Alcázar, 2003). On the other hand, Hsu et al has reported the results of applying 3D Power-Doppler in 141 patients with earlystage cervical cancer. They found blood flow in 85% of the tumors and tumor vascularization was correlated with tumor volume (Hsu et al, 2004). In our experience, vascularization as assessed by 3D power Doppler ultrasonography was higher in invasive

II. Transvaginal color and powerDoppler and its correlation with tumoral features in cervical cancer Initial studies assessing blood flow hemodynamics in cervical cancer in the early 90’s focused on the main feeding cervical vessels: the uterine artery (Enzelsberger et al, 1991) and the cervical branch of the uterine artery (Breyer et al, 1993). This was due basically to the technical limitations of Doppler ultrasound in those days. Enzelsberger et al, (1991) found that mean PI in the uterine arteries of women with cervical cancer was significantly lower than in healthy women. Similar findings were reported by Breyer et al, (1993) when analysing the cervical artery. Tepper et al reported that intratumoral RI was lower in patients with cervical cancer as compared with healthy women (Tepper et al, 1996). Wu et al compared color and power Doppler in assessing intratumoral vascularization in 35 women with cervical cancer 30 healthy women (Wu et al, 2000). They reported color signals in 97% of cancers and found that both PI and a vascular ratio (cross-section area of intratumoral vessels / cross-section area of the tumor) defined by the authors were significantly lower in patients with cervical cancer. They concluded that power Doppler angiography was more useful than color Doppler because the vascular ratio provided more sonographic characteristics among different subclassifications of cervical cancer. Hsieh et al, (1995) found that 46.2% of cervical cancers exhibited blood flow color signals as assessed by transvaginal color Doppler sonography. They reported that in those patients with detectable color signals lymph node involvement was more frequent as compared with those without color signals detectable (33% vs 5.7%, p = 0.005) and this also correlated with a higher cell proliferation index. They did not find differences in tumoral stage, patient’s age, clinical staging, histologic type and DNA ploidy status. These papers indicated that intratumoral blood flow in cervical cancer could be assessed by transvaginal color Doppler. However, blood flow detection rate was very low to be practical from the clinical point of view. Furthermore, Carter challenged these previous studies reporting that no differences would exist in RI or PI between benign and malignant cervical tumors (Carter, 1999). Cheng et al, (1999a) reported a novel vascular index (VI) for the in vivo assessment of angiogenesis in patients with cervical carcinoma. They evaluated 35 patients with stage Ib-IIa cervical carcinoma by transvaginal power Doppler ultrasound, and using image processor software they developed a vascular index (VI = number of colored pixels/number of total pixels) for each tumor. They 142


Cancer Therapy Vol 3, page 143 cancers than in â&#x20AC;&#x153;in situâ&#x20AC;? carcinoma and normal cervix

(Table 1).

Figure 4. Three-dimensional power-Doppler ultrasound from a case of cancer of the cervix. (a) Tumor volume estimation, depicting the lesion contour. (b) Vascular indexes (VI, FI, VFI) within the tumor estimated using this method.

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Alcázar et al: Transvaginal color Doppler in the assessment of cervical carcinoma Table 1. 3D power Doppler vascular indexes in cervical cancer

VI (Vascular index)* FI (Flow index)† VFI (Vascular-flow index) ‡

Normal cervix “In situ” (n=5) carcinoma (n=5) 1.68% (1.89) 6.29% (10.2)

Invasive early-stage Invasive advanced-stage carcinoma (n=7) carcinoma (n=16) 32.50% (10.0) 40.42% (14.1)

21.57 (2.6)

20.69 (3.1)

29.80 (6.3)

31.1 (6.5)

0.49 (0.5)

1.51 (2.1)

10.07 (4.3)

12.44 (6.02)

Data expressed as mean, standard deviation in parentheses. * p < 0.001 † p < 0.05 ‡ p < 0.05

tumors had the better response. These results could be explained by several facts. First, it has been demonstrated that correlation between blood supply and oxygenation is absent in large tumors (Asomov et al, 1984). Second, tumor oxygenation would depend no only on the extent of angiogenesis, but also in the capability of the newly formal vessels to supply oxygen. It has been shown that this capability is impaired in neoangiogenesis vessels (Fenton and Way, 1993). Third, as indicated earlier the higher the vascularization is the more aggressive the tumor is (Ueda et al, 1998). This could indicate that highly vascularized tumors could be more resistant to chemotherapy and radiotherapy (Alcázar and Jurado, 1999). Despite these initially surprising results, we performed a second study in an extended series of patients with locally advanced cervical carcinoma (Alcázar, 2004). The results of this second study confirmed the preliminary results. We found that the likelihood ratio for complete response to chemorradiation for tumors with a PI > 0.45 was 3.3 (95% CI: 2.1-4.5). Chen et al reported that the use of the vascularity index described by their group would be useful to predict the response to neoadjuvant chemotherapy in cervical carcinoma (Chen et al, 2004). They found that nonresponders had higher vascularized tumors than responders do and persisted highly vascularized during treatment these findings are in agreement with our data.

However, as Testa et al, (2004) we did not find any correlation between 3D power Doppler derived vascular indexes and tumor characteristics among invasive cervical cancer.

III. Transvaginal color Doppler for predicting treatment response Several studies have evaluated the role of transvaginal color Doppler for assessing or predicting the response to treatment in women with cervical cancer. Pirhonen and col. analyzed intratumoral vascularization by color mapping in 14 patients with advanced cervical carcinoma treated with external radiotherapy (Pirhonen et al, 1995). They used a subjective color map grading (Grade 1: normal amount of vessels to grade 5: extremely high amount of vessels). They evaluated patients prior to start treatment and then five follow-up examinations during treatment. These authors found that a decrease in tumor vascularization during radiotherapy was associated with a better outcome, whereas persistence of high vascularity was associated with a poor response. They concluded that color Doppler ultrasound might be useful in early assessment of therapeutic response during RT. Similar findings were reported by Greco et al, (1997) in a series of 14 women with advanced cervical cancer. These authors evaluated intratumoral vascularization by means of transvaginal color Doppler before and after neoadyuvant chemotherapy. Ten out of 14 patients had a successful therapeutic response. The authors reported that in these cases a significant increase on intratumoral RI and PI was observed after treatment, whereas no differences were found in those four women who did not respond to chemotherapy. We hypothesized that, since oxygenation plays an essential role in the response to radiotherapy in solid tumors and as tumor oxygenation depends on angiogenesis, the higher vascularized tumors would have a higher oxygen delivery to tissue and, therefore, would have a better response to chemoradiation in locally advanced cervical cancer. We tested this hypothesis in a preliminary study on 10 patients (Alcázar et al, 1999). However, surprisingly, we found that poorly vascularized

IV. Conclusions Transvaginal color or power Doppler allows a noninvasive assessment of tumor vascularization in cervical carcinoma. Although most published studies come from two groups they have shown that tumor vascularization as assessed by this technique correlates with some individual tumor characteristics such as tumor volume, lymph node involvement and tumoral stage. However, there is no consensus about which Doppler parameter or parameters correlate better with tumor characteristics. Regarding the role of this technique for predicting response to treatment all published studies indicate that it may be useful for this purpose. However, most series are small and, therefore, studies in larger series are needed to confirm these results. Should be this confirmed it would

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Cancer Therapy Vol 3, page 145 Foster FS, Burns PN, Simpson DH, Wilson SR, Christopher DA, Goertz DE (2000). Ultrasound for the visualization and quantification of tumor microcirculation. Cancer Mestastasis Rev 19, 131-8 Greco P, Cormio G, Vimercati A, Nacci G, Di Vagnio G, Loverro G, Selvaggi L (1997) Transvaginal color Doppler ultrasound for monitoring the response to neoadjuvant chemotherapy in advanced cervical cancer. Acta Obstet Gynecol Scand 76, 169-72 Guerriero S, Ajossa S, Lai MP, Risalvato A, Paoletti AM, Melis GB. (1999) Clinical applications of colour Doppler energy imaging in the female reproductive tract and pregnancy. Hum Reprod Update 5, 515-29 Hsieh CY, Wu CC, Chen TM, Chen CA, Chen CL, Wang JF, Chang CF, Hsieh FJ. (1995) Significance of intratumoral blood flow in cervical carcinoma assessed by color Doppler ultrasound. Cancer 75, 2518-22. Hsu KF, Su JM, Huang SC, Cheng YM, Kang CY, Shen MR, Chang FM, Chou CY. (2004) Three-dimensional power Doppler imaging of early-stage cervical cancer. Ultrasound Obstet Gynecol 24, 664-71. Kaku T, Hirakawa T, Kamura T, Amada S, Kinukawa N, Kobayashi H, Sakai K, Ariyoshi K, Sonoda K, Nakano H. (1998) Angiogenesis in adenocarcinoma of the uterine cervix. Cancer 83, 1384-90. Maulik D (1997) Doppler in Obstetrics and Gynecology. Springer-Verlag, New York USA Obermair A, Wanner C, Bilgi S, Speiser P, Kaider A, Reinthaller A, Leodolter S, Gitsch G. (1998) Tumor angiogenesis in stage IB cervical cancer: correlation of microvessel density with survival. Am J Obstet Gynecol 178, 314-9. Pirhonen JP, Grenman SA, Bredbacka AB, Bahado-Singh RO, Salmi TA (1995) Effects of external radiotherapy on uterine blood flow in patients with advanced cervical carcinoma assessed by color Doppler ultrasonography Cancer 76, 67-71 Raine-Fenning NJ, Campbell BK, Clewes JS, Kendall NR, Johnson IR. (2004) The interobserver reliability of threedimensional power Doppler data acquisition within the female pelvis. Ultrasound Obstet Gynecol 23, 501-8. Schlenger K, Hockel M, Mitze M, Schaffer U, Weikel W, Knapstein PG, Lambert A. (1995) Tumor vascularity--a novel prognostic factor in advanced cervical carcinoma. Gynecol Oncol 59, 57-66. Suren A, Osmers R, Kuhn W (1998) 3D color power angio imaging: a new method to assess intracervical vascularization in benign and pathological conditions. Ultrasound Obstet Gynecol 11, 133-7 Tepper R, Zalel Y, Altaras M, Ben-Baruch G, Beyth Y (1996) Transvaginal color Doppler ultrasound in the assessment of invasive cervical carcinoma Gynecol Oncol 60, 26-9 Testa AC, Ferrandina G, Distefano M, Fruscella E, mansueto D, Basso D, Salutari V, Scambia G (2004) Color Doppler velocimetry and three-dimensional color power angiography of cervical carcinoma. Ultrasound Obstet Gynecol 24, 44552 Tjalma W, Van Marck E, Weyler J, Dirix L, Van Daele A, Goovaerts G, Albertyn G, van Dam P. (1998) Quantification and prognostic relevance of angiogenic parameters in invasive cervical cancer. Br J Cancer 78, 170-4. Ueda M, Ueki K, Kumagai K, Terai Y, Okamoto Y, Ueki M, Otsuki Y (1998) Apoptosis and tumor angiogenesis incervical cancer after preoperative chemotherapy Cancer Res 58, 2343-6

be very interesting because the use of this technique could allow a more tailored treatment protocol for any given patient. Another problem to be solved is the reproducibility of the technique in order to be introduced into clinical practice. This problem may be solved by the use of 3D power-Doppler ultrasound.

References Asomov IS, Karakulov RK, Sazonova NA, Malygina AI, Morozova TG (1984) Oxigen status and angioarchitectonics of different types of tumors and their changes after radiotherapy Radiobiologiia 24, 630-5 Abulafia O, Sherer DM (2000) Angiogenesis of the uterine cervix Int J Gynecol Cancer 10, 349-57. Alcรกzar JL, Jurado M (1999) Transvaginal color Doppler for predicting pathological response to preoperative chemoradiation in locally advanced cervical carcinoma: a preliminary study Ultrasound Med Biol 25, 1041-5. Alcรกzar JL, Castillo G, Jurado, M, L_pez-Garc_a G (2003) Intratumoral blood flow in cervical cancer as assessed by trasnvaginal color Doppler ultrasonography: correlation with tumor characteristics Int J Gynecol Cancer 13, 510-4. Alcรกzar JL, Castillo G, Martinez-Monge R, Jurado M (2004) Transvaginal color Doppler for predicting response to concurrent chemoradiotherapy for locally advanced cervical carcinoma J Clin Ultrasound 32, 267-72. Breyer B, Despot A, Predanic M, Judin S (1993) Characteristics of blood flow in cancer of the uterine cervix Ultrasound Obstet Gynecol 3, 268-70. Carter JR (1999) Cervical tumor characterization by transvaginal color flow Doppler ultrasound Int J Gynecol Cancer 9, 27984 Chen CA, Cheng WF, Lee CN, Su YN, Hsieh CY, Hsieh FJ (2004) Power Doppler vascularity index for predicting the response to neoadjuvant chemotherapy in cervical carcinoma Acta Obstet Gynecol Scand 83, 591-7 Cheng WF, Le CN, Chu JS, Chen CA, Chen TM, Shau WY, Hsieh CY, Hsieh FJ (1999a) Vascularity index as a novel parameter for the in vivo assessment of angiogenesis in patients with cervical carcinoma Cancer 85, 651-7 Cheng WF, Wei LH, Su YN, Cheng SP, Chu JS, Lee CN (1999b) The possible use of color flow Doppler in planning treatment in early invasive carcinoma of the cervix Br J Obstet Gynecol 106, 1137-42 Cosgrove D (2003) Angiogenesis imaging-ultrasound Br J Radiol 76, S43-S49 Dinh TV, Hannigan EV, Smith ER (1996) Tumor angiogenesis as predictor of recurrence in stage Ib squamous cell carcinoma of the cervix. Gynecol Oncol 87, 751-4 Enzelsberger H, Skodler WD, Vavra N, Reinold E (1991) Ultrasonic Doppler flow studies of the uterine artery in women with cervix cancer Gynecol Obstet Invest 32, 112-4 Fenton BM, Way BA (1993) Vascular morphometry of KHT and RIF-1 murine sarcomas Radiother Oncol 28, 57-62 Fleischer AC, Niermann KJ, Donnelly EF, Yankeelov TE, Canniff KM, Hallahan DE, Rothenberg ME (2004). Sonographic depiction of microvessel perfusion. J Ultrasound Med 23, 1499-506 Folkman J, Watson K, Ingber D, Hanahan D (1989) Induction of angiogenesis during the transition from hyperplasia to neoplasia Nature 339, 58-61

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Juan Luis Alcรกzar

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Cancer Therapy Vol 3, page 147 Cancer Therapy Vol 3, 147-152, 2005

Outcomes of hysteroscopy and hysterectomy in breast cancer patients Research Article

Pedro T. Ramirez 1,*,Charlotte C. Sun 1, Claudia I. Vidal2, Veronica Schimp 3, Brian Slomovitz1, Michael W. Bevers1, and Diane C. Bodurka1 1

Department of Gynecologic Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030 Department of Genitourinary Medical Oncology, The University of Texas Health Science Center, Houston, TX 77030 3 Department of Gynecologic Oncology, Wayne State University, Detroit, MI 48201 2

__________________________________________________________________________________ *Correspondence: Pedro T. Ramirez, M.D., Department of Gynecologic Oncology, Unit 440, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030; Phone: (713) 745-5498; Fax: (713) 792-7586; E-mail: peramire@mdanderson.org Key words: breast cancer, mastectomy, hysteroscopy, hysterectomy Received: 23 December 2004; Accepted: 24 February 2005; electronically published: March 2005

Summary The purpose of this study was to identify symptoms that led to gynecologic consultations and findings on diagnostic studies and pathologic evaluation in breast cancer patients treated with mastectomy that underwent hysteroscopy and hysterectomy. We searched the patient database at The University of Texas M. D. Anderson Cancer Center. Records were reviewed to determine demographic and clinical characteristics. Two hundred and seventeen patients were included in our study. A total of 122 patients (56%) received chemotherapy for their breast cancer, 107 (49%) received radiotherapy, and 99 (46%) received hormones. Of those treated with hormones, 96 (97%) received tamoxifen. The most common reasons for gynecologic consultations were vaginal bleeding (43%) and abnormal ultrasound findings (21%). A total of 176 patients (81%) underwent hysterectomy and 54 (31%) had malignancy in final pathology. Of the 69 patients who used tamoxifen and underwent a hysterectomy, 10 (14%) patients were diagnosed with endometrial cancer.In women who have undergone mastectomy for breast cancer and subsequently undergo hysteroscopy or hysterectomy, the number of abnormal findings on ultrasound studies and endometrial evaluation is high, as is the likelihood of malignant uterine or ovarian lesion. by ultrasonography is not confirmed by hysteroscopic or histologic pathology review. Endometrial sampling by microcurettage in tamoxifen-treated patients has not proved to be as sensitive and accurate as hysteroscopy and curettage (Powles et al, 1998; Neven and Vernaeve, 2000). It has been our observation that in the Gynecologic Oncology Center in our institution, many patients with a history of breast cancer who seek the advice of a gynecologist or gynecologic oncologist do so for symptoms of vaginal bleeding either secondary to anovulatory cycles caused by the chemotherapeutic agents used to treat their breast cancer or secondary to tamoxifen use. There is also significant anxiety in asymptomatic patients undergoing treatment with tamoxifen who are found to have abnormalities on routine transvaginal ultrasonography. The purpose of this study was to identify the symptoms in breast cancer patients who previously underwent mastectomy and subsequently presented to the

I. Introduction An estimated 211,300 new cases of invasive breast cancer were diagnosed among women in the United States during 2003. During the same year, approximately 40,200 women died of this disease (Ries et al, 1975-2000). Most patients with breast cancer are offered mastectomy or breast conservation therapy at the time of diagnosis. For over 20 years, tamoxifen has been widely used in the chemoprevention of breast cancer. It is administered as a potential breast cancer chemopreventive agent because studies have shown that tamoxifen given for 5 years reduces the incidence of recurrent breast cancer by 42% and reduces the incidence of contralateral breast cancer by 47% (Fisher et al, 1998). There is poor correlation between sonographic, hysteroscopic, and histologic findings in most patients using tamoxifen (Hulka and Hall, 1993; Goldstein, 1994; Mourits et al, 1999). In 45-90% of postmenopausal longterm tamoxifen users, an increased endometrial thickness 147


Ramirez et al: Outcomes of hysteroscopy and hysterectomy in breast cancer patients outpatient Gynecologic Oncology Center in our institution. We also sought to identify the diagnostic studies used in the evaluation of these patients prior to clinical disposition. Finally, we analyzed the histopathologic findings of hysteroscopy and hysterectomy.

A. Breast cancer diagnosis and treatment The median age at the time of breast cancer diagnosis was 46.5 years (range, 22 to 82 years). The majority of the patients (146, 67%) had a diagnosis of invasive ductal carcinoma. Location of breast cancer was as follows: left breast, 92, (42%); right breast, 102, (47%); bilateral, 23 (11%). Estrogen receptor status was documented for 140 patients. Of these, 98 (70%) were estrogen receptor positive and 42 (30%) were estrogen receptor negative. Similarly, progesterone receptor status was documented for 128 patients, and 80 (62%) were progesterone receptor positive while 48 (38%) were progesterone receptor negative. Information for HER2-neu receptor was available for 49 patients. Of these, 18 (37%) patients tested positive for the HER2-neu receptor. A total of 122 (56%) of the 217 patients received some form of chemotherapy as treatment for their breast cancer. The most common regimen was 5-fluorouracil, doxorubicin, and cyclophosphamide. In addition, 107 (49%) of 215 patients were treated with radiotherapy and 99 (46%) of 212 patients were treated with hormonal therapy. Ninety-six of the 99 patients (97%) treated with hormones received tamoxifen. The median time elapsed between the breast cancer diagnosis and the gynecologic consultation was 34 months (range, 1 to 338 months).

II. Materials and Methods We conducted a retrospective analysis of all patients with breast carcinoma who underwent mastectomy at The University of Texas M. D. Anderson Cancer Center between January 1990 and December 2001. We then identified patients from this group who had been referred to and evaluated in our outpatient Gynecologic Oncology Center and ultimately underwent either dilatation and curettage (D and C) with hysteroscopy or total hysterectomy. All cases were identified and retrieved by the Department of Medical Informatics at our institution. The investigation was conducted after Institutional Review Board approval was obtained. The medical records of all identified patients were reviewed, and the following information was gathered: patient age, race, family history of breast, ovarian or endometrial cancer, time of diagnosis of breast cancer, history of chemotherapy administration, radiation therapy, and hormonal therapy, reason for gynecologic oncology consultation, imaging studies performed at the time of consultation, results of these studies, type of definitive treatment, and histopathologic findings from D and C and hysteroscopy or hysterectomy. We excluded all patients who presented to the Gynecologic Oncology Center for any of the following reasons: a routine annual examination in an asymptomatic patient, vaginal dryness, infectious vaginal discharge, request for tubal ligation, treatment of endomyometritis, or evaluation of urinary incontinence. Additional exclusion criteria included hysterectomy prior to breast cancer diagnosis, gynecologic consultation or diagnosis of gynecologic malignancy prior to breast cancer diagnosis, hysterectomy and breast cancer surgery performed simultaneously, and missing medical record or no patient information available. We also excluded patients who were advised to undergo a D and C and hysteroscopy or hysterectomy and subsequently were lost to follow-up. Statistical analysis was performed using SPSS version 11.5.1. Descriptive statistics were used to evaluate demographic and clinical characteristic of the patients.

B. Gynecologic treatment

consultations

and

The most common reasons for gynecologic consultations were vaginal bleeding, 92 (43%); abnormal finding on ultrasonography, 45 (21%); recommended by breast oncologist, 16 (7%); evaluation of abnormal Pap smear, 14 (6%); concerns due to positive family history of breast or ovarian cancer, 9 (4%); and self-reported anxiety, 3 (1%). After initial evaluation, a total of 139 (64%) of the 217 patients had sonography performed. Other studies ordered included chest radiography, 164 (76%) and computed tomography scan of the abdomen and pelvis, 53 (24%). Prior to the D and C and hysteroscopy or hysterectomy, 120 (55%) of 217 patients underwent endometrial evaluation by endometrial biopsy. Findings on biopsy were considered abnormal in 35 cases (29%). A total of 64 patients (29%) underwent a D and C and hysteroscopy. Forty-four (69%) of the 64 patients who underwent a D and C also had an endometrial biopsy performed in the office prior to the D and C. The majority of patients underwent at least two diagnostic studies prior to the D and C and hysteroscopy. The most frequently ordered imaging study prior to D and C and hysteroscopy was transvaginal ultrasonography, which was ordered in 84% of patients. A total of 176 patients (81%) underwent a hysterectomy after their consultation in the Gynecologic Oncology Center. The median time from breast cancer diagnosis to hysterectomy was 42 months (range, 1 to 338 months). Findings on pathology review of the hysterectomy specimen were available for 163 patients.

III. Results A total of 262 patients were identified during the initial search. Forty-five patients were excluded according to the criteria outlined above. The remaining 217 patients were included in the final analysis. The median age for this group at presentation to the Gynecologic Oncology Center was 52 years (range, 28 to 83 years). The median follow-up time from the date of the first visit to the Gynecologic Oncology Center to the date of last contact was 25 months (range, 1 to 323 months). The ethnicity of the patients in our study was White, 77%; African American, 11%; Hispanic, 10%; and Asian, 2%. Information regarding family history was available for 199 patients. Of these patients, 69 (35%) had a history of maternal breast cancer, and 17 (8%) had a history of paternal breast cancer. Information regarding family history of ovarian cancer was available for 204 patients. Of these, 14 (7%) had a family history of ovarian cancer.

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Cancer Therapy Vol 3, page 149 The pathology results were malignant in 54 cases (33%) (Table 1). Of the 176 patients who ultimately underwent a hysterectomy, 24 (14%) also underwent a D and C and hysteroscopy prior to the hysterectomy. A median of three (range, 0 to 13) diagnostic studies was performed in the patients who underwent a hysterectomy. Of the 96 patients who had a history of tamoxifen use or were current users of tamoxifen, 62 (64%) underwent an endometrial biopsy. A D and C and hysteroscopy was performed in 47 (49%) of the 96 patients who received tamoxifen. Ultimately, 69 (72%) of the 96 patients who received tamoxifen underwent a hysterectomy. The most common malignancy was endometrial cancer, which was found in 10 patients. All of these tumors were categorized as stage I disease. However, two patients were found to have uterine papillary serous carcinoma, and three patients were diagnosed with uterine sarcomas (carcinosarcoma in 2 patients and leiomyosarcoma in 1).

There seems to be significant debate as to whether patients receiving tamoxifen should undergo routine evaluation of the endometrium. The current recommendations of the American College of Obstetrics and Gynecology are outlined in Table 2. However, it is not uncommon for an asymptomatic patient to consult a gynecologic oncologist following the recommendation of her medical oncologist or on her own initiative to discuss the risk of endometrial cancer in the setting of tamoxifen exposure. The evaluation of patients receiving tamoxifen is often difficult. Findings on ultrasonography alone may be misleading because tamoxifen produces an echogenic, thick, irregular, cystic appearance of the endometrial stroma and the myometrium. Fong et al, (2001) evaluated the performance characteristics of transvaginal ultrasonography and hysterosonography for the diagnosis of endometrial abnormalities in 138 asymptomatic postmenopausal patients with breast cancer who were receiving tamoxifen. The authors concluded that an endometrial thickness of 6 mm should be considered the upper limit of normal in this patient population. In addition, they suggested that hysterosonography improves specificity by reducing the false-positive rate of transvaginal ultrasonography. Others have suggested that office hysteroscopy can also serve as a conclusive diagnostic tool for the evaluation of the endometrium in this group of patients (Timmerman et al, 1998; Garuti et

IV. Discussion Earlier detection and treatment of breast cancer will likely translate into better survival. In addition, continued research in the treatment and surveillance of breast cancer will also likely lead to better survival. Gynecologic oncologists are consultants for many women who routinely inquire about their risks of gynecologic malignancies or who need to be treated for conditions that develop as a result of breast cancer treatment. A significant percentage of the patients in our study were treated with hormones, and the majority of these were treated with tamoxifen. The estrogenic effects of tamoxifen on the vaginal epithelium of postmenopausal women with breast cancer have previously been documented (Ferrazzi et al, 1977). In addition, tamoxifen has a stimulatory effect on the endometrium (Boccardo et al, 1984). In 1985, Killackey et al, were the first to report an association between tamoxifen use and the development of endometrial cancer. Tamoxifen has been shown to increase the risk of developing endometrial cancer regardless of the dose recommended (van Leeuwen et al, 1994; Fisher et al, 1998).

Table 2. American college of obstetrics and gynecology recommendations for evaluation of patients receiving tamoxifen • Women taking tamoxifen should be monitored closely for symptoms of endometrial hyperplasia or cancer and should have a gynecologic examination at least once a year • Women taking tamoxifen should be educated about the risks of endometrial proliferation, endometrial hyperplasia, and endometrial cancer. Women should be encouraged to promptly report any abnormal vaginal symptoms, including bloody discharge, spotting, staining, or leukorrhea • Any abnormal vaginal bleeding, bloody vaginal discharge, staining, or spotting should be investigated • Because screening tests have not been effective in increasing the early detection of endometrial cancer in women using tamoxifen and may lead to more invasive and costly diagnostic procedures, they are not recommended • Tamoxifen use should be limited to 5 years’ duration because a benefit beyond this time has not been documented • If atypical endometrial hyperplasia develops, appropriate gynecologic management should be instituted, and the use of tamoxifen should be reassessed. If tamoxifen therapy must be continued, hysterectomy should be considered in women with atypical endometrial hyperplasia. Tamoxifen use may be reinstituted following hysterectomy for endometrial carcinoma in consultation with the physician responsible for the woman’s breast care

Table 1. Malignant surgical pathology findings on hysterectomy (n=54) Finding Ovarian tumor Primary invasive carcinoma Metastatic breast carcinoma Uterine tumor Primary: Endometrioid Sarcoma Papillary serous carcinoma Metastatic breast carcinoma Primary invasive fallopian tube carcinoma Primary peritoneal carcinoma Primary appendiceal carcinoma

No. of patients

(%)

8 5

15 9

23 5 5 3 1

42 9 9 6 2

3 1

6 2

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Ramirez et al: Outcomes of hysteroscopy and hysterectomy in breast cancer patients al, 2002). It is important to note, however, that the same parameters used for endometrial evaluation in the general population should not be applied to postmenopausal patients who are undergoing treatment with tamoxifen (Achiron et al, 1995). In our study, we found that the majority of patients who developed endometrial carcinoma developed the more common endometrioid type. However, another group of patients developed higher risk histologic subtypes, such as papillary serous carcinoma and sarcoma. This has previously been reported in other studies. From our own institution, Silva et al, (1995) demonstrated that breast cancer patients treated with tamoxifen were at increased risk not only for endometrioid adenocarcinoma but also for other histological subtypes, such as papillary serous and clear cell carcinoma. Not only have there been cases of tamoxifen-associated endometrial cancers that are deeply invasive and of higher grade (Malfetano, 1990), but there have also been a number of reports of tamoxifenrelated uterine carcinosarcomas (Clarke, 1993; Kloos et al, 2002), as well as rare cases of stromal sarcomas, adenosarcomas, and leiomyosarcomas (Clement et al, 1996; Sabatini et al, 1999). We also found that a number of patients in our study were diagnosed with ovarian masses benign tumors, malignant primary tumors, and metastatic tumors from other primary sites. Previous reports (Cohen et al, 1994, 1996) have shown that in a select group of postmenopausal breast cancer patients treated with tamoxifen, the rate of ovarian tumors was 5.7%. This was four to five times higher than the rate of similar pathologic conditions in nonselected, asymptomatic and untreated postmenopausal women. A partial explanation of this finding may be the fact that women with breast malignancies, regardless of tamoxifen use, are more likely to develop benign or malignant ovarian tumors because of genetic factors. A small number of patients in our study also had metastatic breast cancer to the endometrium. This finding has previously been reported by Horn et al, (2000). In summary, we found that women who had undergone a mastectomy and subsequently underwent a hysteroscopy or hysterectomy often had several diagnostic studies before gynecologic surgery. In addition, these patients frequently underwent several invasive procedures before definitive treatment was performed. In our study, a significant number of patients ultimately had a malignancy diagnosed. Although most of these malignancies were diagnosed in the uterus and were the more common endometrioid type, some were of a more aggressive histologic subtype. In addition, a number of invasive ovarian carcinomas were also detected. These findings highlight the importance of prudent and expeditious evaluation in this patient population. Given these data the focus of future studies should address whether patients undergoing mastectomy should be offered a prophylactic hysterectomy and bilateral salpingo-oophorectomy at the same time. Another very important question that remains unanswered is how patients undergoing treatment with tamoxifen should be followed after diagnosis and treatment of their breast cancer. Until prospective studies

show the efficacy of screening tests or diagnostic modalities, we must continue to follow the recommendations of the American College of Obstetrics and Gynecology.

