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Volume 5 Number 2 December, 2007


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

!!!!!!!!!!!!!!!!!!!!!!!! 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:

!!!!!!!!!!!!!!!!!!!!!!!! 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 Der Channing, J. Ph.D, Lineberger Comprehensive Cancer Center, USA Devarajan, Prasad M.D., Cincinnati Children's Hospital, 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-Gutenberg-

University, 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 Ng, Eddie YK, Ph.D., Nanyang Technological University, Singapore 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 Werner, Jochen Alfred M.D., Philipps-University of Marburg, Germany Wieand, Harry Samuel Ph.D., National Surgical Adjuvant Breast and Bowel Project (NSABP), USA Yamada, Akira Ph.D., Kurume University Research Center for Innovative, Japan Yu, Dihua M.D., Ph.D., The Univ. Texas M. D. Anderson Cancer Center, USA Zagon, Ian, Ph.D., The Pennsylvania State University, USA

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

Chen, Jiguo, Ph.D, The University of Texas Health Science Center at San Antonio, USA 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 Taupin, Philippe, Ph.D., National University of Singapore, Singapore 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

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Table of contents Cancer Therapy Vol 5 Number 2, December 2007


Type of Article

Article title

Authors (corresponding author is in boldface)


Review Article

Fernando Arias de la Vega, Miguel A Dominguez Dominguez, Juan I Arraras, Elena Villafranca Iture, Ana Manterola Burgaleta, Enrique Martinez Lopez, Gemma Asin, Lorena del Rio


Review Article


Research Article


Research Article


Research Article


Research Article


Research Article


Case Report


Research Article

Hyperfractionated radiation therapy and cisplatin for locally advanced head and neck cancer. A non-randomized comparison of Quality of Life between two consecutive treatment protocols at a single institution The role of physical activity in the prevention of cancer Metastatic malignant melanoma of the ovary: Case Report and literature review Flow cytometry and KI67 expression in rat`s urinary bladder carcinogenesis treated with Allium Sativum Needle-like morphology of H2K4b polyplexes associated with increases in transfection in vitro Transcriptional repression of antiapoptotic proteins mediated by the tumor suppressor protein p53 Cyclosporine increases in vitro sensitivity to prednisolone in childhood acute lymphoblastic leukemia Multiple myeloma, thrombosis and thromboprophylaxis Targeted therapies in the treatment of advanced Non-Small-Cell Lung Cancer elderly patients


Review Article

Assessment of apoptotic markers as predictors of response to neoadjuvant FEC chemotherapy in locally advanced breast cancer


Research Article


Review Article


Research Article

Management of recurrent high-grade gliomas Factors influencing late rectal toxicity after radical radiotherapy of localized and locally advanced prostate cancer Management of a large, exophytic

Joanna Kruk, Hassan Y. Aboul-Enein Azam sadat Mousavi, Mojgan Karimi Zarchi, Fatemeh Ghaemmaghami, Farokh Tirgari Attalla Farag El-kott

Qixin Leng, Jason Kahn, Jingsong Zhu, Puthapparampil Scaria, James Mixson Józefa Wesierska-Gadek, Gerald Schmid Jan Styczynski, Mariusz Wysockiors

Victor Hugo Jiménez-Zepeda, Virginia Jeanet Domínguez-Martínez Antonio Rossi, Paolo Maione, Filomena Del Gaizo, Giuseppe Colantuoni, Carmine Ferrara, Dario Nicolella, Ciro Guerriero, Daniela Comunale, Alba De Vita, Cesare Gridelli Veerabhadram Garimella, Mark B Watson, Hayley Cairns, Amulya Chaturvedi, Philip J Drew, Anne Hubbard, Michael J Lind, Anthony Maraveyas, Lindsey Turnbull, Lynn Cawkwell !ihomir N. Eftimov, Ivan D. Ivanov, Alexander P. Petkov, Emil Nakov Gerald Lim, Harold Lau, Penny Brasher

James H. Brashears III, David T. Marshall, Katherine Morgan, Seung-Jun

rectal adenocarcinoma with concurrent neoadjuvant chemoradiation, surgery, and adjuvant concurrent hyperfractionated chemoradiation (the modified sandwich approach) Consolidation chemotherapy with maintenance paclitaxel in patients with advanced ovarian cancer after complete response to platinum based chem"therapy Suppression of breast cancer metastasis through the inhibition of VEGFmediated tumor angiogenesis Oral mistletoe lectins: A case for their use in cancer therapy Green tea treatment of ultraviolet-B (UVB) skin carcinogenesis in mice


Review Article


Case Report


Review Article


Review Article


Research Article


Research Article


Research Article

CA 125 behaviour by gender and potential prognostic role in early stage lung cancer


Case Report


Research Article


Case Report


Review Article

Molecular mechanisms of cisplatin and its liposomally encapsulated form, Lipoplatin. Lipoplatin as a chemotherapy and antiangiogenesis drug Lack of response to a cisplatinadriamycin based chemotherapy regimen in a patient affected by adenocarcinoma of the lung with rhabdoid features Recent clinical trials in diffuse intrinsic brainstem glioma Upper limb deep vein thrombosis: update on risk factors in oncological patients


Research Article


Research Article

O, Alberto J. Montero

Azam Sadat Mousavi, Zeinab Nazari, Fereshteh Fakor, Fatemeh Ghaemmaghami, Mitra Modares Gilani, Nadereh Behtash, Gholamreza Tougeh, Mojgan Karimi Zarchi Jun Zhang, Andrew Lu, Derrick Beech, Binghua Jiang, Yi Lu Ian F. Pryme, Truls M. Dale, Pontus Tilrem Mohammed I. El-Sherry, Mahmoud A. Zaher, Mohammad Salah-El-Din M. Youssef, Yasmin O. El-Amir Alberto M. Martelli, Camilla Evangelisti, Francesca Chiarini, William L. Blalock, Veronica Papa, Federica Fal‡

The phosphatidylinositol 3Kinase/Akt/Mammalian target of rapamycin signaling network as a new target for acute myelogenous leukemia therapy Wilbur B. Bowne, Josef Michl, Martin Novel peptides from the RAS-p21 and p53 proteins for the treatment of cancer H. Bluth, Michael E. Zenilman, Matthew

Adjuvant therapy for endometrial cancer: “Sandwich therapy” of carboplatin and paclitaxel with radiation therapy. The Women and Infants’ Hospital experience and review of the literature Study of preoperative focused

R. Pincus Fable Zustovich, Alessandra Buja, Marina Da Prà, Giorgio Farina, Stefania Zovato, Mauro Buttarello, Egle Perissinotto, Giuseppe Cartei Teni Boulikas

Nicola Silvestris, Gianni Simone, Anna Maria Sebastio, Carla Broussard, Salvatore Pisconti, Gianmauro Numico, Giuseppe Pezzella Stanislaw R. Burzynski Pierpaolo Di Micco, Rosanna Di Fiore, Sandro Quaranta, Giuseppe V. Viggiano, Ilaria J. Romano, Alferio Niglio, Andrea Fontanella, Bruno De Simone, Antonella Angiolillo, Giuseppe Castaldo Don S Dizon, Carolyn K McCourt, Terissa Martin-Hanley, Laurent Brard, AnnMarie Bradley, Christina Bandera

Hernan I. Vargas, William C. Dooley,

microwave phased array thermotherapy in combination with neoadjuvant anthracycline-based chemotherapy for large breast carcinomas Evolutionary conserved interaction of TACC2/TACC3 with BARD1 and BRCA1: potential implications for DNA damage response in breast and ovarian cancer A perspective on tyrosine kinase inhibitors in gastrointestinal stromal tumors and cancer: past and present with emphasis on future and cost Comparative pharmacokinetic profile of carboxyamidotriazole and carboxyamidotriazole-orotate Transcutaneous ultrasound guided fine needle aspiration of the esophageal cancer Bilateral hilar intraductal cholangiocarcinoma successfully resected by technical variant liver resection


Research Article


Case Report


Research Article


Case Report


Case Report


Case Report


Case Report


Review Article


Review Article


Research Article

Expression of EGFR and p-EGFR correlates with cisplatin sensitivity in oral squamous cell carcinomas


Review Article


Research Article


Case Report


Case Report

Localized androgen-independent prostate cancer: an emerging disease Alteration in antioxidant defense mechanisms in the small intestines of methotrexate treated rat may contribute to its gastrointestinal toxicity Combining p53 gene therapy with bronchial artery infusion inhibits growth of non-small cell lung cancer, a case report Recurrent multiple myeloma mimicking metastatic pancreatic

Alan J. Fenn, Mary Beth Tomaselli, Jay K. Harness

Brenda Lauffart, Omkaram Gangisetty, Ivan H. Still

Helmy M. Guirgis

Gary J. Grover, Jane Kelly, George Moore, Henry Jacoby, Rashida A. Karmali, Gregory S. Gorman Andrias Hambardzumyan, Anzhela Hayrapetyan John Spiliotis, Athanasios Rogdakis, Anastasios C. Datsis, Evagelia Katsiki, Angeliki Sarantopoulou, Arhodoula Vaxevanidou, Spiros Kekelos, Stylianos Katsaragakis Hiroyasu Yasuda, Katsutoshi Nakayama, Takahiko Sasaki, Masanori Asada, Motoki Yoshida, Kazuhiro Yanagihara, Mutsuo Yamaya

Partial response by nitroglycerin plus amrubicin regimen in patients with refractory and recurrent advanced nonsmall cell lung cancer who had received at least third-line chemotherapy Synovial sarcoma arising in the gastric Sarah Akhunji, Ian Musil, Ada Baisre de Leon, Achyut Bhattacharyya, Lee D. wall: case report and literature review Neutrophil gelatinase-associated lipocalin: new paths for an old shuttle Multiple carcinoids of the intestine

Cranmer Prasad Devarajan

Hatem Sembawa, Esther Lamoureux, Adrian Gologan, Walter Gotlieb, Philip H Gordon Yukihiro Hiraishi, Takeshi Wada, Ken Nakatani, Itaru Tojyo, Takashi Matsumoto, Kenji Negoro, Shigeyuki Fujita Giuseppe Colloca, Antonella Venturino, Franco Checcaglini Viswa Kalyan Kolli, Premila Abraham, Bina Isaac

Yong-Song Guan, Yuan Liu, Qing He, Lin Yang, Long Sun

Alejandro Calvo, Mauricio Carballo, Satheesh Kathula,Veronica Camacho


Review Article


Case Report


Case Report

carcinoma An insight into PET-CT based radiotherapy treatment planning Successfull high-dose chemotherapy of secondary central nervous system anaplastic T-cell Non-Hodgkin$s lymphoma in a patient with a 50-year history of relapsing malignant lymphomas Cardiac metastasis from a clear cell sarcoma of tendons and aponeurosis


Review Article

Designing platinum compounds in cancer: structures and mechanisms

Ramachandran Prabhakar, Pandjatcharam Jagadesan, Goura K Rath Sebastian Fetscher, Jan Schmielau, Osnat Bairey

Marisa Di Seri, Angelo Zullo, Evelina Miele, Gian Paolo Spinelli, Federica Tomao, Linda Cerbone, Francesca Iodice, Silverio Tomao Teni Boulikas, Alexandros Pantos, Evagelos Bellis, Petros Christofis

Cancer Therapy Vol 6, page 1 Cancer Therapy Vol 6, 1-10, 2008

Palladium-based chemotherapeutic agents: Routes toward complexes with good antitumor activity Review Article

Adnan Salim Abu-Surrah1,*, Haitham H. Al-Sa’doni2, Maher Y. Abdalla3 1

Department of Chemistry, Hashemite University, P. O. Box 150459, Zarqa-13115, Jordan Department of Chemistry, Al al-Bayt University, Al-Mafraq, Jordan. 3 Department of biology and Biotechnology, Hashemite University, P. O. Box 150459, Zarqa-13115, Jordan 2

__________________________________________________________________________________ *Correspondence: Adnan Salim Abu-Surrah, Department of Chemistry, Hashemite University, P. O. Box 150459, Zarqa-13115, Jordan; Tel: +962 5 390 3333 (Ext. 4315); Fax: +962 5 390 3349; E-mail: Key words: Palladium complexes, Anticancer agents, Cytotoxicity, Chiral ligands Abbreviations: diethyl-2- quinolmethylphosphonate, (2-dqmp); mercaptopyridines, (MP); monoethyl-2-quinolmethylphosphonate, (2Hmqmp); structure-activity relationships, (SAR) Received: 21 November 2007; Revised: 18 December 2007 Accepted: 18 December 2007; electronically published: January 2008

Summary Chemical-, pharmacological-, and clinical-research on anticancer coordination complexes has yielded remarkable anticancer agents such as cisplatin, carboplatin, and oxaliplatin. Since the discovery of cisplatin, the development of analogue complexes has been an empirical task. Studies have shown that the range of platinum complexes with antitumor activity is not restricted to the structural analogues of cisplatin. The established structure-activity rules have been broken: active platinum complexes without NH groups, trans-platinum complexes, multinuclear complexes, cationic complexes, and several classes of palladium(II) complexes have emerged. The foremost target of most research groups was to find a convenient anticancer drug that can be used efficiently for the treatment of human tumors. This study gives an up to date overview of the anticancer chemistry of palladium compounds with an emphasis on the new strategies used in the development of new palladium antitumor agents.

different from that of cisplatin and its analogues. Their pattern of antitumor activity is also altered with respect to cisplatin. Comparison of common features and differences between different classes may point to some rules for the rational design of complexes with a different spectrum of clinical activity to cisplatin and activity to cisplatinresistant tumors. The significant similarity between the coordination chemistry of palladium(II) and platinum(II) compounds has advocated studies of Pd(II) complexes as antitumor drugs (Rau et al, 1996). A key factor that might explain why platinum is most useful comes from the ligandexchange kinetics. The hydrolysis in palladium complexes is too rapid: 105 times faster than for their corresponding platinum analogues. They dissociate readily in solution leading to very reactive species that are unable to reach their pharmacological targets. Compared to cisplatin, the corresponding cispalladium, cis-[Pd(NH3)2Cl2] and cis-[Pd(DACH)Cl2]

I. Introduction Cis-diaminedichloroplatinum(II), [cis-(NH3)2PtCl2], clinically called cisplatin is one of the most successful anticancer compound (Rosenberg et al, 1969). After the discovery of its activity, thousands of platinum complexes have been synthesized and evaluated for their anticancer activity. Research in the field of platinum-based cancer chemotherapy showed that cisplatin and its analogous compounds exhibit very similar patterns of antitumor sensitivity and susceptibility to resistance which means that most of them produce identical adducts with DNA. The determining factors of cytotoxicity thus do not always follow the original structure-activity relationships (SAR). Possibly, the new clinically useful metal based anticancer agents should have novel structures unrelated to those agents assigned to platinum complexes. Therefore, several unconventional complexes that violate the SAR rules have been synthesized and evaluated (Abu-Surrah, 2007). The mechanism of action of nonclassical complexes is


Abu-Surrah et al: Palladium-based chemotherapeutic agents dqmp)2PdCl2] (1, Figure 1). The complexes of the diester 2dqmp were found to be more active than those of the monoesterbased ligand (2-Hmqmp). This may partly be ascribed to the greater leaving activity of the halogen ligands in the complex bearing the 2-dqmp ligand and to their greater lipophilicity or solubility.

(DACH: (1R,2R)-(–)-1,2-diaminecyclohexane) do not show antitumoral activity. It is well known that the former undergoes an inactive trans-conformation and that the two compounds hydrolyze very fast assuming that they interact in vivo with a lot of molecules particularly proteins preventing them to reach the DNA, their pharmalogical target (Butour et al, 1997, Wimmer, et al, 1989, Zhao, et al, 1999). This considerably higher activity of palladium complexes implies that if an antitumor palladium drug is to be developed, it must somehow be stabilized by a strongly coordinated nitrogen ligand and a suitable leaving group. If this group is reasonably non labile, the drug can maintain its structural integrity in vivo long enough. Several review articles have appeared during recent years dealing with platinum-based anticancer agents (Suzanne et al, 1987; Fuertes et al, 2003; Reedijk, 2003; Wang et al 2005, Kostova, 2006). The biological mechanism of palladium(II) complexes with an emphasis on cyclopalladated complexes has recently been reviewed (Caires, 2007). In this review, the progress in the field of anticancer chemistry of palladium-based transition metal complexes during the last 10 years will be highlighted. Methodologies for application of bulky aromatic or aliphatic nitrogen ligands, chiral organic moieties, chelates containing other donor atoms than nitrogen, and multinuclear palladium complexes will be discussed. The complexes that illustrated the prominent strategies utilized in the development of anticancer palladium-based agents will also be presented.

The synthesis and cytotoxicity evaluation of some trans-[(L)2Pd(X)2] complexes (2) (L = N,N-dimethyl-Oethylthiocarbamate: DMTC or N-methyl-Oethylthiocarbamate: MTC, X = Cl, Br) were reported (Furlani et al, 1996). Other palladium complexes based on 2-mercaptopyridines (MP) were also prepared. The [(MP)3Pd(Br)]+. Br! (3) is of potential therapeutic use since it has lower IC50 values on LoVo cell lines than cisplatin and around the same as its Pt(II) analogue (Carrara et al, 1996). Palladium(II) complexes with alkyl phosphonates ligands derived from aniline and quinoline were reported. Most of the aniline compounds (e.g. 4) showed cytotoxicity in vitro against animal and human tumor cell lines (Curic et al, 1996). Complexes with naturally occurring compounds have also been utilized. The palladium complex which contains the bulky nitrogen ligand harmine (7-methoxy-1-methyl9H-pyrido[3,4-b]indole, trans-[Pd(harmine)(DMSO)Cl2] (5, Figure 1) exhibits a greater cytotoxic activity against P388, L1210 and K562 cell lines than cisplatin (Al-Allaf et al, 1998). Recently, we reported about the synthesis and molecular structure of a new enantiometrically pure, chiral trans-palladium(II) complex, trans-[Pd{(R)-(+)-bornylamine}2Cl2] (6) (Figure 1) that bears the bulky amine ligand R-(+)-bornylamine (endo-(1R)- 1,7,7-trimethylbicyclo[2-2-1]-heptan-2-amine). The complex showed similar antitumor activity against HeLa cells when compared with the activity of the standard references, cisplatin, carboplatin and oxaliplatin (Abu-Surrah et al, 2002). A trans-palladium complex of the general formula, trans-[Pd(L)2Cl2] (L = 2-chloro-6-[(2-methoxybenzyl)amino]-9-isopropylpurine) has been reported (TrĂĄvnĂ­"ek et al, 2007). The complex has been tested in vitro for its cytotoxicity against malignant melanoma (G361) cell lines. Promising in vitro cytotoxic effect has been found (IC50 = 15 #M). Palladium(II) complexes of the form: trans-PdCl2L2, where L=3-hydroxypyridine, 2-hydroxypyridine and 4hydroxypyridine respectively have been investigated for antitumour activity against ovarian cancer cell lines: A2780, A2780cisR and A2780ZD0473R (Huq et al, 2007). The compounds were found to be less active than cisplatin but they are often found to be more active against the resistant cell lines than the parent cell line. The 2-hydroxypyridinebased complex was found to be most active against the three cell lines.

II. Palladium anticancer chemistry Numerous palladium complexes with promising activity against tumor cell lines have been synthesized (Graham et al, 1979; Rau et al, 1996). In general, the strategies that have been applied to design these agents were on the window of reactivity usually employed for the potential platinum antitumor drugs. Different types of monodentate ligands were applied in the synthesis of these complexes. In addition, several research groups have focused on the preparation of Pd(II) complexes bearing bidentate ligands as a way to stabilize these compounds and to prevent any possible cis-trans isomerism (Mansuri et al, 1992).