References Achiron R, Lipitz S, Sivan E, Goldenberg M, Horovitz A, Frenkel Y, Mashiach S (1995) Changes mimicking endometrial neoplasia in postmenopausal, tamoxifen-treated women with breast cancer, a transvaginal Doppler study. Ultras Obstet Gynecol 6, 116-20. Boccardo F, Guarneri D, Rubagotti A, Casertelli GL, Bentivoglio G, Conte N, Campanella G, Gaggero G, Comalli G, Zanardi S, Nicolo G (1984) Endocrine effects of tamoxifen in postmenopausal breast cancer patients. Tumori 70, 61-8. Clarke MR (1993) Uterine malignant mixed mullerian tumor in a patient on long-term tamoxifen therapy for breast cancer. Gynecol Oncol 51, 411-5. Clement PB, Oliva E, Young RH (1996) Mullerian adenosarcoma of the uterine corpus associated with tamoxifen therapy, a report of six cases and a review of tamoxifen-associated endometrial lesions. Int J Gynecol Pathol 15, 222-9. Cohen I, Altaras MM, Lew S, Tepper R, Beyth Y, Ben-Baruch G (1994) Ovarian endometrioid carcinoma and endometriosis developing in a postmenopausal breast cancer patient during tamoxifen therapy, a case report and review of the literature. Gynecol Oncol 55, 443-7. Cohen I, Beyth Y, Tepper R, Shapira J, Zalel Y, Figer A, Cordoba M, Yigael D, Altaras MM (1996) Ovarian tumors in postmenopausal breast cancer patients treated with tamoxifen. Gynecol Oncol 60, 54-8. Ferrazzi E, Cartei G, Mattarazzo R, Fiorentino M (1977) Oestrogen-like effect of tamoxifen on vaginal epithelium. BMJ 1, 1351-2. Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M, Cronin WM, Vogel V, Robidoux A, Dimitrov N, Atkins J, Daly M, Wieand S, Tan-Chiu E, Ford L, Wolmark N (1998) Tamoxifen for prevention of breast cancer, report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 90, 1371-88. Fong K, Kung R, Lytwyn A, Trudeau M, Chapman W, Nugent P, Glanc P, Manchul L, Szabunio D, Myhr T (2001) Endometrial evaluation with transvaginal US and hysterosonography in asymptomatic postmenopausal women with breast cancer receiving tamoxifen. Radiology 220, 76573. Garuti G, Cellani F, Grossi F, Colonnelli M, Centinaio G, Luerti M (2002) Saline infusion sonography and office hysteroscopy to assess endometrial morbidity associated with tamoxifen intake. Gynecol Oncol 86, 323-9. Goldstein SR (1994) Unusual ultrasonographic appearance of the uterus in patients receiving tamoxifen. Am J Obstet Gynecol 170, 447-51. Horn LC, Einenkel J, Baier D (2000) Endometrial metastasis from breast cancer in a patient receiving tamoxifen therapy. Gynecol Obstet Investig 50, 136-8. Hulka CA, Hall DA (1993) Endometrial abnormalities associated with tamoxifen therapy for breast cancer, sonographic and pathologic correlation. AJR Am J Roentgenol 160, 809-12. Killackey MA, Hakes TB, Pierce VK (1985) Endometrial adenocarcinoma in breast cancer patients receiving antiestrogens. Cancer Treat Rep 69, 237-8. Kloos I, Delaloge S, Pautier P, Di Palma M, Goupil A, Duvillard P, Cailleux PE, Lhomme C ( 2002) Tamoxifen-related uterine carcinosarcomas occur under/after prolonged treatment,

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Cancer Therapy Vol 3, page 151 report of five cases and review of the literature. Int J Gynecol Cancer 12, 496-500. Malfetano JH (1990) Tamoxifen-associated endometrial carcinoma in postmenopausal breast cancer patients. Gynecol Oncol 39, 82-4. Mourits MJ, Van der Zee AG, Willemse PH, Ten Hoor KA, Hollema H, De Vries EG (1999) Discrepancy between ultrasonography and hysteroscopy and histology of endometrium in postmenopausal breast cancer patients using tamoxifen. Gynecol Oncol 73, 21-6. Neven P, Vernaeve H (2000) Guidelines for monitoring patients taking tamoxifen treatment. Drug Safety,22, 1-11. Powles TJ, Bourne T, Athanasiou S, Chang J, Grubock K, Ashley S, Oakes L, Tidy A, Davey J, Viggers J, Humphries S, Collins W (1998) The effects of norethisterone on endometrial abnormalities identified by transvaginal ultrasound screening of healthy post-menopausal women on tamoxifen or placebo. Br J Cancer 78, 272-5. Ries LAG, Eisner MP, Kosary CL, Hankey BF, Miller BA, Clegg L, Mariotto A, Fay MP, Feuer EJ, Edwards BK editors. SEER Cancer Statistics Review, 1975-2000. National Cancer Institute. Bethesda, MD, 2003.http,

//seer.cancer.gov/csr/1975 2000) Sabatini R, Di Fazio F, Loizzi P (1999) Uterine leiomyosarcoma in a postmenopausal woman treated with tamoxifen, case report. Eur J Gynaecol Oncol 20, 327-8. Silva EG, Tornos C, Malpica A, Mitchell MF (1995) Uterine neoplasms in patients treated with tamoxifen. J Cell Biochem 3, 179-83. Timmerman D, Deprest J, Bourne T, Van den Berghe I, Collins WP, Vergote I (1998) A randomized trial on the use of ultrasonography or office hysteroscopy for endometrial assessment in postmenopausal patients with breast cancer who were treated with tamoxifen. Am J Obstet Gynecol 179, 62-70. van Leeuwen FE, Benraadt J, Coebergh JW, Kiemeney LA, Gimbrere CH, Otter R, Schouten LJ, Damhuis RA, Bontenbal M, Diepenhorst FW, et al (1994) Risk of endometrial cancer after tamoxifen treatment of breast cancer. Lancet 343, 448-52.

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Cancer Therapy Vol 3, page 153 Cancer Therapy Vol 3, 153-158, 2005

Ovarian mixed germ cell tumor presenting as tuberculosis Case Report

Fatemeh Ghaemmaghami*, Azam Sadat Moosavi, Malihe Hasanzadeh Gynecologist Oncologist, Tehran University of Medical Sciences

__________________________________________________________________________________ *Correspondence: Fatemeh Ghaemmaghami, Associate Professor, Gynecologist Oncologist, Tehran University of Medical Sciences. Address: Gynecology Oncology Department, Vali-e-Asr Hospital, Imam Khomeini Hospital Complex, Keshavarz Blvd., Tehran 14194, Iran. Phone: #98-21-6939320, Fax: #98-21-6937321, E-mail: valrec2@yahoo.com, ftghaemmagh@yahoo.com Key words: germ cell tumor, GTN, Tuberculosis, Endodermal sinus tumor, tumor marker, EMA-EP, MEP Abbreviations: Received: 17 January 2005; Accepted: 28 January 2005; electronically published: March 2005

Summary To introduce a patient with rare ovarian mixed germ cell tumor who presented as miliary tuberculosis.Patient was a 25 year old Afghani woman who had admitted with complains of fever, dyspenea, abdominal pain. She had a fixed pelvic mass in physical examination, abnormal findings in auscultation of the lungs, and chest X-ray.We suggested miliary tuberculosis at the first time due to socio economic nature and chest X-ray appearance. We found positive pregnancy test and echogenic mass with ultrasound in paraclinic work up. We suggested high risk GTN due to pulmonary manifestation (dyspenea and chest X-ray appearance) at the second time. So she received EMA-EP regimen.She found acute abdomen cause to hemorrhage, when she had received EMA part. Mixed germ cell tumor was confirmed by histological examination after laparatomy and removing tumoral mass. Then she received MEP regimen four courses and lived being time. In women of reproductive age who has pulmonary symptoms, GTN and mixed germ cell tumor with choriocarcinoma element should be considered. Approximately 10% of patients presents with an acute abdomen secondary to intracapsular hemorrhage, torsion and/or rupture (Hurteau and Williams, 2001). Ovarian germ cell tumors present at a relatively early stage; stage I (75%); and only few cases present stage IV (5%) (Disaia and Creasman, 2002). Mixed germ cell tumors may secret either AFP, HCG, both or neither depending on components of tumor (Hurteau and Williams, 2001). There are several case reports about peritoneal tuberculosis mimicking advanced ovarian cancer (Straughn et al, 2000; Bilgin et al, 2001; Protopapas et al, 2003). In contrast, ovarian cancer which presented as tuberculosis may be rare.

I. Introduction Mixed germ cell ovarian tumors represent a relatively small proportion of all ovarian tumors (Disaia and Creasman, 2002). Since germ cell tumors of the ovary consist fewer than 5% of ovarian cancer and mixed germ cell tumors accounting for approximately 19% of all cases (Hurteau and Williams, 2001). These ovarian tumors accounts for about 1% of ovarian malignancies. Mixed germ cell tumors contain at least, two components of malignant germ cell tumors (Disaia and Creasman, 2002). In a case series, the most common components of such tumors were reported as; dysgerminoma (80%), endodermal sinus tumor (70%), immature teratoma (%53), choriocarcinoma (20%), and embryonal carcinoma (16%). The most common combination was dysgerminoma and EST (Gershenson et al, 1984). The most presenting symptom in ovarian germ cell tumors is abdominal pain with or without pelvic pain that could be seen in %75 of cases (Disaia and Creasman, 2002).

II. Case A 25 year-old, Gravid 3, Parity 3 Afghani woman was referred to Vali-e-Asr hospital in October 2003 with a history of abdominal pain, low grade fever during 3 months ago. In physical examination a fixed mass in lower abdomen which extends upper the hilus was revealed. In pelvic examination, uterus and ovary could not be distinguished separately and a fixed pelvic mass could be 153


Ghaemmaghami et al: Ovarian mixed germ cell tumor presenting as tuberculosis palpated. Temperature at admission and during hospitalization was about 38°C, in auscultation of the lungs, abnormal sounds could be heard. In urgent chest X-ray showed multiple nodular lesions suggested metastasis lesions, but pulmonary tuberculosis could not rule out by radiologist (Figure 1). In abdominal ultrasound examination a huge mass was revealed. Abdominal and pelvic CT-scan showed abdominal heterogeneous mass but it was not possible distinct uterus and ovaries (Figure 2).

Miliary tuberculosis with pulmonary and peritoneal involvement was suggested due to these signs in Afghani lady at the first time. We found positive pregnancy test in routine laboratory examination. We requested sputum samples, blood cultures, tuberculin test for Acid-Fast bacilli which were negative. Serum tumor markers measurement were done, !â&#x20AC;&#x201C;hCG=815000 mIU/ml, but results of other tumor markers, CA-125 and AFP took about one week to be ready. Chest CT-scan showed multiple metastatic lesions (Figure 3).

Figure 1. Chest X-ray with multiple nodular lesions.

Figure 2. Chest CT-scan with multiple metastatic lesions.

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Figure 3. Abdominal and pelvic CT-scan with heterogeneous mass.

We suggest choriocarcinoma with pulmonary involvement and stage III, score 13, due to sever pulmonary symptoms, dyspenea, and elevated !–hCG. So we considered combined chemotherapy with EMA-EP. When she had received part of EMA, other tumor markers measurement that was taken before chemotherapy was ready as follows (CA-125= 54 IU/ml, AFP=2650 ng/ml), so we suggested mixed germ cell tumors with choriocarcinoma thirdly and we decided to laparatomy so we scatuleated for elective surgery. But she had acute abdomen, urgent laparatomy was performed. A large cystic and solid mass weighted 5000gr arising from right ovary was found, it was firmly adhered to the sigmoid colon. Tumoral mass was removed intact without rupture. Mixed germ call tumor with components of Endodermal sinus tumor and choriocanainoma was reported by histological examination (Figures 4, 5). We considered BEP regimen chemotherapy in patients with germ cell tumors. Patient could not receive Bleomycin due to abnormal pulmonary function test. We considered MEP regimen (Methotrexate, Etoposide, and Cisplatin). Patient received four courses of MEP (once every three weeks) 7 days after surgery. The MEP regimen was given to the patient as follows: Methotrexate Etoposide Cisplatin

40 mg/m2 100 mg/m2 20 mg/m2

III. Discussion Different variables make report of this case an important issue. First, it was a rare case of mixed germ cell tumors which presented at stage IV. Second, it was presenting as miliary tuberculosis due to pulmonary symptoms, signs and fever. Third, it was mimicking to GTN stage III after paraclinic work up due to positive pregnancy test and chest X-ray appearance. Cases of mixed choriocarcinoma and Endodermal sinus tumor are rare and reported just in a few cases reports (Disaia and Creasman, 2002). Also stage IV of germ cell tumors are just in 5% of cases (Disaia and Creasman, 2002). Many points are mimicking this case as miliary tuberculosis. First, a fever which is prevalent in three categories; infections, malignancies, and collagen diseases (Zamir et al, 2003). Second, It is estimated that 8 million new cases of TB occurred world wide in 1997 that 95 percent of them were occurred in developing countries; Asia (5 million), Latin American (0.4 million), the Middle East (0.6 million) (Braunwald et al,2001). It means that in developing countries, tuberculosis remains endemic and present with non-specific symptoms and signs such as pelvic and abdominal pain, mass, ascites and hence mimic ovarian cancer (Straughn et al, 2000; Bilgin et al, 2001; Protopapas et al, 2003). And the last point is that respiratory symptoms, signs and radiological patterns of the lungs in choriocarcinoma are similar to primary pulmonary disease (Hurteau and Williams, 2001). There are many reports of peritoneal tuberculosis mimicking as advanced ovarian cancer. In contrast, we could not find any report that ovarian cancer presented as peritoneal tuberculosis. This patient may be the first case in this matter. Such a mimicking is due to Afghani patient who had fever and pulmonary manifestations.

IV or IM day 1 q 3 weeks IV daily 1-5 q 3 weeks IV daily 1-5 q 3 weeks

After 2 courses of chemotherapy AFP and CA-125 were both decreased to normal measurement. After 3 courses of chemotherapy !–hCG was normal (!–hCG<5). Last course of chemotherapy was received by the patient after negative !–hCG and now about 9 months after surgery, she is under observation and follow up.

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Ghaemmaghami et al: Ovarian mixed germ cell tumor presenting as tuberculosis

Figure 4. elements of Endodermal sinus tumor where are reticular pattern and extra cellular and intra cellular hyaline droplets were detected.

Figure 5. Elements of choriocarcinoma where necrosis and hemorrhage and biphasic neoplastic cell was detected.

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Cancer Therapy Vol 3, page 157 Mixed germ cell tumors containing Endodermal sinus tumor elements have elevated serum AFP levels, ranging from >100 to far higher than 1000ng/ml. The titer of serum AFP in this case was higher than common range (Aoki et al, 2003). The presence or absence of choriocarcinoma is considered to influence overall survival or clinical outcome of the patients with mixed germ cell tumor. Several cases of mixed germ cell tumor were reported with metastasis foci composed of pure choriocarcinoma (Peccatori et al, 1995). EMA-EP regimen as first line chemotherapy in management of high risk GTN has showed good response (Ghaemmaghami et al, 2004a, b). But Cisplatin containing combination chemotherapy such as Bleomycin, Etoposide and cisplatin (BEP) chemotherapy recommend in germ cell tumors. Usually 3 to 4 courses of chemotherapy should be performed .In mixed germ cell tumors another additional courses, after negative results of tumor markers, should be considered (Berek and Hacker, 2000). The prognosis of patients with mixed germ cell tumor can be poor because of the presence of a choriocarcinoma element and these patients to require more aggressive chemotherapy (Peccatori et al, 1995). Methotrexate is the best chemotherapic agent in management of choriocarcinoma. Since MEP (Methotrexate, Etoposide, Cisplatin) may be good regimen in mixed germ cell tumor with choriocarcinoma elements which BEP regimen is not justify due to pulmonary dysfunction. In conclusion, although the diagnosis of gestational trophoblastic neoplasia should be considered in any woman of reproductive age who has pulmonary symptoms, mixed germ cell tumor also should be consider especially when she has pelvic mass. Combined chemotherapy with MEP regimen may be good regimen in mixed germ cell tumor with chorionic elements.

References Aoki Y, Kase H, Fujita K (2003) Dysgerminoma with a slightly elevated alpha-fetoprotein level diagnosed as a mixed germ cell tumor after recurrence. Gynecol Obstet Invest 55, 58-9. Berek JS, Hacker NF (2000) Non epithelial ovarian and fallopian tube cancers Chapter 12, in Practical Gynecologic Oncology, third edition. Lippinkot Williams and Wilkins, 523-551. Bilgin T, Karabay A, Dolar E et al (2001) Peritoneal tuberculosis with pelvic abdominal mass, ascites and elevated CA-125 mimicking advanced ovarian carcinoma, A series of 10 cases. Int J Gynecol Cancer 11, 290-299. Braunwald E,Hauser S,Fauci A et al: Principals of internal medicin,15 th edition, New York McGraw-hill 2001;1:10241025. Disaia PJ, Creasman WT (2002) Germ cell, stromal and other ovarian tumors. In: DiSaia PJ, Creasman WT, editors. Clinical Gynecologic Oncology, 5th ed. St Louis: Mosby Year Book ,351-74 Gershenson DM, Deljunco G, Copelaned LJ, Ruthledge FN (1984) Mixed germ cell tumors of the ovary. Obstet Gynecol 64, 200-206. Ghaemmaghami F, Behtash N, Soleimani K, Hanjani P (2004) Management of patients with metastasic GTN. Gynecol Oncol (in press). Ghaemmaghami F, Modares M, Arab M et al (2004) EMA-EP regimen, as first line of multiple agent chemotherapy in high risk GTN patient (stage II-IV). Int J Gynecol Cancer 14, 360-365. Hurteau JA and Williams JS (2001) Ovarian germ cell tumors chapter 20 in ovarian cancers, Book Edited by Steephen C, Rubin. G and Sutton P. Lippincott Williams & Wilkins. Peccatori F, Bonzzi C, Chiari S et al (1995) Surgical management of malignant ovarian germ cell tumors, 10 years experience of 12 patients. Obstet Gynecol. 86, 367-377. Protopapas A, Milingos S, Diakomanolis E et al (2003) Miliary tuberculosis peritonitis mimicking advanced ovarian cancer. Gynecol Obstet Invest 56, 89-92. Straughn JM, Robertson MW, Partridge E (2000) A patient presenting with a pelvic mass, Elevated CA-125, and fever, a case report. Gynecol Oncology 77, 471-472. Zamir D, Leibovitz I, Polychuck I et al (2003) Fever of unknown origin. Acta Clin Belg 58, 356-9.

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Cancer Therapy Vol 3, page 159 Cancer Therapy Vol 3, 159-166, 2005

Role of somatostatin analogues in the treatment of androgen ablation-refractory prostate adenocarcinoma Review Article

Alessandro Sciarra*, Gianna Mariotti, Anna Maria Autran Gomez, Franco Di Silverio Department of Urology, University La Sapienza, Rome, Italy

__________________________________________________________________________________ *Correspondence: Dr. Sciarra Alessandro, Department of Urology, University La Sapienza, Via Nomentana 233, 00161 Rome, Italy; Tel./Fax +39 6 44 6959 ; e-mail: sciarrajr@hotmail.com Key words: prostate neoplasms, somatostatina analogues, neuroendocrine Abbreviations: antisurvival, (ASF); chromogranin A, (CgA); combined androgen blockade, (CAB); computerized tomography scan, (CT); Growth Hormone, (GH); insuline-like growth factor, (IGF); neuroendocrine, (NE); progression â&#x20AC;&#x201C;free survival, (PFS); prostate specific antigen, (PSA); somatostatin receptors, (SSTR); Received: 15 February 2005; Accepted: 9 March 2005; electronically published: March 2005

Summary The progression to androgen ablation-refractory stage (D3) of prostate cancer corresponds to cancer cell escape from androgen withdrawal-induced apoptosis. Of note, salvage chemotherapy cannot extend the median survival of approximately 10 months for stage D3 patients. Novel therapeutic strategies that target the molecular basis of androgen resistance are therefore required. We reviewed the literature on the use of somatostatin analogues in the treatment of D3 prostate adenocarcinoma and we analysed the rationale and clinical results of our combination therapy using lanreotide and ethinylestradiol. Negative experiences have been reported on the use of somatostatin analogues in monotherapy. On the other hand, interesting results have been obtained as combination therapy and the median progression free survival reported in our experience using lanreotide acetate plus ethinylestradiol, clearly surpassed the 10 months survival historically described for stage D3 patients. The use of somatostatin analogues in combination therapy for the treatment of D3 prostate cancer, sustains the novel concept in cancer treatment in which therapies may target not only cancer cell itself but, in combination, also its microenvironment, which can confer protection from apoptosis.

on the use of somatostatin analogues in the treatment of prostate adenocarcinoma and to analyse the rationale and clinical results of our combination therapy.

I. Introduction The progression to androgen ablation-refractory stage (D3) of prostate cancer corresponds to cancer cell escape from androgen withdrawal-induced apoptosis (Landstrom et al, 1994). Of note, salvage chemotherapy can extend the median survival of approximately 10 months for stage D3 patients (Koutsilieris et al, 1990; Hudes et al, 1992). Novel therapeutic strategies that target the molecular basis of androgen resistance are therefore required. We previously proposed a combination therapy of ethinylestradiol and somatostatin analogue to reintroduce objective clinical responses in metastatic androgen ablation-refractory prostate cancer patients (Di Silverio and Sciarra, 2003). The purpose of this article is to review the literature

II. Somatostatin monotherapy

analogues

in

Native somatostatin is characterised as an inhibitory peptide with exocrine, endocrine and autocrine activity (Newman et al, 1987). The general inhibitory function of somatostatin is wide ranging and affects a number of organ systems.The effect of somatostatin on various organ systems are thought to be mediated via specific somatostatin receptors (SSTR). To date, five different subtypes (SSTR 1-5) have been identified and cloned in human tissue (Hejna et al, 2002). While all five subtypes

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Sciarra et al: Somatostatin analogues in f androgen ablation-refractory prostate adenocarcinoma display a similar affinity to somatostatin, there are major differences in binding of currently available somatostatin analogues (Pollak and Schally, 1998) to various SSTR subtypes. Investigations concerning the exact intracellular mechanisms effected by different SSTRs with regards to cellular proliferation and induction of apoptosis are ongoing. Recently, SSTR subtype expression has been characterized in various neoplastic tissues (Reubi et al, 2001). There appears to be a predominance of only one or two SSTR subtypes in most tumors investigated. There is a clear predominance of SSTR 1 expression in prostate cancers, which may also express SSTR 5. The highly SSTR 2 affine octapeptide somatostatin analogues such as octreotide remains the drugs of choice for application in a majority of pure neuroendocrine tumors, since such tumors most often express predominantly SSTR 2 (Pollak and Schally, 1998). However, other somatostatin derivates, such as lanreotide, which have a good affinity of SSTR 5 in addition to that for SSTR 2, may advantageously identify SSTR 5 expressing tumors, such as prostate adenocarcinoma. Somatostatin analogues, however, seem to interact at tissue level, also through a receptor binding independent mechanism (Hejna et al, 2002). Long-acting somatostatin analogues have been developed, specifically designed for antitumor activity. Schally, (1988) synthesised > 300 analogues using solidphase methods resulted in octapeptide “super analogues”, which are more potent and have longer durations of action than either native somatostatin and octreotide.

Several clinical trials have demonstrated impressive efficacy of somatostatin analogues in a variety of hypersecretory disorders resistant to standard therapy (Hejna et al, 2002). They have also proved useful for the management of symptoms caused by neuroendocrine diseases. The primary effect of somatostatin analogues is not a direct cytotoxic effect of neuroendocrine cells but the inhibition of the release of various peptides hormones secreted by neuroendocrine cells (Hejna et al, 2002). The observation that somatostatin analogues inhibit the release of various neuroendocrine products has stimulated interest in its use as an antiproliferative and proapoptotic agent. Antiproliferative and proapoptotic actions of somatostatin analogues have been demonstrated in various tumor models including breast, prostate, colon, pancreatic (Murphy et al, 1987; Schally, 1988; Smith and Solomon, 1988). Moreover, somatostatin analogues have a wide therapeutic index and are apparently free of major side effects (Schally, 1988). Most of the reported side-effects are gastrointestinal in nature and include minor nausea, diarrhoea, constipation. Clinical trials and experiences on the use of somatostatin analogues as monotherapy for the treatment of prostate cancer, reported negative results (Table 1) (Carteni et al, 1990; Dupont et al, 1990; Logothetis et al, 1994; Verhelst et al, 1994; Figg et al, 1995; Maulard et al, 1995; Vainas et al, 1997; Koutsilieris et al, 2001; Di Silverio and Sciarra, 2003). Octreotide was used to treat patients with advanced hormonal-refractory prostate cancer in a study by Logothetis et al, (1994).

Table 1. Hormone-refractory prostate cancer: clinical experiences with somatostatin analogues Treatment

Dosage

Number cases

Results

Reference

Octreotide

100µg tds s.c.

7

Pain reduction

Carteni et al, 1990

Octreotide

600-1350µg/day s.c.

10

Disease progression after 21 days

Dupont et al, 1990

Octreotide

400-1000µg/day s.c.

5

Temporary halt in PSA rising

Verhelst (28)

Octreotide

100mg qds s.c.

22

Stimulation of prostate tumor growth

Logothetis et al, 1994

Lanreotide

30mg once a week i.m.

30

20% partial response (PSA decrease)

Maulard et al, 1995

40% improvement performance status Lanreotide

4-24 mg/day s.c.

25

No modifications

Figg et al, 1995

Octreotide

Not clarified

14

Symptom-free responses

Vainas et al, 1997

30 mg/14 days i.m. + 4 mg/day os

11

90% objective (PSA decrease) and symptomatic response

Koutsilieris 2001

Lanreotide dexamethasone

plus

et

al,

Progression-free survival =7 months Lanreotide acetate ethinylestradiol

plus

73.9 mg i.m. every 4 weeks + 1 mg/day os

10

90% objective (PSA decrease) and symptomatic response Progression-free survival = 18.5 months

160

Di Silverio Sciarra, 2003

and


Cancer Therapy Vol 3, page 161 The dose of octreotide applied to 24 cases was 0.1 mg s.c. every 8 h for 6 weeks. No patients had objective evidence of tumor regression and in 10 cases serum prostate acid phospatase level rose at an accelerated rate after 1-2 months of treatment. However, 6 patients underwent salvage chemotherapy after octreotide therapy and 5 of whom achieved objective tumor regression. The authors therefore concluded that octreotide monotherapy might stimulate prostatic tumor growth but may also sensitise tumor cells to subsequent chemotherapy. A total of 30 patients with hormone-refractory prostate cancer were treated with a slow-release formulation of lanreotide (30 mg i.m. once a week) by Maulard et al, (1995). Toxicity related to the treatment was minor, performance status and bone pain improved in 40% and 35% of patient respectively, but a PSA decrease by at least 50% was reported only in 20% of cases.

mechanisms (Sciarra et al, 2003a). These mechanisms result from the interaction of the tumor cells with the local microenvironment, either at local or metastatic sites. Major mediators of this interaction are neuropeptides secreted by neuroendocrine (NE) cells in prostate tissue and insuline-like growth factor (IGF) â&#x20AC;&#x201C;1. The locally bioavailability of these peptides and growth factors on prostate cancer cells, activates anti-apoptotic mechanisms more than proliferative direct effects. These represent real survival pathways involved in prostate cancer progression and androgen-deprivation therapy resistancy. The development of survival factor-mediated resistance to anticancer therapies is a major hurdle preventing longlasting clinical responses to conventional or investigational therapies (Koutsilieris et al, 2002). This realisation has led to the novel concept of antisurvival (ASF) therapy for prostate cancer as a component of anticancer treatments and to the concept of a combination therapy for hormone-refractory disease.This approach is novel as instead of attempting to directly induce cancer cell apoptosis, it aims at neutralising the protective effect conferred upon cancer cells by the survival factors. This neutralisation alone may not induce apoptosis, but it can enhance the sensitivity or reverse the resistance of tumors cells to other anticancer strategies with direct cytotoxic effects (Reyes-Moreno et al, 1998; Koutsilieris et al, 2000, 2002). On these basis, Koutisileris et al, (2001) firstly proposed a combination therapy with dexamethasone and long acting somatostatin analogue in stage D3 prostate cancer patients, i.e. patients with metastatic prostate cancer who had become refractory to combined androgen blockade. In this setting, Growth Hormone (GH) â&#x20AC;&#x201C; independent and GH-dependent production of IGF-1 has been implicated in the development of a epigenetic form of cancer cells resistance to pro-apoptotic therapies. Among its diverse pharmacological effects, dexamethasone acts to downregulate the GH-independent production of IGF-1, whereas somatostatin analogue suppresses the level of GH-dependent IGF-1. This paradigm of an ASF therapy, which was practically an anti-IGF-1 therapy, yielded objective responses and major improvement of bone pain and performance status in D3 cases. The treatment schedule includes administration of oral dexamethasone plus long acting somatostatin analogue (lanreotide or octreotide in i.m. injections) in combination with androgen ablation therapy.In the initial cohort of patients receiving the combination therapy the median overall survival clearly surpassed 12 months and also their post-relapse performance status and bone pain were still significantly improved compared to their baseline status, even months after relapse. The stimulating feature of this ASF approach is that its combination with LHRH-analogues can reintroduce clinical responsiveness to LHRH analogues.