A. Trans-palladium(II) complexes Relatively bulky monodentate ligands have been utilized to produce the complexes of this family. Due to the steric effect that results from the bulk on the donor atoms, these ligands could minimize any possible cis-trans isomerism and insure the direct separation of the desired trans- Pd isomers (Abu-Surrah et al, 2002). In general, research results indicated that most of the trans-palladium complexes showed a better activity than the cisplatinum isomers and superior activity than that of the cispalladium isomers. More importantly, they showed activities equal to (or superior than) those of cisplatin, carboplatin, and oxaliplatin (the anti-cancer drugs in clinical use) in vitro. A comparative study on antitumor activity was carried out between the Pd(II) dihalide complexes of monoethyl-2quinolmethylphosphonate (2-Hmqmp) and diethyl-2quinolmethylphosphonate (2-dqmp) (Tusek-Bozic et al, 1991). The diester ligand has two potential donors, the N from quinoline and the O from phosphoryl giving the complex [trans-(2-

B. Palladium(II) complexes containing bidentate nitrogen ligands Navarro-Ranninger and colleagues reported the synthesis of square planar dichloro palladium(II) complexes with spermidine and spermine ligands


Cancer Therapy Vol 6, page 3 (Navarro-Ranninger et al, 1993). These types of chelating ligands have been used because of their relevant biological activity; they are involved in proliferation and differentiation of cells in DNA replication and membrane stabilization. Complexes of spermidine (7, Figure 2) give values of IC50 similar to cisplatin, whereas those of spermine have low antiproliferative activity. Ethylendiamine-based palladium(II) complexes with pyridine (8, Figure 2) or its derivatives were also reported (Zhao et al, 1999). The increase of the electron donor properties of the substituents firstly led to an increase of the donor strength of the coordinated pyridines, which directly led to the increase of the cytotoxic activity of the palladium complexes. An alternative method to the synthesis of the enantiomerically pure DACH-based palladium(II) complexes (DACH: (1R,2R)-(–)-1,2-diaminecyclohexane) was utilized (Abu-Surrah et al, 2002). In this method the desired organic bidentate ligand was allowed to react with [cis-Pd(PhNC)2Cl2], a palladium(II) starting material that is soluble in most organic solvents, in CH2Cl2 at 25°C. Following this procedure, the nucleophilic substitution

reaction of the complex [cis-Pd(PhNC)2Cl2] with (1R,2R)(–)-1,2-diaminecyclohexane afforded the square planar Pd(II) complex [(1R,2R)-(–)-(DACH)PdCl2] (9) in a high yield (Figure 3).The corresponding cationic, aqua complex, [(DACH)Pd(H2O)2]2+. 2NO3! (10) and the oxalate complex (oxalipalladium) [(DACH)Pd(C2O4)] (11) have also been isolated and characterized (Abu-Surrah et al, 2003). A new approach to increase the stability of the palladium(II) complexes by forming two chelate rings around the central atom was applied. L-cysteine derived ligands such as py-CH2-accys (12, Figure 4) (accys: Nacetyl-S-methylene-2-(2-pyridine)-L-cysteine) have been applied (Rau et al, 1998). The S,N-chelating mode of these ligands is of importance, since only the side chain of the amino acid is involved in metal coordination, whereas the amino acid function remains uncoordinated, leaving this functional group accessible for the attachment of other amino acid or peptides. It has been found that the reactivity of these palladium complexes compete with some platinum(II) complexes.

CH2 -PO(OEt) 2

OCON(CH3 ) 2 S


N Pd







PO(OEt) 2







N (3)


(OEt) 2OP CH2








Cl N


Pd N











CH3 (4)



Pd NH 2

O (6)

Cl CH3


Figure 1. Structures of some trans-palladium(II) complexes (1-6).

+ Cl NH3






Pd NH2







Figure 2. Palladium(II) complexes with ethylenediamine nitrogen ligand (7 and 8).

Abu-Surrah et al: Palladium-based chemotherapeutic agents

* *

* *



comparison among [(bipy)Pd(NO3)2] (14), [(AMP)Pd(NO3)2], [(AEP)Pd(NO3)2], [(DACH)Pd(Meorot)] (bipy = 2,2'-bipyridyl, AMP = 2-aminometylpyridine, AEP = 2-aminoethylpyridine, Meorot = 3methylorotate) showed that only [(DACH)Pd(Meorot)] (15) was active, giving a high activity for sarcoma 180 but a low one against P388 leukemia. Similarly, [(DACH)Pd(5-fluroorot)] reported later (Butour et al, 1997), displayed significant antitumor activity. These strong chelating ligands replacing chloro or nitro ligands induce a reduction in the rate of hydrolysis. 2,2'-dipyridylamine-based palladium(II) complexes containing glycine or L-alanine have been prepared and evaluated (Paul et al, 1993). The alanine based complex (16, Figure 4) showed better cytotoxicity against P388 lymphocytic leukemia cells than the glycine based one. The aromatic ligands such as 1,10-phenanthroline, which is one of the most used ligands in coordination chemistry, has been utilized in the field of antitumortransition metal chemistry. Its planar nature enables its participation as a DNA intercalator. Several derivatives of it were prepared and used as tetradentate ligands. The activities of [(N,N-dialkyl-1,10-phenanthroline-2,9dimathanamine)Pd(II)] (17) (alkyl: Me, Ethyl, propyl, cyclohexyl) are significantly dependent on the nature of the alkyl substituents. The complexes bearing the bulkiest groups showed lower IC50 values than cisplatin (Zhao et al, 1998). Palladium(II) complexes containing S-donors (diethyldithiocarbamate: ddtc) in addition to the N-N ligands (bipyridine, phenanthroline, and DACH) have also been investigated (Mital et al, 1989). The most active were the bipyridine and phenanthroline-based complexes (18, Figure 4).

Pd Cl




OH2 Pd



NH2 (10)

O * *






Pd NH2



Figure 3. Palladium(II) complexes with (1R,2R)-(–)-1,2diaminecyclohexane (DACH) (9-11).

Palladium compounds bearing two chelating N-N and O-O ligands were prepared (Mansuri-Torshizi et al, 1991). The N-N ligand did not influence the activity but the oxygen coordinated leaving group did. Selenite complexes were invariably better cytotoxic agents than tellurite complexes and cisplatin. The complex [bipy)Pd(SeO3) (13) was found to bind to DNA through a coordinate covalent bond. The cytotoxic activity of palladium complexes bearing nitrate (NO3) in addition to a bidentate nitrogen ligand (Wimmer et al, 1989) was investigated. A

NH 2

(NO 3)2



Pd NH 2

Pd S







* *



Pd O

NH 2 O








NH 2




CH 3

(12) H N N


(Cl) N



Pd NH 2


Pd O O



S Pd



C H3

C H3





Figure 4. Palladium(II) complexes containing different types of nitrogen ligands (12-18).


C N( CH 2C H 3)2

Cancer Therapy Vol 6, page 5 This was related to the flat structure of the aromatic N-N ligands and the more hydrophobic nature of the complex. Bipy and phen complexes showed IC50 values lower than cisplatin against P388 lymphocytic leukemia cells.

C. Palladium(II) phosphine ligands


(mercapto or amino) have been reported by Khan and colleagues in 1991. Methionene coordinates to Pd(II) through amino nitrogen and sulfur, thus leaving a carboxylic group free. It has been found that the complex [(methionine)Pd(2-merpy)Cl]+. Cl! (21, Figure 6) has in vitro IC50 value lower than 10 Âľg/ml, so it could act as a potential antitumor agent. Heterocyclic thiosemicarbazones are of considerable interest due to their potential beneficial antineoplastic activity. It is assumed that the presence of some metallic ions may enhance their antitumor activity due to their ability to form chelates. The phenyl acetaldehyde thiosemicarbazone-based palladium complex (22, Figure 6) has been found to display an enhanced in vitro activity compared to its platinum analogue (Quiroga et al, 1998). In addition, this complex is active in cisplatin resistant cell lines. Other thiosemicarbazide derivatives have also been studied. [(Benzyl)Pd{bis(thiosemicarbazonate)}] (23, Figure 6) showed IC50 values in a concentration range similar to that of cisplatin and a notable activity in cisplatin-resistant cell lines (Matesanz, 1999). Palladium complexes with 2-benzoylpyridine derived thiosemicarbazones with good antitumor activity were also reported (Rebolledo, et al, 2005). Recently, the antitumor functions and mechanisms of a 1,2-naphthoquinone-2-thiosemi-carbazone-based palladium(II) complex were investigated against MCF-7 human breast cancer cells (Chen et al, 2004). The results revealed that the complex is an effective antitumor agent. The study of mechanism of action showed that the metal complex can only stabilize the single-strand nicked DNA, but not double-strand breakage intermediates. The synthesis of a palladium(II) complex of the general formula [Pd(N-O)2] (N-O: 3-ethanimidoyl-2methoxy-2H-1,2-benzoxa-phospinin-4-ol-2-oxide) has also been reported (Budzisz, et al, 2004). The cytotoxic activity of this complex against the human leukemia cell lines, HL-60 and NALM-6 showed that the effects exhibited by this complex was comparable to those reported for cisplatin and carboplatin.


Some palladium(II) complexes showed a discrete antitumor activity in vitro compared to the platinum based drugs because of their extremely high lability in biological fluids (Navarro-Ranninger et al, 1993). Therefore, it has been suggested that the organometallic biphosphine-based cyclopalladated complexes that are more stable and less toxic could have a more specific antitumor activity in vivo (Caires et al, 1999). Some cyclopalladated complexs based on biphosphine ligands (19 and 20, Figure 5) have been prepared and investigated for their antitumor activity in a syngeneic B16F10 murine melanoma model (Rodrigues et al, 2003). The ionic complex (19) caused 100% tumor cell death at very low concentration (< 1.25 ÂľM). Palladium and a broad series of group VIII transition metal complexes containing bidentate phosphine ligands of the general formula [L2 MXm]n+ nX! [L = Ph2P-A-PPh2, A = (CH2)2, (CH2)3 or cis-CH = CH; M = Fe, Co, Rh, Ir, Ni, Pd; X = Cl, Br, I, NO3, ClO4, CF3SO3; m = 0-2; n = 03] were prepared and evaluated for in vitro cytotoxicity, in vivo antitumor activity in murine tumor model and mechanism of action. The mechanism of these complexes appears different from that of cisplatin based on effects on DNA and lack of cross resistance with L1210/DDP, a line of L1210 murine leukemia resistant to cisplatin (Shurig et al, 1989). Recently, it has been reported on a new palladium(II) complex bearing a bidentate P-N ligand which was formed via the condensation of 2-(diphenylphosphino)benzaldehyde and ethyl hydrazinoacetate (Male$evi% et al, 2006). The cytotoxic activity of the complex was similar to that of cisplatin.

D. Palladium(II) complexes mixed donor atom ligands


Palladium(II) complexes with mixed nitrogen-sulfur ligands such as methionine and substituted pyrimidines

(Cl) P



Pd P



N CH 3


N CH3 CH 3




Figure 5. Palladium(II) complexes bearing 1,2-bis(diphenylphosphino)ethane (dppe) (19 and 20).



Abu-Surrah et al: Palladium-based chemotherapeutic agents








N Cl
















Figure 6. Palladium(II) complexes with mixed donor ligands (21-23).

the putrescine complex is much more active than the spermidine one (Navarro-Ranninger et al, 1993). Zhao and colleagues studied dinuclear palladium complexes containing two functional [Pd(en)(pyridine)Cl]+ units bridged by Se or S (Zhao et al, 1999). The complexes are water soluble. The Se-bridged Pd(II) dimer (26) has a lower IC50 than the S analogue or cisplatin against the HCT8 cancer cells line.

E. Multinuclear Palladium(II) complexes Navarro-Raninger and colleagues reported in 1993 that the synthesis of putrescine (24, Figure 7) and spermine (25)-based dinuclear palladium complexes. The complex (24) is a coordination complex of a dimer nature. In 25, the 4 amino groups of the spermine coordinate to two cis-Pd-centers. The cytotoxicity results showed that


Cl Pd

Cl Pd Cl






Cl Pd




Pd Cl




(24) NH2

Cl Pd







Pd N CH 3

Pt H3N






Cl (27) (CH2) 4 NH2 NH2 OH Pt H3N N

H3N Pd



CH 3 CH3 CH3 N PPh

PPh 2 CH3

(NO3) 2

H 2N




(CH2 )4 (28) Figure 7. Multinuclear palladium-based complexes (24-28).


NH3 Cl


Cancer Therapy Vol 6, page 7 Dinuclear cyclopalladated organometallic complexes containing biphosphine ligands were also reported by Rodrigues and colleagues in 2003). The dimer Pd(II) complex [Pd2(S(-)C2, N-dmpa)2(µ-dppe)Cl2] (27) (dmpa = enantiomer S(-) of N,N-dimethyl-1-phenylethylamine; dppe = 1,2-bis(diphenylphosphino)ethane) showed to be the most active in vivo compared to the corresponding mononuclear complexes. It delays tumor growth and prolongs animal survival. Eddings and colleagues reported the first 2-cyano-2isonitroso-N-morpholinylacetamide (HMCO) based dimeric palladium (II) complex, [Pd(MCO)]2. (Eddings et al, 2004) The complex was tested in vitro on antiproliferating activity using human cervical cancer HeLa cell lines, and cisplatin as a positive control substance. It is found to be active compound inflicting death on 28% of the cells, with 55% value for the cisplatin under the same conditions. Giovagnini and colleagues have reported the synthesis and in vitro cytotoxic activity of new palladium(II) derivatives of methylsarcosinedithiocarbamate and its S-methyl ester (Giovagnini et al, 2005). The biological activity of these compounds, as determined by growth inhibition and apoptosis induction, has been investigated in both human leukemic promyelocites HL60 and human squamous cervical adenocarcinoma HeLa cell lines, and their activity has been compared to the wellknown platinum-based anticancer agent cisplatin. On the basis of these experimental results, [Pd(MSDT)X]n (MSDT = methylsarcosinedithiocarbamate; X = Cl, Br) complexes show a strong dose-dependent growth inhibition of both HL60 and HeLa cells, with IC50 values slightly higher than those recorded for cisplatin. A trinuclear palladium complex has also been reported. The complex [{trans-PtCl(NH3)}2-#-{transPd(NH3)(2-hydroxypyridine)-(H2N(CH2)6NH2)2]4+. 4Cl! (28, Figure 7) was found to exhibit significant anticancer activity against the cell lines A2780 A2780cisR and A2780 (Cheng et al, 2006). The compound is believed to form a range of interstrand GG adducts with duplex DNA that induces global changes in the DNA conformation, unlike cisplatin and ZD0473 ([cis-(2-methylpyridine)(ammine)dichloroplatinum(II)]) that form mainly intrastrand adducts that induces a local kink in a DNA strand.

potential antitumour palladium complexes have been emerged. The foremost target of most research groups was to find a convenient anticancer drug that can be used efficiently for the treatment of human tumors. The most profitable one could be that of good solubility in water and the ability to transport (through the membranes), fortitude in the cell, binding to the DNA, and eventually excretion from the body with minimum side effects. Taking into consideration the similarities between platinum and palladium, the role of thiol compounds in drug resistance should thoroughly be studied. It has been shown that cellular thiols can sequester cisplatin, leading to a reduction in the levels of cisplatin–DNA damage.

Acknowledgment Financial support by the Hashemite University is gratefully acknowledged.

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III. Conclusions With the aid of inorganic- or coordinationchemistry, it is possible to design novel therapeutic and diagnostic agents. Solubility, reactivity, electronic and steric properties, and the geometry of metal complexes can be controlled by simply varying or modifying the ligand around the metal center. It is apparent from the data presented in this review that both the metal and the ligand determine the biological activity. Platinum(II) attacks DNA, but other metal ions may have different target sites, and it will be interesting to follow the progress of the further metals in the clinical trials. Our review provides a perfect example of how small changes in molecular structure could lead to profound differences in biological activity. Several classes of 7

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Cancer Therapy Vol 6, page 9 Cyclopalladated compounds as chemotherapeutic agents: Antitumor activity against a murine melanoma cell line. Int J Cancer 107,498-504. Rosenberg B, Vancamp L, Trosko J, Mansour V (1969) Platinum Compounds: a New Class of Potent Antitumour Agents. Nature 222, 385-386. Shurig JE, Harry HA, Timmer K, Long BH, Casazza AM (1989) Antitumor activity of bis[bis(diphenylphoshino)alkane and alkene] groupVIII metal complexes Prog Clin Biochem Med 10, 205-216. Suzanne E, Sherman and Stephen J, Lippard Sherman SE, Lippard, S. J. (1987) Structural aspects of platinum anticancer drug interactions with DNA. Chem Rev 87, 11531181. Tashiro T, Kawada Y, Sakurai Y, Kidani Y (1989) Antitumor activity of a new platinum complex, oxalato (trans-l-1,2diaminocyclohexane)platinum (II): new experimental data. Biomed Pharmacother 43, 251-260. Trávní"ek z, Sz)"ová L, Popa I (2007) Synthesis, characterization and assessment of the cytotoxic properties of cis and trans-[Pd(L)2Cl2] complexes involving 6benzylamino-9-isopropylpurine derivatives J Inorg Biochem 101, 477-492. Tusek-Bozic LJ, Matijasic I, Bocelli G, Calestani G, Furlani A, Scarcia V, Papaioannou A (1991) Preparation, characterization and activity of palladium(II) halide complexes with diethyl 2-quinolylmethylphosphonate (2dqmp). X-Ray crystal structures of trans-[Pd(2-dqmp)2X 2](X = Cl or Br). J Chem Soc Dalton Trans, 195-201. Wang D, Lippard SJ (2005) Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 4, 307–320. Wimmer FZ, Wimmer S, Castan P, Cros S, Johnson N, ColacioRodrigez E (1989) The antitumor activity of some palladium(II) complexes with chelating ligands Anticancer Res 9, 791-794.

Zhao G, Lin H, Yu P, Sun H, Zhu S, Su X, Chen Y (1999) Ethylenediamine-palladium(II) complexes with pyridine and its derivatives: synthesis, molecular structure and initial antitumor studies. J Inorg Biochem. 73, 145-149. Zhao G, Sun H, Lin H, Zhu S, Su X, Chen Y (1998) Palladium(II) complexes with N,N -Dialkyl-1,10phenanthroline-2,9-dimathanamine: synthesis, characterization, and cytotoxic activity. J Inorg Biochem 72, 173-177. Zhao G, Lin H, Zhu S, Sun H, Chen Y (1998) Dinuclear palladium(II) complexes containing two monofunctional [Pd(en)(pyridine)Cl]+ units bridged by Se or S. Synthesis, characterization, cytotoxicity and kinetic studies of DNAbinding. J Inorg Biochem 70, 219-226.