III. Somatostatin analogues in combination therapy: it exist a rationale The mechanism of action of somatostatin analogues may suggest the use of these drugs not in monotherapy but in combination therapy for tumors such as prostate cancer. Also in breast cancer favourable results have been obtained by the use of somatostatin analogues in combination therapy. Twenty-two post-menopausal patients with metastatic breast cancer were randomised to receive either 40mg/day of tamoxifen or a combination consiting of 40 mg tamoxifen plus 0.2 mg of octreotide tds s.c. (Bontenbal et al, 1998). An objective response was found in 36% of the patients treated with tamoxifen alone and in 55% of patients treated with the combination therapy. The management of metastatic neoplasias has traditionally relied on therapeutic modalities, which almost exclusively aim at directly inducing cancer cell death. However, the in vivo response of malignant cells to anticancer therapies is directly influenced by the local microenvironment in which they reside (or metastasise) (Koutsilieris et al, 2002).Microenvironment factors may attenuate the antitumor activity of several cytotoxic agents on neopalstic cells.In particular, organ sites frequently involved in metastatic advanced diseases, appear to confer to neoplastic cells protection from anticancer drug-induced apoptosis. This protection may be mediated by several mechanisms including growth factors cytokines released by the normal cellular constituents of the host-tissue microenvironment (Koutsilieris et al, 2000). Therefore additional emphasis should be placed on the design of novel treatments that can neutralise the protection that the microenvironment offers to tumors cells. An example of the role of the microenvironment in protecting tumor cells from anticancer therapies is within the setting of hormonerefractory prostate cancer. For years, it has been a widely accepted notion that resistance to hormonal therapy is an outcome exclusively determined at the genetic level and involving mutations that neutralise pro-apoptotic intracellular pathways and/or activates anti-apoptotic ones (Koutsilieris et al, 2002). It is now well documented that this resistance can also be conferred by epigenetic

IV. Somatostatin analogues combination with estrogens

in

We previously analysed (Di Silverio and Sciarra, 2003) for the first time in the literature, whether, in

161


Sciarra et al: Somatostatin analogues in f androgen ablation-refractory prostate adenocarcinoma patients with stage D3 prostate cancer, the combination of ethinylestradiol and lanreotide can offer objective responses and/or symptomatic improvements. We followed the study design used by Koutsilieris et al, (2001). As Koutsilieris et al, (2001) we evaluated patients with metastatic androgen ablation-refractory prostate cancer. However, differently to Koutsilieris et al, we discontinued the LHRH analogue and we started a combination therapy with ethinylestradiol and lanreotide acetate. The rationale for our combination therapy is: 1- to inhibit the protective (antiapoptotic) effect of NE system on prostate adenocarcinoma cells (somatostatin analogue); 2- to use a new mechanism to induce castration (estrogen); 3- to add a direct cytotoxic effect on prostate cells (estrogen) (Figure 1). Some studies have shown that the number of NE tumor cells (25-25) and chromogranin A (CgA) serum levels increase during hormonal therapy (Abrahamsson, 1996; Angelsen et al, 1997; Monti et al, 2000; Sciarra et al, 2003a, b) for prostate adenocarcinoma. As previously underlined, at the cellular level, refractoriness to androgen ablation therapy occurs principally because prostate cancer cells can be rescued from androgen ablation-induced apoptosis. It has been shown that Bcl2 proto-oncogen, which is an antiapoptotic factor, is preferentially expressed in foci of prostate adenocarcinoma cells in the vicinity of NE differentiation (Segal and Cohen, 1994; Jongsma et al, 2000). In hormone- refractory (D3) prostate cancer, NE cells may protect prostate adenocarcinoma cells from anticancer therapies through the neutralization of pro-apoptotic intracellular pathways. The rationale for somatostatin-analogue therapy in D3 prostate tumor is not to directly induce cancer cell apoptosis but to neutralize the protective effect conferred upon cancer cells by the survival factors derived by NE prostate cells. The antigonadotropic effect of estrogens has been exploited therapeutically. Both experimental and clinical evidence suggest that estrogen therapy may be superior to

castration in terms of efficacy for the treatment of advanced prostate cancer (Robinson et al, 1995; Chang et al, 1996; Rosenbaum et al, 2000; Smith et al, 2000). Moreover, analysing prostatectomy specimens of untreated and treated (CAB) prostate cancer patients, Kruithof-Dekker et al, (1996) showed that androgen deprivation leads to an upregulation of estrogen receptor expression in prostate cancer tissue. It has been supposed that the beneficial effect of estrogens is based not only on reduction of the androgen concentration but also on a simultaneous direct cytotoxic effect (Hudes et al, 1992) on prostate cancer cells. All these data support our rationale: to discontinue LHRH-analogue and to substitute it with estrogen therapy. An important question is whether responses achieved in our study most likely constitute an indirect evidence of a potential survival benefit offered by the combination therapy rather than a response to lanreotide only or ethinylestradiol only. As previously showed, in advanced hormonalrefractory prostate cancer, negative experiences have been reported on the use of somatostatin analogues in monotherapy (Carteni et al, 1990; Dupont et al, 1990; Logothetis et al, 1994; Verhelst et al, 1994; Figg et al, 1995; Maulard et al, 1995; Vainas et al, 1997). On the other hand, the median progression free survival reported in our study clearly surpassed the 10 months survival historically described for stage D3 patients, even when estrogen therapy or salvage chemotherapy is administered (Hudes et al, 1992; Chang et al, 1996). However, additional studies will be required to fully elucidate the precise in vivo mechanism of action for the combination of estrogens with somatostatin analogues. As for the study of Koutsilieris et al, (2001) the design of our pilot trial involved a longitudinal methodology, as defined by Spilker (1991), which is appropriate for study of even small cohorts of patients. In our first experience (Di Silverio and Sciarra, 2003), we prospectively evaluated 10 consecutive patients with stage D3 disease, who received a combination therapy

Figure 1. Rationale for our combination therapy with somatostatin analogue and estrogen

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Cancer Therapy Vol 3, page 163 consisting of the following: 1)oral ethinylestradiol (1mg daily); 2) lanreotide (lanreotide acetate 73.9 mg in every 4 weeks). None of these cases had a history of severe cardiovascular diseases, neither a history of other disorders or therapies or conditions known to interfere with CgA levels. All patients had diffuse skeletal metastases (> 3 metastatic foci) documented by radionuclide bone scan and computerized tomography scan (CT). All patients had previously experienced objective clinical responses to combined androgen blockade (CAB) using triptorelin plus antiandrogen (flutamide, bicalutamide) documented by prostate specific antigen (PSA) decline by more than 50% of baseline, which had lasted for less than 24 months. Upon progression, all patients were withdrawan from antiandrogens for at least 6 weeks (no patients responded). Therefore, all patients discontinued CAB and received the combination therapy ethinylestration plus lanreotide (Figure 2). In this first experience, 90% (95%CI =55.599.8%) of cases had objective (complete= PSA < 4 ng/ml, or partial= at least 50% PSA decrease from baseline) clinical response to the combination therapy, corresponding to a statistically significant (in comparison to the baseline refractoriness) rate of re-introduction of responsiveness to the combination with lanreotide and ethinylestradiol (McNemar’s paired !2 test; p<0.01). In responders, median time to PSA nadir was 5 months (range 3-12 months; 95% CI 4-8 months). In all cases, the PSA responses were accompanied by concomitant statistically significant reduction in bone pain score (p=<0.0001), as well as significant improvement in the ECOG performance status score (p<0.0001). The symptomatic improvement of pain and performance status appeared to be temporally associated with the changes in objective response markers and it is suggested that the main mechanism of action of this combination therapy

affects those mechanisms regulating the growth and/or survival of the metastatic cells, rather than involving a non-specific anti-inflammatory or analgesic effect (Koutsilieris et al, 2001). The rates and the time to achieve the symptomatic and objective responses that we described, are comparable to those reported in the study of Koutsilieris et al, (2001). However, with our combination therapy we obtained a longer duration of objective responses. In particular, median duration of bone pain response, ECOG response and progression –free survival (PFS) was 17.5 (95% CI 12-19), 18 (95% CI 12-19) and 18.5 (95% CI 14-21) months respectively in our study and 13 (95% CI 12-14), 19 (95% CI 13-25) and 7 (95% CI 310) months in the study of Koutsilieris et al, (2001) We analysed modifications in serum CgA levels during the combination therapy. Comparison of serum CgA levels at baseline, during follow-up, at maximal response and at relapse from therapy revealed a significant change of CgA levels during the course of the combination therapy (P<0.0001; Friedman’s nonparametric ANOVA). We observed a significant decrease in serum CgA levels during the administration of the combination therapy (median value of maximal CgA decline = 38.4% of baseline levels; 95%CI 33.2-50.3% range 28.6%-64.9%), as compared with the baseline CgA levels. In our patients, time to CgA nadir was lower than time to PSA nadir; therefore it seems that CgA response preceded PSA response. Our baseline levels of CgA were similar to those reported in other experiences on metastatic prostate cancer cases (Jongsma et al, 2000; Sciarra et al, 2003b). The significant reduction of circulating CgA, documented in this cohort of patients suggests that a reduction of NE activity on prostate cancer cells may be a mechanism accounting for at least part of the encouraging responses that were observed. Interestingly, the patients’serum CgA levels were not significantly increased

Figure 2. Study design for our trial (4) on lanreotide acetate and ethinylestradiol combination therapy in D3 prostate cancer cases.

163


Sciarra et al: Somatostatin analogues in f androgen ablation-refractory prostate adenocarcinoma at relapse suggesting that NE activity may be not involved at relapse from this combination therapy. The modifications in CgA levels reported in our study are lower if compared with those observed in pathologically confirmed NE tumors such as small cell carcinoma of the lung. However, we must remember that NE differentiation of prostate adenocarcinoma consists of the presence of NE cells with a focal distribution in the common prostatic adenocarcinoma (Jongsma et al, 2000; Sciarra et al, 2003b). A limit of our analysis may be the determination of only serum expression of CgA. However, none of our cases presented a history of other disorders known to interfere with CgA levels. Some authors reported a significant correlation between serum and tissue expression of CgA in prostate cancer (Jongsma et al, 2000; Sciarra et al, 2003b). Moreover, in 8 cases we had the opportunity to analyse CgA expression at prostate tissue level by immunohistochemistry. Prostate tissue specimens were obtained by transrectal ultrasound-guided prostate biopsy and formalin fixed and paraffin embedded prostate specimens were sectioned to 5Âľm thick prior to the analysis. Diffuse immunohistochemical staining for CgA was found in biopsies obtained in D3 cases at baseline from our therapy. On the contrary a limited and focal staining for CgA was showed in cases with objective clinical response to our combination therapy. No major treatment related side effects were reported during the combination therapy. In particular no serious cardiovascular renal, or liver-gastrointestinal events were found during the follow-up, with the exception of transient mild epigastric discomfort, effectively controlled with antacid regimen. None of our 10 cases discontinued the treatment due to side effects related to the combination therapy. All cases developed gynecomastia and mild breast pain. It is true that none of our cases had a history of severe cardiovascular diseases at baseline, but the dose (1 mg) of ethinylestradiol and the duration of follow-up (no longer than 24 months) may also contribute for differences with other experiences on estrogen therapy (Robinson et al, 1995; Rosenbaum et al, 2000; Smith et al, 2000). At January 2004, 20 D3 cases have been included in our analysis and submitted to the combination therapy with ethinylestradiol and lanreotide (Sciarra et al, 2004). Criteria for inclusion and study protocol were similar to those previously described (Di Silverio and Sciarra, 2003). Results continue to be encouraging and supporting the rational for our combination therapy.In particular, at January 2004 19 out of the 20 cases (95%) showed objective (complete = 5 cases (25%) or partial = 14 cases (70%)) clinical response to the combination therapy demonstrated by at least 50% PSA decrease from baseline. In only one case the biochemical response was accompanied by a reduction in the number of bone metastases at bone scan. Two out of the 20 patients (10%) died, both of prostate cancer (at 10 and 16 months respectively) and 6 cases (30%) developed clinical progression (rising PSA levels to more than 50% of PSA nadir) (mean of 7.8 months, median of 7 months, range 412 months) during the follow-up. All other 14 patients (70%) are still alive without disease progression after a

median of 16.5 months (mean of 13.9 months, range 4-24 months) of follow-up during the combination therapy (Sciarra et al, 2004).

V. Conclusions It should be emphasized that any conclusion regarding the usefulness of this combination therapy, in comparison to other proposed treatment strategies for stage D3 prostate cancer, can only be drawn in randomised controlled clinical trials. The results of our study indicate that such randomised trials are warranted, because the combination of ethinylestradiol and lanreotide had a favourable toxicity profile, offered objective and symptomatic responses in patients with limited treatment options and refractoriness to conventional hormonal therapy strategies and in particular, offered a median overall survival that was superior to the 10 -month median survival for hormone-refractory patients. This combination therapy also sustains the novel concept in cancer treatment in which therapies may target not only cancer cell itself but, in combination, also its microenvironment, which can confer protection from apoptosis.

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Cancer Therapy Vol 3, page 165 Jongsma J, Oomen MHA, Noordzij MA, Romijn JC, Van Der Kwast TH, Schroder FH et al (2000) Androgen independent growth is induced by neuropeptides in human prostate cancer cell lines. Prostate 42, 34-44. Koutsilieris M, Laroche B, Thabet M, Fradet Y (1990) The assessment of disease aggressivity in stage D2 prostate cancer patients. Anticancer Res 10, 333-336.. Koutsilieris M, Mitsiades C, Dimopoulos T, Iannidis A, Ntounis A, Lambou T (2001) A combination therapy of dexamethasone and somatostatin analog reintroduces objective clinical responses to LHRH analog in androgen ablation-refractory prostate cancer patients. J Clin Endocrin Metab 86, 5729-5736. Koutsilieris M, Mitsiades C, Dimopoulos T, Vacalicos J, Lambou T, Tsintavis A (2002) Combination of dexamethasone and a somatostatin analogue in the treatment of advanced prostate cancer. Exp Opin Investig Drugs 11, 283-293. Koutsilieris M, Mitsiades C, Sourla A (2000) Insulin-like growth factors I and urokinase-type plasminogen activator bioregulation system as a survival mechanism of prostate cancer cells in osteoblastic metastases, development of antisurvival factor therapy for hormone-refractory prostate cancer. Mol Med 6, 251-267. Kruithof-Dekker IG, Tetu B, Janssen PJ, Van der Kwast TH (1996) Elevated estrogen receptor expression in human prostate stromal cells by androgen ablation therapy. J Urol 156, 1194-7. Landstrom M, Damber JE, Bergh A (1994) Prostatic tumor regrowth after initially successful castration therapy may be related to a decreased apoptotic cell death rate. Cancer Res 54, 4281-4284. Logothetis CJ, Hossan EA, Smith TL (1994) SMS 201-995 in the treatment of refractory Prostatic cancer. Anticancer Res 14, 2731-2734. Maulard C, Richaud P, Droz JP (1995) Phase I-II study of the somatostatin analogue lanreotide in hormone-refractory prostate cancer. Cancer Chemother Pharmacol 36, 259-62. Monti S, Sciarra A, Falasca P, Di Silverio F (2000) Serum concentrations and prostatic gene expression of chromogranin A and PSA in patients affected by prostate cancer and benign prostatic hyperpalsia. J Endocrinol Invest 23 (suppl 8), 53-54. Murphy WA, Lance VA, Moreau S (1987) Inhibition of rat prostate tumor growth by an ocatpeptide analog of somatostatin. Life Sci 40, 2515-2522. Newman JB, Lluis F, Townsend CM Jr (1987) Somatostatin. In, Thompson JC, Greeley GH Jr, Rayford PL, Townsend CM Jr editors, Gastrointestinal Endocrinology. New York. McGraw-Hill Book Co., 286-299. Pollak MN, Schally AV (1998) Mechanisms of antineoplastic action of somatostatin analogs. Proc Soc Exp Biol Med 217, 143-152.

Reubi JC, Waser B, Schaer JC, Laissue JA (2001) Somatostatin receptor sst1-sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands. Eur J Nucl Med 28, 836-846.. Reyes-Moreno C, Sourla A, Choki I (1998) Osteoblast-derived survival factors protect PC-3 human prostate cancer cells from adriamycin apoptosis. Urology 52, 341-347. Robinson MR, Smith PH, Richards B (1995) The final analysis of the EORTC Genito-Urinary Tract Cancer Co-Operative Group phase III clinical trial (protocol 30805) comparing orchidectomy, orchidectomy plus cyproterone acetate and low dose stilboestrol in the management of metastatic carcinoma of the prostate. Eur Urol 28, 273-283.. Rosenbaum E, Wygoda M, Gips M (2000) Diethylstilbestrol is an active agent in prostate cancer patients after failure to complete androgen blockade. Proc Am Soc Clin Oncol 19, 1372A. Schally AV (1988) Oncological applications of somatostatin analogues. Cancer Res 48, 6977-6985. Sciarra A, Bosman C, Monti, G, Gentile, V, Ciccariello M, Pastore A, Salvatori G, Fattore F, Di Silverio F (2004) Somatostatin analogues and estrogens in the treatment of androgen ablation refractory prostate adenocarcinoma. J Urol 5 (5 Pt 1), 1775-83. Sciarra A, Mariotti G, Gentile V, Voria G, Pastore A, Monti S, Di Silverio F (2003a) Neuroendocrine differentiation in human prostate tissue, is it detectable and treatable? BJU Int 91, 438-45.. Sciarra A, Monti S, Gentile V, Mariotti G, Cardi A, Voria G, Lucera R, Di Silverio F (2003b) Variation in chromogranin A serum levels during intermittent versus continuous androgen deprivation therapy for prostate adenocarcinoma. Prostate 55, 168-79. Segal G, Cohen F (1994) Bcl2 protooncogen expression in prostate cancer and its relationship to the Prostatic neuroendocrine cell. Arch Pathol Med 118, 616-8. Smith DC, Redman BG, Flaherty LE, Li L, Strawderman M, Pienta KJ (1998) A phase II trial of oral diethilstilbestrol as a second-line hormonal agent in advanced prostate cancer. Urology 52, 257-60. Smith JP, Solomon TE ( 1988) Effects of gastrin, proglumide and somatostatin on growth of human colon cancer. Gastroenterology 95, 1541-48. Spilker B (1991) Guide to clinical trias. New York, Raven Press. Vainas G, Pasaitou V, Galaktidou G (1997) The role of somatostatin analogues in complete antiandrogen treatment in patients with prostatic carcinoma. J Exp Clin Cancer Res 16, 119-126. Verhelst J, De Longueville M, Ongena P (1994) Octreotide in advanced prostatic cancer relapsing under hormonal treatment. Acta Urol Belg 62, 83-88

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Cancer Therapy Vol 3, page 167 Cancer Therapy Vol 3, 167-176, 2005

HER-2/neu overexpression in Croatian breast cancer patients: results of one-year multicentric prospective study Research Article

Jasminka Jakic-Razumovic1,*, Jadranka Bozikov2, Bozena Sarcevic3, Viktor Separovic3 Smiljana Kosanovic3, Nives Jonjic4, Elvira Mustac4, Snjezana Tomic5, Josko Bezic5, Bozo Kruslin6, Majda Vucic6, Davor Tomas6, Branko Dmitrovic7, Valerija Blazicevic V7, Tratincica Jakovina8, Drazen Svagelj9, Igor Boric10, Zdenko Njiric10, Vesna Stitic11, Mira Mlinac-Lucijanic M11, Nada Restek-Samarzija12, Hrvoje Predrijevac12, Milan Gosev13 1

Department of pathology, Clinical Hospital Center and Medical School Zagreb Andrija Stampar School of Public Health-Medical School Zagreb 3 Department of pathology Clinic for tumors Zagreb 4 Department of pathology Medical faculty Rijeka 5 Department of pathology Clinical Hospital Split 6 Department of pathology Clinical Hospital â&#x20AC;&#x153; Sestre milosrdniceâ&#x20AC;? Zagreb 7 Department of pathology Clinical Hospital Osijek 8 Department of pathology General Hospital Slavonski Brod 9 Department of pathology General Hospital Vinkovci 10 Department of pathology General Hospital Dubrovnik 11 Department of pathology General Hospital Karlovac 12 Hoffmann-La Roche Zagreb11, Croatia 2

__________________________________________________________________________________ *Correspondence: Jasminka Jakic-Razumovic M.D, Ph.D., Department of Pathology, Clinical Hospital Center Zagreb, Kispaticeva 12, 10000 Zagreb, Croatia; phone: + 385 1 2388 523; fax: + 385 1 2421 861; e-mail: drazumov@hotmail.com Key words: breast carcinoma, HER-2/neu, overexpression, prognostic factors Abbreviations: cyclophosphamide, metothrexate and fluorouracil, (CMF); disease free survival, (DFS); estrogen, (ER); progesterone, (PR); vascular invasion, (VI); Received: 28 February 2005; Accepted: 8 March 2005; electronically published: March 2005

Summary Treatment approaches and prognosis for breast carcinoma patients depend on different prognostic factors. Uniform reporting of breast carcinoma specimens in sense of traditional prognostic and additional factors such as HER-2/neu is very important for oncologists managing breast carcinoma patients. The aim of this study was to uniform reporting of breast carcinoma specimens, to standardize HER-2/neu testing and reporting as part of routine pathological report in multicentric study for Croatian population, and to correlate HER-2/neu overexpression with other prognostic factors. In this study 1442 breast carcinoma patients during one year from 9 Hospitals in Croatia were prospectively collected and analyzed with data of age, menopausal status, tumor size, type and grade, nuclear grade, axillary node status, intratumoral and extratumoral vascular invasion (VI), estrogen (ER) and progesterone (PR) receptor status, and HER-2/neu expression. The standardization of HER-2/neu staining by HercepTest (Dako, Glostrup, Denmark) was made by repeated collective analysis of doubtful slides, and by repeating of staining in one referral center. All traditional prognostic factors were compared with HER-2/neu status. The results showed that there is statistically significant correlation between HER-2/neu expression and histological grade, nuclear grade, tumor size, lymph node status (p<0.001), extratumoral vascular invasion

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Jakic-Razumovic et al: HER-2/neu overexpression in Croatian breast cancer patients (p=0.039), and premenopausal status (p=0.040). Negative correlation was found between HER-2/neu overexpression and steroid receptor positivity (p<0.001). When lymph node status was correlated with other prognostic factors lymph node positivity correlates with extratumoral VI (p<0.001). It seems that this prospective study showed correlation between HER-2/neu and some traditional prognostic factors. Oncologists will follow this prospectively collected group of patients carefully, and prognostic influence of investigated factors will be determined in the future.

II. Materials and methods

I. Introduction

In this study 1442 consecutive breast carcinoma patients during one year (September 2001 to September 2002) from 9 centers in Croatia were collected. For all patients data of age, menopausal status, hystological type, tumor size, grade, nuclear grade, axillary node status, intratumoral and extratumoral VI, ER and PR status by immunohistochemistry were collected (Table 1). The median age was 60 years (range, 23 to 94). Figure 1 shows age distribution in investigated breast carcinoma patients. The tumor size ranged from 0.1 cm to 20.0 cm (median 2.0 cm). Majority of breast carcinomas were of ductal invasive type (77.2%), lobular invasive carcinoma was found in 145 (10.1%) patients, while other types as mixed, mucinous, papillar, medullar, tubular or other rare types were found in 184 (12.7%) patients. Peritumoral and intratumoral lymphatic/vascular invasion was assessed in hematoxillin and eosin stained slides, and were determined as negative and positive. Peritumoral VI was present in 346 (24%) cases, while intratumoral VI was found in 111 (7.7%) cases. There were 544 cases (37.7%) with and 662 cases (45.9%) without axillary lymph node metastases. For 236 (16.4%) patients axillary node status was unknown. Histological grade 1 and 3 were equally distributed, while grade 2 was found in 767 (55.7%) of patients (Elston et al, 1991; Frierson et al, 1995). Majority of patients were postmenopausal (74.0%), while 374 (26.0%) were premenopausal. Imunohistochemistry was carried out in formalin fixed, paraffin-embedded tissue sections by streptavidin-biotin peroxidase complex method using an automatized immunostainer (TechMate, Dako). The antibodies employed in the study were anti-ER (clone ER1D5), diluted at 1/100, Dako), PR (clone 1A6, diluted at 1/100, Dako). Positive scoring of immunohistochemistry for ER and PR was assessed on the basis of the visually estimated percentage of neoplastic cells with nuclear staining. All cases without any staining were considered as negative. Positive nuclear staining was semiquantitatively divided in three groups of positivity as mild staining (<10% cells), moderate (10-50% cells) and strong staining (> 50% cells), but for final statistical analysis any nuclear staining was used as positive reaction. For all patients HER-2/neu status was determined using standard Herceptest (Dako). At the beginning of this study there were some differences in intensity of staining interpretation even all participants were using the same scoring system proposed by the manufacturer. Given the potential clinical importance HER-2/neu status in patients management, interlaboratory variability in HER-2/neu IHC results is a matter of legitimate concern. Because of that, slides were first read in each institution, and after that two experts evaluated only positive stained slides (2+, 3+) in blinded fashion. Expert pathologists after adequate training in HercepTest evaluation could reach excellent intraobserver (!=0.921) and interobserver reproducibility (!=0.832). The experts discussed discordant results and complete agreement was achieved. After that the results from the 9 laboratories and experts were compared. Discrepancy between negative (0.1+) and positive (3+) results occurred in few cases (!=0.96, !=0.98 respectively). Distinguishing weakly (2+) from strongly (3+) positive results showed agreement in only 62% of positive cases (!=0.37). After consensus was agreed by analyzing doubtful slides by all

In Croatian female population the incidence and mortality of breast carcinoma is unfortunately very high. The mammography-screening program for female population in Croatia does not exists, except some sporadic actions in certain regions of country. That is probably reason for detection primary breast carcinomas of larger size and advanced clinical stage in comparison with some other European countries. Despite of that, in recent years mortality rate is slightly dropping down, which is probably result of improvement of therapeutic approaches. It is known that the choice of therapy regiments in primary breast carcinoma patients is based on clinical stage and pathological predictive and prognostic factors such as tumor size, grade, lymph node and steroid receptor status 1. For that reason uniform reporting of breast carcinoma specimens is very important for oncologists making predictions for patient therapy respond and survival. Uniform pathological management and reporting, which among others includes standardization of HER-2/neu testing and reporting, is very important not only for metastatic breast carcinoma patients, than probably also for at least some certain group of newly diagnosed patients (Jacobs et al, 2000; Rampaul et al, 2001; Hsu et al, 2002). The aim of this study was to uniform reporting of breast carcinoma specimens, as well as to standardize HER-2/neu testing and reporting as part of routine pathological report in prospective multicentric study including 9 centers in Croatia. Additionally, verification of association of HER-2/neu expression with other prognostic factors was done. This group of prospectively collected patients will be clinically carefully followed, and effect of combination of prognostic factors in lymph node negative and positive patients on disease free survival (DFS) and overall survival will be investigated in the future. It is known that HER-2/neu positivity is prognostic and predictive factor for therapy respond in patients with metastatic disease, and that selection of patients with strongly HER-2/neu positive tumors is required to obtain clinical benefit from the HER-2/neu targeted therapy in the metastatic breast cancer studies. In some reports HER2/neu is very powerful prognostic factor indicating poor prognosis in primary diagnosed patients, as well as predictive factor for certain adjuvant therapy respond in particular group of patients. Indeed, several studies suggest that HER-2/neu positive patients experience decreased responses to hormonal therapy. Therefore, HER-2/neu status probably should be part of pathologic report in patients with primary breast cancer, as is recently recommended in St. Gallen conference in January 2005.

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Cancer Therapy Vol 3, page 169

Table 1. Distribution of breast carcinoma patients in 9 centers in Croatia during one year (September 2001 to September 2002) Institution Clinical Hospital Split Clinical hospital center Zagreb General Hospital Vinkovci Clinical Hospital Osijek General Hospital Slavonski Brod Medical School Rijeka Clinic for tumors Zagreb Clinical Hospital â&#x20AC;&#x153;Sisters of mercyâ&#x20AC;? Zagreb General Hospital Dubrovnik Total

Number

%

196 219 30 163 44 293 369 85 43

13.6 15.2 2.1 11.3 3.1 20.3 25.6 5.9 3.0 1442

Figure 1. Age distribution in investigated breast carcinoma patients pathologists included in this study, the generalized ! values indicated substantial agreement (!=0.85).

positive, while PR were positive in 972 (59.5%) breast carcinoma patients. Almost equal number of patients was treated by mastectomy and spearing surgery procedure (quadrantectomy and tumorectomy with free margins). There was a difference between 9 centers in type of surgical procedure, e.g. in some centers majority of cases were treated with spearing surgery, while in others simple mastectomy was performed in majority of patients (Table 2). The association of HER-2/neu overexpression with classical prognostic factors is shown in Table 3. Statistically significant association between HER-2/neu expression and lymph node status (p<0.001), tumor size (p<0.001), histological (p<0.001) and nuclear grade (p<0.001), extratumoral VI (p=0.039) and premenopausal status (p=0.040) were found. Statistically significant negative association between HER-2/neu and steroid receptor expression was found (p<0.001).

A. Statistical analysis Analysis was carried out using data analysis software package Statistica ver. 6.0 (StatSoft, 2001). Results were expressed as median and range for quantitative variables and as frequencies and percentages for categorical variables that were grouped following logical classes. Association of categorical variables was tested using the Pearson chi-square test. Results were considered statistically significant at p<0.05.

III. Results Table 2 shows the distribution of all investigated parameters in 1442 breast carcinoma patients. Of 1442 tumors 197 (13.7%) were HER-2/neu positive and 1241 were negative (86.3%). There were 312 (21.8%) patients younger than 50 years, and majority of patients (78.2%) were 50 years or older. In 865 (65.0%) patients ER were

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Jakic-Razumovic et al: HER-2/neu overexpression in Croatian breast cancer patients

Table 2. Distribution of all investigated parameters in 1442 breast carcinoma patients Factors Age < 50 " 50 Menopause no yes Histologic type ductal invasive lobular invasive others Tumor size < 1cm 1 - 2 cm 2 - 5 cm > 5 cm Grade 1 2 3 Nuclear grade 1 2 3 VI (intratumoral) no yes VI (extratumoral) no yes ER negative positive PR negative positive Lymph nodes negative positive unknown HER-2/neu negative positive Type of surgery quadrantectomy mastectomy

Number

%

312 1120

21.8 78.2

374 1067

26.0 74.0

1113 145 184

77.2 10.1 12.7

128 506 674 101

9.1 35.9 47.8 7.2

295 767 316

21.4 55.7 22.9

267 750 326

19.9 55.8 24.3

1330 111

92.3 7.7

1094 346

76.0 24.0

465 865

35.0 65.0

538 972

40.5 59.5

662 544 236

45.9 37.7 16.4

1241 197

86.3 13.7

741 701

51.4 48.6

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Cancer Therapy Vol 3, page 171 Table 3. Crosstabulation of HER-2/neu expression with other prognostic factors in 1442 breast carcinoma patients Factors Lymph nodes negative positive Tumor size < 1cm 1-2 cm 2-5 cm > 5 cm Grade 1 2 3 Nuclear grade 1 2 3 VI (intratumoral) negative positive VI (extratumoral) negative positive ER negative positive PR negative positive Menopause no yes

HER-2/neu (%) negative

positive

Hi2

p

595 (90.1) 455(84.0)

65 (10.9) 87(16.0)

10.37

<0.001

121 (94.5) 449(88.9) 556 (82.7) 84 (84.0)

7 (5.5) 56 (11.1) 116 (17.3) 16 (16.0)

17.67

<0.001

278 (94.2) 665 (87.0) 236 (74.9)

17 (5.8) 99 (13.0) 79 (25.1)

48.83

<0.001

248 (92.9) 657 (88.1) 246 (75.5)

19 (7.1) 89 (11.9) 80 (24.5)

43.14

<0.001

1148 (86.6) 92 (82.9)

178 (13.4) 19 (17.1)

1.18

0.277

952 (87.3) 287 (83.0)

138 (12.7) 59 (17.0)

4.28

0.039

365 (78.7) 785 (90.8)

99 (21.3) 80 (9.2)

37.86

<0.001

420 (78.2) 730 (92.2)

117 (21.8) 62 (7.8)

53.51

<0.001

311 (83.2) 929 (87.4)

63 (16.8) 134 (12.6)

4.20

0.040

Lymph node status is crosstabulated with other investigated parameters and results are shown in Table 4. It is found statistically significant association between lymph node status and tumor size (p<0.001), histological (p<0.001) and nuclear grade (p<0.001) and intra (p=0.038) and extratumoral VI (p<0.001), while association was negative with both, ER and PR positivity (p<0.001). Menopausal status and other investigated parameters were crosstabulated and results are shown on the Table 5. No association was observed between menopausal status and lymph node status, tumor size, intratumoral VI, and PR status. Postmenopausal patients more often had ER negative tumors (p<0.001), higher-grade tumors

(p=0.007), higher tumor nuclear grade (p=0.037), and presence of extratumoral VI (p<0.001) than premenopausal.

IV. Discussion Treatment and prognosis of breast carcinoma patients depend on histological diagnosis including basic prognostic factors and additional markers. Recently, a number of studies have indicated that overexpression of HER-2/neu in breast carcinoma patients indicate worse prognosis and lower probability of responsiveness to some therapy protocols (Ellis et al, 2001; Love et al, 2003; Schiff et al, 2004). Particularly, it is shown that HER171


Jakic-Razumovic et al: HER-2/neu overexpression in Croatian breast cancer patients 2/neu positive breast cancer is resistant to endocrine therapy, and that HER-2/neu positivity is associated with shortened survival in ER-positive breast cancer patients treated with hormonal agents, compared with those who are HER-2/neu negative. It is known that testing of HER2/neu overexpression or amplification is in usage in metastatic breast carcinoma patients because trastuzumab is widely established as an essential and well-tolerated treatment for the management of previously treated HER2/neu positive metastatic breast caracinoma patients. The testing of newly diagnosed breast cancer specimens for HER-2/neu still not has achieved â&#x20AC;&#x153;standard of practiceâ&#x20AC;? status for the management of breast carcinoma patients in the United States and Europe. According to the St. Gallen,s conference highlights the Panel was not ready to accept the information suggesting that overexpression of HER-2/neu may indicate a lower probability of

responsiveness to tamoxifen and perhaps cyclophosphamide, metothrexate and fluorouracil (CMF) (Paik et al, 2000; Osborne et al, 2003) as currently useful for patient care. Therefore, the predictive utility of HER2/neu overxpression in newly diagnosed breast carcinoma patients still awaits confirmation, but it seems that after collecting valuable data the Panel finally accepted HER2/neu testing as a part of routine pathological report at the last St.Gallen conference in January 2005 (data not published yet). Additionally, the discussion as to the best method to determine HER-2/neu status in breast carcinoma samples continuous, with the fluorescenece in situ hybridization method gaining popularity, owing to the recent evidence that in comparison with immunohistochemical analysis this method gives more reliable results.

Table 4. Crosstabulation of lymph node status with other prognostic factors in 1442 breast carcinoma patients Factors Tumor size < 1cm 1-2 cm 2-5 cm > 5 cm Grade 1 2 3 Nuclear grade 1 2 3 VI (intratumoral) negative positive VI (extratumoral) negative positive ER negative positive PR negative positive Menopause no yes

lymph node status (%) negative positive

Hi2

p

81 (63.3) 272(53.8) 282 (41.8) 19 (18.8)

17 (13.3) 149 (29.4) 308 (45.7) 66 (65.3)

94.85

<0.001

174 (69.3) 347 (53.1) 120 (44.6)

77 (30.7) 306 (46.9) 149 (55.14

33.35

<0.001

156 (65.6) 351 (54.5) 122 (45.4)

82 (34.5) 293 (45.5) 147 (54.6)

20.79

<0.001

619 (55.8) 43 (44.8)

491 (44.2) 53 (55.2)

4.29

0.038

550 (60.0) 112 (38.8)

367 (40.0) 177 (61.1)

39.98

<0.001

182 (47.6) 433 (59.0)

200 (52.4) 301 (41.0)

13.08

<0.001

215 (48.9) 400 (59.2)

225 (51.1) 276 (40.8)

11.45

<0.001

162 (52.4) 500 (55.7)

147 (47.6) 397 (44.3)

1.02

0.313

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Cancer Therapy Vol 3, page 173 Table 5. Crosstabulation of menopausal status with other prognostic factors in 1442 breast carcinoma patients Factors Lymph nodes negative positive Tumor size < 1cm 1-2 cm 2-5 cm > 5 cm Grade 1 2 3 Nuclear grade 1 2 3 VI (intratumoral) negative positive VI (extratumoral) negative positive ER negative positive PR negative positive

Menopause (%)

Hi2

p

no

yes

162 (24.5) 147(27.0)

500 (75.5) 397 (73.0)

1.02

0.31

33 (25.8) 132(26.1) 170 (25.2) 30 (29.7)

95 (74.2) 373 (73.9) 504 (74.8) 71 (70.3)

0.94

0.816

84 (28.5) 176 (23.0) 100 (31.7)

211 (71.5) 590 (77.0) 216 (68.3)

9.76

0.007

66 (24.7) 182 (24.30) 103 (31.6)

201 (75.3) 567 (75.7) 223 (68.4)

6.62

0.037

352 (26.5) 22 (19.8)

977 (73.5) 89 (80.2)

2.37

0.124

302 (80.7) 72 (91.7)

791 (19.3) 274 (8.4)

33.14

<0.001

153 (32.9) 187 (21.6)

312 (67.1) 677 (78.4)

20.13

<0.001

143 (26.6) 197 (24.9)

395 (73.4) 594 (75.1)

0.47

0.492

However, at this point it is accepted that HER-2/neu testing by immunohistochemistry is valid method for screening and should be supplemented by FISH method in some cases of doubtful positive reaction (2+ staining intensity) before the trastuzumab treatment (Falo et al, 2003; Goldhirsch et al, 2003). Standardization of these parameters remains an important objective to optimize interlaboratory agreement, and therefore to compare HER2/neu immunohistochemical staining results obtained in 9 pathology departments the evaluation of the reproducibility of staining and assessments was done. After some repeating of staining in one referral center, and discussing of doubtful slides, there was good interlaboratory and interobserver agreement, and valuable data were collected that may be used in the development of quality assurance policies. The present study showed the usefulness of multicentric comparative studies in initiating the development guidelines as has been shown in

some other studies (Jacobs et al, 2000; Hsu et al, 2002; Santinelli et al, 2002; Gunhan et al, 2004). Investigation of adjuvant monoclonal antibody treatment against HER-2/neu for breast carcinoma patients overexpressing HER-2/neu are currently ongoing, but this type of treatment in the adjuvant setting outside of clinical trials is not currently justified. Ongoing prospective studies will show importance of HER-2/neu testing in making decisions of antibody usage in some subgroups of primary breast carcinoma patients with HER-2/neu overexpression in combination with other poor prognostic markers. Well-established diagnosis of breast cancer with all relevant traditional prognostic markers is a basis of good patients treatment. Therefore, we conducted this study to make consensus about histological prognostic factors (tumor size, grade, nuclear grade, vascular invasion), immunohistochemical predictive factors (ER, PR) and

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Jakic-Razumovic et al: HER-2/neu overexpression in Croatian breast cancer patients HER-2/neu overexpression in newly diagnosed breast carcinoma patients during one year in Croatian population. The aim also was to make quality control study for HercepTest in 9 centers in Croatia. Since recently HER2/neu overexpression has gained therapeutic implications and following these developments demand for pathologists to evaluating properly HER-2/neu in breast cancer specimens has been rapidly increasing. For all that we succeeded in great amount, and finally an consensus was made for uniform reporting of prognostic factors in breast carcinoma patients. Particularly, cconsensus was made about using Nottingham model for determination of histological grade in breast carcinoma patients, which is more objective, has excellent reproducibility when used by experienced pathologists (Elston et al, 1991; Frierson et al, 1995; Robbins et al, 1995). Using this scheme incidence of G1-G3 was similar as described in many publications. Additionally, according to the St.Gallen,s recommendation there was agreement to use immunohistochemical method and scale of positivity for determination of ER and PR in breast carcinoma patients (Goldhirsch et al, 2003). It is known that most laboratories worldwide have switched to immunohistochemistry to assess steroid receptors, and it is shown in some studies that PR by immunohistochemistry provided significantly better results than by ligand-binding assay in predicting outcome. It is also shown that ER and PR are codependent variables and PR was a weaker predictor of response to endocrine therapy (Mohsin et al, 2004). Using immunohistochemical method and St.Gallen,s criteria we found 65.0% ER positive and 59.5 % PR positive breast carcinomas, which is very similar to other published studies using the same method (Colon et al, 2002). Controversy surrounds the correlation between HER2/neu expression and other prognostic markers, as has been discussed in preclinical and clinical studies. The objective of the current study was to investigate association of HER-2/neu overexpression with parameters that are assessed routinely in clinical practice (age, hormonal status, cancer grade, nuclear grade, vascular invasion and axilary lymph node status). The results showed that HER-2/neu overexpression was associated significantly with negative ER and PR status, tumor and nuclear high grade, larger tumors, extratumoral VI, positive lymph nodes and menopausal status, as similarly showed some other authors (Coradini and Daidone, 2004). In subgroup of patients presenting with hormoneresponsive Taucher et al, (2003) also showed recently that likelihood of HER-2/neu overexpression in G1/G2 tumors is very small, and therefore, that the assessment of HER2/neu status in this group of patients with breast carcinoma may be considered unnecessary, unless the role of HER2/neu status in adjuvant treatment has been proven. Despite relevant research efforts and identification of many putative good prognostic factors, few of these factors are clinically useful for identifying patients at minimal risk of relapse and with worse prognosis, or patients likely to benefit from specific treatments. Some of them such as HER-2/neu, EGFr, cyclin E, VEGF, urokinase type plasminogen activator-1 and recently discovered anti-apoptosis protein survavin, are suggested

to fit in the category high-level clinico-laboratory effective biomarkers. However, it is known that there is no single biomarker that is able to identify patients with the best (or worse) prognosis or those that would be responsive to a given therapy. Rapid implementation of laboratory findings to clinical practice is followed by many difficulties, including technical statistical concerns, a lack of assay standardization and comparability, and the modern design of studies. Many studies are performed on too small group of patients to provide reliable results. The studies are often heterogeneous in terms of treatment, patients and tumor characteristics, and data may be evaluated using different analytical approaches and thus no easily comparable. Adequately planned prospective studies are required to assess clinical utility of biomarker determinations. The present study showed the usefulness of multicentric comparative studies in initiating the development guidelines, and we hope that collected data will serve as a reference point for future studies of the epidemiological aspect breast cancer among women living in Croatia.

Acknowledgements The authors greatly acknowledge the contribution and support of Ankica Ajdukovic, Smilja BumberBolanca, and Hoffmann-La Roche Zagreb for providing HercepTests for all study participants, and their generous guidance, suggestions and human support during this period. We also appreciate the assistance of the pathology laboratory staff from all pathology departments included in this study.

References Colon E, Reyes JS, Gonzales Keelana C, Climent, Peras C (2002) Prevalence of steroid receptors and HER-2/neu in breast cancer biopsies of women living in Puerto Rico. Health Sci J 21, 299-303. Coradini D, Daidone MG (2004) Biomolecular prognostic factors in breast cancer. Curr Opin Obstet Gynecol 16, 49-55. Ellis MJ, Coop A, Singh B, Muriac L, Llombert-Cussac A, Janicke F, Miller WR, Evans DB, Dugan M, Brady C, Quebe-Fehling E, Borgs M (2001) Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for Erb-1-and/or Erb-2- positive, estrogen receptor-positive primary breast cancer: Evidence from a phase III randomized trial. J Clin Oncol 19,3808-16. Elston CW, Ellis IO (1991) Pathological prognostic factors in breast cancer I. The value of histological grade in breast cancer: experience from large study with long-term followup. Histopathol 19, 403-10. Falo C, Moreno A, Lloveras B, Figueras A, Varela M, Escobedo A (2003) Algorithm for the diagnosis of HER-2/neu status in breast-infiltrating carcinoma. Am J Clin Oncol 26, 465-70. Frierson HF, Wolber RA, Berean KW, Franquemont DW, Gaffey MJ, Boyd JC, Wilbur DC (1995) Interobserver reproducibility of the Nottingham modification of the Bloom and Richardson histological grading scheme for infiltrating ductal carcinoma. Am J Clin Pathol 105, 195-8. Goldhirsch A, Wood WC, Gelber RD, Coates AS, Thurlimann B, Senn HJ (2003) Meeting Highlights: Updated International Expert Consensus on the Primary Therapy of Early Breast Cancer. J Clin Oncol 21, 3357-65. Gunhan O, Kafousi M, Kurt B, Koutsopoulos AV, Karslioglu Y,

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Cancer Therapy Vol 3, page 175 Stathopoulos E, Celasun B. (2004) Comparison of C-erbB2 (HER-2/Neu) immunohistochemistry results on invasive breast carcinomas. Experience of 2 pathology departments in Turkey and Greece. Anal Quant Cytol Histol 26, 251-4. Hsu CY, Ho DM, Yang CF, Lai CR, Yu IT, Chiang H (2002) Interobserver reproducibility of HER-2/neu protein overexpression in invasive breast carcinoma using DAKO HerecepTest. Am J Clin Pathol 118, 693-8. Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ (2000) HER-2/neu protein expression in breast cancer evaluated by immunohistochemistry. A study of interlaboratory agreement. Am J Clin Pathol 113, 251-8. Love RR, Duc NB, Havighurst TC, Mohsin SK, Zhang Q, DeMets DL, Allred DC (2003) HER-2/neu overexpression and response to oophorectomy plus tamoxifen adjuvant therapy in estrogen receptor-positive premenopausal women with operable breast cancer. J Clin Oncol 21, 453-7. Mohsin SK, Weiss H, Havighurst T, Clark GM, Berardo M, Roanh I, To TV, Zho Q, Love RR, Allred DC (2004) Progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: a validation study. Mod Pathol July 23 (advance online publication) Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG, Fuqua SA, Wong J, Allred DC, Clarck GM, Schiff R (2003) Role of the estrogen receptor coactivator AIB1 (SRC3) and HER-2/neu in tamoxifen resistance in breast cancer. J Natl Cancer Inst 95, 53-61. Paik S, Bryant J, Tan-Chiu E, Yothers G, Park C, Wickerham DL, Wolmark N (2000) HER-2 and choice of adjuvant chemotherapy for invasive breast cancer. National Surgical Adjuvant Breast and Bowel Project Protocol B-15. J Natl Cancer Inst 92, 1991-8. Rampaul RS, Pinder SE, Elston CW, Ellis IO (2001) Prognostic and predictive factors in primary breast cancer and their role in patient management: The Nottingham Breast Team. EJSO 27, 229-38. Robbins P, Pinder S, de Klerk, Dawkins H, Harvey J, Sterrett G, Ellis J, Elston C (1995) Histological grading of breast

carcinomas. A study of interobserver agreement. Hum Pathol 26, 873-9. Santinelli A, Baccarini M, Colanzi P, Stramazzotti D, Fabris G (2002) Immunohistochemical evaluation of HER-2/neu expression in infiltrating breast carcinoma: a study of reproducibility. Anal Quant Cytol Histol 24, 54-62. Schiff R, Massarweh SA, Shou J, Bharwani L, Mohsin SK, Osborne CK (2004) Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance. Clin Cancer Res 10, 331S-6S. StatSoft, Inc. Statistica [computer program]. Version 6.0. Tulsa (OK, USA): StatSoft; 2001. Taucher S, Rudas M, Mader RM, Gnant M, Dubsky P, Bachleitner T, Roka S, Fitzal F, Kandioler D, Sporn E, Fridl J, Mittlbock M, Jakes S (2003) Do we need HER-2/neu testing for all patients with primary breast carcinoma? Cancer 15, 2547-53.

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Cancer Therapy Vol 3, page 177 Cancer Therapy Vol 3, 177-184, 2005

Tumor-specific human monoclonal antibody GAH recognizes non-muscle myosin heavy chain type A as a cell surface antigen Research Article

Yoko Hirakawa1,*, Yoshiko Yoshiyama4, Hisae Niki1, Shinsuke Ooike1, Jun Kondo1, Saiko Hosokawa1, Kazuhiro Takahashi2, Kazuhiro Nagaike3, Hideo Nakamura1, Makoto Tsurufuji1 and Toshiaki Tagawa1 1

Pharmaceuticals Research Unit, Mitsubishi Pharma Corporation, Yokohama, Japan Pharmaceuticals Development Unit, Mitsubishi Pharma Corporation, Tokyo, Japan 3 Science & Technology Research Center, Mitsubishi Chemical Corporation, Yokohama, Japan 4 ZOEGENE Corporation, Yokohama, Japan 2

__________________________________________________________________________________ *Correspondence: Yoko Hirakawa, Pharmaceuticals Research Unit, Mitsubishi Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan; Tel: +81-45-963-4724; Fax: +81-45-963-4641; E-mail: Hirakawa.Youko@ma.m-pharma.co.jp Key words: human monoclonal antibody, GAH, non-muscle myosin heavy chain type A, tumor antigen Abbreviations: carcinoembryonic antigen, (CEA); Chinese hamster ovary, (CHO); immunoliposomal doxorubicin, (ILD); monoclonal antibody, (MAb); non muscle myosin heavy chain type B, (nmMHCB); non-muscle myosin heavy chain type A, (nmMHCA); polyethyleneglycol, (PEG); propidium iodide, (PI) Received: 3 March 2005; Accepted: 15 March 2005; electronically published: March 2005

Summary We previously obtained the human monoclonal antibody (MAb) GAH produced by a human-mouse hybridoma derived from lymphocytes of a cancer patient through screening solely based on selective binding to the surface of human tumor cells. The MAb reacts to a high percentage of human solid tumor cells with good selectivity, is internalized by GAH-positive cultured tumor cells in vitro, and when conjugated to polyethyleneglycol-modified immunoliposomal doxorubicin improves the anti-tumor efficacy of the formulation in vitro and in vivo. Here, we report for the first time on the identification of human non-muscle myosin heavy chain type A (nmMHCA) as an unusual tumor cell-surface antigen which GAH recognizes. We found that a chromatographic fraction of extract of GAH-positive MKN45 human stomach tumor cells containing an approximately 200 kDa protein was positive for GAH-staining on a dot-blot assay, and peptide sequences obtained from the same fraction mainly consisted of those of 200 kDa nmMHCA. In addition, GAH could immunoprecipitate nmMHCA from lysates of COS-7 cells transfected with its cDNA or MKN45 cells, and a 200 kDa protein could be recovered from the surface of MKN45 cells labeled with biotin and was found to be nmMHCA using a specific polyclonal antibody. Although the mechanisms by which the epitope of GAH on a cytoskeletal component is selectively exposed on tumor cell surface and internalized is unknown, this finding emphasizes the potential of such â&#x20AC;&#x153;non-membrane proteinsâ&#x20AC;? as new targets for antibody therapy to treat cancer.

MAb technology is anticipated to provide safe and efficacious therapies for these patients, primarily based upon the strict specificity of antigen recognition by antibody. Our laboratory has been conducting research on MAb for the treatment of solid tumors, especially focusing on its application in liposome delivery, mainly because efficacious anti-cancer activity with a broad spectrum should be possible when nanoparticles containing potent

I. Introduction Almost 30 years of biomedical research on MAbs for cancer treatment have resulted in the discovery of significant benefits to some patients using drugs such as rituximab, gemtuzumab, and trastuzumab among others (Baselga, 2001; King and Younes, 2001; Giles et al, 2003). However, there are still unresolved needs for cancer therapy, especially for that against solid tumors. 177


Hirakawa et al: Non-muscle myosin heavy chain type A as a tumor antigen with P3U1 mouse myeloma cells to produce hybridomas. They were then screened for the production of human antibody which reacted against the human tumor cell surface by Cell-ELISA and flowcytometric analysis. One of the selected human monoclonal antibodies was named GAH. Its cDNA was cloned and recombinant GAH (IgG 1) was obtained from established Chinese hamster ovary (CHO) cells transfected with GAH expression vectors (Hosokawa et al, 2004). As a control antibody, human immunoglobulin huIgG was purified from human serum (SCANTIBODIES Laboratory, Inc., Santee, CA) or purchased from Cappel (West Chester, PA). Anti-nmMHC rabbit polyclonal antibody (BT-561) and normal rabbit IgG were purchased from Biomedical Ltd, (Acacia Ridge, Queensland, Australia) and Biogenesis Ltd. (Poole, UK), respectively, and horseradish peroxidase (HRP)-labeled anti-human immunoglobulin and HRP-labeled anti-rabbit IgG were acquired from Cappel. FITC-labeled antibody was prepared as previously described (Hosokawa et al, 2004).

cytotoxic agent is conjugated with a MAb that can recognize “pan-tumor” antigens selectively expressed on tumor cell surface. For this purpose, we previously screened a panel of human-mouse hybridomas derived from lymphocytes cells of cancer patients based on the binding reactivity to the surface of tumor cells freshly isolated from human cancer tissue, and obtained a tumor-specific human monoclonal antibody named GAH (Hosokawa et al, 2004). GAH selectively stains cancer cells in human tissue sections from 13/14 stomach, 4/11 colon, 5/11 mammary, and 0/7 lung cancers, while no positive staining was observed for those of non-tumor and various normal specimens (Hosokawa et al, 2004). Furthermore, polyethyleneglycol (PEG)-modified immunoliposomal doxorubicin (ILD) with GAH has in vivo anti-tumor efficacy selectively against GAH-reactive stomach and colorectal cancer xenografts (Hamaguchi et al, 2004; Hosokawa et al, 2004). Additionally, we reported that ILD exhibits selective antitumor efficacy in vitro and in vivo against tumor cell lines with a positive correlation between the number of GAH-binding sites on tumor cell surfaces and the ILD therapeutic index (Hosokawa et al, 2003). While the usefulness of GAH for liposome delivery to tumor cells was examined, the nature of the antigen on the tumor cell surface recognized by the antibody remains to be clarified since GAH is selected solely dependent on the binding activity to the tumor cell surface without knowing or assuming what antigen the antibody recognizes. We have been pursuing the molecular nature of the antigen expressed on the cell surfaces of a wide range of human solid tumors to obtain deeper insight into the mechanism responsible for this binding and the behavior of GAH selectively observed against tumor cells. Consequently, we found that non-muscle myosin heavy chain type A, which is a cytoskeletal component inside cells, is a component of the antigen exposed on the tumor cell surface recognized by GAH using biochemical approaches and confirmatory experiments involving gene transfer.

C. Flow cytometric analysis of antibody reactivity against the cell surface of viable cells The cells were incubated with FITC-labeled antibody diluted with human serum, and after washing they were analyzed in the presence of propidium iodide (PI) with a flow cytometer (FACScan, Becton Dickinson, San Jose, CA). For the PInegative cell population, the mean fluorescence intensity of FITC was measured and converted to the total amount of bound FITC per cell using a Quantitative kit (Flow Cytometry Standards Co., San Juan, PR) as a calibration standard (Hosokawa et al, 2004).

D. Partial purification of GAH antigen from MKN45sc To prepare a crude membrane fraction of MKN45sc, a resected tumor mass of MKN45sc was first homogenized in TNE buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA and protease inhibitors) with an ultrasonic homogenizer. The supernatant after centrifugation at 400 g for 5 min was then layered on top of 45% sucrose in TNE buffer and was ultracentrifuged (104,000 g) for 1 hour. The interface fraction was subsequently collected and solublized by adding sodium deoxycholate to a final concentration of 1%, and the cleared extract was applied to a Q-Sepharose-HP column. After a linear concentration gradient was developed from 50 mM to 1 M NaCl, it was further eluted with 20 mM Tris-HCl (pH 7.5) containing 8 M urea. The obtained fractions were analyzed by 7.5% SDSPAGE under reduced conditions and dot-blot assay, where each fraction was spotted on a PVDF membrane and incubated with GAH followed by HRP-labeled anti-human immunoglobulin. The binding reaction of GAH to the spotted materials was visualized by Konica Immunostain HRP-1000 (Konica Corp., Tokyo, Japan).

II. Materials and methods A. Cell lines Human stomach tumor cell line MKN45 cells were purchased from IBL Co., (Fujioka, Japan) and human colon cancer cell line HCT-15 and monkey cell line COS-7 cells were obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). All cell lines were cultured in DMEM supplemented with 10% FBS at 37°C in humidified 5% CO2 and 95% air. To obtain cells from transplanted tumor tissues, MKN45 xenografts subcutaneously grown on the backs of BALB/C nude mice (CLEA Inc., Tokyo, Japan) were processed as previously described (Hosokawa et al, 2004). In brief, the tissues were minced and passed through nylon mesh and then centrifuged to remove cell debris. Cells derived from the transplanted tumor were designated MKN45sc, while cultured MKN45 cells were designated MKN45cul.

E. Peptide sequence analysis Chromatographic fractions which were positive for GAH binding on the dot-blot assay or CBB-stained 200 kDa protein derived from GAH-immunoprecipitates within SDS-PAGE gel fragments were subjected to conventional peptide sequence analysis including degradation by CNBr (Nakarai Tesque, Kyoto, Japan) or treatment with lysyl endopeptidase (Wako Pure Chemicals, Osaka, Japan), respectively. The resulting peptides were separated using reversed phase HPLC followed by peptide sequence analysis with a peptide sequencer. Sequence homology was then searched for using FASTA (Sequence Similarity Search by GenomeNet).

B. Antibodies GAH (IgG1, !)-producing cells were established by a conventional hybridoma technique (Hosokawa et al, 2004). Lymphocytes obtained from a colon cancer patient were fused

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F. Analysis of cell surface protein

binding sites for GAH than the same cell line cultured in vitro does. For example, the number of GAH molecules bound to the subcutaneous transplanted tumor cells (MKN45sc) was 1.2 x 105/cell, while the number against cultured MKN45 (MKN45cul) was 1 x 104/cell. On the other hand, this increase was not observed for antibody against carcinoembryonic antigen (CEA). This phenomenon was observed not only for the MKN45 cell line, but also for other human cancer cell lines such as HCT-15, a colon cancer cell line (data not shown). We further used MKN45sc for the purification of antigen because of this higher binding of GAH. In our pilot study involving flow cytometry, the binding reactivity of GAH to the cell surface was decreased to approximately 50% by trypsin treatment, but not by periodic acid treatment, which inactivated the sugar moieties. This suggests that proteins might be a component of the antigen molecule and that glycolipid or carbohydrate is not likely an antigenic determinant. Based upon these observations, we tried to purify the antigen from the crude membrane fraction of MKN45sc solublized with sodium deoxycholate by Q-Sepharose HP ion-exchange column chromatography. Since GAH antigen could not retain its antigenesity upon SDS treatment, conventional SDS-PAGE/Western blotting analysis of tumor cell lysate probed with GAH was not performed. A dot-blot analysis as described in “Materials and methods” was therefore employed for tracing GAH binding reactivity among the fractions. As a result, fractions eluted with 8 M urea containing a 200 kDa protein on SDS-PAGE in common were found to be positive for GAH-staining (Figure 1). Then, these GAHpositive fractions were pooled and treated with CNBr, and each of the chromatographically isolated peptide fragments was analyzed for its amino acid sequence. The peptide sequences were found to be identical to that of some regions of the human non-muscle myosin heavy chain type A protein sequence (nmMHCA; SWISS-PROT P35579, Figure 2), for which the deduced molecular weight was 227 kDa.

The surface molecules on intact cells were chemically labeled with biotin as described by Yanase et al, 1997. Cell suspensions of MKN45cul and MKN45sc were labeled by incubation for 30 minutes in 1 mg/ml sulfo-NHS-biotin (PIERCE, Rockford, IL) in PBS at 4°C. The labeled cells were next lysed by adding TNE buffer with 1% NP40 and homogenized with an ultrasonic homogenizer, and soluble supernatant was applied to Protein A Sepharose CL-4B (Amersham Biosciences) bound with GAH or huIgG, and incubated overnight rotating at 4°C. Then the resins were washed and bound materials were run on SDS-PAGE using 4-12 % gradient gels under reduced conditions, transferred to a PVDF membrane and probed with HRP-labeled avidin (Vector Laboratories, Burlingame, CA) or BT-561 followed by HRP labeled anti-rabbit IgG. After this, bound HRP-conjugate was visualized by ECL (Amersham Biosciences).

G. Preparation of nmMHCA expression vector and transfection into COS-7 cells The nmMHCA 5' cDNA clone HA1.0 ,GenBank M81105, (Toothaker et al, 1991) and the 3' cDNA clone HALES, GenBank M31013, (Saez et al, 1990) were kindly provided by Dr. Robert S. Adelstein (National Institutes of Health, Bethesda, MD). The expression vector of the full-length nmMHCA was constructed from 5' and 3' fragments of nmMHCA cDNA and recloned into appropreate sites of the pEF1B-HALES. The obtained expression vector was then transfected into COS-7 cells with PolyFect (QIAGEN, Hilden, Germany), and after 48 hours culture, the cells were processed and subjected to immunoprecipitation as described above.

III. Results A. Characterization purification of GAH antigen

and

partial

While we have already evaluated and reported on the antitumor efficacy of ILD conjugated with GAH (Hosokawa et al, 2003, 2004; Hamaguchi et al, 2004), efforts have also been made to identify the antigen which GAH recognizes. When we looked for the most efficient material for the purification of GAH antigen, we found by flow cytometric analysis that tumor cells isolated from xenotransplants in nude mice usually possesses more

Figure 1. Isolation of antigen from a crude membrane fraction of MKN45sc. A sodium deoxycholate soluble crude membrane fraction of MKN45sc was separated using QSepharose HP with a linear gradient of NaCl (dotted line) followed by 8 M urea, and the elution was monitored at 280 nm (solid line). The peak eluted with 8 M urea (bar) was then subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining (insert).

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Figure 2. Amino acid sequence of human nmMHCA. The amino acid sequence of nmMHCA is shown (SWISS-PROT P35579). The protein from Q-Sepharose chromatography was fragmented by CNBr and resulting peptide sequences were determined (shaded). The 200 kDa band of immunoprecipitation was treated with lysyl endopeptidase and the sequences were analyzed (outlined).

antigenic complex containing the protein. The first case implies that the nmMHCA should be exposed on the tumor cell surface, although the molecule is regarded an intracellular cytoskeltal protein (Herman and Pollard, 1981). To test this, the cell surface of intact MKN45sc or MKN45cul cells, among which cell surface reactivity against GAH is different, was labeled with sulfo-NHSbiotin, and the immunoprecipitates with GAH or control human IgG were probed with HRP-conjugated avidin on a SDS-PAGE/Western blot. A prominent 200 kDa band was detected only for the case of the GAH-immunoprecipitate from MKN45sc (Figure 4, Lane 1), but hardly any biotinylated band was detected for the MKN45cul preparation (Figure 4, Lane 4). The control human IgG did not recover any detectable molecule from either cell preparation (Figure 4, Lanes 7, 8). In contrast, when immunoprecipitates prepared in the same manner were probed with BT-561 and anti-rabbit Ig-HRP instead of HRP-labeled avidin, the 200 kDa band was detected not

B. Immunoprecipitation of nmMHCA by GAH To examine whether GAH could recognize and immunoprecipitate nmMHCA protein, we transfected an expression vector of its cDNA into COS-7 cells which express the type B non muscle myosin heavy chain (nmMHCB) but not nmMHCA (Sellers, 2000). nmMHCA expression in whole cell lysate was confirmed by Western blot analysis using an anti-nmMHC rabbit polyclonal antibody (BT-561), where a 200 kDa band was observed in COS-7 transfected with the nmMHCA expression vector (Figure 3, Lane 1), but not in mock-treated cells as a negative control (Figure 3, Lane 4). When immunoprecipitates with GAH from the lysate of cells transfected with the expression vector were analyzed by Western blotting with BT-561, the 200 kDa band was detected (Figure 3, Lane 2), but was not seen in the immunoprecipitate with control human IgG (Figure 3, Lane 3). These results suggest that GAH directly binds to the 200 kDa nmMHCA, or alternatively binds to the 180


Cancer Therapy Vol 3, page 181 Figure 3. Immunoprecipitation of transiently expressed nmMHCA. The soluble supernatant of the nmMHCA transient transformant of COS-7 cells and mock cells were immunoprecipitated with GAH or huIgG and then stained with BT-561. Lanes 1-3: nmMHCA transformants, Lane 4: mock cells, Lane 2: immunoprecipitation with GAH, Lane 3: immunoprecipitation with huIgG, Lanes 1 and 4: cell soluble supernatant stained with BT-561.

only for MKN45sc but also for MKN45cul (Figure 4, Lane 2, 5). These results show that the nmMHCA molecule was present in MKN45cul and MKN45sc but was exposed on the cell surface of the latter, which might explain the difference in GAH-reactivity against MKN45sc and MKN45cul as seen by FCM analysis. Whereas the results above showed that GAH reactivity coincided with the expression of nmMHCA on the tumor cell surface, there remains the possibility that GAH recognized the antigenic complex containing nmMHCA, coimmunoprecipitating nmMHCA together with the target molecule. We therefore analyzed the 200 kDa band as well as other bands detected on the 4-12% SDS-PAGE of GAH-immunoprecipitates from MKN45sc cells. As a result of peptide sequence analysis, the 200 kDa band was again identified as human nmMHCA (SWISS-PROT P35579) (Figure 2). The faint bands A and B shown in Figure 4 were determined to be the Nterminus fragments of nmMHCA, and band C was identified as human "-actin. Although actin might be associated with nmMHCA, it was shown that GAH did not directly bind to an authentic sample of human plateletderived actin containing ":85% and #:15% (Cytoskeleton, Inc. Denver, USA ) by dot blot analysis (data not shown). From these results showing GAH could recover nmMHCA, we concluded that it was likely that the antibody recognized a portion of nmMHCA selectively exposed on the MKN45 tumor cell surface.

tumor cell lysate, and that this coincides with tumor cell surface expression of the protein. Although the direct binding of GAH to nmMHCA should be investigated, these results imply that nmMHCA works as a cell surface

Figure 4. Immunoprecipitation of cell surface protein. The cell surface of MKN45sc (lanes 1-3, 7) and MKN45cul (lanes 4-6, 8) was biotinylated and the solubilized supernatant was then immunoprecipitated with GAH (lanes 1-6) or huIgG (lanes 7 and 8), and analyzed by SDSâ&#x20AC;&#x201C;PAGE and Western blotting (4-12% polyacrylamide gradient gel) using HRP-labeled avidin (lanes 1, 4, 7, and 8), BT-561 (lanes 2 and 5) or normal rabbit IgG (lanes 3 and 6). Bands A and B are N-terminus fragments of nmMHCA and band C is "-actin.