From left to right: Maher Abdalla, Adnan Abu-Surrah, Haitham Al-Sa’doni


Abu-Surrah et al: Palladium-based chemotherapeutic agents


Cancer Therapy Vol 6, page 11 Cancer Therapy Vol 6, 11-24, 2008

New directions in the management of renal cell carcinoma Review Article

Marco Antonio Arap1, Ariel Galapo Kann2, Gustavo dos Santos Fernandes2, Antonio Carlos Buzaid2, Sami Arap1, Fernando Cotait Maluf2,* 1 2

Department of Urology, University of Sao Paulo Medical School, São Paulo, Brazil Oncology Center, Sirio Libanês Hospital, São Paulo, Brazil

__________________________________________________________________________________ *Correspondence: Fernando Cotait Maluf, Rua Adma Jafet 91 CEP 01308-050, São Paulo, São Paulo, Brazil; Tel/Fax: 55-11-31550210; e-mail: Key words: Molecular target therapy, Motzer criteria, good, poor and intermediate prognosis, RCC patients, novel agents, Adjuvant therapy, cytoreductive nephrectomy Abbreviations: central nervous system, (CNS); Eastern Cooperative Oncology Group, (ECOG); interleukin 6, (IL-6); mammalian target of rapamycin, (mTOR); platelet-derived growth factor, (PDGF); Renal clear cell carcinoma, (RCCC); vascular endothelial growth factor, (VEGF); von-Hippel-Lindau, (VHL) Received: 29 October 2007; Revised: 24 December 2007 Accepted: 31 December 2007; electronically published: January 2008

Summary Renal cell carcinoma represents nearly 3% of all cancers, frequently affecting patients at the age of 50 to 70 years. Few treatments options were available until recently for metastatic renal cell carcinoma. The 5-year median survival for those patients was estimated to be less than 10%. This review explored the data of the most relevant trials focused on new approaches with novel agents with several mechanisms of action such as: sunitinib, sorafenib, bevacizumab, temsirolimus and their combinations with traditional agents. We describe mechanism of action, activity and toxicity profile of all those agents as well as administration schedule. The surgical treatment was also revised, highlighting the data about nephrectomy in metastatic disease.

outcome in this aggressive disease that is generally chemo and immunotherapeutic-resistant. A relevant knowledge was provided by a study that showed that there is a similar genetic profile between sporadic renal clear cell carcinoma (non inherited and responsible for 60 to 70% of all renal tumors) and RCC secondary to the von-Hippel-Lindau (VHL) syndrome (hereditary disease characterized by vascular tumors including RCC representing 1% to 4% of all renal tumors) (Kondo et al, 2001, 2002). In the VHL syndrome, the mutation, deletion or chemical modifications of the VHL gene lead to lower protein levels or even inactivation of this protein with consequent higher levels of hypoxia inductible factor !. As a result, the expression of growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are markedly increased. Both VEGF and PDGF are intrinsically related to the promotion of angiogenesis, cancer invasiveness, and metastasis. Supporting prior findings, recent studies suggest that more than 60% of sporadic RCC have, as a critical point in their pathogenesis, somatically acquired mutations or methylation of the VHL gene (Kim WY et al, 2004), suggesting that may exist link between the VHL

I. Introduction Renal cell carcinoma represents nearly 3% of all cancers, frequently affecting patients at the age of 50 to 70 years. Renal clear cell carcinoma (RCCC) represents 60 to 70% of primary malignant renal tumors. In 2006, almost 35710 people in the United States developed renal cell carcinoma and 12480 died because of progression of disease (Cancer facts and figures, 1996). Epidemiologic data shows that in the last two decades the incidence of renal tumors has increased 2% to 4% each year (Kosary et al, 1993). Few treatments options were available until recently for metastatic renal cell carcinoma. The 5-year median survival for those patients was estimated to be less than 10%. The objective response rate obtained with chemotherapy was only 5%. Higher objective responses were reported with interferon-! (objective response rate of 12%) as well as with high-dose interleukin-2 (objective response rate of 19%). After failure to prior cytokine based-therapy, the overall median survival was only 10 to 13 months (Motzer et al, 2004). Therefore, newer therapies were clearly necessary in order to improve


Arap et al: New directions in the management of renal cell carcinoma syndrome and sporadic RCC. In this review article we discuss the mechanisms of action, safety and role of novel agents both in the first-line and salvage, setting as well as the role of surgery in the treatment of advanced renal carcinoma (Schemes 1-3).

II. Molecular target therapy previously untreated patients

acceptable toxicity profile, easy schedule, and antitumoral activity. The use of sunitinib in patients with central nervous system (CNS) metastases was sometimes avoided because the potential risk of bleeding. However, this year one retrospective study suggested that anti-VEGF agents including sunitinib (13 patients) and sorafenib (10 patients) does not increase the risk of either intratumoral bleeding or any CNS bleeding when a local treatment, involving particularly radiation, was previously administered (Unnithan et al, 2007). Recently, two randomized phase III studies (Escudier et al, 2007b; Motzer et al, 2007b) and one randomized phase II study (Yang et al, 2003b) have been conducted comparing agents capable of VEGF inhibition (experimental arm) versus interferon-! (control arm) in metastatic renal cell


A. Patients with good or intermediate prognosis according to Motzer criteria Sunitinib is an oral drug that inhibits VEGF (types 13) and PDGF (! e !) receptors, as well as other tyrosinekinase proteins. Sunitinib according to a phase I study (Faivre et al, 2006), Sunitinib was associated with an

Scheme 1. Management of metastatic renal cell carcinoma regarding the role of surgery.

Scheme 2. Management of metastatic renal cell carcinoma in the first-line setting.


Cancer Therapy Vol 6, page 13

Scheme 3. Management of metastatic renal cell carcinoma in the second-line setting.

carcinoma patients with good or intermediate prognosis according to Motzer criteria. This muticenter phase III study conducted by Motzer and colleagues included 750 patients with metastatic or unresectable renal cell carcinoma and no prior systemic treatment (Motzer et al, 2007b). Patients were randomized to receive interferon-! or sunitinib. Treatment with subcutaneous interferon-! was administered at the initial dose of 3MU three times a week (tiw) with drug escalation, if no toxicity, until 9MU tiw. Treatment with oral sunitinib was given daily at the dose of 50mg for four consecutive weeks followed by two weeks of rest with cycles administered every six weeks. Patients were randomly assigned according to LDH (1.5 " versus > 1.5 x normal superior limits), Eastern Cooperative Oncology Group (ECOG) performance status (0 versus 1), presence or absence of prior nephrectomy. The primary end point of this study, with an statistical power of 90%, was to demonstrate if the experimental arm would increase progression-free survival by 35% over to the control arm (from 4.6 to 6.2 months). Secondary endpoints included: overall survival, response rate and toxicity. The efficacy and safety data was evaluated by an independent committee in addition to the investigators. Patient characteristics were similar in both arms. Nearly 90% of the patients had prior nephrectomy, 14% had prior radiotherapy, 60% had ECOG 0, and 75% of patients had lung involvement. According to Motzer criteria nearly 36% of patients had good prognostic factors, 58%

intermediate and only 6% had poor prognostic factors. Patients assigned to sunitinib had superior response rates (31% versus 6%, p < 0.000001) as well as progression-free survival rates (11 months versus 5 months, HR 0.41, 95% CI, 0.32-0.53, p < 0.000001) compared to the control arm (Figure 1). Also, patients on sunitinib had a trend towards improvement in overall survival, though the OS has not been reached in either groups (HR 0.65, 95% CI, 0.440.94, p = 0.02). Subgroup analysis revealed an advantage in progression-free survival both in good (not reached versus 8 months, HR 0.37, 95% CI, 0.21-0.64) and intermediate (11 months versus 4 months, HR 0.39, 95% CI, 0.28-0.54) prognostic groups treated with sunitinib. Update of these results confirmed prior findings (Motzer et al, 2007a). Sunitinib was associated with more frequent grade III and IV toxicities including diarrhea (5%), erythrodysesthesia (5%), fatigue (7%) and hypertension (8%). On the other hand, interferon-! was associated with more often grade III and IV fatigue (11%). It is important to recognize that sunitinib has been also associated with hypothyroidism. In one series fifteen of 42 patients (36%) who were receiving sunitinib developed clinically significant hypothyroidism during the course of treatment. Abnormal results on blood tests indicating at least some level of hypothyroidism occurred in a total of 26 of 42 patients (62%). The longer sunitinib was administered, the greater was the chance of developing suppression of thyroid function (Desai et al, 2006).


Arap et al: New directions in the management of renal cell carcinoma Figure 1. Kaplan-Meier analysis of progression-free survival of sunitinib and interferon-#.

Another study reported similar results (12 out of 55 patients) (Shaheen et al, 2006). Hypothyroidism can contribute to fatigue associated with sunitinib. Therefore, it is advised to monitor thyroid hormone levels in patients treated with this agent. A randomized phase II trial (Yang et al, 2003b), including 116 previously treated patients evaluated the impact on progression-free survival of two different schedules of bevacizumab administered intravenously at 3mg/kg and 10mg/kg every two weeks in comparison to placebo. This trial demonstrated an advantage on progression-free survival rates for patients treated with high-dose bevacizumab compared to placebo (4.8 months versus 2.5 months, p < 0.001). More recently a phase III trial (Escudier et al, 2007a) accrued 649 metastatic renal cell carcinoma patients (including patients with predominant clear-cell component) not exposed to prior systemic therapy and who had prior nephrectomy, 322 patients were randomized to subcutaneous interferon-# (9MUI tiw) and 327 were assigned to receive interferon-# (9MUI tiw) plus bevacizumab (10mg/kg every 2 weeks). Patients were stratified according to registration center and Motzer prognostic criteria. Primary end point of this study was overall survival. The aim of this study was to evaluate if the experimental arm would increase overall survival over interferon-# from 13 to 17 months with a statistical power of 80% (p < 0.05). Secondary end points included progression-free survival, time to progression, response rate and toxicity. Exclusion criteria included CNS involvement, cord compression and use of anti-coagulants. Both arms were well balanced. Progression-free survival was higher in the interferon-# plus bevacizumab arm (10.2 versus 5.4 months, HR 0.63, p < 0.0001). A trend toward improved overall survival was observed with the addition of bevacizumab to interferon-# (p=0.0670) An advantage in progression-free survival was observed in almost all subgroups including those with favorable (12.9 months versus 7.6 months, HR 0.60, p = 0.004) and intermediate prognostic score (10.2 months versus 4.5 months, HR 0.55, p < 0.0001). Nevertheless, the addition of bevacizumab appeared not to favorably impact patients

with poor prognostic features (2.2 months versus 2.1 months, HR 0.81, p < 0.45). The addition of bevacizumab to interferon-# was associated with an advantage in progression-free survival regardless of age (<40 versus 4064 versus $ 65), gender and number of metastatic sites (" 2 versus > 2). Patients receiving the combination regimen had more grade III/IV toxicity (60%) when compared to the control group (45%) including fatigue (23% versus 15%), proteinuria (6.5% versus 0%), hypertension, (3.9% versus 0.7%) and hemorrhage (3.3% versus 0.3%). Despite that the combination arm was considered generally well tolerated. Based on these results, both sunitinib alone and bevacizumab plus interferon-# can be considered standard first-line therapy options in patients with unresectable or metastatic RCC with favorable or intermediate prognostic factors. Treatment costs, schedule profile, and patientâ&#x20AC;&#x2122;s and physicianâ&#x20AC;&#x2122;s preferences must influence the regimen of choice between these two regimens. Is important to mention that the combination of bevacizumab and interferon-# has not yet been approved for the Food and Drug Administration, limiting the use of this therapy in some countries including the United States. Sorafenib is a bi-aryl urea orally active characterized by Raf-1 serine-threonine kinase inhibition wich has inhibitory properties against VEGF (type 1-3) and PDGF receptors (! e !) (Mancuso et al, 2006). The activity of sorafenib was demonstrated in a phase III study including patients who failed prior systemic therapy (Escudier et al, 2007b). A randomized phase II study, including 189 patients, compared subcutaneous interferon-! at dose of 9MU tiw to oral sorafenib at the continuous dose of 400mg twice a day (bid) as first therapeutic line for RCC line. Primary end point included evaluation and comparison of progression-free survival between the two arms. Patient characteristics were similar in both arms. Despite the fact that sorafenib arm had superior quality of life outcomes over interferon-! (p = 0.02), there were no differences in response rates (5% versus 9%) and progression-free survival (5.7 months versus 5.6 months, HR 0.88, 95% CI, 0.61-1.27, p = 0.5). Sorafenib was associated with greater grade III and IV non-hematologic


Cancer Therapy Vol 6, page 15 toxicities including diarrhea (6%), erythrodysesthesia (11%), and skin rash (6%). On the other hand, interferon-! was associated with more frequent grade III and IV fatigue (11%), nausea (6%), constitutional symptoms (6%) and confusion (4%) (Escudier et al, 2006). An update of this trial, recently presented, evaluated the role of sorafenib dose-escalation to 600mg bid for 44 patients who progressed on the standard dose dose (400mg bid). Surprisingly, patients on higher doses had a median progression-free survival of 4.1 months, any degree of tumor shrinkage in 44% of the cases, and reasonable tolerance (Szczylik et al, 2007). A phase II trial with intrapatient sorafenib dose-escalation up to 1200-1600mg a day (Amato et al, 2007) was presented this year. A total of 44 patients were included, 25 of them was not treated before, 91% of all patients could receive higher doses and the authors reported objective responses of 55%, including 19% of complete responses. These encouraging results suggest that higher doses of sorafenib might circumvent drug resistance of this agent administered at the conventional schedule, this trial also showed an increased dose dependent toxicity, the most common treatment related adverse events were: hand/foot syndrome, skin rash, diarrhea, alopecia, fatigue, hypertension, hypophosphatemia, and elevated amylase/lipase. These findings also raises the question regarding that the initial sorafenib doses evaluated in both phase II and III trials might be suboptimal and if this is true new studies should evaluate higher doses schedules.

intravenously at the dose of 25mg. In the combination arm interferon-! was administered initially at the dose of 6MU tiw associated to weekly temsirolimus at the dose of 15mg. Patients were stratified according to registration center and absence or presence of prior nephrectomy. Patients had to have at least three of the six unfavorable prognostic factors that included: performance status 60-70 (according to the Karnofsky scale), LDH > 1.5 x the superior normal limits, hemoglobin level lower than inferior normal limit, corrected serum calcium > 10mg/dL, time from the diagnosis to the study entry < 1 year, multiple sites of metastasis. The primary end point of this study was overall survival. With a statistical power of 90%, the design of this trial planned to show an increase in overall survival by 40%, in favor of temsirolimus arms (from 4.9 to 6.9 months, two-sided, p value < 0.025). Secondary end points included: progression-free survival, response rates (including stable disease for the clinical benefit analysis), and toxicity. This study also had an independent review committee for reviewing the efficacy and safety data. The three arms were well balanced. Nearly 67% of the patients had prior nephrectomy, 80% had clear cell histology, and more than 70% of the patients had unfavorable prognostic factors according to Motzer criteria. Overall survival in the combination arm was not superior compared to control arm (HR 0.96, 95% CI, 0.761.20; p = 0.70). Median overall survival rates in the interferon-!, temsirolimus, and combination arms were 7.3 months, 10.9 months, and 8.4 months, respectively (Table 1). Temsirolimus alone was associated with an advantage in overall survival (HR, 0.73, 95% CI, 0.580.92; p = 0.008) and progression-free survival (p < 0.001) over interferon-! alone. Single agent temsirolimus was well tolerated. Grade III or IV adverse events occurred in 67% of patients in the temsirolimus group, as compared with 78% of patients in the interferon group (P=0.02) and 87% of patients in the combination-therapy group (P=0.02). Most common grade III or IV toxicities from temsirolimus included hyperglycemia (10%) and anemia (12%) dyspnea and asthenia. Asthenia was most common in the two groups receiving interferon alone or in combination. Grade III or IV asthenia was reported in 11% of patients in the temsirolimus group, in 26% of those in the interferon group (P<0.001), and in 28% of those in the combination-therapy group (P<0.001). When compared with patients in the interferon group, mild to moderate rash, peripheral edema, and stomatitis affected more patients who received temsirolimus, either alone or in combination with interferon. Anemia, neutropenia, and thrombocytopenia were more common in the combinationtherapy group than in the interferon group (P<0.001 for anemia, neutropenia, and thrombocytopenia) or in the temsirolimus group (P<0.001 for neutropenia and thrombocytopenia, and P=0.002 for anemia). Hyperglycemia, hypercholesterolemia, and hyperlipidemia were more common in the temsirolimus group and the combination-therapy group, reflecting inhibition of mTOR-regulated glucose and lipid metabolism. Based on these results single agent temsirolimus should be considered the standard first-line therapy for patients with poor prognosis characteristics according to Motzer criteria.

B. Patients with poor and intermediate prognosis according to Motzer criteria Other important pathway involved in the biological cascade in RCC carcinoma is represented by a serine/threonine protein kinase knowed as mammalian target of rapamycin (mTOR). Overexpression of mTOR is associated with prolonged tumor cell survival as well as high cell proliferation rates. Temsirolimus (CCI-779) is an mTOR inhibithor, such action is explained by the interaction of the drug with mTOR forming a complex that inactivates the kinase through phosphorylation phosphorilation inhibition. This effect induces apoptosis, cell cycle arrest at the G1 phase as well as cell response to growth factors and nutrients. Temsirolimus also inhibits one of the most important mTOR promoters, the AKT (serine-threonine kinase), as well as their regulators (PI3 kinase and tumor suppressor gene PTEN). Several studies have demonstrated that both AKT and mTOR play a critical role in renal cell carcinoma tumorigenesis and biologic behavior. Also, temsirolimus is known as an inhibitor of the hypoxia induced-! factor that is associated with direct effects on both VEGF and PDGF (Smolewski, 2006; Rubio-Viqueira et al, 2007). A phase III trial was conducted (Hudes et al, 2007), including 626 treatmentna誰ve patients with metastatic renal cancer associated with poor and intermediate prognostic characteristics. Patients were randomized among interferon-! versus temsirolimus versus the combination of both agents. Treatment with subcutaneous interferon-! was administered initially at the dose of 3MU tiw with dose escalation, if no toxicity, until 18MU tiw. Single-agent weekly temsirolimus was given 15

Arap et al: New directions in the management of renal cell carcinoma Table 1. Overall survival by treatment arm comparing interferon-#, temsirolimus, or both.

N Deaths, n (%) OS, median (95% CI), months Protocol-defined comparisons OS, hazard ratio (95% CI) OS, log-rank p

Overall Survival by Treatment Arm IFN (Arm 1) TEMSR (Arm 2) 207 209 149 (34) 141 (32) 7.3 (6.1, 8.9) 10.9 (8.6, 12.7)

TEMSR + IFN (Arm 3) 210 152 (34) 8.4 (6.6, 10.2)


Arm 2: Arm 1

Arm 3: Arm 1


0.73 (0.57, 0.92) 0.0069

0.95 (0.76, 1.20) 0.6912

INF = Interferon-#; TEMSR = Temsirolimus; OS = Overall Survival; N = number of patients; NA: Not Achieved

Confirming the activity of this class of agents, a phase II study evaluated everolimus at dose of 10mg orally and reported partial responses in 9 of 28 metastatic renal cell carcinoma patients exposed to prior systemic therapies (Amato et al, 2006). Based on the fact that agents that inhibit multiple kinases involved on the VEGF and PDGF pathways have established activity in advanced disease, it is plausible as part of future strategies to evaluate synergistic interactions among them with other class of agents such as immunotherapeutic drugs. A phase II study, including 62 patients, evaluated the role of combining sorafenib 400mg orally bid continuously with interferon-! 10MU tiw as first-line therapy. Eligible patients had metastatic or unresectable renal carcinoma with a clear-cell component, no prior systemic therapy, performance status 0 to 1, and measurable disease. The primary end point was objective response. Twelve (19%) of 62 assessable patients achieved an objective confirmed response. An additional 31 patients (50%) had an unconfirmed partial response or stable disease as best response. The median progression-free survival was 7 months (95% CI, 4-11 months). The most common adverse events were fatigue, anorexia, anemia, diarrhea, nausea, rigors/chills, leukopenia, fever, and transaminases elevation (Ryan, 2007). Another phase II study was conducted including 40 patients who were treated with sorafenib and interferon-#, as first- or secondline therapy in metastatic renal cell cancer, on the same schedule of the previous study have the following results: response rate 33% (95% CI, 19% - 49%; 13 of 40 patients), including 28% partial responses (n = 11) and 5% complete responses (n = 2). The median duration of response was 12 months. With a median follow-up time of 14 months, median progression-free survival time was 10 months (95% CI, 8 - 18 months). Fatigue, anorexia, anemia, diarrhea, hypophosphatemia, rash, nausea, and weight loss were the most common toxicities. Grade IV toxicities were uncommon but included hypophosphatemia, neutropenia, rash, fatigue, and anemia. Dose reductions were required in 65% of patients (Gollob et al, 2007). A phase III randomized study is ongoing, planning to accrual 499 patients with RCC, comparing daily sunitinib 50mg for 4 weeks followed by 2 weeks of rest versus daily sunitinib 37.5mg continuously for 6

weeks versus sunitinib at the conventional schedule combined with interferon-!. The primary goal of this trial is to evaluate if the combination arm will be associated with an improvement in time to progression from 8 to 12 months compared to the sunitinib alone arms (statistical power of 85%). This study will answer important questions regarding potential synergisms among different agents with distinct mechanisms of action as well as delineate the optimal sunitinib schedule. Another randomized phase II study has being conducted by ECOG, including 360 patients with RCC, comparing intravenous bevacizumab 10mg/kg given every 2 weeks versus bevacizumab at the same schedule associated with intravenous temsirolimus administered at the dose of 25mg weekly on days 1, 8, 15, and 22 or bevacizumab associated with sorafenibe 400mg bid continuously. The fourth arm of this trial consists of the combination of sorafenibe 400mg bid plus temsirolimus 25mg on days 1, 8, 15, and 22. The study primary end point includes the evaluation and comparison of progression free survival among the four arms.