IV. Discussion In this report, we showed that the new human monoclonal IgG1 antibody GAH, which selectively binds to tumor cell surfaces, can recover nmMHCA protein from 181


Hirakawa et al: Non-muscle myosin heavy chain type A as a tumor antigen antigen which GAH recognizes. We are currently trying to examine whether GAH can directly bind to highly purified recombinant nmMHCA protein and to identify its epitope. While nmMHCA is a cytoskeletal protein, several previous reports (Willingham et al, 1974; Olden et al, 1976) investigated myosin expression on the surface of some non-muscle, non-tumor cells including mouse L929 fibroblast, although the type of myosin among the family of myosin molecules was not specified (Sellers, 2000). While the mechanism by which nmMHCA can be located and exposed on the cell surface is unknown, our finding that nmMHCA is present on the cell surface of tumor cells including MKN45 grown in vivo to a much more extent than for those cultured in vitro, should help clarify this mechanism. Moreover, in conjunction with our previous data on GAH reactivity (Hamaguchi et al, 2004; Hosokawa et al, 2004), which is limited to tumor cells, it seems that cell surface expression of nmMHCA is required but not sufficient for the explanation of the tumor specific reactivity. It is possible that some accessory molecules may have to be associated with nmMHCA to permit the binding by GAH. Several minor bands were also visualized by the immunoprecipitation results; two of them were nmMHCA fragments, and one of them was "-actin, which GAH can not directly bind. Further detailed analysis of other minor bands might be needed to understand the precise mechanism of GAH-tumor cell interaction. Additionally, this GAH-reactivity of nmMHCA on the tumor cell surface might reflect the cells malignant behavior, since it is the high percentage of reactivity against human malignant tumors that distinguishes GAH from other human MAbs. Several cancer-reactive human monoclonal antibodies derived from the lymphocytes of cancer patients recognize such cytoskeletal proteins; CLNIgG recognizes a portion of vimentin (Hagiwara et al, 2001), while AE6F4 and COU-1 bind to cytokeratin (Borup-Christensen et al, 1990; Ichikawa et al, 1997). The epitope for COU-1, which binds to surface of cancer cells, is a cancer associated cleavage product of the cytokeratin 8/18 complex (Ditzel et al, 2002). Together with our present study, these reports suggest that even ubiquitously distributed cytoskeletal proteins act as surface antigens in a tumor-specific manner, which might need structural changes or the association of other molecules. We believe that our approach by which GAH was successfully obtained should be significant for finding new targets for cancer treatment that can not be obtained by conventional approaches, where researchers have postulated dealing with a category of â&#x20AC;&#x153;plasma membrane proteinâ&#x20AC;? predicted from primary structural similarity. We have prepared anti-nmMHCA polyclonal antibodies using a set of deca-peptides originated from nmMHCA sequence. One of the antibodies, raised against a peptide on the carboxy-terminal side, showed cancer specific reactivity when normal and cancerous tissue sections were immunostained; the specificity was similar to that of GAH. The polyclonal antibody, however, did not share all of its properties with GAH, it reacted against non-epidermal cells, such as vascular endothelial cells

unlike GAH (Niki, unpublished observation). These differences in reactivity might be due to the difference in the epitopes that they recognize, as observed in the case of above-mentioned COU-1, which showed an abnormal staining pattern compared to conventional anti-cytokeratin 8 and cytokeratin 18 antibodies (Ditzel et al, 2002). In summary, this is the first report to reveal that nmMHCA can be a new tumor-associated cell surface antigen and thus a new target of monoclonal antibody therapy for cancer. GAH-conjugated ILD is being evaluated in a phase I clinical study in Japan involving cancer patients with digestive organ tumors.

Acknowledgements We are grateful to Dr. Robert Adelstein for his generous gift of the nmMHCA cDNA clones HA1.0 and HALES. We also thank Dr. Takayuki Miyanishi at Nagasaki University for his review of the manuscript.

References Baselga J (2001) Clinical trials of Herceptin R (trastuzumab). Eur J Cancer 37, Suppl 1, S18-24. Borup-Christensen P, Erb K, Ditzel H, Nielsen B, Larsen JK, Svehag SE, and Jensenius JC (1990) Human-human hybridoma producing monoclonal antibodies against colorectal cancer-associated antigens. APMIS 98, 674-684. Ditzel HJ, Strik MC, Larsen MK, Willis AC, Waseem A, Kejling K, and Jensenius JC (2002) Cancer-associated cleavage of cytokeratin 8/18 heterotypic complexes exposes a neoepitope in human adenocarcinomas. J Biol Chem 277, 21712-21722. Giles F, Estey E, and O'Brien S (2003) Gemtuzumab ozogamicin in the treatment of acute myeloid leukemia. Cancer 98, 2095-2104. Hagiwara H, Aotsuka Y, Yamamoto Y, Miyahara J, and Mitoh Y (2001) Determination of the antigen/epitope that is recognized by human monoclonal antibody CLN-IgG. Hum Antibodies 10, 77-82. Hamaguchi T, Matsumura Y, Nakanishi Y, Muro K, Yamada Y, Shimada Y, Shirao K, Niki H, Hosokawa S, Tagawa T, and Kakizoe T (2004) Antitumor effect of MCC-465, pegylated liposomal doxorubicin tagged with newly developed monoclonal antibody GAH, in colorectal cancer xenografts. Cancer Sci 95, 608-613. Herman IM, and Pollard TD (1981) Electron microscopic localization of cytoplasmic myosin with ferritin-labeled antibodies. J Cell Biol 88, 346-351. Hosokawa S, Tagawa T, Niki H, Hirakawa Y, Ito N, Nohga K, and Nagaike K. (2004) Establishment and evaluation of cancer-specific human monoclonal antibody GAH for targeting chemotherapy using immunoliposomes. Hybrid Hybridomics 23, 109-120. Hosokawa S, Tagawa T, Niki H, Hirakawa Y, Nohga K, and Nagaike K (2003) Efficacy of immunoliposomes on cancer models in a cell-surface-antigen-density-dependent manner. Br J Cancer 89, 1545-1551. Ichikawa A, Tachibana H, Kawamoto S, Kamei M, Honjoh T, Hashizume S, and Shirahata S (1997) Cytokeratin 8 and 19 as antigens recognized by adenocarcinoma-reactive human monoclonal antibody AE6F4. Hum Antibodies 8, 195-202. King KM, and Younes A (2001) Rituximab: review and clinical applications focusing on non-Hodgkin's lymphoma. Expert Rev. Anticancer Ther 12, 177-186. Olden K, Willingham M, and Pastan I (1976) Cell surface myosin in cultured fibroblasts. Cell 8, 383-390.

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Cancer Therapy Vol 3, page 183 Saez CG., Myers JC, Shows TB, and Leinwand LA (1990) Human nonmuscle myosin heavy chain mRNA: generation of diversity through alternative polyadenylylation. Proc Natl Acad Sci USA 87, 1164-1168. Sellers JR (2000) Myosins: a diverse superfamily. Biochimica et Biophysica Acta 1496, 3-22. Toothaker LE, Gonzalez DA, Tung N, Lemons RS, Le Beau MM, Arnaout MA, Clayton LK, and Tenen DG (1991) Cellular myosin heavy chain in human leukocytes: isolation of 5' cDNA clones, characterization of the protein, chromosomal localization, and upregulation during myeloid differentiation. Blood 78, 1826-1833. Willingham MC, Ostlund RE, and Pastan I (1974) Myosin is a component of the cell surface of cultured cells. Proc Natl Acad Sci USA 71, 4144-4148. Yanase K, Smith RM, Puccetti A, Jarett L, and Madaio MP (1997) Receptor-mediated cellular entry of nuclear localizing anti-DNA antibodies via myosin 1. J. Clin Invest 100, 2531.

Yoko Hirakawa

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Cancer Therapy Vol 3, page 185 Cancer Therapy Vol 3, 185-188, 2005

Radical parametrectomy in the treatment of invasive cervical cancer after simple hysterectomy Research Article

Nadereh Behtash1,*, Haleh Ayatollahi1, Hasanzadeh, Fatemeh Esfehani2

Fatemeh

Ghaemmaghami1,Malihe

1

Gynecology Oncology, Tehran University of Medical Sciences, Tehran, Iran. Epidemiology, Tehran University of Medical Sciences, Tehran, Iran.

2

__________________________________________________________________________________ *Correspondence: Nadereh Behtash, Associate Professor, Tehran University of Medical Sciences. Gynecology Oncology Department, Vali-e-Asr Hospital, Keshavarz Blvd., Tehran 14194, Iran. Phone: #98-21-6939320, Fax: #98-21-6937321, E-mail: nadbehtash@yahoo.com Key words: Radical Parametrectomy, Invasive cervical cancer Abbreviations: neoadjuvant chemotherapy, (NACT); radical parametrectomy (RP) Received: 24 December 2004; Revised: 31 January 2005 Accepted: 8 February 2005; electronically published: March 2005

Summary To assess the morbidity and efficacy of radical parametrectomy (RP) following simple hysterectomy in patients with invasive cervical carcinoma. Seven year retrospective chart review identified 5 patients that underwent RP with pelvic and paraaortic lymphadenectomy and upper vaginectomy. Data were collected on demographics, tumor stage, histology and survival. One patient had stage IA lesion, one stage IB1, 2patients had stage IIA, and one with unknown stage. One of these patients had adenocarcinoma. Median age was 41.6 years. The most indication for hysterectomy was abnormal vaginal bleeding (3 out of 5, 60%). Two patients had pelvic node metastases. Surgical margins in all 5 patients were tumor free at the time of RP. Two patients with positive pelvic nodes received adjuvant radiotherapy. Mean follow up time was 48.8 months. Four patients are alive without disease, and one patient who had been node positive, died 12 months after receiving radiation. RP is an acceptable option for patients diagnosed with incidental finding of invasive cervical cancer at the time of simple hysterectomy. Careful selection of RP for patients not having residual tumor, will obviate adjuvant radiotherapy in most cases.

carcinoma as incidental finding in simple hysterectomy specimen (Behtash et al, 2003). Overall survival in cervical carcinoma treated with simple hysterectomy is less than 50% at 5 years (Jones and Jones, 1943; Daniel and Brunschwig, 1961; Barber et al, 1968). Additional therapy in these patients include radiation therapy (Cosbie, 1963; Green Jr and Morse Jr, 1969; Andras et al, 1973; Davy et al, 1977; Papavasiliou et al, 1980; Perkins et al, 1984; Behtash et al, 2003), or additional surgery (Barber et al, 1968; Green Jr and Morse Jr, 1969; Orr et al, 1986; Chapman et al, 1992; Kinney et al, 1992; Behtash et al, 2003). Described in 1961 by Daniel and Brunschwig, RP is a surgical procedure that allows one to complete the evaluation of the tissues of concern, namely upper vagina, parametrium and the regional lymphatics.

I. Introduction The management of early stage cervical carcinoma is primarily surgical in the majority of patients. Invasive cervical carcinoma after simple hysterectomy, can be treated with radiotherapy or reoperation involving a pelvic and/or paraaortic lymphadenectomy, radical Parametrectomy, and upper vaginectomy (Orr et al, 1986; Hopkins et al, 1990; Chapman et al, 1992; Roman et al, 1993; Crane and Schneider, 1999). Since radiation therapy results in loss of ovarian function and greater frequency of sexual dysfunction than operative techniques, RP seems more beneficial option. It can be performed safely in most patients, who have an early stage invasive carcinoma of the cervix with the expectation of an acceptable rate of long term disease free survival (Kinney et al, 1992). Due to inadequate screening and diagnostic work up, we have frequent cases of cervical

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Behtash et al: Radical parametrectomy for cervical cancer after simple hysterectomy The objective of our study is to evaluate the morbidity and efficacy of RP in patients with cervical carcinoma in their simple hysterectomy specimen.

Chemotherapy treatment consisted of Cisplatinum 20 mg/q 10 days/ for 3 courses, Vincristine 1 mg/q 10 days/ for 3 courses. After radical surgery, both of these patients had positive lymph nodes in pelvis, residual tumor in vaginal specimen with clear margins. They received post operative external beam radiotherapy (6800-7000 cGy). One of these patients is alive without disease after 89 months. But the other one died about 15 months after completing adjuvant radiotherapy with progressive disease. Two patients (stage IA2, IB1) had no residual disease in preoperative and pathologic evaluation following RP. Although one of these two patients, in preoperative colposcopic exam, showed moderate dysplasia in cuff. Both of these patients are alive without disease in 65 months and 23 months follow up. One patient, who underwent subtotal hysterectomy due to abnormal vaginal bleeding, had stage IB2 adenocarcinoma of cervix and received 5400 cGy external radiotherapy before RP. After 6 weeks, she underwent radical cervicectomy plus RP plus upper vaginectomy plus pelvic and paraaortic lymphadenectomy. She had no residual tumor in pathologic specimens, and is alive without disease 48 months after radical surgery. Overall, the 5 year survival for our patients with completed RP is 80% with a median follow up of 48 months. With a median follow up of 48 months (17-89) four patients are still alive without disease (23, 48, 65, and 89 months). One patient died due to progressive disease at 17 months after surgery.

II. Materials and methods A retrospective chart review identified 150 patients with early stage cervical carcinomas (FIGO stage IA-IIA) treated at Vali-e-Asr University Hospital of Tehran University of Medical Sciences, Tehran, Iran from 1997-2003. Five of these patients were evaluated for RP secondary to the diagnosis of invasive cervical cancer in cervical specimen following simple hysterectomy. To be considered eligible for RP patients were required to have a normal pelvic exam before surgery with no evidence of residual disease in the parametrium (stage IA-IIA). RP was performed on 2 months, 4months, 4months, 18 months, and 10 years after simple hysterectomy. Clinicopathologic information including demographics, indication for hysterectomy, tumor stage, histology, nodal status, operative complications, length of stay, recurrence and survival was collected. One patient had subtotal hysterectomy in the first procedure. Follow up data were obtained by review of the medical records or by the patient correspondence.

III. Results Two patients had stage IIA lesions; one patient had stage IB1; one patient IA2 and one stage IB2. Demographic information is listed in Tables 1 and 2. Four patients underwent RP, upper vaginectomy, pelvic and/or paraaortic lymphadenectomy, and the fifth one had an additional cervicectomy due to previous subtotal hysterectomy. Median number of dissected pelvic lymph nodes was 12. We had no major operative and post operative complications. Mean post operative length of stay was 5 days. Two patients had cuff biopsy (large cell nonkeratinizing SCC) in preoperative evaluation. They received 3 courses of neoadjuvant chemotherapy (NACT) before operation (RP).

IV. Discussion Incidental finding of cervical carcinoma is rather frequent following simple hysterectomy for apparently benign diseases. In our series, the most common primary diagnosis was CIN and abnormal uterine bleeding. Based on our previous study, inadequate preoperative (prehysterectomy) evaluation for patients with abnormal Papsmear and vaginal bleeding, were the main reasons of inappropriate management (Behtash et al, 2003). Unfortunately, in our series (Behtash et al, 2003), even patients with biopsy proven SCC of cervix underwent simple hysterectomy, although even adequate preoperative evaluation could not find occult cervical carcinoma. Simple hysterectomy is considered to be adequate surgery only for patients with microinvasion (stage IA1) (Jones and Jones, 1943; Daniel and Brunschwig, 1961; Barber et al, 1968). Additional treatment for more invasive carcinoma finding after simple hysterectomy is radiation therapy with or without chemotherapy or RP with lymphadenectomy. In appropriate candidates, it seems radical reoperation, especially in a young patient, allows ovarian preservation and functional vagina (Orr et al, 1986; Chapman et al, 1992; Kinney et al, 1992). Selection of an eligible patient for RP is critical. Leath III, et al, (2004) showed patients with no clinical evidence of residual at the vaginal apex and parametrium have an excellent overall survival. They had a rather high operative morbidity (30%) including 2 incidental

Table 1. Age Gravidity Para Menarche First intercourse

Mean 41.6 4.75 3.7 12.5 15.75

Range 35-47 1-9 1-6 12-13 12-21

Table 2.

Stage

Pathology Indications simple hysterectomy

for

IA2 IB1 IB2 IIA SCC Adenocarcinoma CIN Abnormal uterine bleeding

Number of patients 1 1 1 2 4 1 2 3

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Cancer Therapy Vol 3, page 187 cystectomy and massive transfusion in 4 patients. In other series, there are rate of morbidity following RP (Orr et al, 1986; Chapman et al, 1992; Kinney et al, 1992). We had no massive transfusion, no major surgical or postoperative morbidity in these five patients. In a study Leath III et al, (2004), the length of hospital stay was 3.6 days, which is much shorter than our series, although different insurance problems in two countries may influence this point. Postoperative adjuvant radiotherapy may increase morbidity dramatically, which has been reported in radical hysterectomy (Martimbeau et al, 1978; Barter et al, 1989; Fiorica et al, 1990). We planned NACT before radical surgery in two patients with disease in central culf, to minimize the likelihood of postoperative radiotherapy. Due to positive pelvic node and residual tumor in vaginal specimen (with clear margin), they received adjuvant radiotherapy. As Orr et al, (1986) noted the decision for planning radiation can only be made after surgery. Four largest series that evaluated RP in the management of cervical cancer included 18-27 patients (RP (Orr et al, 1986; Chapman et al, 1992; Kinney et al, 1992; Leath III et al, 2004). Seventeen patients out of 23 in Orr series (Orr et al, 1986) had negative RP specimen and all were with no evidence of recurrence with a modest 36 month follow up. With a median follow up of 72 months, Chapman et al (1992) reported 89% five year survival. Their study also confirmed that extended survival can be obtained in patients with a negative RP specimen. Furthermore, the absence of long term morbidities, such as urinary fistula, was encouraging. Kinney et al, (1992) reported 82% survival with a median follow up of 8.4 years. In their series, all the recurrences occurred within 4 years. The incidence of positive nodes in these four large series was 10% (9/93), while the incidence of positive vaginal margin or positive parametrium is 7% (7/93). Eighty percent of patients were able to have a completed RP without evidence of residual disease. Cosbie, (1963) reported 116 patients with cervical cancer over 3 decades that received radiotherapy following simple hysterectomy. In spite of 16 patients with microinvasive disease, their 5 year survival rate was only 71%. Green and Morse, (1969) evaluated outcome of 84 patients that received either surgery or radiation following extrafacial or subtotal hysterectomy in cervical cancer. Five year overall survival in radiation group was 30% while 67% in radical operation patients. Importantly, the authors demonstrated that a delay in further therapy of greater than 4 months was associated with an overall 5 year survival of 18% as compared to 42% when patients were treated within 4 months (Andras et al, 1973; Davy et al, 1977). Survival data from the University of Virginia utilizing radiation therapy were 93% at 5 years (Crane and Schneider, 1999). However, 12/18 patients suffered either grade 1 or 2 acute toxicities and 2 had long term complications consisting of chronic diarrhea and small bowel obstruction

that necessitated surgical exploration. Survival statistics in these series may be influenced by inclusion of patients with higher risk factors such as large diameter tumors, tumors at the cervical margin or involving the parametrium and/or vagina and nodal metastasis. Although radiotherapy is quite effective for patientsâ&#x20AC;&#x2122; early invasive carcinoma of cervix after simple hysterectomy, its serious potential morbidity and costs should be considered. In our country, long waiting list for radiotherapy, frequent cases of inappropriately managed, common incidence of cervical carcinoma are additional reasons that make RP as an acceptable and safe alternative for carefully selected patients. To our knowledge this is the first report of radical parametrectomy in our country.

References Andras EJ, Fletcher GH, Rutledge F (1973) Radiotherapy of carcinoma of the cervix following simple hysterectomy. Am J Obstet Gynecol 115, 647-55. Barber HR, Pece GV, Brunschwig A (1968) Operative management of patients previously operated upon for a benign lesion with cervical cancer as surprise finding. Am J Obstet Gynecol 101, 959-65. Barter JF, Soong SJ, Shingleton HM, Hatch KD, Orr Jr JW (1989) Complications of combined radical hysterectomypostoperative radiation therapy in women with early stage cervical cancer. Gynecol Oncol 32, 292-6. Behtash N, Mousavi A, Mohit M, Modares M, Khanafshar N, Hanjani P (2003) Simple hysterectomy in the presence of invasive cervical cancer in Iran. Int J Gynecol Cancer 13, 177-81. Chapman JA, Mannel RS, Disaia PJ, Walker JL, Berman ML (1992) Surgical treatment of unexpected invasive cervical cancer found at total hysterectomy. Obstet Gynecol 80, 9314. Cosbie W (1963) Radiotherapy following hysterectomy performed for or in the presence of cancer of the cervix. Am J Obstet Gynecol 85, 332-7. Crane CH, Schneider BF (1999) Occult carcinoma discovered after simple hysterectomy treated with postoperative radiotherapy. Int J Radiot Oncolo Biol Phys 43, 1049-53. Daniel W, Brunschwig A (1961) The management of recurrent carcinoma of cervix following simple total hysterectomy. Cancer 14, 582-6. Davy M, Bentzen H, Jahren R (1977) Simple hysterectomy in the presence of invasive cervical cancer. Acta Obstet Gynecol Scand 56, 105-8. Fiorica JV, Roberts WS, Greeberg H, Hoffman MS, Lapolla JP, Cavangh D (1990) Morbidity and survival patterns in patients after radical hysterectomy and postoperative adjuvant pelvic radiotherapy. Gynecol Oncol 36, 343-7. Green TH Jr, Morse WJ Jr (1969) Management of invasive cervical cancer following inadvertent simple hysterectomy. Obstet Gynecol 33, 763-9. Hopkins MP, Peters WAIII, Anderson W, Morley GW (1990) Invasive cervical cancer treated initially by standard hysterectomy. Gynecol Oncol 36, 7-12. Jones H, Jones G (1943) Panhysterectomy versus irradiation in early cancer of cervix. JAMA 122, 930-2. Kinney WK, Egorshin EU, Ballard DJ, Podratz KC (1992) Long term survival and sequelae after surgical management of invasive cervical carcinoma diagnosed at the time of simple hysterectomy. Gynecol Oncol 44, 24-7.

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Behtash et al: Radical parametrectomy for cervical cancer after simple hysterectomy Leath III CA, Straughn Jr JM, Bhoola Snehal M, Patridge EE et al (2004) The role of radical Parametrectomy in the treatment of occult cervical carcinoma after extrafascial hysterectomy. Gynecol Oncol 92, 215-19. Martimbeau PW, kjorstad KE, Kolstad P (1978) Stage IB carcinoma of the cervix, the Norwegian Radium Hospital, 1968-1970, results of treatment and major complications, Lymphedema. Am J Obstet Gynecol 131, 389-94. Orr JW, Ball GC, Soong SJ, Hatch KD, Partridge EE, Austin JM (1986) Surgical treatment of women found to have invasive cervix cancer at the time of total hysterectomy. Obstet Gynecol 68, 353-6.

Papavasiliou C, Yiogarakis D, Pappaskeramopoulos A (1980) Treatment of cervical carcinoma by total hysterectomy and post operative external irradiation. Int J Radiot Oncol biol Phys 6, 871-4. Perkins PL, Chu AM, Jose B, Achino E, Tobin DA (1984) Posthysterectomy mega voltage irradiation in the treatment of cervical carcinoma. Gynecol Oncol 17, 340-8. Roman LD, Morris M, Mitchell MF, Eifel PJ, Burke TW, Atkinson EN (1993) Prognostic factor for patients undergoing simple hysterectomy in the presence of invasive cancer of cervix. Gynecol Oncol 50, 179-83.

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Cancer Therapy Vol 3, page 189 Cancer Therapy Vol 3, 189-192, 2005

A summary on lamivudine primary prophylaxis of hepatitis B virus reactivation in chronic HBsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy Research Article

Viroj Wiwanitkit Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok Thailand 10330

__________________________________________________________________________________ *Correspondence: Viroj Wiwanitkit, M.D., Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 10330; Tel: 662 256 4136; Fax: 662 218 3640; e-mail: Viroj.W@Chula.ac.th Key words: lamivudine, primary prevention, chemotherapy, Hodgkin’s lymphoma Abbreviations: hepatitis B virus surface antigen, (HbsAg); hepatitis B virus, (HBV) Received: 1 March 2005; Revised: 10 March 2005 Accepted: 07 April 2005; electronically published: April 2005

Summary Several hepatitis viruses have been mentioned as a risk factor for development of Hodgkin’s lymphoma. In addition, the reactivation of hepatitis in Hodgkin’s lymph patients co-infected with hepatitis virus treated with chemotherapy is reported. It is noted that hepatitis B virus (HBV) reactivation of various degrees of severity. Lamivudine , a nucleoside analogue that can directly suppress HBV replication, is tried as a prophylaxis and treatment of the HBV reactivation. Here, the author summarized the recent report on using lamivudine primary prophylaxis of hepatitis B virus reactivation in chronic HbsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy. A literature review to find the previous reports about using lamivudine primary prophylaxis of hepatitis B virus reactivation in chronic HbsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy was performed. According to this study, there are 4 recruited reports covering 31 cases of using lamivudine primary prophylaxis of hepatitis B virus reactivation in chronic HBsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy. Of these cases, 10 cases were prescribed for lamivudine primary prophylaxis and the results showed no reactivation of fulminant hepatitis while in the other 21 cases without prophylaxis the reactivation were noted in 15 cases. Therefore, the preventive effect of having lamivudine primary prophylaxis is 100 % (odds ratio is uncountable high). Here, the author concluded that lamivudine prophylaxis might be effective, when chemotherapy is given to an HBsAg-positive patient with non-Hodgkin's lymphoma. However, before further implication, a larger case-control study is required for making a final conclusion. Hodgkin’s lymph patients co-infected with hepatitis virus treated with chemotherapy is reported (Rossi et al, 2001). Rossi et al noted that hepatitis B virus (HBV) reactivation of various degrees of severity, including fulminant hepatitis, might develop in 20-50% of hepatitis B virus surface antigen (HbsAg)-positive patients undergoing immunosuppressive or cytostatic treatment (Rossi et al, 2001). Management of the reactivation of HBV becomes a new insight for the present therapy of any cases with Hodgkin’s lymphoma. Lamivudine, a nucleoside analogue that can directly suppress HBV replication, is tried as a prophylaxis and treatment of the HBV reactivation. Here, the author summarized the recent report on using lamivudine primary prophylaxis of

I. Introduction Keresztes et al, (2003) said that numerous observations implied that the pathogenesis of malignant lymphomas is multifactorial and that viruses probably play an important etiologic role (Keresztes et al, 2003). Lymphoma is a common hematological malignancy. Fisher and Fisher said that several pathogens have been linked to the risk of lymphoma, including Epstein-Barr virus, human immunodeficiency virus, hepatitis virus, and simian virus 40 (Fisher and Fisher, 2004). Several hepatitis viruses have been mentioned as a risk factor for development of Hodgkin’s lymphoma (Chow, 1993; Takada, 1999). In addition, the reactivation of hepatitis in

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Wiwanitkit: Lamivudine primary prophylaxis of HBV reactivation in chronic HbsAg Table 1. Previous reports on using lamivudine primary prophylaxis of hepatitis B virus reactivation in chronic HbsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy Reports Tsutsumi et al, 2004a Silvestri et al, 2000 Nakagawa et al, 2002

Number of patients 4 4 2

Dosage lamivudine regimen 100 mg/day 100 mg/day 100 mg/day

Number of patients with reactivation/ number of patients receive prophylaxis 0/1 0/4 0/2

* Indeed, there is another additional report (Shibolet et al, 2002;) on using lamivudine primary prophylaxis of hepatitis B virus reactivation in chronic HBsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy, however, there is no complete data on the outcome specifically to non Hodgkin’s lymphoma in this study.

hepatitis B virus reactivation in chronic HbsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy.

with the episomal form of HBV. This can also be a problem in chemotherapy. Prophylaxis of lamivudine treatment for hepatitis B patients undergoing chemotherapy is therefore recommended. Lau et al (2003) noted that preemptive lamivudine therapy more effectively prevented the development of hepatitis than deferred lamivudine treatment in HBsAg-positive cancerous patients undergoing chemotherapy. Chemotherapy administration to patients with lymphoproliferative diseases and lymphoma that are carriers of hepatitis B can be complicated by reactivation of Hepatitis B and this may lead to morbidity and mortality due to liver failure (Shtalrid et al, 2001). Shtalrid et al noted that lamivudine inhibited replication of HBV and probably ameliorated the severity of already reactivated hepatitis (Shtalrid et al, 2001). In 1999, Maguire et al said that treatment with lamivudine resulted in rapid loss of hepatitis B virus-DNA, resolution of hepatitis and clinical recovery (Maguire et al, 1999). After administration of lamivudine, the patients gradually recovered from liver failure (Tsutsumi et al, 2004b). Therefore, the lamivudine is recommended for treatment of HBV reactivation in chronic HBsAg carriers with Hodgkin’s lymphoma treated with chemotherapy. Presently, there is a new attempt to use lamivudine for primary prophylaxis of HBV reactivation in chronic HBsAg carriers with lymphoma treated with chemotherapy. Indeed, there are many reports on the use of lamivudine for treatment of HBV reactivation in chronic HBsAg carriers with lymphoma treated with chemotherapy but there are only a few reports on the use of lamivudine for primary prophylaxis purpose. In addition, there is no previous report on the preventive property of this regimen. A metanalysis to assess the preventive property is warranted. Here the author performed a retrospective study to summarize the previous reports on use lamivudine for primary prophylaxis of HBV reactivation in chronic HBsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy. According to this study, there are some recent studies on this topic. According to the summative analysis, the effectiveness or prophylaxis rate is very high. In addition, the preventive odds ratio is very high (infinity). Of interest, this is the first report proving the good preventive property of the regimen by case-control analysis. Here, the author concluded that lamivudine prophylaxis might be effective, when chemotherapy is given to an HBsAg-positive patient with non-Hodgkin's

II. Materials and methods A literature review to find the previous reports about using lamivudine primary prophylaxis of hepatitis B virus reactivation in chronic HBsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy was performed. The author used the electronic search engine PubMed in searching for the literatures. Any reports that did not present complete details or contain English details were excluded. The available reports were collected and extracted for the clinical data. Those primary data were used for further metanalysis study. Concerning the metanalysis study, the summative descriptive analysis was performed where appropriate. The SPSS 11.0 for Windows was used for statistical analysis in this study.

III. Results According to this study, there are 4 recruited reports (Silvestri et al, 2000; Nakagawa et al, 2002; Persico et al, 2000; Tsutsumi et al, 2004a) covering 31 cases of using lamivudine primary prophylaxis of hepatitis B virus reactivation in chronic HBsAg carriers with non Hodgkin’s lymphoma treated with chemotherapy. Of these cases, 10 cases were prescribed for lamivudine primary prophylaxis and the results showed no reactivation of fulminant hepatitis while in the other 21 cases without prophylaxis the reactivation were noted in 15 cases. Therefore, the preventive effect of having lamivudine primary prophylaxis is 100 % (odds ratio is uncountable high).