III. Molecular target therapy previously treated RCC patients


Patients with metastatic renal cell carcinoma who failed immunotherapy have a poor prognosis (Flanigan RC et al, 2007). The activity of other immunological agents as well as cytotoxic chemotherapy is minimal and clearly new agents are necessary in order to increase overall survival, palliate symptoms and improve quality of life. Two phase II studies including 168 patients were conducted to evaluate the activity of sunitinib in patients with RCC who failed prior cytokine therapy (interferon-!, interleukin-2, or both). Inclusion criteria included: ECOG 0 or 1, measurable disease, and absence of CNS involvement. Approximately 97% of patients had undergone prior nephrectomy. The response rates were classified according to the RECIST criteria and revised by an independent commission. The objective response rate was 42% with a median progression-free survival of 8.2 months (95% CI, 7.8-10.4 months). The toxicity profile of both trials evaluating sunitinib as second-line therapy is


Cancer Therapy Vol 6, page 17 comparable to that observed with this agent in the first-line setting. (Motzer et al, 2006a,b). An important phase III trial (Escudier et al, 2007b), in treatment refractory RCC, evaluated sorafenib at the standard schedule (400 mg BID) continuous orally) versus placebo. This study included 903 patients and the primary end point was overall survival. Secondary end points included the evaluation and comparison of progressionfree survival, response rate, toxicity and quality of life. Inclusion criteria included: metastatic RCC, disease progression after one prior systemic treatment within the 8 months, ECOG 0 or 1, and absence of CNS involvement metastasis. Poor-risk patients according to Motzer prognostic score (Motzer et al 2004) were excluded from the trial. Patients were stratified according to Motzer criteria (low and intermediate) and country. Patients in both arms had a median age of 58 years, 77% had lung involvement, and 82% had received previous cytokinebased therapy. Prior radiotherapy and nephrectomy were performed in 25% and 94% of the patients, respectively. Approximately 51% of the patients were low and 49%

intermediate-risk. Update of this trial showed an overall survival benefit of sorafenib over placebo, however those results were limited only to patients who did not crossover to receive sorafenib after progression to placebo (17.8 months versus 14.3 months, HR 0.78, 95% CI, 0.62-0.97, p = 0.028) (Figure 2) (Bukowski et al 2007). The diseasefree survival in the sorafenib group was also superior compared to placebo (5.5 versus 2.8 months; HR 0.44, 95% CI, 0.43-0.60, p < 0.001) (Figure 3). Sorafenib was associated with an advantage in progression-free survival among all subgroups including age (< 65 years or $ 65 years), Motzer criteria (low or intermediate-risk), previous systemic treatment (cytokine or others), presence or absence of lung or liver metastasis, and time since diagnosis (< 1.5 years or $ 1.5 years).The most common adverse effects (all grades) were diarrhea (43%), cutaneous reactions (40%), erythrodysesthesia (30%), fatigue (37%), nausea (23%), and hypertension (17%). Grade III and IV hematologic and non-hematological toxicities were uncommon (3%).

Figure 2. Kaplan-Meier analysis of overall survival sorafenib and placebo.

Figure 3. Kaplan-Meier analysis of progression-free survival sorafenib and placebo.


Arap et al: New directions in the management of renal cell carcinoma sorafenib were similar despite the degree of tumor reduction. On the other hand, patients who had no tumor reduction (assessed by the sum of tumor axis) showed inferior progression-free survival rates. Similarly, a phase II trial (Ratain et al, 2006) was designed to evaluate the outcome of patients based on the response according to sorafenib treatment. Three groups of patients were classified according to the response as assessed by RECIST criteria and managed accordingly. Sorafenib was maintained for patients who had tumor reduction $ 25%. On the other hand, sorafenib was discontinued for patients with tumor growth $ 25%. Patients who had tumor reduction < 25% or tumor increase < 25% were randomly assigned to sorafenib maintenance (32 patients) or placebo (33 patients) for an additional 12 weeks. Analyzing this last group of patients who were randomized, a total of 50% of patients on the sorafenib arm versus 18% on the placebo arm were progression-free at 6 months (p=0.007), respectively. These results reinforce previous findings that VEGF inhibitors should be probably maintained in patients without gross tumor progression or unacceptable toxicity, even if tumor size does not decrease dramatically. Some preliminary data suggest the appearance of necrotic component during VEGF inhibitors treatment in prior solid lesions may be useful to evaluate the efficacy of these agents (Figure 4). Other authors suggest that changes in tumor vascularization during treatment as assessed by MRI (De Bazelaire et al, 2003; Morgan et al, 2003) or doppler ultrasonography (Lamuraglia et al, 2005) may also be useful as a criteria of response. These parameters must be evaluated prospectively and compared to the standard response criteria in order to better establish a relationship between image response and clinical benefit with these novel agents. Lastly, as sunitinib, bevacizumab plus interferon-#, sorafenib, and temsirolimus have a major impact both in the first- and second-line setting, it is crucial to evaluate the activity of these agents in patients who failed other VEGF-inhibitor. A retrospective study (Sablin et al, 2007) evaluated the role of sunitinib in patients who progressed on sorafenib (68 patients) or vice-versa (22 patients). The objective response rate in these two groups of patients was 22.7% and 17.6%, respectively, suggesting that both agents may target different pathways rather than the VEGF cascade. As a result, both agents can be considered second-line options after failure to a VEGF inhibitor. In order to better evaluate prospectively the non-cross resistance activity of these agents, dose-response schedules, and potential synergism among these novel agents to revert resistance a phase II randomized trial is ongoing. In this trial, patients who had disease progression on sunitinib at the standard dose are randomized to increase the sunitinib dose versus association of bevacizumab to sunitinib versus switch to sorafenib at standard doses. Similarly, randomization for patients who had disease progression on sorafenib at standard doses includes increasing the sorafenib dose versus association of bevacizumab to sorafenib versus switching to sunitinib at standard doses. Also, based on the two standard firstline therapies, it is extremely important to evaluate the

IV. Unresolved questions raised by novel agents in renal cell carcinoma Despite the encouraging results both in the first- and second-line setting, clinical studies with sunitinib and sorafenib raise several questions regarding optimal doses and intervals, durability of response, impact on long-term survival, definition of biomarkers of response, efficacy in other histologies, safety of administration in patients with CNS metastasis, and whether the standard response criteria, such as RECIST, should be applied to this class of agents. Despite daily dose of sunitinib at 50mg for 4 weeks every 6 weeks was reasonably well tolerated, fatigue occurred in 51% of the patients. Possibly, dose modifications may be necessary in order to maintain the safety drug profile in patients with a lower body surface area than the average patient population included in the sunitinib trials. Also, monitoring serum levels of this agent might help individualize the optimal dose for each patient (Houk et al, 2007). We agree with the reviewer comment and we re-wrote the sentence as a hypothesis. Although a high number of patients achieved a response with sunitinib (and in a minor proportion with sorafenib), it seems that responses are not as durable as those observed with high-dose interleukin 2. Approximately 8% of patients treated with high-dose interleukin-2 achieve complete responses (Yang et al, 2003a), and in 80% of those patients response duration lasts over 8 years. Recently, it was demonstrated that the carbonic anhydrase IX over-expression evaluated by immuno-histochemical analysis (Atkins et al, 2005) and the alveolar component (Upton et al, 2005) in RCC predict the response rates in patients treated with high-dose interleukin 2. Thus high-dose interleukin-2 should still be considered the standard first-line treatment for patients " 60 years, without significant co-morbidities and absence of CNS involvement in association with a favorable histological and molecular profile. Unfortunately, only a few patients meet these criteria. Prognostic features are under evaluation to predict the efficacy of agents that target VEGF and PDGF receptor tyrosine kinases. The role of sunitinib and sorafenib in non clear-cell renal carcinoma and the safety of both agents in patients with brain metastasis are still uncertain. In a recent abstract (Plantade A et al, 2007), sunitinib (38%) and sorafenib (62%) were evaluated in patients with chromophobe (12 patients) and papillary (41 patients) renal cell carcinoma. The response rates achieved with both VEGF inhibitors in chromophobe and papillary subtypes were 25% and 4.8%, respectively. The standard response criteria based on measurable disease (e.g. RECIST) were validated in large studies using cytotoxic agents and generally correlates with clinical benefit. However, it is uncertain whether these criteria are optimal to evaluate efficacy of these novel antiangiogenic agents who have both cytostatic and cytotoxic properties (Gore et al, 2006). In a retrospective analysis (Escudier et al, 2007a), patients who had tumor reduction > 0% and " 10% versus > 10% and " 20% versus > 20% had similar progression-free survival rates. As an example, progression-free survival curves in patients treated with 18

Cancer Therapy Vol 6, page 19 activity of bevacizumab (plus or minus interferon-#) in patients who have progressed on sunitinib and vice-versa. A phase II trial (George et al, 2007) involving 61 bevacizumab-resistant patients evaluated the efficacy of sunitinib and reported objective responses and stable disease rates of 23% and 57%, respectively. The median duration of response and progression-free survival were 30 weeks and 36 weeks, respectively. A retrospective study (Drabkin et al, 2007) evaluated the activity of sorafenib in patients previously treated with bevacizumab. The objective response and stable disease were 2.5% and 77%, respectively. There is no data available regarding the activity of bevacizumab-based regimens sunitinib- or sorafenib-refractory patients. How do we compare the results of these novel agents to immunotherapy or chemotherapy in the second-line setting? Escudier and colleagues reported a response rate with interferon-! of only 2% in 48 patients previously treated with interleukin-2. Similarly, the response rate to

interleukin-2 in 65 patients previously treated with interferon-! was only 4% (Escudier et al, 1999). Another study including 251 patients treated with second-line agents (including immunotherapy and chemotherapy) reported a response rate of only 4% (Motzer et al, 2002). In summary, these novel agents that inhibit important pathways related to the mutation or methylation of the VHL gene represent a new paradigm in the treatment of metastatic RCC. The disease-free survival observed with sunitinib (8.2 months) or sorafenib (5.5 months) in prior treated patients are significantly better than those observed with other â&#x20AC;&#x153;oldâ&#x20AC;? agents (2.4 months). Therefore, these novel agents represent an important progress in the treatment of metastatic RCC (Table 2). Surprisingly, the response rates and progression-free survival seems not to correlate whether patients received prior treatment or not. These findings suggest that these agents may have noncross resistance properties with other agents such as immunotherapy and chemotherapy.

Figure 4. Patient with metastatic renal clear cell carcinoma involving the pancreas. Pre- and post-treatment with sunitinib showing a significant difference of density among the lesions due to necrosis/liquefaction after treatment despite the fact that no change in tumor diameter.

Table 2. Summary of results of anti-VEGF therapies and conventional therapy as second-line treatment (Escudier et al, 1999, 2007; Motzer and Russo, 2000; Yang et al, 2003b; Motzer et al, 2006a,b, 2007b). Treatment



Progression-free survival

Motzer Escudier Yang

Number of patients 168 451 39

Sunitinib Sorafenib Bevacuzimab (high dose) Several (historic data) Immunotherapy (second-line) Interferon-! (first-line)

40% 10% 10%

8.2 months 5.5 months 4.8 months




2.4 months








4.7 months


Arap et al: New directions in the management of renal cell carcinoma immunologic responses may be impaired by the overproduction of interleukin 6 (IL-6), IL-8, IL-10, GMCFS, and other cytokines. Other hypothetical benefits of nephrectomy before systemic treatment are palliation of paraneoplastic syndromes, prevention of complications caused by locally advanced disease, the shed of tumor cells and the interruption of cytokine release by the primary tumor, which might be also involved in the growth of metastases. Many retrospective studies of pre-immunotherapy cytoreductive nephrectomy before systemic treatment have been reported. As the response to cytoreductive nephrectomy is variable, most authors reinforce the importance of patient selection and preoperative evaluation of different criteria believed to be predictive of good outcome, such as ECOG performance status and the presence of liver, bone or central nervous system metastasis (Fallick et al, 1997) Other studies showed that multiple organ metastasis (Han et al, 2003). low Karnofsky performance status, hemoglobin level lower than the normal limits, corrected serum calcium level >10 mg/dL (Motzer et al, 1999), tumor grade, preoperative white blood cell count, partial thromboplastin time, and lymph node metastases are also prognostic (Pantuck et al, 2003). The most important problems associated to surgery are the peri-operative mortality rate (that varies from 0 to 17%) and the inability to receive post-operative systemic therapy due to poor performance status (Bennett et al, 1995). The largest retrospective series is from the National Cancer Institute and included 195 patients who underwent nephrectomy before undergoing interleukin-2 therapy. The overall response was 18%, mortality was 1% and 38% of patients were unable to undergo interleukin-2 treatment due to poor performance status, post-operative complications and tumor progression (Walther et al, 1997). Two important prospective and randomized trials were reported regarding cytoreductive nephrectomy (Flanigan et al, 2001; Mickisch et al, 2001). The trials conducted by SWOG and EORTC used the same protocol (cytoreductive nephrectomy followed by interferon-! or interferon-! alone) and both demonstrated longer survival in the nephrectomy groups. In addition, the trials definitively showed in a well-selected patient population that surgery was rarely a limitation to start systemic therapy, and that the response rate to systemic therapy was similar in both groups. There is no data regarding a potential role for cytoreductive nephrectomy for patients with metastatic disease treated with VEGF tyrosine kinase inhibitors. However, all positive trials that showed a benefit of VEGF, PDGF, and mTOR kinase inhibitors over immunotherapy or placebo included mostly patients who had prior nephrectomy. In conclusion, the role of nephrectomy before systemic therapy for metastatic renal cancer is still to be defined. However, nephrectomy is justified in the palliation of local symptoms, in patients with solitary metastases or low disease burden and in patients with good performance status and limited disease who candidates to receive systemic therapy including newer agents.

V. Adjuvant therapy with molecular target therapy According to the established activity of the VEGF and PGDF tyrosine kinase inhibitors in metastatic disease, clinical studies are ongoing to explore the role of these agents in reducing tumor recurrence, improving survival, and changing (for the first time) the natural history of this disease for high-risk patients in the adjuvant setting. The study, coordinated by the ECOG, plans to accrual 1332 patients with $T1B renal cell carcinoma and randomized them among three arms: sorafenib (400 mg bid on days 142) versus sunitinib (50 mg qd on days 1-28 every 42 days) versus placebo. In all three arms, treatment was given for a total of 9 cycles. The primary end point of this study is to evaluate and compare the disease-free survival curve rates among the three arms. Patients have being stratified by pathologic staging and histological subtype (clear cell or non-clear cell). This trial aim to answer two important questions: if these novel agents are superior to placebo and define the VEGF inhibitor of choice. Another phase III three-arm trial ongoing, plan to accrue 1420 high or intermediate risk patients. The three arms of this trial include continuous three-year sorafenib (400 mg bid) versus one-year sorafenib (400 mg bid) versus placebo. The primary end point of this study is to evaluate and compare the disease-free survival rates among the three arms. This study raises two different questions: whether the target agent impacts on the natural history of disease and the optimal treatment duration these novels agents. A third randomized trial involving another molecular target agent is ongoing, with a planned accrual of 600 patients, comparing the role of a chimeric antibody against carbonic anhydrase protein given intravenously weekly for 24 weeks versus placebo for patients with intermediateand high-risk of relapse. The carbonic anhydrase protein is overexpressed in more than 80 to 95% of the renal tumors and has direct biologic implications in renal clear cell carcinoma biology and behavior. A recent phase II trial (Bleumer et al, 2004) reported response rate and stable disease in 33% of treated patients. The primary end point includes the evaluation and comparison of disease-free survival and overall survival rates between the two arms.

VI. The role of cytoreductive nephrectomy in metastatic renal cell carcinoma To date, cytokine-based immunotherapy is still considered an option for patients with metastatic renal clear cell carcinoma. The role of nephrectomy in this situation, either before or after systemic treatment, remains controversial. The rationale for cytoreduction before immunotherapy is based on the ability of renal cell carcinoma to manipulate hostâ&#x20AC;&#x2122;s natural immunity. It is known that the primary lesion rarely responds to systemic treatment, even in patients who present with regression of metastases (Rackley et al, 1994). Lymphocytes from patients with metastatic renal cancer have increased apoptosis (Cardi et al, 1998; Uzzo et al, 1999) defective Tcell receptors (Finke et al, 1993) and poor signal transduction (Li et al, 1994; Ng et al, 2002). In addition, 20

Cancer Therapy Vol 6, page 21 Escudier B, Chevreau C, Lasset C, Douillard JY, Ravaud A, Fabbro M, Caty A, Rossi JF, Viens P, Bergerat JP, Savary J, NĂŠgrier S (1999) Cytokines in metastatic renal cell carcinoma, is it useful to switch to interleukin-2 or interferon after failure of a first treatment? Groupe Francais d'Immunotherape. J Clin Oncol 17, 2039-43. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, Negrier S, Chevreau C, Solska E, Desai AA, Rolland F, Demkow T, Hutson TE, Gore M, Freeman S, Schwartz B, Shan M, Simantov R, Bukowski RM; TARGET Study Group (2007) Sorafenib in Advanced Clear-Cell Renal-Cell Carcinoma. N Engl J Med 356, 125-134. Escudier B, Koralewski P, Pluzanska A (2007) A randomized, controlled, double-blind phase III study (AVOREN) of bevacizumab/interferon-#2a vs placebo/interferon-#2a as first-line therapy in metastatic renal cell carcinoma (Abst 3a). J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No 18S (June 20 Supplement). Escudier B, Szczylik C, Eisen T, Stadler WM, Schwartz B, Shan M, Bukowski RM (2005) Randomized Phase III trial of the Raf kinase and VEGFR inhibitor sorafenib (BAY 43-9006) in patients with advanced renal cell carcinoma (RCC) (Abst 4510). J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No 18S (June 20 Supplement). Faivre S, Delbaldo C, Vera K, Robert C, Lozahic S, Lassau N, Bello C, Deprimo S, Brega N, Massimini G, Armand JP, Scigalla P, Raymond E (2006) Safety, pharmacokinetic, and antitumor activity of SU11248, a novel oral multitarget tyrosine kinase inhibitor, in patients with cancer. J Clin Oncol 24, 25-35. Fallick ML, McDermott DF, LaRock D, Long JP, Atkins MB (1997) Nephrectomy before interleukin-2 therapy for patients with metastatic renal cell carcinoma. J Urol 158, 1691. Finke JH, Zea AH, Stanley J, Longo DL, Mizoguchi H, Tubbs RR, Wiltrout RH, O'Shea JJ, Kudoh S, Klein E, et al (1993) Loss of T-cell receptor zeta chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res 53, 5613-6. Flanigan RC, Salmon SE, Blumenstein BA, Bearman SI, Roy V, McGrath PC, Caton JR Jr, Munshi N, Crawford ED (2001) Nephrectomy followed by interferon alfa-2b compared with interferon alfa-2b alone for metastatic renal-cell cancer. N Engl J Med 345, 1655-9. Folkman J (1990a) What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 82, 4-6. Folkman J (1990b) Endothelial cells and angiogenic growth factors in cancer growth and metastasis. Introduction. Cancer Metastasis Rev 9, 171-174. Folkman J (1971) Tumor angiogenesis, Therapeutic implications. N Engl J Med 285, 1182-1186. George DJ, Michaelson MD, Rosenberg JE (2007) Phase II trial of sunitinib in bevacizumab-refractory metastatic renal cell carcinoma (mRCC), Updated results and analysis of circulating biomarkers (Abst 5035). J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No 18S (June 20 Supplement). Gollob JA, Rathmell WK, Richmond TM, Marino CB, Miller EK, Grigson G, Watkins C, Gu L, Peterson BL, Wright JJ (2007) Phase II trial of sorafenib plus interferon alfa-2b as first- or second-line therapy in patients with metastatic renal cell cancer. J Clin Oncol 25, 3288-95. Gore ME, Escudier B (2006) Number of metastatic sites rather than location dictates overall survival of patients with nodenegative metastatic renal cell carcinoma. Urology 61, 314-9 Han KR, Pantuck AJ, Bui MH, Shvarts O, Freitas DG, Zisman A, Leibovich BC, Dorey FJ, Gitlitz BJ, Figlin RA, Belldegrun AS (2003) Exposure-response of sunitinib in metastatic renal cell carcinoma (mRCC), A population

VII. Conclusions Recently several advances have been made on both basic and clinical knowledge about renal cell carcinoma, such data allows to see knew horizons and build a more positive future for those patients achieved for this severe disease. In spite of those advances, metastatic RCC is considered incurable and the continue development of knew strategies is mandatory.