IV. Discussion HBV reactivation has been reported in cancer patients following administration of chemotherapy or immunosuppressive therapy and may result in liver damage of varying degrees of severity (Saif et al, 2001). Vento et al (2002) said that, in carriers of HBV, liver damage due to reactivation of viral replication could occur after withdrawal of immunosuppressive drugs. They also noted that there were difficulties in drug prevention and treatment for the reactivation (Vento et al, 2002). In addition, latent HBV infection in healthy individuals with antibodies to hepatitis B core antigen (anti-HBc) can be seen (Murasawa et al, 2000). Murasawa et al (2000) indicated that the majority of healthy individuals positive for anti-HBc, which had been assumed to denote a past history of transient HBV infection, were latently infected 190


Cancer Therapy Vol 3, page 191 of hepatitis B following chemotherapy. Gastroenterol Hepatol 14, 801-3. Murasawa H, Uemoto S, Hijikata M, Ueda Y, Tanaka K, Shimotohno K, Chiba T. (2000) Latent hepatitis B virus infection in healthy individuals with antibodies to hepatitis B core antigen. Hepatology 31, 488-495. Nakagawa M, Simizu Y, Suemura M, Sato B (2002) Successful long-term control with lamivudine against reactivated hepatitis B infection following intensive chemotherapy and autologous peripheral blood stem cell transplantation in nonHodgkin's lymphoma, experience of 2 cases. Am J Hematol 70, 60-3. Pelizzari AM, , Motta M, Cariani E, Turconi P, Borlenghi E, Rossi G (2004) Frequency of hepatitis B virus mutant in asymptomatic hepatitis B virus carriers receiving prophylactic lamivudine during chemotherapy for hematologic malignancies. Hematology J 5, 325-328. Persico M, De Marino F, Russo GDG, Severino A, Palmentieri B, Picardi M, Morante A, Rotoli B, Torella R, De Renzo A (2002) Efficacy of lamivudine to prevent hepatitis reactivation in hepatitis B virus-infected patients treated for non-Hodgkin lymphoma. Blood 99, 724-725. Rossi G, Pelizzari A, Motta M, Puoti M (2001) Primary prophylaxis with lamivudine of hepatitis B virus reactivation in chronic HbsAg carriers with lymphoid malignancies treated with chemotherapy. Br J Haematol 115, 58-62. Saif MW, Little RF, Hamilton JM, Allegra CJ, Wilson WH (2001) Reactivation of chronic hepatitis B infection following intensive chemotherapy and successful treatment with lamivudine, a case report and review of the literature. Ann Oncol 12, 123-9. Shtalrid M, Haran M, Klepfish A, Lurie Y, Malnick S (2001) Effective treatment with Lamivudine of patients with reactivation of hepatitis B following chemotherapy administration. Harefuah 140, 1159-62. Shibolet O, Ilan Y, Gillis S, Hubert A, Shouval D, Safadi R. (2002) Lamivudine therapy for prevention of immunosuppressive-induced hepatitis B virus reactivation in hepatitis B surface antigen carriers. Blood 100, 391-396. Silvestri F, Ermacora A, Sperotto A, Patriarca F, Zaja F, Damiani D, Fanin R, Baccarani M (2000) Lamivudine allows completion of chemotherapy in lymphoma patients with hepatitis B reactivation. Br J Haematol 108, 394-6. Takada K (1999) Virus-associated malignancies. Ryoikibetsu Shokogun Shirizu (25 Pt 3), 421-4. Tsutsumi Y, Kawamura T, Saitoh S, Yamada M, Obara S, Miura T, Kanamori H, Tanaka J, Asaka M, Imamura M, Masauzi N (2004a) Hepatitis B virus reactivation in a case of nonHodgkin's lymphoma treated with chemotherapy and rituximab, necessity of prophylaxis for hepatitis B virus reactivation in rituximab therapy. Leuk Lymphoma 45, 6279. Tsutsumi Y, Tanaka J, Kawamura T, Miura T, Kanamori H, Obara S, Asaka M, Imamura M, Masauzi N ( 2004b) Possible efficacy of lamivudine treatment to prevent hepatitis B virus reactivation due to rituximab therapy in a patient with nonHodgkin's lymphoma. Ann Hematol 83, 58-60. Vento S, Cainelli F, Longhi MS (2002) Reactivation of replication of hepatitis B and C viruses afterimmunosuppressive therapy: an unresolved issue. Lancet Oncol 3, 333-340.

lymphoma. However, before further implication, a larger case-control study is required for making a final conclusion. In addition, the problems of the prophylaxis of lamivudine treatment for hepatitis B patients must be discussed. One is resistance to lamivudine, and the other is the treatment duration of the prophylactic administration of lamivudine. Lok et al, (2004) recommended that lamivudine prophylaxis be continued for six months after completion of chemotherapy or immunosuppressive therapy. On the other hand, several durations of the administration of lamivudine prophylaxis, which reportedly delayed hepatitis B reactivation, were observed after lamivudine prophylaxis treatments were discontinued one to seven months after the end of chemotherapy with rituximab (Dai et al, 2004; Pelizzari et al, 2004). Dai et al, (2004) noted that delayed HBV reactivation could occur in lymphoma patients receiving R+CHOP after withdrawal of preemptive lamivudine. They noted that more protracted lamivudine therapy may be an alternative to close monitoring following chemotherapy, and further studies were needed to define optimal duration of lamivudine therapy (Dai et al, 2004). Pelizzari et al (2004) noted that among HBV carriers treated with chemotherapy for haematologic malignancies, the emergence of HBV YMMD mutant occurred in 3.1% of prophylactic lamivudine courses and was of little clinical relevance. Treatment duration is mentioned for its association with the resistance of lamivudine (Lok AS et al, 2001; Lok ASF et al, 2001). This caution should also be bewared.

References Chow WT (1993) Cancer and viruses. Ann Acad Med Singapore 22, 163-9. Dai MS, Chao TY, Kao WY, Shyu RY, Liu TM (2004) Delayed hepatitis B virus reactivation after cessation of preemptive lamivudine in lymphoma patients treated with rituximab plus CHOP. Ann Hematol 83, 769-774. Fisher SG, Fisher RI (2004) The epidemiology of Hodgkin's lymphoma. Oncogene 23, 6524-34. Keresztes K, Takacs M, Horanyi M, Miltenyi Z, Illes A (2003) HCV and HGV infection in Hodgkin's disease. Pathol Oncol Res 9, 222-5. Lau GK, Yiu HH, Fong DY, Cheng HC, Au WY, Lai LS, Cheung M, Zhang HY, Lie A, Ngan R, Liang R (2003) Early is superior to deferred preemptive lamivudine therapy for hepatitis B patients undergoing chemotherapy. Gastroenterology 125, 1742-1749. Lok AS, Heathcote EJ, Hoofnagle JH (2001) Management of hepatitis B: 2000--summary of a workshop. Gastroenterology 120, 1828-1853. Lok ASF, McMahon BJ (2001) Chronic hepatitis B. Hepatology 34, 1225-1241. Lok ASF, McMahon BJ (2004) Chronic hepatitis B: update of recommendations. Hepatology 39, 857-861. Maguire CM, Crawford DH, Hourigan LF, Clouston AD, Walpole ET, Powell EE (1999) Case report, lamivudine therapy for submassive hepatic necrosis due to reactivation

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Wiwanitkit: Lamivudine primary prophylaxis of HBV reactivation in chronic HbsAg

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Cancer Therapy Vol 3, page 193 Cancer Therapy Vol 3, 193-200, 2005

Meat consumption and risk of colorectal cancer: a case-control study in Uruguay Research Article

Hugo Deneo-Pellegrini 1, Paolo Boffetta2, Eduardo De Stefani1,*, Alvaro L. Ronco3, Pelayo Correa4 and María Mendilaharsu1 1

Departamento de Patología, Instituto Nacional de Oncología, Montevideo, Uruguay. Unit of Environmental Cancer Epidemiology, International Agency for Research on Cancer, Lyon, France. 3 División de Epidemiología, Instituto de Radiología y Centro de Lucha contra el Cáncer, Hospital Pereira Rossell, Montevideo, Uruguay. 4 Department of Pathology, Louisiana State University Health Sciences, New Orleans, Louisiana, United States. 2

__________________________________________________________________________________ *Correspondence: Dr. Eduardo De Stefani, Avenida Brasil 3080 dep. 402, Montevideo, Uruguay.; Tel.: (598) 2 708 23 14; E-Mail: estefani@adinet.com.uy Key words: Meat consumption, colorectal cancer, fried meat, barbecued meat, heterocyclic amines Abbreviations: Age-standardized incidence rates, (ASIR); food frequency questionnaire, (FFQ); heterocyclic amines, (HCA); odds ratios, (OR’s); ninety five percent intervals (95 % CI).

Supported by a grant from the International Agency for Research on Cancer, Lyon, France Received: 29 March 2005; Accepted: 06 April 2005; electronically published: April 2005

Summary In the time period 1996-2003 a case-control study on the relation between meat consumption and colorectal cancer risk was conducted in Montevideo, Uruguay. The study included 556 newly diagnosed cases and 1112 hospitalized controls. Red meat was directly associated with a significant risk of colorectal cancer (OR for men 3.1, 95 % CI 1.85.3 and OR for women 2.0, 95 % CI 1.1-3.6). Also fried meat was positively associated with colon cancer in men (OR 2.2, 95 % CI 1.3-3.5), whereas barbecued meat displayed an elevated risk for women with rectal cancer (OR 2.3, 95 % CI 1.2-4.4). Finally, estimated intake of total heterocyclic amines was consistently associated with colorectal carcinoma (OR 2.0, 95 % CI 1.4-2.8, p-value for trend=0.0001). According to these results, red meat consumption (and estimated exposure to total HCAs) appears to be a strong risk factors for colorectal cancer. plant foods (Comisión Honoraria de Lucha contra el Cáncer, 1993; Matos and Brandani, 2002). This dietary pattern is probably responsible for the high rates observed in Uruguay. Thus, Uruguay is a rather convenient country to perform observational studies. Most case-control studies reported an increased risk of colorectal cancer associated with high consumption of meat, particularly red meat (Manousos et al, 1983; Marchand et al, 1997; La Vecchia et al, 1988; Tuyns et al, 1988; Benito et al, 1990; Gerhardsson de Verdier et al, 1991). Nevertheless, some prospective studies failed to confirm this effect (Phillips and Snowdon, 1985; Bostick et al, 1994; Goldbohm et al, 1994; Knekt et al, 1994). It has been suggested that recall bias could be responsible for this discrepancy between retrospective and prospective studies (Goldbohm et al, 1994). On the other hand, Potter has suggested that case-control studies have the advantage

I. Introduction Colorectal cancer is the second malignancy in frequency among Uruguayan population (Parkin et al, 2002). Age-standardized incidence rates (ASIR) are of 36.1 per 100,000 men and 25.7 per 100,000 women (Parkin et al, 1997). Moreover, in international comparisons among Latin American registries, Uruguayan rates occupy the first place, both in men and women (Parkin et al, 2002). According to several reviews (Potter, 1996; World Cancer Research Fund, 1997), diet is probably the major risk factor in colorectal cancer. In particular, high consumption of red meat and fat and low intake of vegetables and fruits are strongly associated with high risk of colorectal cancer (Potter, 1996; World Cancer Research Fund, 1997). Uruguayan population is characterized by high consumption of red meat and low consumption of 193


Deneo-Pellegrini et al: Meat consumption and risk of colorectal cancer eggs (boiled eggs, fried eggs, mayonnaise), desserts (milk with sugar, rice pudding, custard, marmalade, cake), grains (white rice, maize, polenta, pasta, white bread), high-fat foods (red meat, processed meat, dairy foods, eggs, desserts), raw vegetables (carrot, tomato, lettuce, onion), cooked vegetables (garlic, swiss chard, spinach, potato, sweet potato, beetroot, winter squash, cabbage, cauliflower, zucchini, red pepper), total vegetables (raw vegetables, cooked vegetables), citrus fruits (orange, tangerine), other fruits (apple, pear, grape, peach, banana, plum, fig, fruit cocktail), total fruits (citrus fruits, other fruits), total vegetables and fruits (total vegetables, total fruits), all tubers (potato, sweet potato), legumes (chickpea, kidney bean, lentil). Also red meat was analyzed by the cooking method: fried, barbecued and boiled (stewed) red meat. Finally, estimation of heterocyclic amines exposure was calculated according to the method of Sinha and Rothman (1997).

over prospective ones of a larger age range (Potter, 2000). This age range allowed both genetically and nongenetically participants in retrospective studies (Potter, 2000). Taking into account this discrepancy and the dietary pattern observed in Uruguay, we decided to conduct a case-control study on the relationship between meat consumption and colorectal cancer risk in this high-risk area.

II. Material and methods A. Selection of cases In the time period 1996-2003, all newly diagnosed and microscopically confirmed adenocarcinomas of the colon and rectum were considered eligible for this study. Five thousand and sixty five patients so diagnosed were identified in the four major hospitals of Montevideo, Uruguay. Nine patients refused the interview, leaving a final total of 556 cases of colorectal carcinomas (response rate 98.4 %). Cases were distributed by sex as follows: men (330, 59.3 %) and women (226, 40.7 %). Concerning subsite, 69 patients presented lesions of the right colon (12.4 %), 222 showed tumors of the left colon (39.9 %) and 265 patients presented carcinomas of the rectum (47.7 %).

D. Statistical analysis Relative risks of colorectal cancer, approximated by the odds ratios (OR’s) and its corresponding ninety five per cent confidence intervals (95 % CI), were estimated by multiple unconditional logistic regression (Breslow and Day, 1980). Comparisons between colonic and rectal carcinomas were carried out by polytomous (multinomial) logistic regression (Hosmer and and Lemeshow, 1989). The basic model included terms for age (categorical, 6 strata), sex (ordinal), residence (ordinal), urban/rural status (ordinal), education (categorical, 3 strata), body mass index (categorical, 4 strata), tobacco smoking (categorical, 5 strata), alcohol drinking (categorical, 5 strata), total energy intake (continuous), total vegetables and fruits (categorical, 4 strata) and total fat intake (categorical). Tests for trend were performed after entering categorical variables as ordinal (continuous) in the same model. Departure from the multiplicative model was determined by assessing the likelihood ratio test statistic. An alpha of 0.05 was used as the indicator of statistical significance and, accordingly 95 % CI’s were reported. All p-values were derived from two-sided statistical tests. All the calculations were done with the STATA programme (1999).

B. Selection of controls In the same time period and in the same hospitals, 1375 patients hospitalized for non-neoplastic diseases were considered eligible for the study. These patients presented diseases not related with tobacco smoking, alcohol drinking and without recent changes in their diets. Nineteen patients refused the interview, leaving a final number of 1356 (response rate 98.6 %). From this pool of potential controls, 1112 patients were frequency matched to the cases on age (in ten-years intervals), sex and residence (Montevideo, other counties). These patients presented the following diseases: eye disorders (317 patients, 28.5 %), abdominal hernia (235, 21.1 %), skin diseases (95, 8.5 %), acute appendicitis (75, 6.7 %), varicose veins (82, 7.4 %), urinary stones (62, 5.6 %), injuries (81, 7.3 %), blood disorders (55, 4.9 %), hydatid cyst (57, 5.1 %), fractures (36, 3.2 %) and bone diseases (17, 1.5 %).

III. Results The distribution of cases and controls by sociodemographic variables and selected risk factors is shown in Table 1. As expected from the frequency matched design, age, sex and residence were rather similar. Also the percentage of cases living in rural areas was similar to the percentage of rural controls. Education and income were similar in both series of patients. The proportion of cases with family history of colon cancer was significantly higher compared with controls (OR 4.7, 95 % CI 2.1-10.6). The estimates were similar for each tumor subsite (results not shown). Body mass index was similar in both series of patients and total energy intake was slightly higher among controls compared with cases, but the differences were not significant. Finally, cases and controls smoked and drank alcohol in similar amounts. Odds ratios of colorectal cancer for meat consumption in men are shown in Table 2. Beef consumption was positively associated with colon cancer risk (OR 3.4, 95 % CI 1.8-6.4). Similar estimates were observed for rectal cancer and for both tumor sites together (p-value for trend <0.0001). On the contrary, lamb intake was not associated with colorectal cancer risk. Red meat (beef plus lamb) was significantly associated with increased risk of colorectal adenocarcinoma (OR 3.1,

C. Questionnaire All participants were interviewed face-to-face in the hospitals by two trained social workers. They were administered a questionnaire which included the following sections: sociodemographics, a complete occupational history based in jobs and their duration, self-reported height and weight five years before the date of the interview, family history of cancer in firstdegree relatives, a complete tobacco smoking history, a complete alcohol drinking history, a complete maté drinking history (maté is the folk name of a herbal tea which is drunk every hot), menstrual and reproductive events and a food frequency questionnaire (FFQ) on 64 food items.This FFQ allowed the calculation of total energy intake and is considered as representative of the usual Uruguayan diet. Although it was not validated, it was tested for reproducibility with good results.

D. Definition of food groups The following food groups were created: red meat (beef, lamb), white meat (poultry, fish), processed meat (bacon, sausage, blood pudding, mortadella, salami, saucisson, hot dog, ham, salted meat), total meat (red meat, white meat, processed meat, liver), dairy foods (cheese, butter, whole milk, ice cream),

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Cancer Therapy Vol 3, page 195 95 % CI 1.8-5.3, p-value for trend <0.0001). Poultry consumption was directly associated with rectal cancer risk, but the trend was only marginally significant (OR 1.5, 0.9-2.3, p-value for trend=0.07). Both colon cancer and colorectal cancer were not associated with poultry

intake. Fish consumption, an uncommon item in the Uruguayan diet, was not associated with colorectal cancer risk. The same was observed for white meat consumption (poultry plus fish). Unexpectedly processed meat displayed an inverse association with colorectal

Table 1. Distribution of cases and controls by sociodemographic variables and selected risk factors. Cases Variable Age (in years)

Sex Residence Urban/rural status Education (years)

Income (US dollars)

Family history of colon cancer Body mass index

Total calories

Cigarettes per day

Alcohol drinking

Nยบ patients

Controls

Category 30-39 40-49 50-59 60-69 70-79 80-89 Males Females Montevideo Other counties Urban Rural 0-2 3-5 6+

Nยบ 10 47 92 179 187 41 330 226 285 271 461 95 141 232 183

% 1.8 8.4 16.5 32.2 33.6 7.4 59.4 40.6 51.3 48.7 82.9 17.1 25.4 41.7 32.9

Nยบ 20 94 185 358 374 81 660 452 565 547 903 209 307 386 419

% 1.8 8.4 16.6 32.2 33.6 7.3 59.4 40.6 50.8 49.2 81.2 18.8 27.6 34.7 37.7

<=140 141+ Missing

220 228 108

39.6 41.0 19.4

449 442 221

40.4 39.7 19.9

No Yes <=23.0 23.1-25.3 25.4-27.9 28.0+ <=1857 1858-2283 2284-2688 2689+ Never smokers 1-9 10-19 20-29 30+ Never drinkers 1-60 61-120 121-240 241+

517 39 141 142 137 136 124 144 127 167 124 30 38 59 55 163 65 37 30 11 556

93.0 7.0 25.4 25.5 24.6 24.5 22.3 25.9 22.8 29.0 40.5 9.8 12.4 19.3 18.0 53.3 21.2 12.1 9.8 3.6 100.0

1090 22 278 288 269 277 278 278 278 278 251 56 108 92 81 319 110 71 51 37 1112

98.0 2.0 25.0 25.9 24.2 24.9 25.0 25.0 25.0 25.0 42.7 9.5 18.4 15.6 13.8 54.2 18.7 12.1 8.7 6.3 100.0

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Deneo-Pellegrini et al: Meat consumption and risk of colorectal cancer Table 2. Odds ratios of colorectal cancer for meat consumption in men1.

Meat variable Colon/Rectum (cutpoints)2 /Controls3 Beef 24/34/199 208 51/61/212 365 76/84/249 p-value trend Lamb 54/59/201 0 46/46/213 12 51/74/246 p-value trend Red meat 28/33/182 260 52/63/220 377 71/83/258 p-value trend Poultry 54/58/262 24 63/66/204 52 34/55/194 p-value trend Fish 50/56/254 12 57/67/211 52 44/56/195 p-value trend White meat 58/65/263 52 53/64/203 104 40/50/194 p-value trend Processed meat 50/62/199 70 51/56/219 182 50/61/242 p-value trend Total meat 34/48/197 481 51/48/218 682 66/83/245 p-value trend

OR 1.0 2.1 3.4 0.0001 1.0 0.8 0.9 0.59 1.0 2.0 3.1 0.001 1.0 1.5 0.8 0.60 1.0 1.2 1.0 0.96 1.0 1.1 0.9 0.71 1.0 0.8 0.7 0.13 1.0 1.4 1.7 0.10

Colon 95 % CI

OR 1.0 1.8 2.6 0.001 1.0 0.7 0.9 0.94 1.0 2.1 3.1 0.001 1.0 1.5 1.5 0.07 1.0 1.4 1.3 0.28 1.0 1.3 1.2 0.31 1.0 0.8 0.7 0.16 1.0 0.9 1.5 0.07

1.2-3.8 1.5-4.5

0.5-1.3 0.5-1.4

1.1-3.6 1.6-6.2

0.9-2.2 0.5-1.3

0.8-1.9 0.6-1.6

0.7-1.7 0.5-1.4

0.5-1.2 0.4-1.1

0.8-2.4 0.9-3.0

Rectum 95 % CI

1.3-2.9 1.9-4.6

0.5-1.2 0.6-1.5

1.2-3.7 1.6-6.0

1.0-2.3 0.9-2.3

0.9-2.2 0.8-1.9

0.9-2.0 0.8-1.9

0.5-1.2 0.5-1.1

0.5-1.5 0.9-2.6

Both sites OR 95 % CI 1.0 1.9 2.9 <0.0001 1.0 0.8 0.9 0.52 1.0 2.0 3.1 <0.0001 1.0 1.5 1.1 0.35 1.0 1.3 1.1 0.44 1.0 1.2 1.1 0.63 1.0 0.8 0.7 0.06 1.0 1.1 1.6 0.03

1.3-2.9 1.9-4.6

0.5-1.1 0.7-1.3

1.3-3.2 1.8-5.3

1.1-2.1 0.8-1.6

0.9-1.9 0.8-1.6

0.9-1.7 0.7-1.5

0.6-1.1 0.5-1.0

0.7-1.7 1.0-2.4

1

Adjusted for age, residence, urban/rural status, education, family history of colon cancer in first-degree relatives, body mass index, tobacco smoking, alcohol drinking, total energy intake, total fat and total vegetables and fruits. 2 Cut-off points in servings per year. 3 Number of cases of colon, rectum and number of controls.

adenocarcinoma, which was marginally significant (pvalue for trend=0.06). Finally, total meat consumption displayed a moderate increase in risk of colorectal cancer (OR 1.6, 95 % CI 1.0-2.4, p-value for trend=0.03). Odds ratios of colorectal cancer for meat consumption in women are shown in Table 3. Beef consumption was positively associated with colorectal cancer risk (OR 1.7, 95 % CI 0.9-2.8, p-value for trend=0.05). As was observed in men, lamb intake was not associated with risk of colorectal adenocarcinoma. Red meat intake was associated with a moderate increase in

risk for female colon cancer. On the other hand, rectal carcinoma displayed an OR of 2.3 for high intake of red meat intake (p-value for trend=0.03). Also, ORâ&#x20AC;&#x2122;s for both sites together (colon and rectum) displayed an increased risk of 2.0 (95 % CI 1.1-3.6, p-value for trend=0.01). Poultry consumption increased the risk of female colon cancer (OR 1.7, 95 % CI 0.9-2.9, p-value for trend=0.03). This food item was not associated with rectal cancer. Also fish and white meat intakes were not associated with colorectal cancer risk. Processed meat consumption was associated with a modest increase in risk of female

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Cancer Therapy Vol 3, page 197

Table 3. Odds ratios of colorectal cancer for meat consumption in women1.

Meat variable (cutpoints)2 Beef 208 365 Lamb 0 12 Red meat 260 377 Poultry 24 52 Fish 12 52 White meat 52 104 Processed meat 70 182 Total meat 481 682

Colon/Rectum/ Controls3 41/24/171 55/30/159 45/31/122 p-value trend 49/28/169 58/35/158 34/22/125 p-value trend 45/24/188 54/32/151 42/29/113 p-value trend 27/26/108 45/25/167 69/34/177 p-value trend 41/29/116 55/26/160 45/30/176 p-value trend 31/26/108 52/25/167 58/34/177 p-value trend 46/30/171 45/23/152 50/32/129 p-value trend 40/23/173 57/33/153 44/29/126 p-value trend

OR 1.0 1.3 1.6 0.15 1.0 1.5 1.0 0.98 1.0 1.5 1.8 0.13 1.0 1.1 1.7 0.03 1.0 1.1 0.8 0.40 1.0 1.1 1.3 0.29 1.0 0.9 1.4 0.15 1.0 1.7 1.8 0.09

Colon 95 % CI

0.8-2.2 0.8-2.9

0.9-2.6 0.6-1.8

0.8-2.5 0.9-3.6

0.6-1.9 0.9-2.9

0.7-1.9 0.5-1.4

0.6-1.9 0.8-2.2

0.6-1.6 0.9-2.4

1.0-2.8 0.9-3.3

OR 1.0 1.2 1.9 0.06 1.0 1.6 0.9 0.76 1.0 1.6 2.3 0.03 1.0 0.6 0.8 0.65 1.0 0.7 0.7 0.30 1.0 0.5 0.8 0.69 1.0 0.8 1.3 0.15 1.0 1.5 1.7 0.06

Rectum % CI

0.6-2.4 0.9-3.9 0.05 0.8-2.8 0.5-1.7

0.8-3.2 1.0-5.3

0.3-1.1 0.4-1.5

0.4-1.3 0.4-1.3

0.3-1.0 0.4-1.4

0.4-1.4 0.7-2.4

0.8-2.9 0.8-3.5

Both sites OR95 % CI 1.0 1.3 1.7 1.0 1.5 0.9 0.85 1.0 1.5 2.0 0.01 1.0 0.8 1.2 0.21 1.0 0.9 0.8 0.28 1.0 0.8 1.1 0.63 1.0 0.9 1.4 0.11 1.0 1.6 1.7 0.03

0.8-2.0 0.9-2.8

1.0-2.4 0.6-1.5

0.9-2.4 1.1-3.6

0.5-1.3 0.8-1.9

0.6-1.5 0.5-1.2

0.5-1.3 0.7-1.6

0.6-1.4 0.9-2.1

1.1-2.5 1.1-2.9

1

Adjusted for age, residence, urban/rural status, education, family history of colon cancer in first-degree relatives, body mass index, tobacco smoking, alcohol drinking, total energy intake, total fat and total vegetables and fruits. 2 Cut-off points in servings per year. 3 Number of cases of colon, rectum and number of controls.

colorectal cancer, whereas total meat consumption showed a direct association with colorectal cancer risk (OR 1.7, 95 % CI 1.1-2.9, p-value for trend=0.03). The effect of fried meat in colorectal cancer is shown in Table 4. This cooking method of red meat was positively associated with colon cancer in men (OR 2.1, 95 % CI 1.3-3.1, p-value for trend=0.001). Fried meat was not associated with colon cancer among females. The difference between sexes was significant (p-value for

heterogeneity=0.04). Rectal carcinoma, at difference with colon cancer risk, was not associated with fried meat (pvalue for heterogeneity=0.0005). This heterogeneity between colon and rectal cancers precluded a reliable estimate for fried meat consumption. Odds ratios of colorectal cancer for barbecued meat consumption are shown in Table 4. Females with colon cancer displayed an increased risk of 1.5 (95 % CI 0.9-2.7, p-value for trend=0.08). Also, women were directly

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Deneo-Pellegrini et al: Meat consumption and risk of colorectal cancer associated with rectal carcinoma risk (OR 2.5, 95 % CI 1.3-5.0, p-value for trend=0.006). As expected from the previous results, women were directly associated with colorectal cancer (OR 1.9, 95 % CI 1.2-2.9, p-value for trend=0.005). Men with colon, rectum and colorectal cancers were not associated with risk for barbecued meat. The effect of boiled red meat is also shown in Table 4. Men presented lack of association in colon, rectal and colorectal cancers. Females afflicted with colon carcinoma displayed a modest increase in risk which was nonsignificant, whereas those who presented adenocarcinoma of the rectum showed an inverse association. The p-value

for heterogeneity between both tumor sites was significant (p=0.03). Finally, boiled meat consumption was not associated with colorectal carcinoma risk. Odds ratios of colorectal cancer for estimated heterocyclic amine intake are shown in Table 5. Total heterocyclic amines (HCAâ&#x20AC;&#x2122;s) were consistently associated with risks of colon, rectal, and colorectal cancers. Moreover, high intake of HCAâ&#x20AC;&#x2122;s were similar across genders (OR 1.9, 95 % CI 1.4-2.7, p-value for trend=0.0001). Further adjustment for protein and iron left the results unchanged (results not shown).

Table 4. Odds ratios of colorectal carcinoma for cooking method of red meat1. Males Method (cutpoints) Fried 2 78 130 Barbecued 24 64 Boiled 104 208 Females Method (cutpoints)2 Fried 78 130 Barbecued 24 64 Boiled 104 208

Colon/Rectum/ Controls 36/67/223 40/56/226 75/56/211 p-value trend 42/46/191 57/62/211 52/71/371 p-value trend 52/58/205 50/66/224 49/55/231 p-value trend Colon/Rectum /Controls 43/34/147 48/25/145 50/26/160 p-value trend 44/20/179 57/36/160 40/29/113 p-value trend 34/35/166 59/28/146 48/22/140 p-value trend

OR 1.0 1.0 2.1 0.001 1.0 1.2 0.9 0.47 1.0 0.9 1.0 0.96 Colon OR 1.0 0.9 0.9 0.85 1.0 1.5 1.5 0.008 1.0 1.7 1.5 0.14

Colon 95 % CI

0.6-1.7 1.5-3.4

0.7-1.9 0.5-1.4

0.6-1.4 0.6-1.6

95 % CI

0.6-1.5 0.5-1.5

0.9-2.4 0.9-2.7

1.0-2.9 0.9-2.6

1

OR 1.0 0.7 0.8 0.28 1.0 1.2 1.1 0.59 1.0 1.0 0.9 0.93 Rectum OR 1.0 0.6 0.6 0.09 1.0 2.2 2.5 0.006 1.0 0.7 0.6 0.17

Rectum 95 % CI

OR

Both sites 95 % CI

0.7-1.6 0.6-1.5

1.0 0.8 -3 1.0 1.2 1.0 0.93 1.0 0.9 0.9

95 % CI

0.98 Both sites OR 95 % CI

0.5-1.1 0.5-1.2

0.7-1.8 0.7-1.8

0.6-1.2

0.8-1.7 0.7-1.5

0.7-1.4 0.7-1.4

1.0 0.3-1.1 0.3-1.0

1.2-4.1 1.3-5.0

0.4-1.3 0.3-1.2

0.8 0.8 0.29 1.0 1.7 1.9 0.005 1.0 -

0.5-1.2 0.5-1.2

1.1-2.6 1.2-2.9

- 4

Adjusted for age, residence, urban/rural status, education, family history of colon cancer among first-degree relatives, tobacco smoking, alcohol drinking, total energy intake, total vegetables and fruits, total fat intake and for each other. 2-In servings per year. 3 p-value for heterogeneity 0.0005 4 p-value for heterogeneity 0.03

198


Cancer Therapy Vol 3, page 199 Table 5. Odds ratios of colorectal cancer for estimated intake of heterocyclic amines1. Colon Males Cutpoints 160.5 2 258.6

Colon/Rectum/ Controls 68/203 112/216 150/241 p-value trend

Females Cutpoints 160.5 2 258.6

Colon/Rectum /Controls 59/168 81/154 86/130 p-value trend

OR 1.0 1.5 1.8 0.06

Rectum

95 % CI 0.9-2.6 0.9-3.4

OR 1.0 1.4 2.0 0.009

Colon OR 1.0 1.7 2.3 0.01

95 % CI 0.8-2.3 1.2-3.6 Rectum

95 % CI 0.9-2.9 1.2-4.3

OR 1.0 1.7 1.7 0.17

95 % CI 0.9-3.1 0.8-3.5

Both sites OR 95 % CI 1.0 1.5 0.9-2.2 1.9 1.2-3.0 0.004 Both sites OR 1.0 1.7 2.0 0.01

95 % CI 1.1-2.6 1.2-3.4

1

Adjusted for age, residence, urban/rural status, education, family history of colon cancer among first-degree relatives, tobacco smoking, alcohol drinking, total energy intake, total vegetables and fruits and total fat intake. 2 Nanograms/day.