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Motzer RJ, Hutson TE, Tomczak P, Michaelson MD, Bukowski RM, Rixe O, Oudard S, Negrier S, Szczylik C, Kim ST, Chen I, Bycott PW, Baum CM, Figlin RA (2007) Interferonalfa as a comparative treatment for clinical trials of new therapies against advanced renal cell carcinoma. J Clin Oncol 20, 289-96. Motzer RJ, Mazumdar M, Bacik J, Berg W, Amsterdam A, Ferrara J (1999) Survival and prognostic stratification of 670 patients with advanced renal cell carcinoma.J Clin Oncol 17, 2530-40. Motzer RJ, Michaelson MD, Redman BG, Hudes GR, Wilding G, Figlin RA, Ginsberg MS, Kim ST, Baum CM, DePrimo SE, Li JZ, Bello CL, Theuer CP, George DJ, Rini BI (2006) Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma. J Clin Oncol 24, 16-24. Motzer RJ, Rini BI, Bukowski RM, Curti BD, George DJ, Hudes GR, Redman BG, Margolin KA, Merchan JR, Wilding G, Ginsberg MS, Bacik J, Kim ST, Baum CM, Michaelson MD (2006) Sunitinib in patients with metastatic renal cell carcinoma. JAMA 295, 2516-24. Motzer RJ, Russo P (2000) Systemic therapy for renal cell carcinoma. J Urol 163, 408-417. Ng CS, Novick AC, Tannenbaum CS, Bukowski RM, Finke JH (2002) Mechanisms of immune evasion by renal cell carcinoma, tumor-induced T-lymphocyte apoptosis and NF"B suppression. Urology 59, 9-14. PEShaheen, IRTamaskar, RNSalas, BIRini, JGarcia, LWood, RDreicer, RMBukowski (2006) Thyroid function tests (TFTs) abnormalities in patients (pts) with metastatic renal cell carcinoma (mRCC) treated with sunitinib (Abst 5048). J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No 18S (June 20 Supplement). Plantade A, Choueiri T, Escudier B, Rini B, Negrier S, Ravaud A, Oudard S, Elson P, Bukowski R (2007) Treatment outcome for metastatic papillary and cromophobe renal cell carcinoma patients treated with tyrosine-kinase inhibitors (TKIs) sunitinib and sorafenib (Abst 5037). J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No 18S (June 20 Supplement). Rackley R, Novick A, Klein E, Bukowski R, McLain D, Goldfarb D (1994) The impact of adjuvant nephrectomy on multimodality treatment of metastatic renal cell carcinoma. J Urol 152(5 Pt 1), 1399-403.s Ratain MJ, Eisen T, Stadler WM, Flaherty KT, Kaye SB, Rosner GL, Gore M, Desai AA, Patnaik A, Xiong HQ, Rowinsky E, Abbruzzese JL, Xia C, Simantov R, Schwartz B, O'Dwyer PJ (2006) Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma. J Clin Oncol 24, 2505-12. Rubio-Viqueira B, Hidalgo M (2006) Targeting mTOR for cancer treatment. Curr Opin Investig Drugs 7, 501-12. Ryan CW, Goldman BH, Lara PN (2007) Sorafenib with interferon alfa-2b as first-line treatment of advanced renal carcinoma, a phase II study of the Southwest Oncology Group. J Clin Oncol 25, 3296-301. Sablin MP, Bouiata L, Balleyguier C, Gautier J, Celier C, Balcaceres L, Oudrad S, Ravaud A, Negrier S, Escudier B (2007) Sequential use of sorafenib and sunitinib in renal cancer, Retrospective analysis in 90 patients (Abst 5038). J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No 18S (June 20 Supplement). Smolewski P (2006) Recent developments in targeting the mammalian target of rapamycin (mTOR) kinase pathway. Anticancer Drugs 17, 487-94. Szczylik C, Demkow T, Staehler M, Rolland F, Negrier S, Hutson TE, Bukowski RM, Scheuring UJ, Burk K, Escudier


Cancer Therapy Vol 6, page 23 B (2007) Randomized phase II trial of first-line treatment with sorafenib versus interferon in patients with advanced renal cell carcinoma, Final results (Abst 5025). J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No 18S (June 20 Supplement). Szczylik C, Demkow T, Staehler M, Rolland F, Negrier S, Hutson TE, Scheuring UJ, Schwartz B, Bukowski RM (2006) Randomized phase II trial of the multi-kinase inhibitor sorafenib versus interferon (IFN) in treatment-na誰ve patients with metastatic renal cell carcinoma (mRCC) (Abst 4501). J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No 18S (June 20 Supplement). Unnithan JS, Choueiri T, Garcia J, Dreicer R, Laura W, Bukowski R, Rini B (2007) Safety of VEGF-targeted tyrosine kinase inhibitors in patients (Pts) with metastatic renal cell carcinoma (mRCC) and central nervous system (CNS) metastases (Abst 5047). J Clin Oncol, 2007 ASCO Annual Meeting Proceedings Part I. Upton MP, Parker RA, Youmans A, McDermott DF, Atkins MB (2005) Histologic predictors of renal cell carcinoma response to interleukin-2-based therapy. J Immunother 28, 488-95. Uzzo RG, Rayman P, Kolenko V, Clark PE, Bloom T, Ward AM, Molto L, Tannenbaum C, Worford LJ, Bukowski R, Tubbs R, Hsi ED, Bander NH, Novick AC, Finke JH (1999) Mechanisms of apoptosis in T cells from patients with renal

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Cancer Therapy Vol 6, page 25 Cancer Therapy Vol 6, 25-34, 2008

Thiazolidinediones as anti-cancer agents Review Article

Carmelo Blanquicett*, Jesse Roman, C. Michael Hart Department of Medicine, Emory University and Atlanta Veterans Affairs Medical Centers, Atlanta, GA 30033

__________________________________________________________________________________ *Correspondence: Carmelo Blanquicett, M.D., Ph.D., Dept. of Medicine, Emory University, Division of Pulmonary and Critical Care Medicine, 1670 Clairmont Rd. (151-P), Decatur, GA 30033, USA; Phone: 404-321-6111 ext 7394; Fax: 404-728-7750; e-mail: Key words: Apoptosis, breast cancer, colon cancer, growth/cell cycle arrest mechanism of action in neoplasia, PPAR-!, tumor suppression, TZDs, lung cancer Abbreviations: AMP-activated protein kinase, (AMPK); cyclooxygenase-2, (COX-2); insulin-like growth factor-I, (IGF-I); mammalian target of rapamycin, (mTOR); phosphatase and tensin homolog, (PTEN); prostaglandin-E2, (PGE2); retinoid X receptor, (RXR); Thiazolidinediones, (TZDs); vascular endothelial growth factor, (VEGF) Received: 2 October 2007; Revised: 12 December 2007 Accepted: 20 December 2007; electronically published: February 2008

Summary The PPAR-gamma (PPAR-!) activating thiazolidinedione (TZD) medications are a class of drugs used to improve lipid and glucose metabolism in type-2 diabetes. In addition to their known insulin sensitization action, these drugs have been shown to suppress tumor development in several in vitro and in vivo models. Among the proposed mechanisms for the anti-tumor effects of TZDs, apoptosis induction, cell cycle arrest, and differentiation have been extensively reported. Interestingly, some of the observed anti-tumor effects are independent of PPAR-! activation. The following review will discuss studies employing TZDs as anti-cancer therapies for the most common types of cancers including, lung, breast, and colon and will explore the principal PPAR-!-dependent and -independent mechanisms by which TZDs exert their anti-tumor effects.

The pattern of PPAR-! expression is relatively wellcharacterized. Two PPAR-! isoforms exist that are derived from the alternate promoters, PPAR-!1 and PPAR- !2. The PPAR-!2 isoform is 30 amino acids longer than PPAR-!1 (Mueller et al, 2002) and is less abundant. PPAR-!2 is predominantly expressed in adipose tissue where it exerts pleiotropic effects on metabolism, insulin sensitization, and inflammation. PPAR-!2 is also expressed in vascular endothelium, suggesting a role for this protein in vascular biology as well as in alveolar macrophages (Gervois et al, 2007; Staels et al, 1998; Standiford et al, 2005). In contrast, PPAR-!1 is expressed in a broad variety of tissues including large intestine, kidney, liver (Fajas et al, 1997), and particularly relevant to this article, PPAR-! has been detected in cancer cells (Vamecq and Latruffe, 1999). Several reports have demonstrated that PPAR-! activation has anti-cancer properties. For example, TZDs suppress tumor development in several animal models, and PPAR-! activation arrests malignant cell growth (Sato et al, 2000; Takashima et al, 2001). In addition, treatment of cancer cells with PPAR-!-activating TZDs induces cell differentiation and apoptosis (Lu et al, 2005a). These events are depicted schematically in Figure 1.

I. Introduction Thiazolidinediones (TZDs) are a class of antidiabetic drugs which include pioglitazone, rosiglitazone, ciglitazone and troglitazone, although troglitazone was removed from the market in 2000 because of hepatoxicity. These drugs improve insulin sensitivity through mechanisms that have yet to be clearly defined and therefore, constitute an area of active investigation. TZDs bind with high affinity to the PPAR-gamma (PPAR-!) subtype of peroxisome-proliferator activated receptors (PPARs). PPARs are members of the steroid receptor superfamily of ligand-activated transcription factors. Upon activation, either by synthetic ligands such as TZDs, or endogenous ligands such as natural lipophilic ligands (e.g. fatty acids), PPAR-! forms a heterodimer with the retinoid X receptor (RXR) and binds to PPAR response elements (PPRE) that regulate the transcription of select target genes. Although more than seventy PPAR target genes with PPREs have been identified (Yu et al, 2005), it is likely that this list of PPAR regulated genes will continue to grow. Further, the complexity of PPAR-! -regulated gene expression is enhanced by various co-activators and co-repressors that are recruited to the transcriptional complex by the activated PPAR-! heterodimer. 25

Blanquicett et al: TZDs and Cancer

Figure 1. Activation of PPAR-! by TZDs causes apoptosis, growth arrest, and differentiation in the cancer cell. TZDs activate PPAR-!, resulting in various anti-neoplastic effects in the cancer cell including apoptosis, growth arrest, and differentiation.

Investigation into the molecular mechanisms that underlie PPAR-!-induced anti-cancer effects constitutes an area of active research. Nevertheless, the role of PPAR! in carcinogenesis remains controversial due in part to anti-neoplastic effects of TZDs that are independent of PPAR-!. This review will examine current research evaluating TZDs as anti-tumor compounds in lung, breast and colon carcinoma, 3 of the most common cancers in the U.S., and will describe the proposed mechanisms by which TZDs exert their anti-cancer properties.

induced PPAR-! expression and apoptosis in two human lung cancer cell lines, but not in normal cells (Li et al, 2005). These results suggest the potential for TZDs to target malignant cells without affecting normal cells (the goal of anti-cancer chemotherapy). In nude mice, direct injection of ciglitazone into A549-induced tumors suppressed the rate of tumor growth by 36% (Zhang et al, 2006). While the modest anti-neoplastic effects of TZDs may preclude their use as monotherapy for treatment of lung cancer, combining TZDs with other anti-neoplastic agents could potentially enhance therapeutic efficacy. For example, the inhibitory effect of rosiglitazone on NSCLC cell growth was enhanced by the mammalian target of rapamycin (mTOR) inhibitor, rapamycin (Han and Roman, 2006). mTOR is a serine/threonine kinase that is activated by Akt and regulates protein synthesis, and rapamycin has been shown to inhibit cell growth by blocking the action of mTOR. In addition to the above study, rosiglitazone potentiated gefitinib's antiproliferative effects by increasing the expression of the tumor suppressor, apoptotic gene, phosphatase and tensin homolog (PTEN) (Lee et al, 2006). The combination of the low-dose apoptosis inhibitor, MK886, ciglitazone, and 13-cis-retinoic acid, produced synergistic growth inhibition of lung cancer cells (A549 and H1299) suggesting that targeting PPAR-! and retinoic acid action could be a promising approach to suppress lung cancer growth (Avis et al, 2005). In vitro synergistic antiproliferative and apoptotic effects were also observed by

II. TZDs and lung cancer Non small cell lung carcinoma (NSCLC), the most common cancer in the U.S. is associated with a poor prognosis, indicating that novel therapeutic approaches are urgently needed. In a recent retrospective database analysis from 10 Veterans Affairs medical centers, lung cancer risk among TZD users compared with nonusers was reduced by 33%, after adjustment for confounding interactions (Govindarajan et al, 2007). Although clinical data supporting the efficacy of TZDs in lung cancer is limited, in vitro studies as well as reports in experimental animals support this concept. In vitro studies available supporting the efficacy of TZDs in lung cancer are numerous (Tsubouchi et al, 2000; Satoh et al, 2002; Han et al, 2005). For example, the proliferation of A549 lung cancer cells was significantly inhibited by ciglitazone in a dose- and time-dependent manner both in vivo and in vitro, and PPAR-! expression was markedly upregulated by ciglitazone treatment (Zhang et al, 2006). Troglitazone 26

Cancer Therapy Vol 6, page 27 combining a novel TZD with imatinib (Gleevec速) in various malignant cell lines (Zang et al, 2006). The opportunities to optimize anti-cancer efficacy with various chemotherapy permutations involving TZDs are numerous and suggest their potential benefit. Further, the experimental efficacy of low doses of these agents in these studies suggests potential clinical efficacy with reduced toxic side effects, a major concern in cancer chemotherapy. However, there are a few conflicting studies that suggest that TZD treatment may promote carcinogenesis or tumor development (Lefebvre et al, 1998; Saez et al, 2004). It may be the case that the specific tissue or type of cancer and its stage could contribute to the efficacy or failure of TZDs as anti-neoplastic agents. In support, rosiglitazone, ciglitazone, and prostaglandin J2 (15d-PGJ2) were all potent agonists of PPAR-! transactivation in lung adenocarcinoma cell lines. However, these same ligands had no effect in squamous cell or large cell carcinomas of the lung (Allred and Kilgore, 2005). Prognostic indicators such as the ratio of PPAR-! to RXR-", for example, may be useful in predicting how cells may respond to specific combination treatments such as TZD and 9-cis-retinoic acid, an RXR-" ligand/agonist (Allred and Kilgore, 2005). In vivo and in vitro studies employing TZDs as anti-cancer agents (either alone or as combinatorial agents) for lung cancer are encouraging, however, clinical efficacy remains to be determined.

PPAR-! ligands have been demonstrated to inhibit tumor angiogenesis and invasion in breast cancer (Fenner and Elstner, 2005). Particularly relevant information is expected to come from human studies. In patient tissue samples, PPAR-! immunoreactivity was significantly associated with improved clinical outcome in breast carcinoma patients by univariate analysis (Suzuki et al, 2006). In addition, a recent pilot trial examined short-term (2-6 weeks) treatment with rosiglitazone in 38 women with early-stage breast cancer. Rosiglitazone (8 mg/d), administered between the time of diagnostic biopsy and definitive surgery did not elicit significant effects on breast tumor cell proliferation analyzed as expression of Ki67, a marker of tumor growth and progression as well as proliferation (Yee et al 2007). In pretreatment tumors notable for nuclear expression of PPAR-! as determined by immunohistochemistry, down-regulation of nuclear PPAR-! expression occurred following rosiglitazone administration, contrary to what is expected. This TZD regimen was well tolerated and without serious adverse events (Yee et al, 2007). A prior trial reported in 2003 suggested that in patients with metastatic breast cancer, troglitizone failed to show any clinical benefits (Burstein et al, 2003). Overall, clinical studies are not encouraging. Nevertheless, more and longer term clinical studies are warranted to determine if the promising results obtained in pre-clinical and in-vitro studies can be extrapolated to humans.

III. TZDs and breast cancer

IV. TZDs and colon cancer

Numerous studies have also examined the effects of TZDs in breast cancer. Breast cancer is the most frequent cancer in women and represents the second leading cause of cancer death in this population after lung cancer (Edwards et al, 2005; Al-Mansouri and Alokail, 2006). PPAR-! is expressed in normal and malignant mammary epithelial cells, and TZDs suppress breast carcinoma proliferation in vitro and in experimental animal models (Suh et al, 1999; Nwankwo and Robbins, 2001; Jarrar and Baranova, 2007). Recently, PPAR-! activation by conjugated linoleic acid was shown to have an antiproliferative effect in MCF7 breast cancer cells (Bocca et al, 2007). Breast cancer cells, along with prostate cancer and melanoma cells were shown to undergo apoptosis with PPAR-! ligands (Nunez et al, 2006). Additionally, PPAR! was shown to induce differentiation of malignant breast epithelial cells (Mueller et al, 1998). Interestingly, PPAR! acts as a tumor suppressor not only in breast, but also in skin and ovarian cancers (Nicol et al, 2004). However, PPAR-! was also shown to act as a tumor promoter in breast carcinogenesis (Saez et al, 2004). This seemingly paradoxical outcome was proposed either to occur after a cancer initiation event had already progressed, or to possibly occur following PPAR-!-mediated activation of a cytokine or growth factor such as TGF-#, which depending on the stage of breast cancer, can either be tumor suppressing or tumor promoting (Roberts and Wakefield, 2003). Nevertheless, the preponderance of evidence among studies examining TZDs in breast cancer cells suggests that PPAR-! ligands inhibit proliferation and induce apoptosis both in vitro and in vivo. In addition,

In colon cancer, TZDs may also be of benefit (Kitamura et al, 1999). PPAR-! mRNA and protein expression has been demonstrated in HT-29 colon cancer cells by RT-PCR and western blots, respectively (Sato et al, 2000; Chen et al, 2005), and PPAR-! activation was associated with inhibition of cell growth through induction of apoptosis and suppression of the cell cycle (Takashima et al, 2001). Similarly both the synthetic TZD, pioglitazone, and the natural ligand, 15d-PGJ2, inhibited the proliferation of colon cancer cell lines in a dosedependent manner that was reversed by the TZD antagonist GW9662 (Cerbone et al, 2007; Martinasso et al, 2007; Zhang et al, 2007). In animal studies, a deficiency in intestinal PPAR-! was associated with enhanced tumorigenicity in mouse small intestine and colon (McAlpine et al, 2006). Similarly, in mouse models of colon cancer, PPAR-! agonists inhibited tumor growth or colon carcinogenesis (Yoshizumi et al, 2004; Chintharlapalli et al, 2006; Marin et al, 2006), although isolated studies suggest that PPAR-! agonists induce colon tumors in mice (Yang et al, 2005c). Studies investigating PPAR-! in human subjects with colon cancer are limited. In specimens from colon cancer patients, immunohistochemical analysis demonstrated a correlation between PPAR-! and cell cycle-related molecules but no association was detected between PPAR! and patient survival (Theocharis et al, 2007). Furthermore, the risk reduction for colorectal cancers among diabetes patients taking TZDs did not reach statistical significance (Govindarajan et al, 2007), clearly 27

Blanquicett et al: TZDs and Cancer indicating that more investigation will be required to establish a role for PPAR-! in human colon carcinogenesis.

! and ERK (Li et al, 2006a). Similarly, treating colon cancer cells with rosiglitazone caused apoptosis, detected with TUNEL staining and flow cytometry (Chen et al, 2005). Although the exact mechanisms by which apoptosis was induced in that study were not defined, bcl-2/bcl-x (anti-apoptotic proteins), p53, bad (pro-apoptotic proteins), and the transcription factor NF-"B were implicated in PPAR-! ligand-induced apoptosis. Rosiglitazone treatment also increased PTEN protein levels which can lead to apoptosis through the negative regulation of Akt (Li et al, 1998). PTEN induction by PPAR ligands has also been reported to arrest tumor growth through PTENâ&#x20AC;&#x2122;s tumor suppressor effects. TZD treatment not only induced apoptosis and reduced the antiapoptotic protein, bcl-2, in vitro, but also increased apoptotic bax expression in another study (Yang et al, 2005b). Further, TZDs reduced the anti-apoptotic protein, survivin (Figure 2), and caused dramatic sensitization of human breast cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and caspase activation (Lu et al, 2005b).

V. TZD mechanism of action in neoplasia The molecular basis for the anti-tumor actions of TZDs remains incompletely elucidated. Recent studies employing gene expression analysis of TZD-treated tissues have provided new insights. For example, a recent study in ovarian cancer cells determined that ciglitazone upregulated genes predominantly associated with metabolic, differentiation and tumor suppressor pathways, whereas genes that were downregulated by ciglitazone were mainly involved in cell proliferation, cell cycle, cell organization, and steroid biosynthesis genes (Vignati et al, 2006). These findings are consistent with a genome-wide search for high-scoring PPREs in conserved regions of all human reference genes that found that genes with higher priority scores were those involved in cell cycle arrest, apoptosis and DNA damage response rather than those involved in fatty acid metabolism (Yu et al, 2005; Jarrar and Baranova, 2007). Thus, while distinct mechanisms for the anti-tumor actions of TZDs continue to be uncovered, a large proportion of the studies indicate that TZD-induced PPAR-! activation promotes anti-tumor actions through apoptosis induction, differentiation, and growth (proliferation) arrest. The molecular evidence linking PPAR-! activation and each of these mechanisms is discussed below.

B. Growth/cell cycle arrest In addition to apoptosis, PPAR-! activation may reduce tumor development through the arrest of cancer cell proliferation, through effects on cell cycle checkpoints or growth factor inhibition. Several studies demonstrate that PPAR-! activation suppresses proliferation rates in many cancer types (Weng et al, 2006). For instance, PPAR-! activation not only caused apoptosis and reduced bcl-2 protein, but also inhibited the proliferation of lung cancer cells (Avis et al, 2005). The authors suggested that the observed apoptosis was mediated through the antiproliferative effects of PPAR-!-RXR interactions. In agreement, Elstner and colleagues showed that troglitazone combined with 9-cis-retinoic acid caused apoptosis in breast cancer cell lines, particularly in those in which bcl-2 expression levels were highest (Elstner et al, 2002). One particularly well-known manner of suppressing proliferation rates involves cell cycle progression arrest. Cyclins are cell cycle regulators. Specifically, they are regulatory subunits of cell-cycle-specific kinases, and their

A. Apoptosis One of the main mechanisms of action by which TZDs act as anti-cancer agents involves the induction of apoptosis, an effect that complements the growth suppressive properties of TZDs. A schematic representation of these effects is shown in Figure 2. Numerous studies support the notion that PPAR-! activation induces apoptosis and thus exerts anti-cancer effects (Clay et al, 1999). For instance, in lung cancer cells troglitazone induced apoptotic activity that was PPAR-!and ERK1/2-dependent (Li et al, 2006a). Troglitazone treatment reduced the anti-apoptotic protein, bcl-2, and caused nuclear accumulation and co-localization of PPAR-

Figure 2. Mechanisms of TZD-induced apoptosis. TZDs activate PPAR-!, stimulating heterodimerization with the retinoid X receptor, recruitment of coactivators, and the dissociation of corepressors which ultimately causes apoptosis by decreasing anti-apoptotic proteins such as bcl-2/bcl-x and survivin, while increasing the levels of the pro-apoptotic proteins, p53, bad and phosphatase and tensin homolog (PTEN).


Cancer Therapy Vol 6, page 29 activation is thought to regulate progress through the cell cycle. Cyclins are therefore potential oncogenes; and in fact, cyclin D1 overexpression and/or amplification are common features of several human cancers, thus promoting G1 phase progression (Musgrove et al, 1994; Chen and Harrison, 2005). Exposure to TZD for 24 h caused G0/G1 cell cycle arrest (Kawakami et al, 2002; Yang et al, 2005b). TZD treatment not only decreased protein levels of cyclin D1, but also reduced proliferating cell nuclear antigen, pRb, and Cdk4 and increased the cyclin-dependent kinase inhibitors p21 and p27, in a timedependent manner (Yang et al, 2005b). Because the p21 and p27 kinase inhibitors inhibit CDK2/4 and CDK2 respectively, this can result in cell cycle arrest. In particular, increases in p21 expression levels have been attributed to Sp1 transcriptional activation (Han et al, 2004), as PPAR-! has been shown to interact directly with transcription factors including Sp1 (Krey et al, 1995). In addition to p21, p18 was also demonstrated to be regulated by PPAR-! (Morrison and Farmer, 1999). A schematic representation of some of these events is shown in Figure 3. These data are in agreement with other studies showing that levels of cyclins, specifically cyclin E, cyclin D2 and CDK 2, are decreased following TZD treatment (Zang et al, 2006). Lu and colleagues also demonstrated that TZD treatment profoundly reduced protein levels of cyclin D3 (Lu et al, 2005b). Conversely, p27 expression was increased and degradation was decreased in colon cancer cells following ciglitazone treatment (Chen and Harrison, 2005). Therefore, TZD downregulation or suppression of cell-cycle progression caused by cyclins represents an opportunity to halt uncontrolled cellular growth (Figure 3). Furthermore, ciglitazone inhibited A549 proliferation in mice, in a dose and time-dependent fashion and decreased the expression of cyclin D1. In the same study, it was further concluded that ciglitazone induced differentiation that was associated with cell cycle arrest (Zhang et al, 2006).

in the treatment of a variety of tumors as differentiationinducing agents (Tontonoz et al, 1997; Betz et al, 2005; Li et al, 2006b). PPAR-! was demonstrated to induce differentiation in solid tumors both in vitro and in vivo (Kawamata et al, 2006). For example, TZD-induced differentiation of human cancer cells, defined as a shift toward a more steroidogenic phenotype, was mediated through activation of PPAR-!-dependent pathways (Betz et al, 2005). TZD treatment in pancreatic cancer cells significantly inhibited growth through PPAR-dependent induction of pancreatic ductal differentiation without any increase in apoptosis (Ceni et al, 2005). In HT-29 colorectal cancer cells, TZD treatment inhibited growth and metastasis through differentiation-promoting effects (Yoshizumi et al, 2004). The ability of PPAR-! activation to promote differentiation can be enhanced by the use of TZDs as combinatorial agents. For instance, the combination of the RXR agonist, bexarotene, with the PPAR-! agonist, rosiglitazone, in colon cancer cells caused increased expression of the differentiation marker, CEA, while also decreasing cyclooxygenase-2 (COX-2) expression and prostaglandin-E2 (PGE2) synthesis (Cesario et al, 2006). These findings were later confirmed in vivo in a Moser xenograft tumor model, by the same group (Cesario et al, 2006). These reports and others suggest a potential role for combination regimens of RXR and PPAR-! agonists in the treatment of colon and other cancers (Konopleva et al, 2004; Cesario et al, 2006). In cultured breast cancer cells, PPAR-! ligands caused extensive lipid accumulation and changes in epithelial gene expression associated with a more differentiated, less malignant state (Mueller et al, 1998). In lung cancer cells, ciglitazone induced differentiation (Zhang et al, 2006), and PPAR-! expression inhibited tumorigenesis by reversing the undifferentiated phenotype of metastatic non-smallcell lung cancer cells and activating pathways that promoted a more differentiated epithelial phenotype (Bren-Mattison et al, 2005).

C. Differentiation

D. Additional considerations

Another mechanism by which PPAR-! activation may exert anti-neoplastic effects is through the promotion of cellular differentiation, and early as well as recent evidence indicates that TZDs might have favorable effects

The evaluation of other PPAR isoforms may also prove to be valuable in order to clarify the effect of PPARs on cellular differentiation. PPAR-# increased colonocyte differentiation as well as apoptosis in ligand-treated

Figure 3. Mechanisms of TZD-induced growth arrest. In addition to apoptosis, PPAR-! activation may reduce tumor development through the arrest of cancer cell proliferation and effects on cell cycle checkpoints. TZD treatment activates PPAR-!, stimulating heterodimerization with the retinoid X receptor, recruitment of coactivators, and the dissociation of corepressors resulting in decreased protein levels of activated cyclins that regulate progress through the cell cycle. These include: cyclin D1 (Cd1), as well as Cdk4, Cyclin E, CD2, and CDK2. Conversely, TZDs increase the cyclin-dependent kinase inhibitors p21 and p27 that can inhibit CDK2/4 and CDK2 respectively, ultimately causing cell cycle arrest.


Blanquicett et al: TZDs and Cancer PPAR-# +/+ mice, whereas these effects were not found in PPAR-# -/- mice (Marin et al, 2006). Further, PPAR-# and PPAR-! agonists altered mammary tumorigenesis and produced distinctive histopathologic patterns of tumor differentiation and development (Yin et al, 2005). Consistent with isoform-specific effects of PPARs, the specific TZD or PPAR ligand used may be of significant importance as differentiation effects seen with one ligand may not be observed in another. Clay et al, for example, reported that treating cells with 15dPGJ2 did not increase cellular differentiation, as had been seen in other neoplastic cells, but rather induced cellular events associated with programmed cell death or apoptosis (Clay et al, 1999).

VII. Additional mechanisms of tumor suppression In addition to apoptosis, differentiation induction, or growth arrest, other anti-tumor effects linked with PPAR-! have been reported. Inhibition of invasion and metastasis is one such additional mechanism by which TZDs halt the cancer process. Metastasis was markedly promoted in diabetic and obese mice (Mori et al, 2006). Interestingly, obesity and diabetes have been associated with reduced PPAR-! expression (Itoh et al, 1999; El Midaoui et al, 2006), suggesting that levels of PPAR-! expression may be inversely related to metastatic potential. Angiogenesis is also an important mechanism in the progression and metastasis of tumors. Vascular endothelial growth factor (VEGF) is an important signaling protein involved in angiogenesis, and its overexpression has been associated with the process of metastasis. Pioglitazone not only normalized serum insulin and serum glucose but also serum VEGF levels in hyperinsulinemic, hyperglycemic rats (Yang et al, 2005a). Other studies have also confirmed the anti-angiogenic effects of PPAR-! activation in tumors. These include studies reporting the inhibition of angiogenesis through suppression of chemokine production (Keshamouni et al, 2005), through PPAR-!mediated NO production and subsequent maxi-K channel opening (Kim and Cheon, 2006), as well as VEGFmediated suppression of angiogenesis via VEGF promoter regulation (Peeters et al, 2005). PPAR-! activation has also been shown to counteract effects of the pro-inflammatory cytokines TNF-" and IL1, and He and colleagues demonstrated that troglitazone inhibited insulin-like growth factor-I (IGF-I) through AMP-activated protein kinase (AMPK), an event which was thought to occur through a PPAR-!-independent mechanism (He et al, 2006). In non-small cell lung carcinoma cells, PPAR-! activation promoted ROS formation via proline oxidase induction, which ultimately caused cell death (Kim et al, 2007). And in breast cancer cells, where estrogen receptor status has a significant role in treatment outcome, Bonofiglio and colelagues showed that the estrogen receptor alpha binds to PPAR response elements and represses its transactivation. Physical interactions between the estrogen receptor and PPAR-! that result in PPAR-! signaling interference were also documented and thought to involve PI3K (Bonofiglio et al, 2005). These results are not entirely surprising, considering that it was recently shown that PPARs have a significant effect on many genes, including steroid biosynthesis genes (Vignati et al, 2006). The selected examples used to illustrate additional mechanisms of tumor suppression by TZD actions emphasize the diversity and complexity of the mechanisms which render TZDs anti-neoplastic

VI. PPAR-!-independent mechanisms of tumor suppression In addition to PPAR-!-dependent actions, TZDs demonstrate a number of important PPAR-!-independent effects (Shiau et al, 2005; Han and Roman, 2006; Jarrar and Baranova, 2007). TZDs have been shown to stimulate the proteosomal degradation of cyclins D1 and D3 (Huang et al, 2005; Lu et al, 2005b), to block the G(1)-S transition through translation initiation inhibition (Palakurthi et al, 2001), and to scavenge toxic reactive oxygen species (ROS) (Inoue et al, 1997) through PPAR-!-independent mechanisms. Additional PPAR-!-independent actions of TZDs include the induction of cellular acidosis through inhibition of the Na+/H+ exchanger (de Dios et al, 2001; Turturro et al, 2004), calcium storage depletion (Palakurthi et al, 2001), and release of apoptotic factors from the mitochondria through the production of ROS (Pandhare et al, 2006). Other cited mechanisms by which TZDs exert anti-tumor effects in a PPAR-!-independent manner include upregulation of PTEN/AMPK and down regulation of Akt/mTOR/p70S6 signaling cascades (Han and Roman, 2006). Because of these PPAR-!-independent actions of TZDs, it must be emphasized that mechanistic investigations of individual PPAR-! ligands in cancer therapy must account for both PPAR-!-dependent and independent pathways. The ultimate anti-cancer effect of a specific ligand may also depend on the target cell, the hostâ&#x20AC;&#x2122;s nutritional status, and the availability of coactivators or co-repressors in the cell of interest. Examples of ligand specific effects, independent of PPAR-! activation, abound and further illustrate the need to thoroughly investigate PPAR-!-independent effects. For example, many tumors are resistant to TRAIL, a promising agent that preferentially induces apoptosis in cancer cells, and Lu and colleagues suggest that both TRAIL sensitization and reduction in cyclin D3 protein levels induced by TZDs are likely PPAR-! -independent because neither a dominant negative mutant of PPAR-! nor PPAR-! expression antagonized these effects (Lu et al, 2005). With the high frequency of dysfunctional apoptotic pathways in cancer cells, the degree to which a specific TZD can sensitize cancer cells to apoptosis-inducing agents, or the degree to which a TZD itself can induce apoptosis (independent of PPAR-! status) will require further examination.

VIII. Future directions The most convincing evidence demonstrating TZD efficacy in cancer therapy will undoubtedly come from clinical trials. Despite the numerous pre-clinical studies showing promise of TZDs as anti-cancer agents, currently available results of the few clinical trials that have already


Cancer Therapy Vol 6, page 31 been performed are neither uniformly positive nor conclusive. For example, the effect of TZDs on the likelihood of colon, prostate or breast cancer development appears to be neutral, with the effect of TZDs being neither beneficial nor deleterious (Koro et al, 2007). A phase II trial examining rosiglitazone administration in patients with thyroid cancer found no relationship between PPAR-! mRNA or protein in the neoplasms and radioiodine uptake status after rosiglitazone therapy (Kebebew et al, 2006). Furthermore, a diagnosis of cancer was significantly associated with TZD use, even after correcting for potential confounding variables (RamosNino et al, 2007). However, the evaluation of the effectiveness of TZDs in cancer therapeutics must integrate potential differences among ligands within the same TZD class as identified in the pre-clinical studies discussed above. Additional clinical trials are currently ongoing ( that could clarify the role of TZDs in cancer, and results may not be available for some time. Cancer, as a complex, multifactorial disease will undoubtedly require the combination of agents in order to produce clinical efficacy. TZDs may provide more benefit when combined with other agents. The exploration of drug combinations at low doses may represent a promising strategy (Avis et al, 2005) as suggested by the potentiation of gefitinib by the coadministration of rosiglitazone (Yee et al, 2007). It has also been suggested that in addition to cancer treatment, TZDs may have a role in cancer chemoprevention (Brown and Lippman, 2000; Badawi and Badr, 2002; Fan et al, 2003). However, other studies have provided evidence that PPAR-! ligands may actually foster carcinogenesis. For example, the use of PPAR-! ligands increased the development of colon tumors (Lefebvre et al, 1998). In another report, constitutive expression of PPAR-! in the mammary glands accelerated tumor development (Saez et al, 2004), although it may be the case that expression of PPAR-! without normal, physiological regulation may promote carcinogenesis. These conflicting results emphasize the need to increase the number and length of well-designed clinical studies that will carefully define the role of these promising agents.

Acknowledgments This work was supported by grants from the National Institutes of Health and the Veterans Affairs Research Service (AA16080, CMH; HL080543, JR).

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IX. Conclusions A rapidly expanding body of literature has examined the ability of PPAR-!-activating TZD ligands to contribute to cancer therapy as evidenced by numerous in vitro and in vivo studies. The results of these studies, while mixed, have fostered sufficient interest to stimulate the investigation of TZDs in clinical trials. Additional studies of PPAR-! ligands in combination with other agents or in chemopreventive strategies merit further consideration. Whether the results obtained from in vitro and preclinical studies investigating the anti-cancer potential of PPAR-! ligands will extrapolate to efficacy in human trials, remains to be determined. Clinical trials of adequate power and duration are required to clarify the role that PPAR-! activation may have in the treatment of cancer.


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From left to right: Jesse Roman, Carmelo Blanquicett, C. Michael Hart


Cancer Therapy Vol 6, page 35 Cancer Therapy Vol 6, 35-46, 2008

Gene therapy for esophageal cancer Review Article

Jong Chul Park1, Chulso Moon1,2,â&#x20AC; ,* 1

Department of Otolaryngology and Head and Neck Cancer Research Institute, Department of Oncology, The Johns Hopkins University School of Medicine and the Sidney Kimmel Cancer Center, Baltimore, MD 21218 2

__________________________________________________________________________________ *Correspondence: Chulso Moon, M.D., Ph.D., The Head and Neck Cancer Research Division, Department of Otolaryngology, The Johns Hopkins University, CRBII, 5M03, 1550 Orleans St. Baltimore MD 21231,USA; Tel: (410) 502 5153; Fax: (410) 614 1411: Email: â&#x20AC;  Present address: Cangen Biotechnologies, Inc., 300 E. Lombard Street, Suite 840 Baltimore, MD 21202 Key words: esophageal cancer, molecular biology, gene therapy Abbreviations: 5-fluoro-2'-deoxyuridine 5'-monophosphate, (5-FdUMP); 5-fluorouracil triphosphate, (5-FUTP); 5-fluorouracil, (5-FU); adenocarcinoma, (ADC); adenovirus-expressing wild-type p53, (Ad-p53); cell growth ratio, (CGR); cyclin-dependent kinases, (CDKs); cyclooxygenase-2, (COX-2); cytotoxic T lymphocyte, (CTL); enhanced green fluorescent protein, (EGFP); epidermal growth factor receptor, (EGFR); esophageal adenocarcinoma, (EAC); esophageal squamous cell carcinoma, (ESCC); ganciclovir, (GCV); gap junctional intercellular communication, (GJIC); gastroesophageal reflux disease, (GERD); loss of hetrozygosity, (LOH); onditionally replicative adenoviruses, (CRAd); secretory leukoprotease inhibitor, (SLPI); squamous cell carcinoma, (SCC); Terminal Deoxynucleotidyl Transferase-MediatedNick End-Labeling, (TUNEL); thymidine kinase, (HSV- TK); Uracil phosphoribosyltransferase, (UPRT)

The article was supported in part by the SPORE grant P50 CA96784-01 (to C.M.), Cancer Research Grant from Pyung-Ya Foundation (to C.M.)