1991; Sinha et al, 1998, 1999). Most of these studies consistently showed a significant increase in risk of colorectal and breast carcinomas. In the present study fried meat was associated with an increased risk of colon cancer, whereas rectal carcinoma displayed a null effect. On the other hand, barbecued meat was a strong risk factor for female rectal carcinoma, whereas the effect on colon cancer was rather small. This discrepancy between fried and barbecued meat, two proxy variables of HCA’s exposure, could be due to unknown factors or, taking into account the numerous comparisons made, be a chance finding. In fact, doneness of red meat is a better proxy variable of HCA’s exposure than cooking methods (Sinha et al, 1998). We also employed an estimated amount of total heterocyclic amines (HCA’s). High exposure to HCA’s was associated with a two-fold increase in risk. This finding, albeit subject to misclassification, replicates findings in the recent review of Sinha (2002). Like other case-control studies, our report has limitations. Selection bias is almost impossible to eliminate. In order to minimize its effect, we frequency matched cases and controls on age, sex and residence. Recall bias is always a potential problem in retrospective studies on diet and cancer. Since cases and controls were hospitalized it is probable that both series of participants were subjected to similar forces of recall. Furthermore, cases and controls were drawn from a low socioeconomic strata of the Uruguayan population. This segment of the population is mostly unawere of the potential danger of high consumption of red meat. Also, both interviewers were participating in a large multisite case-control study on environmental factors and several cancer sites. Therefore, it is unlikely that the interviewers have a precise knowledge of the role of diet in colorectal cancer. Our study has also strengths. Perhaps the most important strength is the high response rate observed both in cases

V. Discussion According to the results of our study, beef and red meat were associated with a significant increase in risk for colorectal cancer in men and women. These results were rather similar in colon and rectal cancer. These findings are in accordance with most of the previous case-control and prospective studies (Manousos et al, 1983; Tuyns et al, 1988; La Vecchia et al, 1988; Benito et al, 1990; Gerhardsson de Verdier et al, 1991; Potter, 1996; Le Marchand et al, 1997; World Cancer Research Fund, 1997; Potter, 2000). Moreover, we included a term for total fat intake in all models and, whereas fat displayed a null effect, red meat increased its risk. Aside from its content in fat, red meat is a rich source of iron and protein. Adjustment for these variables left the OR’s for red meat without any change, Thus, other components of red meat should be responsible for the deleterious effect of this food item. Strong candidates for explaining the carcinogenic effect of red meat in colorectal cancer risk are the heterocyclic amines (HCA). These chemicals are formed in muscle meat cooked by frying or barbecuing (Weisburger, 2002). More precisely, the presence of creatine is required in the formation of HCA’s. The carcinogenicity was discovered by Sugimura and Sato in experimental animal studies (Sugimura 2000). Since then, numerous experimental and epidemiological studies have increased the consistence of the noxious effect of HCA’s in colorectal cancer (De Stefani et al, 1997; Sinha et al, 1999, 2001; Le Marchand et al, 2002; Nowell et al, 2002). Moreover, HCA’s have been suggested as carcinogens for several cancer sites, like breast and lung (De Stefani et al, 1997; Sinha et al, 1998). In fact, some case-control studies have found an increased risk for colorectal cancer and breast cancer, after using an estimated variable for PhIP (25, 28). Other studies have employed proxy variables like type of cooking and doneness of meat consumed (Gerhardsson de Verdier et al, 199


Deneo-Pellegrini et al: Meat consumption and risk of colorectal cancer Le Marchand L, Hankin J, Pierce LM et al (2002) Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii. Mutat Res 506-507, 205-214. Le Marchand L, Wilkens L, Hankin J et al (1997) A case-control study of diet and colorectal cancer in a multiethnic population in Hawaii (United States): lipids and foods of animal origin. Cancer Causes Control 8, 637-648. Manousos O, Day NE, Trichopoulos D et al (1983) Diet and colorectal cancer: A case-control study in Greece. Int J Cancer 32, 1-5. Matos E and Brandani A (2002) Review on meat consumption and cancer in South America. Mutat Res 506-507, 243-249. Nowell S, Coles B, Sinha R et al (2002) Analysis of total meat intake and exposure to individual heterocyclic amines in a case-control study of colorectal cancer: contribution of metabolic variation to risk. Mutat Res 506-507, 175-185. Parkin DM, Whelan SL, Ferlay J, Teppo, L and Thomas DB) Cancer Incidence in Five Continents. Volume VIII. (2002) IARC scientific publications N° 155. Lyon, IARC. Phillips RL and Snowdon DA (1985) Dietary relationships with fatal colorectal cancer among Seventh-Day Adventists. J Natl Cancer Inst. 74, 307-317. Potter JD (1996) Nutrition and colorectal cancer. Cancer Causes Control 7, 127-146. Potter JD (2000) Colorectal neoplasia and meat: Epidemiology and mechanisms. In I.T. Johnson and G.R.Fenwick (editors): Dietary anticarcinogens and antimutagens. Chemical and Biological Aspects. Royal Society of Chemistry. Sinha R (2002) An epidemiologic approach to studying heterocyclic amines. Mutat Res 506-507, 197-204. Sinha R and Rothman N (1997) Exposure assessment of heterocyclic amines (HCAs) in epidemiologic studies. Mutat Res 376, 195-202. Sinha R Kulldorff M, Chow WH, Denobile J and Rothman N (2001) Dietary intake of heterocyclic amines, meat derived mutagenic activity, and risk of colorectal adenoms. Cancer Epidemiol Biomarkers Prev 10, 559-562. Sinha R, Chow WH, Kulldorff M et al (1999) Well-done grilled red meat increases the risk of colorectal adenomas. Cancer Res 59, 4320-4324. Sinha R, Kulldorff M, Curtin J et al (1998) Fried, well-done red meat and risk of lung cancer in women (United States). Cancer Causes Control 9, 621-630. Stata Reference Manual Release (1999) Version 6 Stata Press College Station Texas Sugimura T (2000) Nutrition and dietary carcinogens. Carcinogenesis 21, 387-395. Tuyns AJ, Kaaks R and Haelterman M (1988) Colorectal cancer and the consumption of foods: a case-control study in Belgium. Nutr Cancer 11, 189-204. Weisburger JH (2002) Comments on the history and importance of aromatic and heterocyclic amines in public heath. Mutat Res 506-507, 9-20. World Cancer Research Fund (1997) Food nutrition and the prevention of cancer: a global perspective American Institute for Cancer Research Washington DC.

and controls. In summary, the present study gives further support to the noxious effect of red meat in colorectal carcinoma. This strong association was observed for high consumption of beef and red meat. The positive association was observed in colon and rectal cancers and in men and women. Also, the cooking method, that is the exposure to fried and barbecued meat, was associated with increased risks for colorectal cancer. Nevertheless, these associations were inconsistent by tumor site and by gender, suggesting that doneness is a better proxy variable of HCA’s than cooking method. Finally, estimated exposure to total HCA’s was consistently associated with high OR’s for colorectal cancer. Further studies on effect modifiers of the carcinogenic risk of red meat and HCA’s are needed.

References Benito E, Obrador A, Stiggelbout A, et al (1990) Nutritional factors in colorectal cancer risk: A case-control study in Majorca: I Dietary factors. Int J Cancer 45, 69-76. Bostick RM, Potter JD, Kushi LH, et al (1994) Sugar, meat, and fat intake, and non-dietary risk factors for colon cancer incidence in Iowa women (United States). Cancer Causes Control 5, 38-52. Breslow NE and Day NE (1980) Statistical methods in cancer research. Volume 1-The analysis of case-control studies. IARC Scientific Publications N° 32. Lyon, IARC. Comisión Honoraria de Lucha contra el Cáncer (1993) Conocimientos creencias actitud y prácticas sobre cáncer Encuesta de población Cooperación técnica OPP/BID/PNUD Comisión Honoraria de Lucha contra el Cáncer (In Spanish) De Stefani E, Deneo-Pellegrini H, Mendilaharsu M and Ronco A (1997) Meat intake, heterocyclic amines and risk of colorectal cancer: a case-control study in Uruguay. Int J Oncol 10, 573-580. De Stefani E, Ronco A, Mendilaharsu M et al (1997) Meat intake, heterocyclic amines, and risk of breast cancer: a casecontrol study in Uruguay. Cancer Epidemiol Biomarkers Prev 6, 573-581. Gerhardsson de Verdier M, Hagman U, Peters RK et al (1991) Meat, cooking methods and colorectal cancer: a case-referent study in Stockholm. Int J Cancer 49, 520-525. Goldbohm RA, van den Brandt PA, van’t Veer P et al (1994) A prospective cohort study on the relation between meat consumption and the risk of colon cancer. Cancer Res. 54, 718-723. Hosmer DW, and Lemeshow S (1989) Applied logistic regression. New York: John Wiley & Sons. Knekt P, Steineck G, Jaervinen R et al (1994) Intake of fried meat and risk of cancer: a follow-up study in Finland. Int J Cancer 59, 756-760. La Vecchia C, Negri E, Decarli A et al (1988) A case-control study of diet and and colorectal cancer in northern Italy. Int J Cancer 41, 492-498.

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Cancer Therapy Vol 3, page 201 Cancer Therapy Vol 3, 201-218, 2005

FDG PET and PET-CT in uterine cancers Review Article

Tarik Z Belhocine1,* and Perry W Grigsby2 1

Department of Nuclear Medicine, Jules Bordet Cancer Institute, PET-CT Unit, Brussels, Belgium Department of Radiation Oncology/Nuclear Medicine, Mallinckrodt Institute of Radiology, St.Louis, MO, USA

2

__________________________________________________________________________________ *Correspondence: Tarik Z Belhocine MD., Ph.D., Department of Nuclear Medicine, Jules Bordet Cancer Institute, PET-CT Unit, Brussels, Belgium; Tel: 322-541-3093; Fax: 322-541-3094; e-mail: tarikbelhocine@yahoo.fr Key words: FDG, PET, PET/CT, cervical cancer, endometrial cancer Abbreviations: 18F-fluoro-2-deoxy-D-glucose positron emission tomography, (FDG PET); body weight, (BW); gynecologic oncology group, (GOG); intensity modulated radiation therapy, (IMRT); lymphatic mapping and sentinel lymphadenectomy, (LM/SL); magnetic resonance imaging, (MRI); ordered-subsets expectation expectation maximization (OSEM); positron emission tomography, (PET); positron emission tomography: computed tomography, (PET/CT) Received: 28 February 2005; Accepted: 04 April 2005; electronically published: April 2005

Summary Despite early diagnosis of uterine cancers and more efficient combined therapy modality, the optimal management of such gynecological malignancies remains a challenging debate. In the last few years, increasing data highlighted the potential of positron emission tomography (PET) using 18F-fluoro-2-deoxy-D-glucose (FDG) in various cancer types including cervical and endometrial cancers. Accordingly, metabolic imaging was found useful for localization of primary tumor, staging of nodal and distant metastases, and detection of symptomatic and asymptomatic recurrences. Besides, pilot studies pointed out on the prognostic value of FDG PET in treated and untreated cervical cancers. In the present review, we evaluated the clinical added-value that may offer a high-end imaging modality such as FDG PET, eventually combined with CT/MRI, in pre-treatment staging and post-treatment surveillance of uterine cancers. FDG tracer (Kikuchi Y et al, 1972; Marshall MJ et al, 1978; Marshall MJ et al, 1979; Mendez LE et al, 2002; Yen TC et al, 2004). Accordingly, increasing evidence showed the clinical advantages offered by metabolic imaging in the diagnosis, staging, re-staging, and treatment monitoring of such gynecological malignancies. In the present chapter, we summarize the current knowledge on FDG PET in cervical and endometrial cancer. We also highlight the perspectives expected from PET/CT image fusion in the management of women suffering from uterine cancers.

I. Introduction 18

During the last decade, the introduction of F-fluoro2-deoxy-D-glucose positron emission tomography (FDG PET) in the management of various cancers has been shown to significantly improve the pre-treatment staging as well as the post-treatment follow-up of disease (Gambhir et al, 2001; Maisey et al, 2002). For instance, metabolic imaging findings may precede by several weeks conventional imaging results. Growing data also indicate that FDG PET is not just a valuable diagnostic tool, but it may yield unique prognostic information. Moreover, FDG PET may detect active disease in asymptomatic patients, which is critical in terms of treatment efficacy and prolonged survival. The recent development of combined PET/CT devices opened new perspectives in nuclear oncology by allowing an anatometabolic characterization of cancers (Townsend DW et al, 2003). Of particular interest is the clinical added-value of PET/CT in terms of diagnostic accuracy and the potential expected for targeted therapy (Hany TF et al, 2002). Because uterine cancers exhibit an overexpression of glucose transporter-1 (Glut-1) and increased activity of key-enzymes of the glycolytic pathway (Hexokinases I and II), they avidly take up the

II. Cervical cancer Cervical cancer is a public health problem. Worldwide, the disease ranks second to breast cancer in incidence (Parkin et al, 2001). In the United States, about 10.370 cases of invasive cervical cancer will be diagnosed in 2005 with a related mortality estimated to 3710 women (ACS key-statistics, 2005). Besides early screening of cervical cancer, the optimal management of women with invasive disease may nowadays benefit from more efficient treatments (Keys et al, 1999; Rose et al, 1999; Peters et al, 2000; Chi et al, 2001; Grigsby et al, 2001). To this end, we addressed the 201


Belhocine and Grigsby: 18FDG PET and PET-CT in uterine cancers actual potential of metabolic imaging, eventually combined with morphological imaging, in the clinical work-up of cervical cancer.

study, GLUT-1 overexpression was intimately correlated to the malignant transformation of cervical tissues from pre-cancerous dysplastic patterns (i.e. CIN 1, CIN 2, and CIN 3) to cancerous forms. Conversely, in benign cervical epithelium, only weak to moderate GLUT-1 expression was noted, which was related to the proliferating basal cell layers of the mucosa, inflammatory changes, squamous atypia, immature metaplasia, or basal cell hyperplasia (Rudlowski et al, 2003). Thus, human cervical carcinomas and their metastases selectively overexpress GLUT-1, thereby, providing a strong rationale to the feasibility of FDG imaging (Mendez et al, 2002; Rudlowski et al, 2003; Yen et al, 2004). Although pre-neoplastic patterns overexpress GLUT-1, they are not detected by FDG PET; these dysplastic forms likely escape the spatial resolution of current PET devices. Other biological and morphological factors may actually contribute to the high FDG-avidity of invasive cervical cancers such as tumor vascularization, hexokinase expression, hypoxia, proliferative activity, and tumor size (Marshall et al, 1978; Dellas et al, 1996; Bar et al, 2001; Morgan et al, 1998). Metabolic imaging may detect all tumor types including squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, small-cell carcinoma, and clear-cell carcinoma. As one may expect, FDG PET is most often limited by its spatial resolution, which prevents the detection of FIGO stage 0 in situ tumors and FIGO stage IA lesions. Besides the tumor size and the FDG PET methodology, the few false negatives reported in stages IIIII disease may be eventually explained by a low GLUT-1 expression (Table 2). To improve the signal-tobackground ratio, some authors suggested a continuous bladder irrigation using a Foley urinary catheter (Sugawara et al, 1999; Koyama et al, 2003). Others have showed the utility of post-void images to better localize

A. Pre-treatment staging In pre-treatment setting, the International Federation of Gynecology and Obstetrics (FIGO classification) (Table 1) is commonly used for the staging of cervical cancer (Benedet et al, 2000). Such a staging system provides practical guidelines to assess the disease extent locally as well as to adjacent and distant organs. However, the FIGO clinical staging has major limitations. Firstly, the lack of diagnostic accuracy in comparison to the surgical staging may lead to underestimate the scale of disease (Van Nagell et al, 1971; Hricak et al, 1996; Yu et al, 1997). Secondly, nodal involvement, an important prognostic factor, is not taken into account by the FIGO clinical staging, which may affect the treatment planning (Kupets et al, 2001). Below, we have analyzed the performances of FDG PET for assessing the key-parameters that may actually affect the treatment and the prognosis of women with newly diagnosed cervical cancers.

1. T staging As a rule, cervical cancers avidly take up the glucose tracer with a sensitivity ranging from 85% to 100% (Table 2). Moreover, FDG PET was found as accurate as magnetic resonance imaging (MRI) (MRI) in localizing the primary tumor (Narayan et al, 2001; Belhocine et al, 2002; Narayan et al, 2003). Surprisingly, in a prospective study of 219 cervical cancers, the accuracy of metabolic imaging to localize primary tumors (n=75) was not significantly impaired by the patients' glycemia at the time of the PET study (Chang et al, 2005). In a recent basic

Table 1. Carcinoma of the Cervix Uteri: FIGO Staging Classification (September 2000). FIGO Stages 0 I IA IA1 IA2 IB IB1 IB2 II IIA IIB III IIIA IIIB IVA IVB

Definitions Carcinoma in situ (pre-invasive carcinoma) Cervical carcinoma confined to uterus Invasive carcinoma diagnosed only by microscopy. All macroscopically visible lesions – even with superficial invasion – are Stage IB. Stromal invasion ! 3.0 mm in depth and ! 7.0 mm in horizontal spread. Stromal invasion > 3.0 mm and ! 5.0 mm with horizontal spread ! 7.0 mm. Clinically visible lesion confined to the cervix or microscopic lesion > IA2. Clinically visible lesion ! 4.0 cm in greatest dimension. Clinically visible lesion > 4.0 cm in greatest dimension. Tumor invades beyond the uterus but not to pelvic wall or to lower third of the vagina. Without parametrial invasion. With parametrial invasion. Tumor extends to pelvic wall and/or involves lower third of vagina and/or causes hydronephrosis or nonfunctioning kidney. Tumor involves lower third of vagina – no extension to pelvic wall. Tumor extends to pelvic wall and/or causes hydronephrosis or non-functioning kidney. Tumor invades mucosa of bladder or rectum and/or extends beyond true pelvis. Distant metastasis

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Cancer Therapy Vol 3, page 203 Table 2. T Staging of cervical cancer with FDG PET: Literature Data Authors

Years

Primary Staging

FIGO Stages

Tumor Types

Rose et al

1999

32/32

IIB-IVA

Sugawar a et al Umesaki et al Reinhard t et al Kerr et al Kühnel et al Grigsby et al

1999

17/21

IB-IVA

2000

09/13

IA-IIIB

SqCC, ASC, ADC SqCC, ASC, ADC SqCC, ADC

2001

35/35

IB-IIA

2001 2001

10/13 15/15

IB-IVB IB-IIB

2001

101/101

IA-IVB

Narayan et al Nakamot o et al Belhocine et al Singh et al Narayan et al Tsai et al

2001

24/27

IB-IVB

2002

20/20

IB-IVA

2002

22/60

IA-IVA

2003

47/47

IIIB

2003

69/70

IB-IVB

2004

19/19

IB-IIIB

SqCC, ADC, non-SqCC SqCC SqCC, ASC, ADC SqCC, ASC, ADC, CCA, SmCC SqCC, ADC, CCA SqCC, ASC, ADC SqCC, ADC SqCC, ASC, ADC SqCC, ADC SqCC, ASC, ADC

Primary Tumors Detection Rates (SUV-BW max) 30/32 = 93% 16/17 = 94% * (mean SUV = 10.31) 09/09 = 100% (mean SUV = 7.7) 35/35 = 100%

False-Negative Results 2 FN: SqCC, IIB, IIIB 1 FN: SqCC, IIB FN: none FN: none

10/10 = 100% 15/15 = 100%

FN: none FN: none

100/101 = 99%

1 FN: SqCC, IA2

24/24 = 100%

FN: none

17/20 = 85% * (mean SUL = 7.77) 20/22 = 91% (mean SUV = 9.0) 47/47 = 100%

3 FN: SqCC, IB, IIB, IIIB 2 FN: SqCC, IA

69/69 = 100%

FN: none

18/19 = 95% (mean SUV > 8) †

1 FN: IIA

FN: none

Abbreviations: SqCC: squamous cell carcinoma, ASC: adenosquamous carcinoma, ADC: adenocarcinoma, CCA: clear-cell carcinoma, SmCC: small-cell carcinoma, SUV-BW: standardized uptake value-body weight (or decay-corrected tissue activity divided by the injected dose per patient body weight). SUL: SUV-BW corrected by predicted lean body mass. † Moderately differentiated tumors had mean SUV max =11.56 (8.0-18.0) and poorly differentiated tumors had mean SUV max = 17.96 (8.1-41.7). * Overall results including post-void images and semi-quantitative analysis.

poorly FDG-avid primary tumors (Sugawara et al, 1999; Nakamoto et al, 2002). In our experience, however, no particular acquisition (i.e. dynamic sequences, late images, abdominal and pelvic fields of view) was needed to accurately localize the primary tumor. Rather, we routinely use a static whole-body acquisition with eventually the administration of diuretics. Such a protocol allows the detection of primary FDG-avid sites with a high confident using a simple qualitative interpretation, especially from the transaxial and sagittal slices (Figure 1). Importantly, the use of iterative methods for data reconstruction (i.e. OSEM) helps reduce streak artifacts, especially in the vicinity of the bladder. Additionally, semi-quantitative analysis of FDG uptake showed that cervical cancers mostly present with a high mean standardized uptake value-body weight (or decay-corrected tissue activity divided by the injected dose per patient body weight (SUV-BW) ranging from 7.7 to 10.3. Interestingly, in a series of 53 newly diagnosed squamous cell type cervical cancers, the peak SUV values (pSUV) were measured from the primary tumor mass and compared to patients’ survival (Jang et al, 2002). As a

result, the survival of patients with high pSUV (>13) was worse than the low pSUV group (<13) to a p value of 0.053 (Hazard ratio=6.8; 95% CI=0.97-68.7). Overall, the 2-year survivals were 76.0% and 92.3% for the high and the low pSUV groups, respectively. Based on our experience and others, however, no statistical correlation was found between the SUV-BW and the FIGO stage of disease (Sugawara et al, 1999; Belhocine et al, 2002). Similarly, in a recent study, no statistical correlation was found between the FDG uptake and the primary tumor size measured by MRI/CT. So far, a relationship was evidenced between the SUV-BW and the degree of differentiation with no statistical significance (Narayan et al, 2003). Data from literature regarding the performances of FDG PET for T staging are summarized in Table 2. Owing to its sub-centimetric spatial resolution, metabolic imaging cannot assess key-prognostic factors such as tumor volume, stromal invasion, and parametrial involvement. Similarly, FDG PET is inherently limited for the detection of microscopic tumor spreading to adjacent organs (i.e. vagina, uterine corpus, vulva, bladder, rectum). On the other hand, a number of normal and 203


Belhocine and Grigsby: 18FDG PET and PET-CT in uterine cancers

Figure 1. A typical aspect of cervical cancer in FDG PET. Metabolic imaging showed an intense FDG uptake at the level of the primary tumor and no other FDG-avid abnormality on the rest of the body. Tm: tumor; B: bladder; T: Transaxial; S: sagittal; C: coronal.

abnormal conditions may impair the diagnostic accuracy of FDG PET for loco-regional staging of uterine cancers; diagnostic pitfalls including ureteral stasis, bowel retention, as well as inflammatory or infectious changes secondary to cervical stenosis, endometriosis, and irradiation, which may be falsely interpreted as FDG tumor uptake (Nakamoto et al, 2002; Jeffry et al, 2004; Lerman et al, 2004). Of note, endometrial and ovarian FDG uptakes may be seen around the menstrual and ovulatory phases (Lerman et al, 2004). Therefore, physiological and benign FDG uptake patterns in the gynecological sphere (ovaries, endometrium, and cervix) should be kept in mind when performing a PET study for a uterine cancer, especially in pre-menopausal women. Also, in pre-treatment setting, MRI remains the modality of choice for staging loco-regional extension of disease. Beyond the FIGO clinical staging, FDG PET and MRI may provide complementary information on primary tumor status, thereby, optimizing the clinical utility of each modality (Belhocine et al, 2002, Narayan et al, 2003). Also, in pre-treatment setting, MRI remains the modality of choice for staging loco-regional extension of disease. Beyond the FIGO clinical staging, FDG PET and MRI may provide complementary information on primary tumor status, thereby, optimizing the clinical utility of each modality (Belhocine et al, 2002, Narayan et al, 2003). As a practical consequence, for in situ and localized cervical cancers (stages IA-IIB), FDG PET cannot meet the current FIGO staging criteria. Nonetheless, in these patients with early FIGO stages, a negative whole-body FDG PET outside the pelvis, especially for nodal staging (Grigsby et al, 2001; Grigsby et al, 2005), may be of the utmost importance for treatment planning and prognosis. Because the pre-test likelihood of tumor spreading is higher in FIGO stages " IIB, the contribution of metabolic imaging may be particularly significant in more advanced forms of disease by staging suspected and unsuspected nodal and visceral metastases (Narayan et al, 2003). Overall, FDG PET is likely more suitable for UICC/TNM staging system, at least for N and M staging (Table 3). Fused images using either PET/MRI software systems or

PET/CT hybrid devices, may help better assess T staging (Lemke et al, 2004; Yap et al, 2004). So far, beyond the anatomical tumor characterization, metabolic imaging may provide functional insights on tumor behavior (Miller et al, 2001, 2002, 2003).

2. N staging In lymphophilic malignancies such as cervical cancer, nodal status is a key-step in pre-treatment staging (Anderson et al, 1997; Chi et al, 2001). Accordingly, the 5-year overall survival dramatically drops from 87-92% in N0 patients to 42-67% in those of women classified as N1 (Buchsbaum et al, 1979; Tanaka et al, 1984). Also critical is the level of nodal involvement (i.e. pelvic vs. para-aortic vs. supra-clavicular) for treatment planning and prognosis (Benedetti-Panici et al, 1996; Michel et al, 1998; Sakuragi et al, 1999). For instance, low-risk patients with localized cervical cancer but no extra-pelvic nodal involvement may be efficiently managed by pelvic surgery and/or irradiation; thus, avoiding the side effects of systemic chemotherapy. On the other hand, those of patients with FDG-avid para-aortic sites may best benefit from combined therapy modality including platinum-based chemotherapy plus radiation (Landoni et al, 1997; Varia et al, 1998; Rose et al, 1999; Sedlis et al, 1999; Whitney et al, 1999; Grigsby et al, 2001). Also important is the utility of whole-body FDG PET to re-orient the treatment intent from curative to palliative, especially in patients with disseminated disease including supra-clavicular sites (Grigsby et al, 2001; Tran et al, 2003). Hence, an accurate imaging work-up is needed for staging the full extent of nodal disease, thereby, determining the patients' prognosis and selecting the most appropriate treatments. Cumulative data from literature including prospective and retrospective series with variable levels of confidence highlighted the added-value of FDG PET for detecting nodal metastases at each stage of disease spreading (Table 4). The strength of metabolic imaging primarily relies upon its capability to assess, in one session, pelvic, paraaortic, mediastinal, axillary, and

204


Cancer Therapy Vol 3, page 205 supra-clavicular lymph node metastases with a high sensitivity (Figure 2). So far, the performances of FDG PET for detection of nodal involvement may vary from a level to another. For instance, the sensitivity of metabolic imaging is optimal for localizing supra-clavicular metastases (Tran et al, 2003), and most often excellent for the detection of para-aortic metastases (Rose et al, 1999; Grigsby et al, 2001; Yeh et al, 2002; Belhocine et al, 2003; Lin et al, 2003; Ma et al, 2003; Tsai et al, 2004). At the pelvic level, the sensitivity of FDG PET is also high for staging nodal metastases, knowing that microscopic involvement may escape the spatial resolution of current PET devices (Benedetti-Panici et al, 1996; Reinhardt et al,

2001; Kühnel et al, 2001; Williams et al, 2001; Belhocine et al, 2002). Interestingly enough, the use of late imaging (3 hours post-injection) was found particularly useful for detection of additional lymph node metastases compared to early imaging (40 min post-injection), especially at the level of lower para-aortic, iliac, and pelvic chains (Ma et al, 2003; Yen et al, 2003). Such a dual-time protocol may also help reduce false positive results (i.e. ureteral stasis, inflammatory nodes). Nonetheless, in patients with a low pre-test likelihood of nodal involvement, especially in early stage disease, the use of lymphatic mapping and sentinel lymphadenectomy (LM/SL) may be an attractive

Table 3. Carcinoma of the Cervix Uteri: FIGO Stage Grouping and UICC/TNM Classification FIGO Stages 0 IA1 IA2 IB1 IB2 IIA IIB IIIA IIIB

IVA IVB

UICC Classification N N0 N0 N0 N0 N0 N0 N0 N0 N1 N1 N1 Any N Any N Any N

T Tis T1A1 T1A2 T1B1 T1B2 T2A T2B T3A T1 T2 T3A T3B T4 Any T

M M0 M0 M0 M0 M0 M0 M0 M0 M0 M0 M0 M0 M0 M1

Table 4. N Staging of Cervical Cancer with FDG PET: Literature Data Authors

Years

FIGO Stages

1999 1999

Primary Staging 32/32 11/23

Rose et al Grigsby et al Sugawara et al Umesaki et al Reinhardt et al Kerr et al Kühnel et al Grigsby et al Narayan et al Belhocine et al Yeh et al Lin et al Ma et al, Yen et al,

1999 2000 2001 2001 2001 2001 2001 2002 2002 2003 2003 2003

17/21 09/13 35/35 10/13 15/15 101/101 24/27 22/60 42/42 50/50 104/104 47/135

IB-IVA IA-IIIB IB-IIA IB-IVB IB-IIB IA-IVB IB-IVB IA-IVA IIB-IVA IIB-IVA IB-IVB IB2-IVB

Tsai et al,

2004

19/19

IB-IIIB

IIB-IVA IA-IVA

FDG-avid Lymph FDG-avid Metastases Nodes missed my CT/MRI Pv – Pa Pv – Pa Pv – Pa – Sc Pv – Pa – Sc Pv – Pa Pv Pv – Pa Pv – Pa – Ax Pv – Pa Pv – Pa – Sc Pv – Pa Pv – Pa – Md – Sc Pa Pa Pa Ig – Pv – Ic – Pa Md – Sc Ig – Pv – Pa Md – Sc – Nc

Pv – Pa Pv Pv – Pa Pv Pv – Pa Pv – Pa – Sc Pv – Pa Pa – Md – Sc Pa Pa Pa Ig – Pv – Ic – Pa Md – Sc Ig – Pv – Pa Md – Sc – Nc

Abbreviations: Axillary, (Ax); Iliac, (Ic); inguinal, (Ig); Mediastinal, (Md); Neck, (Nc); Para-aortic, (Pa); Pelvic, (Pv); Supra-clavicular, (Sc)

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Belhocine and Grigsby: 18FDG PET and PET-CT in uterine cancers

Figure 2. Pre-treatment staging of cervical cancer with FDG PET. In one session, metabolic imaging revealed multiple FDG-avid sites including the primary tumor and bilateral inguinal, iliac, para-aortic, and supra-clavicular nodal metastases. The patient was upstaged from FIGO stage IB to PET stage IVB (T+N+M+), thereby, re-orienting the treatment from hysterectomy with pelvic lymphadenectomy to concomitant chemo-radiation. Tm: tumor; Ig: inguinal; Il: iliac; Pa: para-aortic; Sc: supra-clavicular.

alternative; such a minimally invasive technique may either detect microscopic lymph node metastases eventually missed by FDG PET or spare the patients unnecessary lymphadenectomies in cases of uninvolved sentinel lymph nodes (Levenback et al, 2002; Belhocine et al, 2003). Importantly, compelling evidence indicate that metabolic imaging is more sensitive and specific than conventional imaging. Not infrequently, FDG PET may detect unsuspected nodal metastases, which are missed by CT/MRI (Rose et al, 1999; Grigsby et al, 2001; Yeh et al, 2002; Belhocine et al, 2003; Ma et al, 2003; Tsai et al, 2004). Therefore, metabolic imaging may significantly impact the treatment choices (i.e. field/volume of irradiation, concurrent cisplatin-based chemotherapy), although the FIGO clinical staging is not taking into account nodal involvement (Grigsby et al, 2001; Narayan et al, 2001; Talbot et al, 2001; Yeh et al, 2002; Belhocine et al, 2002; Grigsby et al, 2005). In accordance with the body corpus of literature, the initial staging of cervical cancer was recently approved as a new reimbursed PET indication in the United States (http://www.cms.hhs.gov/manuals/). In the expectation of extended US medicare coverage, the performance of FDG PET was restricted to the patients with a negative conventional work-up. The inclusion of FDG PET into the routine staging of newly diagnosed cervical cancers appears to be particularly useful in locally advanced disease (FIGO IIB-IVA); a subset of women with a nonnegligible likelihood of extra-pelvic involvement, which may best benefit from whole-body FDG PET (Rose et al, 1999; Narayan et al, 2001; Yeh et al, 2002; Lin et al, 2003; Yen et al, 2003, Grigsby et al, 2003). Because FDG PET may provide prognostic information about patients' outcomes and treatment planning, its incorporation into the initial work-up of early stage disease should be also considered (Grigsby et al, 2001; Reinhardt et al, 2001; K端hnel et al, 2001; Grigsby et al, 2005). Further studies remain needed however, to refine stage-by-stage the optimal indications of metabolic imaging in pre-treatment staging of cervical cancer. Data from literature regarding

the performances of FDG PET for N staging are summarized in Table 4.