Received: 19 November 2007; Revised: 13 December 2007 Accepted: 16 January 2008; electronically published: February 2008

Summary Esophageal cancer is the ninth most common malignancy and the seventh leading cause of cancer death worldwide. Its incidence is dramatically increasing especially that of esophageal adenocarcinoma. A variety of therapeutic strategies have been developed to improve survival for the patients with esophageal cancer but overall survival still remains poor with 5 year survival around 15%. Recent advances in molecular biology of esophageal cancer have documented the role of genetic alterations in tumorigenesis and have led to the development of potential new therapeutic approaches designed to target those genetic alterations. This article will review the molecular biology of esophageal cancer and the recent studies in the gene therapy for esophageal cancer.

decades in the United States. Until the 1970s, esophageal squamous cell carcinoma (ESCC) was the most common type of esophageal cancer and affected mostly African American men who had a long history of smoking and alcohol consumption. Over the last 2 decades, the incidence of adenocarcinoma of the distal esophagus and gastroesophageal junction has progressively increased and currently it accounts for more than 50% of all new cases of esophageal cancer. It affects mostly white men, and its pathogenesis is linked to gastroesophageal reflux disease (GERD) and the development of Barrett epithelium. Surgical resection alone can be curative for localized tumors without evidence of distant metastases. However, only 20-30 % of the patients are candidates for a

I. Introduction Esophageal cancer is the ninth most common malignancy and the seventh leading cause of cancer death worldwide. The incidence of esophageal cancer varies widely according to geographical region and racial background. In the United States, the incidence is dramatically increasing especially that of esophageal adenocarcinoma (EAC), which has the fastest growing incidence rate of all cancers in the United States (DeWeese et al, 2001). In 2007 in the United States, 15,560 people are expected to be diagnosed with esophageal cancer and 13,940 will die of their disease (Jemal et al, 2007). The epidemiology of esophageal carcinoma has changed markedly over the past several 35

Park and Moon: Gene therapy for esophageal cancer potentially curative surgery and overall survival remains poor (Epperly et al, 2001). Recently, adjuvant modality, a combination of chemotherapy and radiation therapy, is often used for downstaging of the tumor and improving the prognosis after surgery (Geh et al, 2001). Despite surgery or chemoradiotherapy, prognosis of esophageal cancer still remains poor, with 5-year survival around 10 % (Enzinger and Mayer, 2003). The failure of conventional therapy occurs mostly because tumors develop resistance to chemotherapy or radiation and attempts to overcome resistance with higher doses of radiation and chemotherapeutics inevitably result in an unacceptable degree of toxicity and bystander damage to normal tissues. The major limitation of all these treatments and their combinations is the lack of specificity for the tumor cell and toxicity to the patient. Recent advances in molecular biology of esophageal cancer have documented the role of genetic alterations in tumorigenesis and have led to the development of potential new therapeutic approaches designed to target those genetic alterations. This article will review the molecular biology of esophageal cancer and the recent studies in the gene therapy for esophageal cancer.

abnormal transcripts are common events that can serve as a potential diagnostic tool. Gene amplification of cyclin D is found preferentially in esophageal cancer, whereas gene amplification of cyclin E and c-met is frequently associated with gastric cancer. Mutations of the cyclindependent kinase inhibitor genes also occur in esophageal and gastric cancers. Cancer of the esophagus exists in 2 main forms with different etiological and pathological characteristics-squamous cell carcinoma (SCC) and adenocarcinoma (ADC) and as described in Table 1, both types shares common genetic changes. Overall, the most common alterations found in esophageal cancers include allelic losses at chromosomes 3p, 5q, 9p, 9q, 13q, 17p, 17q and 18q, as well as mutations of p53 (mostly missense). Rb (deletions), cyclin DI (amplifications) and c-myc (amplifications). The sequence of occurrence of these alterations with respect to histopathological tumor progression has been well characterized. Key checkpoints of cell cycle exist at the G1-S and G2-M phase transitions. Cell cycle is regulated by the complex of cyclin-dependent kinases (CDKs) and their association with specific regulatory subunits, the cyclins via the phosphorylation of its proper target; the cyclin A/CDK2 and cyclin B/CDK2 complexes manage the G2M phase transition, whereas the cyclin D1/CDK4 and cyclin E/CDK2 complexes maintain the G1-S checkpoint (Morgan, 1995). A number of factors are involved in the regulation of the G1- S phase transition. The c-myc protein enhances the progression into S phase and DNA replication while the retinoblastoma protein (Robert et al, 2000) sequesters the E2F transcription factor preventing the process. The p16 protein causes cell cycle arrest by inhibiting the association of CDK4 and cyclin D1, while p53 acts through p21WAF1 to sequester CDKs (Harper et al, 1995). Altered expression in any of these factors can lead to disruption in cell cycle regulation and eventually cause development of esophageal carcinoma (Table 1).

II. Molecular biology of esophageal cancer When developing gene therapy, it is essential to have precise information about the genetic etiology of the disease. The information can be very complex in the case of cancer because several mutational hits may have taken place during carcinogenesis. Many tumor suppressor genes and the oncogenes control the expression of proteins that function as cell cycle regulators. Esophageal cancer involve genetic alterations in several oncogenes, tumor suppressor genes, and potentially DNA repair genes (Montesano et al, 1998) and shares certain common features with gastric cancer. Inactivation of the p53 gene and expression of CD44

Table 1. Oncogenes and tumor suppressor genes commonly altered in esophageal cancer. Tumor Related Gene Oncogene

Gene Cyclin D1 (11q13) ErbB-1 (7q12) ErbB-2 (17q21)

Tumor suppressor gene

p53 (17q13)

p21 (6p21)

p16 (9p21)

Reference ESCC EAC Metzger et al, 2004; Metzger et al, 2004; Kawakubo et al, 2005 Izzo et al, 2007 Hanawa et al, 2006; Sudo et Beilstein et al, 2002 al, 2007 al-Kasspooles et al, 1993; al-Kasspooles et al, Friess et al., 1999 1993; Friess et al, 1999 Audrezet et al, 1993; Putz et al, 2002 Kuwabara et al, 1998; Robert et al, 2000 Bahl et al, 2000; Nakashima et al, 2000; Heeren et al, 2004 Tokugawa et al, 2002 Kwong et al, 2004; Ishii et al, 2007

ESCC: Esophageal squamous cell carcinoma EAC: Esophageal adenocarcinoma


Cancer Therapy Vol 6, page 37 On top of the genetic changes directly involved in cell cycle, pathways related with apoptosis are also important in understanding molecular biology of esophageal cancer. For example, overexpression of E2F-1 can activate CPP32, one of the most important apoptosisinducing molecules, and can cause cleavage of the death substrate poly(ADP-ribose) polymerase, which suggests that activation of the caspase cascade may be a pivotal mechanism in E2F-1-mediated apoptosis (Fueyo et al, 1998). Moreover, this pathway seems separate from the p53-mediated pathway, because E2F-1 dose not increase the expression level of bax protein (a putative p53mediated apoptotic pathway. Also, for the precise understanding for the molecular mechanisms of p53, both detailed information for each interaction between p53 and other key players like RB or MDM2 (Bates et al, 1998) and conceptual understanding that dynamic equilibrium and functional stability of p53 rather than p53 expression itself determines the outcome of p53 are critically important (Kubbutat et al, 1997). For example, a proto-oncogene MDM2, which can be induced by wt-p53 activity, binds to p53 and masks the p53 transcriptional activation domain (Oliner et al, 1993). MDM2 targets p53 protein for degradation in the ubiquitin pathway, which results in abrogation of its antiproliferative and apoptosis-promoting effects. In addition, it has been shown that p14 binds to and induces the degradation of the proto-oncogene MDM2, which results in stabilization of p53 (Kubbutat et al, 1997). Furthermore, cell-cycle arrest mediated by p14 can be abolished in cells lacking functional p53 (Pomerantz et al, 1993), which indicates that p14 may act upstream of p53.

apoptosis (Thompson, 1995). According to several studies, p53 gene alteration is observed in about half of ESCC (Audrezet et al, 1993; Kuwabara et al, 1998; Robert et al, 2000), and more than 100 mutations of ESCC have been reported in the IRAC p53 mutation database (Putz et al, 2002). Most of these mutations are located in four evolutionary conserved domains of the p53 gene, from exon 5 to exon 8, and more than 80% are point mutations. Alterations in p53 are found in 60% of Barrettâ&#x20AC;&#x2122;s metaplasias and 95% of EAC (Putz et al, 2002). Its relevance as a prognostic factor is controversial; some studies suggest that the presence of mutant type p53 is correlated with a poor prognosis, while other studies found a lack of such correlation with a poor prognosis (Ito et al, 2001; Matsumoto et al, 2001; Shibata and Matsubara, 2001; Mathew et al, 2002).

C. p21WAF1 The functions of CDKs are inhibited by two families of CDK inhibitors. The first class comprises the CIP/KIP family (p21WAF1/CIP1, p27KIP1, and p57KIP2), and the other comprises the INK4 family proteins (p16INK4a, p15INK4b, p18INK4c, and p19INK4d). The product of p21WAF1 gene is a cyclin-dependent kinase inhibitor and is thought to play a central role in tumor suppression. The p21WAF1 gene contains a p53binding site in its promoter, and the expression of p21WAF1 was directly regulated by p53; cells with loss of p53 activity due to mutational alteration were unable to induce p21WAF1 (Ohashi et al, 1997). Mutations and deletions of the p21WAF1 gene are rare in human cancers, although p21WAF1 polymorphisms have been seen in certain cancers. In ESCC, polymorphisms have been identified at codon 31 and codon 149 in exon 2 of the p21WAF1 gene, and may play an important role in esophageal tumorigenesis (Bahl et al, 2000). Some reports suggest that wild-type p53 concomitant with p21WAF1 expression is a predictor of a good response to chemotherapy or chemoradiotherapy in ESCC in vivo (Nakashima et al, 2000; Heeren et al, 2004).

A. Cyclins D1 Cyclin D1 is one of the most essential proteins for controlling the cell cycle in the p16-pRb pathway (Roncalli et al, 1998). Cyclin D1/CDK4 regulates mid to late G1 by phosphorylating Rb which allows E2F to activate genes for S-phase (Alt et al, 2000). Amplification of the Cyclin D1 gene or overexpression of the cyclin D1 protein can result in transformation to a malignant phenotype. In ESCC, cyclin D1 overexpression was found to start early in dysplasia and early cancers and play an important role in cell transformation (Kawakubo et al, 2005). Cyclin D1 protein is also increased significantly in Barrettâ&#x20AC;&#x2122;s metaplasia with nuclear accumulation and it increases the risk of progression to EAC by six- to sevenfold (Izzo et al, 2007). Overexpression and genomic amplification of cyclin D1 is found in 22-73% of ESCC and 22-64% of EAC tissues (Metzger et al, 2004). Overexpression of cyclin D1 was also shown to be associated with poor prognosis such as lymph nodes/distant metastases, high tumor grade, poor response to chemotherapy and decreased survival (Lin et al, 2004).

D. p16INK4a p16INK4a is a tumor suppressor protein that inhibits the function of the cyclin D1/CDK4 and CDK6 complex, and causes p53-independent G1 arrest through the phosphorylation of pRb (Bruce et al, 2000). In human cancers, inactivation of the p16INK4a gene is a frequent event associated with homozygous deletion, genetic mutation, or aberrant DNA methylation (Guo et al, 2007; Jie et al, 2007; Milyavsky et al, 2007). Inactivation of the p16INK4a gene in ESCC is common, and caused by homozygous deletion and de novo methylation (Tokugawa et al, 2002). On the other hand, promoter hypermethylation with loss of hetrozygosity (LOH) is a common mechanisms for inactivation of p16INK4a in EAC (Kwong et al, 2004; Ishii et al, 2007).

B. p53 The p53 gene is the most frequently mutated gene identified in human esophageal cancer (Metzger et al, 2004). The p53 gene leads to cell cycle arrest through p21WAF1 induction which sequesters CDKs and by downregulating bcl-2 while upregulating Bax, it induces

E. Growth factors and their receptors The epidermal growth factor receptor (EGFR) family comprises erbB-1 (EGFR), erbB-2 (HER2), erbB-3, and erbB-4, all of which are tyrosine kinase receptors. These 37

Park and Moon: Gene therapy for esophageal cancer receptors translate proteins on the cell membrane, which act as receptors, and have intracellular tyrosine kinase activity and extracellular binding domains. Overexpression of erbB-1 has been reported in many ESCC tumor samples and cell lines (29%-92%) (Hanawa et al, 2006; Sudo et al, 2007). The overexpression of erbB1 in patients with ESCC and EAC was correlated with a poor prognosis and poor response to chemoradiotherapy (Beilstein and Silberg, 2002; Gibson et al, 2003; Gotoh et al, 2007; Wang et al, 2007). The erbB-2 oncogene encodes a truncated form of EGFR, which contains continuously active tyrosine kinase. Cells expressing this oncogene behave as if they are being constantly stimulated by growth factors. While overexpression of erbB-2 at the protein level is seen in EAC, it is less frequent in ESCC and was found in 0% to 38% of tumors (al-Kasspooles et al, 1993; Friess et al, 1999).

p53 in esophageal cancer cells. Thus, overexpression of ectopic E2F-1 protein may stabilize endogenous as well as ectopic p53 protein via the E2F-1/ARF/MDM2/p53 regulatory pathway and, in this way, render cells more sensitive to apoptosis, an outcome that has important implications for the treatment of human esophageal cancers.

III. Corrective gene therapy A number of specific genetic alterations have been identified in esophageal cancer. These genetic events include amplification and/or overexpression of oncogenes, mutations and deletions leading to inactivation of tumor suppressor genes. The identification of these molecular alterations has allowed the development of new therapeutic approaches targeting specific differences between normal and malignant cells (Table 2).

F. E2F-1

A. p53-replacement gene therapy

Mutations that directly perturb the pRB3-mediated G1 restriction point. pRb is thought to exert its cell cycle regulatory effects by binding and sequestering the transcription factor, E2F-1 (Bates et al, 1998). E2F-1 has unique and somewhat paradoxical activities. E2F-1 promotes cellular proliferation by stimulating expression of a number of genes that promote transition from G1 to the S phase. For example, overexpression of E2F-1 stimulates quiescent cells to enter into the S phase, whereas inhibition of E2F-1 prevents entry into the S phase (Itoshima et al, 2000). However, overexpression of E2F-1 has also been shown to induce apoptosis in several cell types including esophageal cancer cells, indicating that E2F-1 plays a role not only in regulating cell growth, but in coordinating programmed cell death (Yang et al, 2000). Furthermore, recent studies with E2F-1 knockout mice suggested that E2F-1 functions as a tumor suppressor gene

Transfection of wt-p53 is an appealing therapeutic strategy for esophageal cancer because of the high frequency of p53 mutations in both squamous cell carcinoma and adenocarcinoma developed in Barrett's esophagus and the central role of p53 in regulating growth and apoptosis (Metzger et al, 2004). Tagawa and his colleagues in Chiba university in Japan demonstrated that when wild-type p53 gene was transduced to human esophageal cancer cells (T.Tn) bearing mutated p53 gene, the transduced cells (T.Tn/p53) which stably expressed wild-type p53 were markedly susceptible to radiation and chemotherapeutics (cisplatin and etoposide) compared with parental cells (Matsubara et al, 1999b). They evaluated the transduction efficacy and in vivo and in vitro antitumor efficacy of the recombinant adenoviral vector Ad5CMV-p53 on human ESCC (Shimada et al, 2001). Ad5CMV-p53 is replicationdeficient (del E1, del E3) adenovirus type 5 in which the E1 region is replaced with the cDNA of the p53 gene and is driven by a cytomegalovirus promoter. Ad5CMV-p53 has been extensively studied in various tumors, especially lung and head and neck cancer (Moon et al, 2003). Ad5CMV-p53 was observed to successfully deliver and express p53 protein followed by p21 protein induction and apoptotic cell death was demonstrated by Terminal Deoxynucleotidyl Transferase-MediatedNick EndLabeling (TUNEL) staining. A significant growth suppression following an Ad5CMV-p53 infection was observed in human esophageal cancer cell lines with a p53 alteration (ECGI-10 and T.Tn) and in T.Tn xenografts in nude mice (Shimada et al, 2001). However, the transduction efficiency was lower than that of other cell lines such as glioblastoma, breast cancer, and cervical cancer probably due to receptor variations and differences in membrane characteristics among different cell lines. So they also investigated the feasibility of plasmid-based p53 gene transfer using electric pulses (electroporation) (Matsubara et al, 2001b). Plasmid vectors are composed entirely of covalently closed circles of double-stranded DNA are substantially easier to massproduce and quality control than are viral vectors. Gene delivery with plasmid vectors do not depend on cell

G. E2F-1 and p53 combinations: E2F-1 as an apoptosis inducer Transfer of the wild-type p53 and E2F-1 genes efficiently induces apoptosis in human esophageal cancer cells and that E2F-1 overexpression directly activates expression of p14 (ARF), which inhibits MDM2-mediated p53 degradation, resulting in the stabilization of p53. Infection of human three esophageal cancer cell lines with adenovirus vector-expressing E2F-1 (Ad-E2F-1) enhanced mRNA and protein expression of ARF and decreased MDM2 protein expression (Itoshima et al, 2000, Yang et al, 2000, Wang et al, 2000). Transfection of ARF plasmid decreased MDM2 protein expression, which in turn increased p53 protein expression. Infection of esophageal cancer cells first with adenovirus-expressing wild-type p53 (Ad-p53) and then with Ad-E2F-1 resulted in rapid induction of apoptosis; in contrast, simultaneous infection with Ad-E2F-1 and Ad-p53 had no significant antitumor effect. Furthermore, infection with suboptimal concentrations of Ad-E2F-1 induced the accumulation of exogenous p53 transduced by suboptimal concentrations of Ad-p53. Moreover, Ad-E2F-1-mediated ARF expression inhibited the up-regulation of MDM2 by overexpressed 38

Cancer Therapy Vol 6, page 39 Table 2. Gene therapy for esophageal cancer. Strategy Gene replacement therapy

Suicide gene therapy

Immunogene therapy

Suicide/ Immunogene therapy Oncolytic therapy

Target gene p53 p53

Gene delivery Retrovirus Adenovirus

Modality +/- Radiation/chemotherapy Monotherapy

p53 p53

Electroporation Adenovirus

p21WAF1 p21WAF1 HSV-TK HSV-TK UPRT UPRT, HSV-TK (Double Suicide gene therapy) IL-2 GM-CSF IL-21, IL-23 TNF-! IFN-!

Adenovirus Gene gun Retrovirus Retrovirus Adenovirus Adenovirus (AxCA.UT)

+/- Chemotherapy +/- Heavy carbonionradiation Monotherapy +/- Chemotherapy Monotherapy Monotherapy Monotherapy Monotherapy


Retrovirus Retrovirus Retrovirus Adenovirus Cationic multilamellar liposome Electroporation Electroporation

Virus HSV type-1 (NV1066) Adenovirus (Ad5/Ad3-chimeric Cox-2 promoter-driven conditionally replicative adenovirus)

surface receptors like viral vectors, however, gene delivery with plasmid vectors is highly inefficient unless the DNA is either associated with other molecules and/or physical energy is applied to aid cell entry. Electroporation increased gene expression by 100- to 1000-fold compared to injection of naked plasmid DNA (Aihara and Miyazaki, 1998). The exact mechanism by which delivery of plasmid into cells is enhanced is not certain although it is clear that membranes become effectively permeable once a critical voltage has been achieved (Neumann and Kakorin, 2002). The growth of esophageal tumors was suppressed with electroporation-mediated chemotherapy (electrochemotherapy) with bleomycin compared with the treatment with bleomycin or electroporation alone. Intratumoral injection of the wild-type p53 gene into p53mutated esophageal tumors followed by electroporation also inhibited tumor growth. When mice were administered with the wild-type p53 gene and an anticancer agent, subsequent electroporation produced a synergistic therapeutic effect (Matsubara et al, 2001b). This group also reported that growth suppression was significantly potentiated by combined treatment with heavy carbon-ion beams and Ad5CMV-p53 as compared to that treated with either of them alone (Oohira et al, 2004). The first clinical trial of Ad5CMV-p53 in patients with chemoradiation resistant advanced esophageal carcinoma was initiated in 2000 in Japan. The primary objective was to evaluate the antitumor effects, to observe

Ref s Shimada et al, 2001 Shimada et al, 2006 Matsubara et al, 2001b Oohira et al, 2004 Fujii et al, 2001 Tanaka et al, 2004 Matsubara et al, 1999a Matono et al, 2003 Nakamura et al, 2001 Shimizu et al, 2001

Monotherapy Monotherapy Monotherapy +/- Radiation +/- Chemotherapy

Matsubara et al, 1998 Sugaya et al, 1998 Shan et al, 2004 Gupta et al, 2002 Tsunoo et al, 2002

Monotherapy Combination therapy

Matsubara et al, 2001a Hanari et al, 2007

Monotherapy Monotherapy

Stiles et al, 2003 Davydova et al, 2004

the biological responses to p53 gene transduction, and to evaluate the safety of this therapy. On a 28-day cycle, intratumoral injections of Ad5CMV-p53 (INGN 201; ADVEXIN) were administered on days 1 and 3 at four dose levels (10 x 10(11) particles to 25 x 10(11) particles) and treated for up to five cycles (Shimada et al, 2006). Administration of multiple courses was feasible and welltolerated. Out of ten patients who received a total of 26 cycles, nine showed local tumor responses and one had progressive disease. The treatment was well-tolerated with no dose-limiting toxicity (Shimada et al, 2006).