3. M staging Because FDG PET is a whole-body and non-invasive imaging technique, it allows the detection of distant metastases in one session. This is particularly relevant for M staging of cervical cancers, especially in advanced stages of disease. From our experience and others, metabolic imaging was able to detect suspected and unsuspected visceral metastases including liver, peritoneal, lung, and bone localizations. Moreover, whole-body FDG PET was found more accurate than routinely used protocols (FIGO staging including CT/MRI) for detection of distant sites (Grigsby et al, 1999; Kerr et al, 2001; Belhocine et al, 2002; Yen et al, 2003). So far, as observed for other cancer types, false positive results may occur in a particular context (i.e. inflammation, infection, ureteral stasis, bowel retention). Causes of misinterpretation are summarized in Table 9. A dual-phase protocol (40 min and 3 hours) may be useful to distinguish either benign disease or physiological uptake from malignancy (Yen et al, 2003; Tsai et al, 2004). The recent introduction of combined PET/CT may significantly improve the diagnostic accuracy of both PET and CT either by detecting non FDG-avid sites (i.e. infracentimetric lung metastases) or by precising the anatomic localization of FDG-avid sites. Last but not least, a single and efficient examination such as whole-body FDG PET, and more so PET/CT, is most often well tolerated in the initial work-up of patients. Data from literature regarding the performances of FDG PET for M staging are summarized in Table 5.

B. Post-treatment surveillance The major issue in women previously treated for cervical cancer is the lack of standardized guidelines (Chi et al, 2001; Olaitan et al, 2001). Hence, the post-treatment surveillance may considerably vary from one center to another. Also critical is the suboptimal sensitivity of routine protocols including the clinical examination at control visits and conventional imaging procedures for the 206


Cancer Therapy Vol 3, page 207 Table 5. M Staging of Cervical Cancer with FDG PET: Literature Data Authors

Years

Primary Staging

FIGO Stages

FDG-avid distant Metastases

Grigsby et al,

1999

11/23

IA-IVA

Lung

Kerr et al,

2001

10/13

IB-IVB

Lung – Liver

Belhocine et al,

2002

22/60

IA-IVA

Bone – Lung

Yen et al,

2003

47/135

IB2-IVB

Peritoneum – Bone – Liver – Lung

Tsai et al,

2004

19/19

IB-IIIB

Bone – Lung

detection of recurrent disease (Ansink et al, 1996; Salvesen et al, 2001). On the other hand, the efficacy of new chemo-radiation regimens stresses the need for early recognition of recurrences, especially in asymptomatic women (Bodurka-Bevers et al, 2000; Maiman, 2002). Compelling data from literature indicate the ability of FDG PET to detect local (i.e. cervix, vagina, rectal, pelvic wall), nodal (local and distant sites), and distant recurrences (lung, bone, liver, peritoneum). Also, FDG PET may play a useful role in the surveillance work-up of treated cervical cancers (Belhocine et al, 2004). For instance, metabolic imaging may distinguish localized recurrences (i.e. pelvic or extra-pelvic sites) from disseminated disease (Grigsby et al, 1999; Park et al, 2000; Sun et al, 2001; Belhocine et al, 2002; Havrilesky et al, 2003; Tsai et al, 2004; Lai et al, 2004; Wong et al, 2004). Therefore, whole-body FDG PET may impact the treatment choices in terms of surgery (i.e. pelvic exenteration) ± radiation versus systemic chemotherapy ± radiation. Because metabolic imaging has the ability to assess the tumor biology, it may reveal recurrences early in the course of disease, before the onset of clinical symptoms and radiological evidence; such FDG PET precession might be used to give the patient the best chance of salvage therapies and prolonged survival. In the largest series of women with previously treated cervical cancers but no evidence of recurrence at the time of the PET study (n = 249), metabolic imaging revealed FDGavid abnormalities in 32.1% of patients; among them, 11.2% had clinically or histologically confirmed recurrences (Ryu et al, 2003). In another series (n = 44), FDG PET detected asymptomatic recurrences in 30.8% of patients (Unger et al, 2004). Similarly, in 38 patients with cervical cancer, we showed the added-value of FDG PET for detecting unsuspected disease in 11 of out 25 patients (44%) with confirmed recurrences (Belhocine et al, 2002) (Figure 3). On the other hand, a negative FDG PET was able to reliably rule out macroscopic recurrences with no evidence of disease after a minimal follow-up of 12 months. Therefore, the costs and the side effects of unnecessary treatments were avoided. In line with our results, recent data from a large study group of 381 patients with cervical cancer confirmed the high negative predictive value of metabolic imaging when performed 9

to 14 months after completion of therapy; accordingly, ‘no evidence of disease’ state on PET corresponded to a disease-free period > 2 years with a recurrence rate of 2.5% (1.06%/year) after a negative PET (Kim et al, 2004). Remarkably, in another large sample of patients (n=152) treated by radiation and concurrent weekly chemotherapy, those of patients who were free of FDG-avid sites, on average 3 months post-therapy, had a 5-year overall survival of 92% (Grigsby et al, 2004). As reported for many types of cancers, this initial study also highlighted the feasibility of FDG PET to monitor women previously treated by concurrent chemo-radiation, thereby, allowing the classification of patients into three groups: complete metabolic response (no FDG uptake), partial metabolic response (persistent abnormal FDG uptake in the irradiated cervix or lymph nodes), and progressive disease (new sites of abnormal FDG uptake outside the irradiated volume). The impact of FDG PET findings on treatment strategy and patients’ survival are detailed below. To optimize the surveillance of women with treated cervical cancers, one should note the complementary information provided by a highly sensitive in vivo imaging such as FDG PET and a highly specific in vitro marker such as SCC-Ag (Belhocine, 2003). In a phase II study including 27 cases of cervical cancer explored by FDG PET for unexplained elevation of SCC-Ag serum levels in the follow-up, metabolic imaging was able to detect FDGavid lesions in 17 of out 18 patients with recurrent disease. In this series, not only FDG PET helped avoid futile therapeutic intervention but it significantly increased the patients’ overall survival (Chang et al, 2004). In another pilot study, the same group showed the utility of FDG PET to detect recurrent disease in asymptomatic patients with elevated SCC-Ag serum levels (Chang et al, 2004). Further studies are needed to confirm these promising data, and to refine the combination of FDG PET and PET/CT with SCC-Ag, especially in terms of marker cutoff and control intervals in the follow-up. Based on our experience and others, the following indications may be proposed in the work-up of previously treated cervical cancers: 1) the localization of recurrences in symptomatic patients as well as in cases of elevated tumor markers (SCC-Ag) and radiological suspicion, 2) the early detection of asymptomatic recurrences in the

207


Belhocine and Grigsby: 18FDG PET and PET-CT in uterine cancers

Figure 3. Post-treatment re-staging of cervical cancer with FDG PET. In a patient with a cervical cancer previously treated by hysterectomy and radiotherapy, the routine follow-up work-up was normal. Metabolic imaging showed peri-hepatic FDG-avid sites, which were consistent with recurrences. Histology concluded to peritoneal involvement from cervical cancer. T: Transaxial; S: sagittal; C: coronal.

post-therapy surveillance, especially in high-risk patients, 3) the exclusion of active disease in the routine follow-up, 4) the treatment monitoring, especially after chemotherapy and/or radiotherapy. Recent data also highlighted the prognostic significance of FDG uptake in terms of 5yoverall survival and 5y-cause-specific survival in patients previously treated by radiotherapy and concurrent weekly chemotherapy (Grigsby et al, 2003; Grigsby et al, 2004). Besides well-known prognostic variables (clinical status, tumor markers, treatment modality), FDG PET has been proposed as another parameter that might be incorporated into the treatment-decision making of previously treated cervical cancers (Yen et al, 2004; Lai et al, 2004). So far, cost-effective studies are still needed to best incorporate FDG PET into routine surveillance of treated cervical cancer. Similarly, the optimal timing for monitoring chemo-radiation remains to be defined yet (Nakamoto et al, 2002; Laking et al, 2003; Yen et al, 2003). Because most recurrences from cervical cancers occur during the two first years following the primary therapy, the use of FDG PET, and nowadays of PET/CT, eventually combined with SCC-Ag should be focused over this follow-up period (Bodurska-Bevers et al, 2000; Olaitan et al, 2001). Importantly, early after radiation therapy (< 12 weeks) or surgery (< 6 weeks), the inflammatory changes (or “flare phenomenon”) surrounding the resected primary tumor and/or the irradiated nodes may dramatically hamper the interpretation of PET images (Nakamoto et al, 2002). Besides, no clear data are available regarding the minimal timing for the performance of PET controls in patients with treated cervical cancers without compromising the sensitivity of the metabolic technique; of note, a temporary “metabolic shut-down” was previously reported within the 2 first weeks after chemotherapy in patients with germ cell cancer (Cremerius et al, 1998). Also, in the expectation of clinical evidence, a rational schedule for the post-therapy

surveillance of cervical cancer may include a PET study every 3-4 months the first year, and every 6 months the second year, and then once a year on a patient-by-patient basis (Maiman et al, 2002; Belhocine et al. 2003, Grigsby et al, 2003; Grigsby et al, 2004). Dedicated prospective studies are needed to best define the place of FDG PET and PET/CT for monitoring cervical cancers. Data from literature regarding the performances of FDG PET for detection of recurrences are summarized in Table 6.

C. Treatment impact The use of FDG PET for pre-treatment clinical staging has had a significant impact on the therapeutic management of patients with advanced stage disease (Belhocine et al, 2003). Standard therapy for advanced stage cervical cancer is irradiation with concurrent chemotherapy (Grigsby et al, 2001). Irradiation is directed to the pelvis to include the primary cervical cancer and the pelvic lymph nodes. Para-aortic irradiation is given only to those patients that have positive para-aortic lymph nodes. Patients that have tumor spread beyond the para-aortic region at the time of initial diagnosis have little chance for cure. Therefore, from the treatment perspective, the extent of disease at the time of diagnosis is crucial. Palliative therapy should be administered to patients with spread of disease beyond the para-aortic lymph nodes chain. In our patients population, 6% of patients were found to have biopsy-proven clinically occult lymph node metastases to the supra-clavicular lymph node region. With current best therapy, these patients are not curable and should be administered palliative therapy directed toward symptomatic sites of disease. In our study of the comparison of CT and total body FDG PET, we found that 6% of patients had abnormal lymph nodes in the paraaortic region by CT of the abdomen. These abnormal lymph nodes were confirmed by FDG PET to have increased FDG uptake and an additional 12% of patients 208


Cancer Therapy Vol 3, page 209 Table 6. Detection of Recurrences from Cervical Cancer with FDG PET: Literature Data Authors

Years Patients

Grigsby et al, Park et al, Sun et al, Belhocine et al, Havrilesky et al, Ryu et al, Lai et al, Wong et al, Unger et al,

1999 2000 2001 2002 2003 2003 2004 2004 2004

12 36 20 38 28 249 40 35 44

Suspicion of Recurrence - Clinical - Clinical, Ag-SCC or CEA - Clinical, Radiological, Ag-SCC - Clinical, Radiological, None * - Clinical, Radiological - None * - Histology, Clinical, Radiological - Clinical, Radiological - Clinical, None *

Sites of FDG-avid Recurrences - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis

FDG-avid Recurrences missed by CT/MRI -Pelvis, Extra-pelvis -Pelvis, Extra-pelvis - Not Available - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis - Pelvis, Extra-pelvis

Abbreviations: * None: Women with no clinical, radiological, and biological evidence of recurrent disease at the time of the PET study.

were found to have abnormal FDG uptake in the paraaortic lymph nodes. Thus, these patients with FDG avid para-aortic lymph nodes received curative irradiation to this region whereas no irradiation would have been administered to the para-aortic lymph node region in the absence of increased FDG uptake (Grigsby et al, 2001; Esthappan et al, 2003). Our study also demonstrated similar findings in the pelvis. Abnormal lymph nodes by CT were present in 22% of patients but 60% had abnormal FDG uptake. Thus, 38% of patients received additional irradiation dose to the pelvis due to positive pelvic lymph nodes. In our population of over 300 patients undergoing FDG PET for initial staging evaluation for cervical cancer the patient specific treatment management was changed in over one-half of the patients. Curative therapy was not administered to those with disease beyond the para-aortic lymph nodes and in the remaining patients either the irradiation portal or the irradiation was altered in the majority. One consideration with regard to the treatment of patients with presumed advanced stage disease is the use of concurrent chemotherapy with irradiation. A recent study from the Gynecologic Oncology Group (GOG 109) administered concurrent chemotherapy and irradiation in patients with pathologically positive lymph nodes (Peters et al, 2000). This study was updated and demonstrated no benefit to the use of concurrent chemotherapy in patients with only one positive lymph node (Monk et al, 2005). We demonstrated a similar finding with FDG PET (Grigsby et al, 2005). In this study, there was no apparent clinical benefit to the use of concurrent chemotherapy with irradiation in patients who do not have metastatic lymph node involvement as evaluated by FDG PET. Therefore, FDG PET may prove useful in selecting a subgroup of patients with locally advanced cervical cancer without lymph node metastases that will not benefit from the administration of concurrent chemotherapy.

population the only significant pre-treatment prognostic factor, as evaluated by Cox Multivariate Logistic Regression, for the development of recurrent cervical cancer and for death from cervical cancer was the extent of lymph node metastases as determined by pre-treatment whole body FDG PET (Grigsby et al, 2001; Singh et al, 2003). In Figure 4, survival curves related to FDG nodal uptake illustrate the prognostic information brought by metabolic imaging in untreated cervical cancers. Although pre-treatment extent of metastatic disease as determined by FDG PET is the most significant prognostic factor, the post-treatment response to therapy has proven to be of greater clinical significance. We have demonstrated that a post-treatment total body FDG PET performed at three months after the completion of irradiation with or without concurrent chemotherapy can show no FDG uptake, persistent FDG uptake, or new abnormal foci of FDG uptake (Grigsby et al, 2004). About three-forths of patients will have no abnormal foci of FDG uptake and their long term cancer survival is about 90%. Conversely, those of patients who presented with any FDG uptake (persistent or new foci) after the completion of their initial therapy had a very poor prognosis. Current and future clinical indications that may derive a benefit from FDG PET and PET/CT in the workup of cervical cancers are summarized in Table 7.

III. Endometrial cancer Endometrial cancer is primarily a malignancy of post-menopausal women (Rose, 1996; Barakat et al, 2001). In the United States, the disease is the most common gynecological cancer. According to the American Cancer Society, about 40.880 new cases of uterine cancers (body of the uterus) will be diagnosed during 2005 with a related mortality estimated to 7310 women (ACS keystatistics, 2005). So far, only few data are available on the contribution of FDG PET in the management of endometrial cancer. Herewith, we report our own experience in pre-treatment nodal staging and posttreatment follow-up of such gynecological cancer.

D. Prognosis Multiple prognostic factors have been identified in patients with advanced stage carcinoma of the cervix. Some of the pre-treatment factors that are considered to be of importance are patient age, tumor size, lymph node metastases, and clinical stage of the disease. In our patient

A. Pre-treatment nodal staging In newly diagnosed endometrial cancer, various pretreatment imaging studies have been performed to identify 209


Belhocine and Grigsby: 18FDG PET and PET-CT in uterine cancers

Figure 4. Survival curves related to FDG nodal uptake in pre-treatment staging of cervical cancer

Table 7. Cervical Cancer and FDG PET or PET/CT: Current and Future Clinical Indications Clinical Indications Initial Staging

Re-Staging

Added-Value of FDG PET and PET/CT - PET/CT measurement of primary tumor volume - Nodal staging - Distant staging - PET/CT guided-biopsy - Treatment planning : ! Definition of fields/volumes of irradiation to pelvic and/or para-aortic chains ! 3D PET/CT-based intracavitary brachytherapy ! Selection of patients for surgery and/or irradiation ! Selection of patients for concurrent chemo-radiation * - Prognosis Detection of suspected recurrences based on TM, symptoms, and CI Simple follow-up surveillance Treatment monitoring following chemo-radiation Prognosis

Abbreviations: * A baseline study is warranted in patients scheduled for chemo-radiation; TM: tumor markers (SCC-Ag); CI: conventional imaging.

extent of disease. However, none have proven to be particularly useful. The current FIGO staging system calls for a lymph node dissection to complete their surgical staging but there are complications associated with this procedure. Conventional imaging methods such as CT and MRI have been unreliable in evaluating pelvic and paraaortic lymph nodes (Connor et al, 2000; Hricak et al, 1991). We performed a prospective pilot study to evaluate the performance of total body FDG PET pre-operatively to detect pelvic and para-aortic lymph node metastases. In our study, twenty patients underwent total body FDG PET before surgical staging. The sensitivity and specificity were 67% and 94% respectively for FDG detection of tumor spread to lymph nodes (Horowitz et al, 2004).

B. Post-treatment surveillance In a retrospective series of 34 women with previously treated endometrial carcinomas (FIGO stages IB-IVA), FDG PET was compared to routine protocols including the clinical examination and conventional imaging procedures (Belhocine et al, 2002). In this seminal study, metabolic imaging was able to detect all recurrences suspected by the routine follow-up but one case of lung micrometastases, which were detected on thoracic CT. FDG-avid recurrences were located above and below the diaphragm in 50% of patients, which included lung, pleural, liver, peritoneal, central pelvic, nodal, ureteral, and bone localizations. In 35% of cases, FDG-avid recurrences were located below the diaphragm including central pelvic,

210


Cancer Therapy Vol 3, page 211 pelvic wall, abdominal and pelvic lymph nodes, liver, and peritoneal sites. In 15% of patients, FDG-avid recurrences were located above the diaphragm only, which included lung metastases, mediastinal and supra-clavicular nodes. Importantly, 85% and 15% of FDG-avid recurrences were detected in patients with advanced stages (FIGO II-IV) and early stages of disease (FIGO IB), respectively (Figure 5). Interestingly enough, in 12% of patients, FDG PET detected unsuspected recurrences in women with asymptomatic disease. Overall, FDG PET had an actual impact on treatment choices in 35% of patients (9/26); 6 patients being symptomatic while 3 had no symptoms at the time of the PET study. These data are in line with those published by Saga et al, in a series of 21 patients with treated endometrial carcinomas (FIGO IA-IVB) explored by FDG PET either for detecting a recurrence or for treatment monitoring (Saga et al, 2003). The study group included women with endometrioid carcinoma (n=11) and non-endometriod tumors including serous papillary carcinoma (n=6), small cell neuroendocrine carcinomas (n=1), mixed endometrioid and serous carcinoma (n=2), and mixed endometriod and clear cell carcinoma (n=1). Interestingly, FDG PET was found more sensitive than abdominal CT

plus pelvic MRI for detecting loco-regional recurrences. In addition, metabolic imaging was able to detect unknown lesions in 19% of patients. As reported in our series, FDG PET was most often positive in women with advanced stage of disease (III-IV), non-endometrioid tumors, and high grade endometrioid tumors (G 3). Equally, metabolic imaging findings influenced the treatment choices in 33.3% of patients (7/21). Last but not least, FDG PET had a high negative predictive value to rule out recurrence (no false negative result after a minimal follow-up of 5 months); this is also in line with our data demonstrating only 1 false negative result (lung micrometastases) among 11 patients with a negative FDG PET and a minimum follow-up of 6 months (Table 8). In another series of 9 women with treated endometrial carcinomas, Grigsby et al, also showed the usefulness of metabolic imaging for detection of recurrences, which were unsuspected by the routine protocol (Grigsby et al, 1999). In this preliminary study, histologic confirmation of clinically suspected recurrent disease was obtained in all cases. Among the 7 patients with local recurrence in the vaginal apex, a focal abnormality was detected in 4 patients by FDG PET

Figure 5. Post-treatment surveillance of endometrial cancer with FDG PET. In the follow-up of a patient with previously treated endometrial carcinoma, metabolic imaging was able to detect disseminated recurrent disease. FDG-avid sites included lung, liver, nodal, and peritoneal metastases.

Table 8. Detection of Recurrences from Endometrial Cancer with FDG PET: Literature Data Authors Years Number of patients Prevalence of Recurrences Patterns of PET Recurrences Types of Recurrences Detection of unknown Recurrences (%patients) False Negative Results Sensitivity Specificity Diagnostic Accuracy Treatment Impact

Belhocine et al, 2002 34 Stage III-IV LocalDistantLocal and Distant NodalVisceral 12%

Saga et al, 2003 21 Stage III-IVHigh grade endometrioid tumors (G3) Non-endometrioid tumors LocalDistantLocal and Distant Nodal Visceral 19%

Lung micrometastases 96% 78% 90% 35%

None 100% 88.2% 93% 33.3%

211


Belhocine and Grigsby: 18FDG PET and PET-CT in uterine cancers Table 9. Causes of Inaccuracies with FDG PET in Uterine Cancers: Literature Data Causes of False Positive Results - Ureteral stasis - Bowel retention - Post-radiation changes - Post-surgery changes - Recent traumas - Inflammation / Infection: reactive nodes, granulation tissue, anthracosis, endometriosis, tuberculosis - Functional ovarian cysts - Ovulatory and menstruating phases versus 2 patients by CT. Vaginal recurrences missed by metabolic imaging were ! 2 cm. In 1 patient, both FDG PET and CT missed brain metastases, while detecting clinically unknown metastases in the mediastinum. In 3 of out 7 women with a vaginal recurrence, metabolic imaging also detected pelvic lymph node, umbilical, and lung metastases, respectively. In another patient with known para-aortic nodal recurrence, only FDG PET was able to detect lung metastases. Based on the few data from literature, FDG PET appears to be feasible in the assessment of treated endometrial carcinomas. Moreover, metabolic imaging may significantly impact the patient's management by detecting recurrent foci on the entire body, thereby, differentiating localized from disseminated recurrence patterns. Also critical is the ability of FDG PET to detect clinically and radiologically unsuspected recurrences, which may help initiate the appropriate treatment early in the course of disease. From this perspective, metabolic imaging may be a substantial adjunct to the routine followup of endometrial cancer (Shumsky, 1994). Large prospective studies are warranted to confirm these promising results.

Causes of False Negative Results - Small (< 1cm) or microscopic disease : cervix, nodes, lung, bladder, rectum - Stromal invasion - Parametrial involvement - Indolent malignant disease - Previous radiotherapy - Low GLUT-1 expression - Lack of anatomic landmarks

from FDG PET plus CT in treatment planning and survival prediction.

A. PET/CT software image fusion 1. Diagnosis In a recent study, Tsai et al, assessed the added-value of side-by-side interpretation of FDG PET and CT/MRI in gynecological cancers (Tsai CC et al, 2003). Their analysis was focused on the imaging discrepancies, which were correlated to pathology results in 32 patients (22 cervical cancers, 7 ovarian cancer, 2 endometrial cancers, and 1 leiomyosarcoma). Among the 24 women with uterine cancers, the software fusion contribution was found significant for localization of biopsy sites (12 patients), treatment planning (5 patients), accurate diagnosis (9 patients: change of FDG PET results in 5 and MRI/CT results in 4), differentiation between pathological and physiological uptake (11 patients). As a conclusion, the authors showed the clinical benefit derived from software image fusion in gynecological cancers based on amore confident and more precise interpretation of FDG PET findings and MRI/CT results as well.

2. Treatment outcomes

IV. Perspectives Although FDG PET may significantly improve the management of patients with gynecological cancers, inherent technical and biological limitations have been shown to impair both the sensitivity and specificity of metabolic imaging. Causes of inaccuracies with FDG PET in uterine cancers are summarized in Table 9 In recent years, combined imaging modalities have allowed both metabolic and anatomic characterization of cancer. By using either software image fusion (i.e. sideby-side PET and CT/MRI systems) or hardware image fusion (i.e. hybrid PET/CT devices), FDG uptakes may be precisely located into diverse organs (Schaffler et al, 2000; Wahl, 2004). In cervical and endometrial cancers, initial results highlighted the incremental clinical value of anatometabolic imaging, especially in terms of diagnostic accuracy. Pilot studies also suggest the potential expected

planning

and

patientsâ&#x20AC;&#x2122;

Current treatment planning in radiation therapy for patients with gynecologic cancers is based on the patient specific extent of the known disease. We have shown that FDG PET clearly identifies more sites of disease than any other current imaging method. Radiation treatment planning is now often administered utilizing intensity modulated radiation therapy (IMRT) to increase the irradiation dose to the tumor while decreasing the irradiation dose to normal tissue. This aspect of irradiation therapy and treatment planning is most evident in patients with cervical cancer with positive para-aortic lymph nodes (Figure 6). Fused PET/CT images can be imported into current radiation therapy treatment planning systems. Increased doses to positive lymph nodes can be administered. We are currently performing a phase I/II study to evaluate this treatment planning process.

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Cancer Therapy Vol 3, page 213

Figure 6. Treatment planning in a patient with FDG-avid para-aortic involvement. Fused PET-CT images can be imported into current radiation therapy treatment planning systems. Increased doses to positive lymph nodes can be administered, while decreasing doses to normal tissues.

An interesting area of development has been the use of FDG PET as a guide to the evaluation and planning of intracavitary brachytherapy for patients with cervical cancer. We have developed a process whereby the brachytherapy irradiation applicators can be visualized within the metabolic tumor volume (Mutic et al, 2001). Isodose distributions can then be generated to demonstrate the irradiation dose distributions to the metabolic tumor volumes. This process has great potential to increase the irradiation dose to the primary cervical tumor while decreasing the dose to normal structures. In another pilot study, the metabolic volume as determined by FDG PET and CT software image fusion allowed to selecting the patients who may best benefit from radiation therapy; these patients had a tumor volume < 60cm3 and no lymph node disease. Alternatively, patients with other combinations suggestive of poor prognosis (V " 60cm3 and/or nodal involvement) may be treated by more aggressive therapies (Miller and Grigsby, 2002).

physiological endometrial and ovarian patterns may have been falsely interpreted as tumor uptake by using FDG PET alone (Lerman et al, 2004). Hence, FDG PET may benefit from the anatomic details provided by a concomitant CT; a "one-stop-shop" imaging aimed at increasing the diagnostic accuracy of both techniques, especially in treatment planning. Additionally, whole-body PET/CT may reduce the number, and then the cost and irradiation, of separated CT and PET studies. In a recent study, Blodgett et al compared the performances of PET/CT versus CT alone in staging and re-staging of 18 patients with cervical cancers (Blodgett et al, 2002). In 50% of cases (9/18), PET/CT detected additional tumor sites, which were missed by CT. Besides, PET/CT significantly altered the stages of disease in 33% of women (6/18). Moreover, in 33% of patients (6/18), PET/CT modified the treatment options from observation or surgery to chemotherapy or IMRT to cover para-aortic and pelvic chains. In another study, Israel et al, compared the performances of PET/CT versus PET and/or CT alone in 57 patients with gynecological cancers, including 38 cervical cancers, 6 endometrial cancers, and 13 ovarian cancers (Israel et al, 2003). As a main conclusion, PET/CT improved image interpretation over PET and/or CT in 51% of patients. Combined imaging technique led to a change in management of 11 patients with uterine cancers (25%) including 10/38 patients with cervical cancer and 1/6 patients with endometrial cancer. These results are in line with those obtained by Cohade et al, who directly compared PET and PET/CT in a series of 15 patients with

B. Hardware PET/CT image fusion With the advent of combined PET/CT devices, a single multimodality imaging is nowadays feasible in clinical routine. In gynecological cancers, this allows to avoiding many artifacts due to internal organs motion (i.e. abdominal peristalsis, urine stasis) and patients' repositioning between PET and MRI/CT (Wahl, 2004). In a reference study including 285 consecutive female patients, Lerman et al. also showed the usefulness of PET/CT for differentiating normal from abnormal FDG uptakes within the gynecological sphere; otherwise, benign and 213


Belhocine and Grigsby: 18FDG PET and PET-CT in uterine cancers endometrial cancer. In this later study, 12 of out 49 tumor sites (+24.5%) detected by PET/CT and PET were mislocalized or misdiagnosed by PET alone (Cohade et al, 2003). In particular, PET/CT helped to correctly reclassify 5 equivocal FDG-avid foci by PET alone as either benign or malignant lesions. The same group also evaluated the added-value of PET/CT over PET in 13 patients with cervical cancer. Similarly, combined imaging was found particularly contributive for a better anatomic localization (physiological versus pathological) and characterization (benign versus malignant) of foci with increased FDG uptake, which were difficult either to localize (>1/3 foci) or to diagnose (# 1/5 foci) on PET alone (Tatsumi et al, 2003). More recently, Grisaru et al, compared the value of PET/CT versus standard imaging procedures (STD = CT/MRI) in 53 patients with gynecological cancers including 21 cervical cancers, 8 endometrial cancers, 19 ovarian cancers, 1 primary peritoneal tumor, 1 vulvar cancer, 1 vaginal cancer, 1 tubar cancer, and 1 gestational trophoblastic neoplasia (Grisaru et al, 2004). In pretreatment staging, the diagnostic accuracy of PET/CT was optimal in 15 patients with either cervical cancers (n=9) or uterine cancers (n=6). Conversely, STD had 2 false positive results (1 CT and 1 MRI) and 3 false negative results (3 CT). Hence, in 5 of out 15 patients (33%), PET/CT accurately staged the extent of disease versus CT/MRI. In post-treatment staging, PET/CT was also more sensitive and specific than STD. Among 14 patients who were previously treated for either cervical cancer (n=12) or uterine cancer (n=2), PET/CT detected additional recurrences in 8 patients (8 false negatives from STD) and ruled out a recurrence suspected by CT in 1 patient (1 false positive from STD). In this clinical setting, PET/CT was contributive in 9 of out 14 patients (64%) versus standard conventional imaging. The added-value of PET/CT was particularly significant in localizing primary uterine tumor missed by CT as well as in detecting lung metastases missed by STD protocol, spinal cord compression missed by MRI, local and distant lymph nodes missed by CT. Based on the current data, the clinical added-value of PET/CT primarily relies upon its ability to accurately localize FDG-avid sites compared to either PET alone or CT alone. So far, the role of PET/CT remains limited for the detection of non FDG-avid sites, especially normalsized metastatic nodes (i.e. microscopic tumors). In a recent study by Sironi et al, including 35 patients with early stage cervical cancer (FIGO IA-IIA), integrated PET/CT was found a reliable modality for pre-operative lymph node staging except for lesions smaller than 0.5 cm (micrometastases) (Sironi et al, 2004). These findings are in line with previous data showing the limitation of FDG PET for detecting microscopically involved pelvic nodes (Reinhardt et al, 2001; Kühnel et al, 2001; Williams et al, 2001; Belhocine et al, 2002). On the other hand, recent data showed that (un)treated cervical cancers with no FDG-avid nodal sites presented with a better prognosis than those with lymph node metastases detected by FDG PET (Grigsby et al, 2001; Grigsby et al, 2004; Yen et al, 2004). Besides, initial results from a prospective study

showed the lack of benefit of concurrent irradiation with weekly cisplatin chemotherapy (versus radiotherapy alone) in patients with cervical cancer and negative lymph nodes by FDG PET (Grigsby et al, 2005). Although the size/volume is a key-factor in the lesion detectability by PET and PET/CT, the metabolic activity of tumor is another important parameter to be considered. For instance, < 1cm nodes that are metabolically active may be detected by PET and PET/CT, while " 1cm (nodes) with a low FDG uptake (i.e. metastases with