B. p21WAF1-replacement gene therapy Fujii and colleagues in Kurume University evaluated the antitumor effect of exogenous expression p21WAF1 in esophageal cancer cells (Fujii et al, 2001). Adenovirusmediated expression of exogenous p21WAF-1 effectively reduced cell growth in cell lines with high cell growth ratio (CGR) KE3 and TE9, but not in low CGR cell lines (KE4 and TE11). p21WAF1-mediated growth suppression was associated with the induction of involucrin, a marker of squamous cell differentiation. This study suggested that the basal level, but not the stimulated level, of p21WAF1 expression play a pivotal role in abnormal growth in human squamous cell carcinoma of the esophagus. Based on their previous study, they evaluated the effect of p21WAF1 gene therapy using gene gun technology (Tanaka et al, 2004). Gene delivery by gene gun utilizes a shock wave to accelerate plasmid-coated


Park and Moon: Gene therapy for esophageal cancer microparticles into target cells or tissues. As this method is cell surface receptor-independent, it can successfully deliver genes into a wide spectrum of mammalian cell types and has been used most extensively for the delivery of DNA vaccines to the skin. It was reported that the gene gun is less invasive and simpler than other gene delivery systems (Zhang et al, 2007). p21WAF1 transfection to KE3 and YES2 cells (weakly expressed p21Waf1 protein cells) showed a high expression of p21WAF1 protein after applying this gene gun technique and caused statistically significant growth inhibition in those cells (Tanaka et al, 2004). In KE3 and YES2 cells, significant growth inhibition was observed after combination therapy using p21WAF1 transfection and anticancer drug 5-FU compared with 5-FU alone (Tanaka et al, 2004).

disappeared. They suggested the p53 gene of tumor cells thereby may influence the efficacy of the HSV-TK/GCV system (Matsubara et al, 1999a). Matono and colleagues demonstrated the correlation of gap junctional intercellular communication (GJIC) and the extent of the bystander effect, suggesting that the degree of GJIC was predictive to identify a tumor as suitable for gene therapy with the HSV-TK/GCV system (Matono et al, 2003). They also showed that treatment with retinoic acid was associated with increased GJIC and augmented bystander killing, indicating that GJIC chemically-enhanced with retinoic acid might be useful to improve response in suicide gene therapy (Matono et al, 2003).

B. Uracil phosphoribosyltransferase/5fluorocytosine

IV. Suicide gene therapy

5-fluorouracil (5-FU) is one of the most widely applied chemotherapy agents used in the treatment of a variety of human cancers, such as cancer of the colon, breast, stomach, pancreas and lung. In mammalian cells, 5-FU is metabolized into either 5-fluorouracil triphosphate (5-FUTP) or 5-fluoro-2'-deoxyuridine 5'-monophosphate (5-FdUMP). 5-FUTP is then incorporated into RNA and interferes with RNA processing, while 5-FdUMP irreversibly inhibits thymidylate synthase and hence DNA synthesis (Evrard et al, 1999). However, the role of systemic 5-FU in cancer treatment has been limited by a low local concentration of the agent and systemic toxicities. Thus, a prodrug/enzyme suicide gene therapy approach was employed to overcome this limitation by increasing the sensitivity of 5-FU. Uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that synthesizes a pyrimidine nucleotide precursor, uridine monophosphate, from uracil or phosphoribosylpyrophosphate (PRPP). This enzyme is present in bacteria Escherichia coli, but is absent in mammalian cells. UPRT can directly covert 5-FU to FUMP by bypassing rate-limiting reactions controlled by the cellular enzymes and greatly enhances the cytotoxicity of 5-FU. Nakamura and colleagues constructed a recombinant adenovirus containing the UPRT gene driven by a CAG (composed of a CMV immediate early enhancer and a modified chicken !-actin) promoter (AdCA-UPRT) (Nakamura et al, 2001). When the UPRT gene was transduced into cultured esophageal cancer cell lines, the sensitivities of all cultured cell lines to 5-FU were increased. When an AdCA-UPRT was directly injected into the tumors inoculated in nude mice followed by 5-FU administration, tumor proliferation was markedly inhibited compared with that in the group treated with 5-FU alone (Nakamura et al, 2001).

The suicide gene therapy involves transduction of tumor cells with a suicide gene followed by systemic administration of prodrug, usually the antiviral agent. The enzyme product encoded by the suicide gene converts potentially non-toxic prodrugs to highly toxic metabolites that subsequently kill the tumor cells.

A. HSV-tk/gnaciclovir The most frequently used suicide gene therapy protocol is the Herpes simplex virus type 1 thymidine kinase (HSV- TK)/ganciclovir (GCV) system. HSV- TK converts prodrug GCV to GCV monophosphate, which is further phosphorylated by cellular kinases, forming toxic GCV triphosphate. GCV triphosphate incorporates into the DNA and inhibits cellular DNA polymerase, leading to cell killing (Aghi et al, 2000). Mammalian cells lack HSVTK, thus GCV causes toxic effects only after cells are transfected with HSV- TK gene. Along with the HSV- TK gene transduced cells, neighboring non-transduced tumor cells are also observed to be killed thereby enhancing the efficacy of this system (Freeman et al, 1993). This â&#x20AC;&#x153;bystander effectâ&#x20AC;? involves various mechanisms including the direct cell-to-cell passage of toxic drug metabolites through the gap junctions, induction of apoptosis, and killing of tumor endothelial cells (Denning and Pitts, 1997). The applicability of the HSV- TK /GCV approach in cancer therapy was first demonstrated by Culver and colleagues who documented the regression of tumors in rats with a cerebral glioma after intratumoral injection of retrovirus-mediated HSV- TK, followed by treatment with GCV (Ram et al, 1993). Matsubara and colleagues first examined the antitumor effect of HSV- TK /GCV suicide gene therapy on human esophageal cancer model (Matsubara et al, 1999a). Two human esophageal cancer cell lines, T.Tn cells which bears truncated p53 and TE2 cells with wildtype p53, were transduced with the HSV- TK gene (T.Tn/TK and TE2/TK) followed by GCV treatment. Both transduced cells, T.Tn/TK and TE2/TK, showed increased sensitivity to GCV compared with that of respective wildtype cells in vitro. However, the growth suppression of T.Tn/TK tumors was marginal in nude mice model and the tumors regrew thereafter while the growth of TE2/TK tumors was significantly inhibited and all the tumors

C. Double-suicide gene therapy The same group evaluated the antitumor activity and bystander effect of double gene transfer, the addition of HSV-TK to UPRT, using recombinant adenovirus vector (AxCA.UT) containing a UPRT/HSV-TK fusion gene (UT) (Shimizu et al, 2001). AxCA.UT treatment significantly enhanced the sensitivity of human esophageal cancer cells to and significantly enhanced the growth 40

Cancer Therapy Vol 6, page 41 inhibition effects of UPRT gene therapy in vitro. Moreover, both 5-FU and GCV showed bystander effects on growth inhibition. In an in vivo study, the therapeutic outcome of AxCA.UT treatment significantly enhanced the antitumor activity of AdCA-UPRT treatment (Shimizu et al, 2001).

VI. Oncolytic therapy To overcome the low efficacy of tumor cell infection by replication-incompetent viral vectors, replicationcompetent viruses were exploited to proliferate specifically in malignant cells and spread within the tumor. These viruses act as direct cytotoxic agents using their ability to kill infected cells by cellular lysis. Their replication leads to amplification of the viral particles and subsequent release of newly made progeny to infect surrounding transformed cells after killing the infected cells, leaving normal cells unaffected. Thus, this process effectively amplifies gene transduction and achieves highlevel gene expression within tumor cells

V. Immunogene therapy The goal of cancer immunotherapy is to induce antibody and/or cytotoxic T lymphocyte (CTL) immune responses against cancer cells. Immunotherapy can be classified into two main categories; active immunotherapies (the stimulation of the immune system) and passive immunotherapies (the creation of immune cells). Active immunotherapies include specific active immunotherapy or vaccine therapy which elicits hostspecific anti-tumor immune responses. Non-specific active immunotherapy or immunomodulatory therapy, on the other hand, elicits a general immune system response to cancer using cytokines, such as GM-SCF and interleukins. Passive immunotherapies include direct administration of monoclonal antibodies designed to target a specific receptor on the surface of a cancer cell. Most of attempts made for the therapeutic application of immune mechanisms for the treatment of esophageal cancer are through the use of immune-stimulatory cytokines. The immunogene therapy is one of immunotherapeutic methods, which uses recombinant DNA constructs which encode cytokines, tumor antigens and accessory molecules. Numerous cytokines that modulate immune responses have been identified and evaluated for their antitumor effects in esophageal cancer. The use of direct recombinant cytokine therapy, although highly effective at eradicating tumors, is generally of limited applicability due to its toxicity or subtherapeutic protein half-life. As a result, strategies using genetic methodology have been applied including cytokine gene transfection using viral vectors (Gupta et al, 2002; Matsubara et al, 1998; Shan et al, 2004; Sugaya et al, 1998) and cytokine plasmid DNA transfer using electroporation (Matsubara et al, 2001a) or cationic multilamellar liposomes (Tsunoo et al, 2002). IL-2 (Matsubara et al, 1998, 2001a), GM-CSF (Sugaya et al, 1998; Matsubara et al, 2001a), IFN-" (Tsunoo et al, 2002), IL-21 and -23 (Shan et al, 2004), and TNF-! (Gupta et al, 2002), either alone or in combination with other strategies, have shown an antitumor immune response which can be demonstrated by reduced tumor volume, intracranial T lymphocytes infiltration or a survival advantage in vivo. Recently, Hanari and colleagues at Chiba University published the results of the combination of HSV- TK /GCV suicide gene therapy and IL-21 immune gene therapy. Plasmid DNA containing HSV-TK-suicide gene or IL-21 gene was injected into TE2 and Colon26 tumors developed in nude mice using in vivo electroporation. They showed that IL-21-transduced tumors disappeared completely in syngeneic BALB/c mice but not in T-celldepleted nude mice, suggesting IL-21 induces T- and NKcell-dependent antitumor effects (Hanari et al, 2007).

A. Herpes simplex virus type-1 HSV-1 was described as the first genetically engineered virus for virotherapy for the treatment of esophageal cancer. HSV-1 has cytolytic property by nature and can infect a broad range of cell types. It has the large genome that can be replaced with multiple therapeutic transgenes without eliminating its oncolytic ability. In addition, many anti-herpetic drugs are available as a safeguard against unfavorable replication of the virus. NV1066 is attenuated HSV mutant derived from strain F that has a deletion of the internal repeats. NV1066 also contains an insertion of the gene encoding enhanced green fluorescent protein (EGFP) under the control of a cytomegalovirus promoter, facilitating visualization of infected cells. NV1066 is attenuated primarily due to deletion of one copy of the diploid "neurovirulence" gene encoding ICP34.5. NV1066 effectively replicated within and killed BE3 esophageal adenocarcinoma cells in vitro and in subcutaneous and intraperitoneal mouse xenograft models (Stiles et al, 2003). Apart from its therapeutic effect, NV1066 showed the potential of localization of metastatic or locally advanced esophageal cancer. The expression of EGFP in infected cells can be used to visualize the virus and thus localize tumor deposits with the use of fluorescent laparoscopy and thoracoscopy (Stiles et al, 2003).

B. Adenovirus Adenovirus has been intensely investigated as a direct oncolytic agent against various types of human cancers and a variety of Phase I and II clinical trials have demonstrated the tolerability and safety of conditionally replicative adenoviruses (CRAd) administration; CV706 for prostate cancer (DeWeese et al, 2001), ONYX-015 for head and neck cancer (Nemunaitis et al, 2001), malignant glioma (Chiocca et al, 2004) and pancreatic cancer (Hecht et al, 2003). One common approach to target virus replication to a specific subset of cells is through the use of tissue- or tumor-specific promoters such as midkine, secretory leukoprotease inhibitor (SLPI), and cyclooxygenase-2 (COX-2) promoters. COX-2 is frequently expressed in neoplastic tissues and plays an essential role in many aspects of cancer progression in gastrointestinal cancers both directly and indirectly (Ono et al, 2005). Yamamoto and colleagues have constructed CRAd by controlling E1 gene expression with the COX-2


Park and Moon: Gene therapy for esophageal cancer promoter (Davydova et al, 2004). They also used infectivity enhancement based on incorporation of an RGD-4C motif into the HI loop of the adenoviral fiber knob domain as well as replacement of the Ad5 knob with the Ad3 knob (Ad5/Ad3-chimeric Cox-2 promoter-driven CRAds). Adenoviruses with the RGD-4C motif configured in the HI loop of the fiber-knob region showed superior infectivity in cells showing low CAR expression compared with vectors with wild-type Ad5 fiber. This enhancement is mainly mediated by the binding of the RGD motif onto integrins that are frequently overexpressed on the surface of the target cancer cells (Davydova et al, 2004). Ad5/Ad3-chimeric Cox-2 promoter-driven CRAds selectively infected esophageal tumor cells and exhibited significantly improved oncolysis and progeny production compared with unmodified and RGD-modified vectors without sacrificing tumor selectivity in vivo and in vitro (Davydova et al, 2004).

promoter design and envelope modification (Ono et al, 2005), it remains to be seen whether any of these systems can be used for systemic delivery. On the other hand, a recent study has shown that liposomes have the potential for systemic delivery of genes to distant sites with minimal toxicity and it may be possible to use a liposome-based delivery system in conjunction with the other vector systems to effectively treat human cancers in vivo (Moon et al, 2003). Other key factors in ensuring the success of gene therapy will be to develop a clear understanding of how it can best play a role in the clinic. For example, immunogene therapies are only ever likely to be effective in clinical situations where patients are at or have been returned to a state of low tumor burden and still have effective, functioning immune systems. Moreover, gene therapy is likely to be very effective in combination with pre-existing clinical regimens, such as chemotherapy and radiotherapy. A large number of studies are now showing great potential for combining gene therapy and pharmaceutical, immunological, and radiotherapeutic approaches to kill cells more effectively and in greater numbers. In summary, there is still a long road ahead before the results gleaned from preclinical and clinical studies of gene therapy strategies approach their full potential in esophageal cancer and any other solid tumors, however, these strategies are already proving their worth as the next step in cancer treatment and, because gene therapy targets the etiology of the disease, may eventually have a role in cancer prevention.

VII. Conclusions Significant advances in understanding the molecular biology of esophageal cancer have led to the application of gene therapeutic methods where genetic material is transferred into human cells and expressed in those cells for a therapeutic purpose. However, the gene therapy in the field of esophageal cancer is in primitive stage compared to those in lung, head and neck and brain cancers. Majority of studies have been performed in Asian countries especially in Japan. That is mostly because the incidence of esophageal cancer varies among different regions and has been reported high in Asian regions (Asian esophageal cancer belt); 2-3 per million in North America and Europe, and 11.1 per million in Japan. There has been no reported result of clinical trial of gene therapy in patients with esophageal cancer yet except one report of a Phase I/II study of Ad5CMV-p53 therapy from Chiba University in Japan. Although limitations still exist, the preclinical findings have shown the great potential of gene therapeutic methods for the treatment of esophageal cancer. Initial concerns that the existence of multiple genetic lesions in cancer cells would prevent the application of gene therapy to cancer appear to be unfounded. Correction of a single genetic lesion has yielded significant tumor regression. Clinical trials of p53 gene replacement have provided information that will be useful in the design of future gene therapy strategies. The ability to target delivery systems to tumor cells (targeting) distributed widely throughout a patientâ&#x20AC;&#x2122;s body would simultaneously increase efficacy and decrease potential toxicity; thus far, however, no such systemically targeted vectors exist (Neumann et al, 2002; Moon et al, 2003). Surface targeting would be optimal to prevent nonproductive binding and sequestration of vectors before they reach their target cells. It is now possible to activate infection through retroviral envelope binding only in tissues that express, for example, tumor-associated proteases and surface targeting is now also possible for adenoviral vectors. Promoters for transcriptional targeting of tumors need to be active in tumor cells and quiescent in normal cells. In the ideal case, the promoter would be tumor specific and despite impressive advances in both

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Park and Moon: Gene therapy for esophageal cancer


Cancer Therapy Vol 6, page 47 Cancer Therapy Vol 6, 47-54, 2008

Experience in the management of 52 patients with soft tissue sarcoma. The results of a median followup of seven years Research Article

E. George Elias1,*, Sally D. Brown1, William Joel Culpepper II2 1

Surgical Oncology Center, The Harry and Jeanette Weinberg Cancer Institute at Franklin Square, Baltimore, Maryland. Department of Epidemiology and Preventive Medicine, Division of Healthcare outcomes Research, University of Maryland, School of Medicine, Baltimore, Maryland. 2

__________________________________________________________________________________ *Correspondence: E. George Elias, M.D., The Harry and Jeanette Weinberg Cancer Institute Franklin Square, 9103 Franklin Square Drive, Suite 2300, Baltimore, Maryland 21237, USA; Telephone: 443-777-7911; FAX: 443-777-6311; E-Mail: Key words: Soft tissue sarcoma Abbreviations: malignant fibrous histiocytoma, (MFH); malignant peripheral nerve sheath tumor, (MPNST); mesna + doxorubicin + ifosfamide + dacarbazine, (MAID); primitive neuro-ectodermal tumor, (PNET); radiation therapy, (RT); Soft tissue sarcomas, (STS) Received: 3 October 2007; Revised: 17 December 2007; Accepted: 17 December 2007; electronically published: February 2008

Summary The prognosis of soft tissue sarcoma depends on several factors, which include the tumor-grade (cell differentiation), tumor size, margins of resection, and the site of the primary. Patients with well-differentiated tumors are managed by radical resection only. On the other hand, those with moderately differentiated and poorly differentiated sarcomas are managed by radical resections followed by postoperative adjuvant radiation therapy. This has been our standard treatment. This study was carried out to determine the efficacy of this therapy on the survival. This is a retrospective study of patients with sarcomas of somatic soft tissues, retroperitoneal, and visceral origins. All the patients were managed by radical resections. The majority also received post-operative radiation therapy for moderately and poorly differentiated sarcomas. The duration of follow-up ranged from 3 months (due to early death) to 22 years with a median of seven years. Fifty-two consecutive patients were studied. Thirty-three patients had high-grade, nine had intermediate, and 10 had low-grade sarcomas. Twenty-one patients (40.4%) died, 18 of them succumbed to their disease. Six patients with high-grade sarcoma underwent resection of the tumor, received adjuvant systemic chemotherapy followed by radiation therapy. Four are alive free of disease at 4 - 7 years, and 2 died of their disease at 24 and 34 months. Radical resection and post-operative irradiation (two local therapeutic modalities) may be sufficient for local control. However, peri-operative adjuvant systemic therapy should be considered in patients with high-grade sarcomas who could succumb to systemic metastases.

lines (Mills, 1995; Rossai, 1996). The prognosis of these tumors depends on the adequate margins of resection, the site of origin, and the stage of the disease. The stage includes the tumor size (with T1 = ! 5 cm, and T2 = > 5 cm) and, more importantly, the tumor grade (G), i.e., cell differentiation. Patients with well-differentiated tumors, i.e., low-grade (G1) are managed by radical resection or wide excision. However, those with moderatelydifferentiated, i.e., intermediate grade (G2) and poorly differentiated, i.e., high-grade (G3) sarcomas are managed by radical resection and post-operative irradiation. This is to reduce the rate of local recurrence with the intent of improving survival. This study was carried out to determine the efficacy of this therapeutic approach and to

I. Introduction Soft tissue sarcomas (STS) are relatively rare malignancies. It is estimated that 9,220 new cases will be diagnosed and 3,560 will die of the disease in the U.S. in the year 2007 (Jemal et al, 2006). These tumors constitute a heterogeneous group of neoplasms with wide variation in propensity for aggressive behavior. At one time, it was thought that STS were related to normal soft tissues and arose from fat, fascia, smooth muscle, fibrous tissue, blood vessels, or nerves. However, it has been shown that the majority of STS arise from primitive multipotential mesenchymal cells, which during the malignant transformation undergo differentiation along one or more


Elias et al: Management of soft tissue sarcoma assess correlates of survival: tumor size, grade, cell type and the primary site of the tumor.

space, 2 in the viscera, and 2 in the pelvis. Sarcomas that arose from buttocks or breast were considered truncal and were included in the torso region numbers.

II. Patients and Methods

C. Surgical management

This is a retrospective study of patients with STS of different cell types and various sites of origin. Patients with dermatofibrosarcoma protuberans, Kaposiâ&#x20AC;&#x2122;s sarcoma, gastrointestinal stromal tumors, uterine sarcoma, desmoid tumors, and sarcomas arising in subcutaneous tissues were excluded. Each case was microscopically diagnosed, identifying the cell type and grade on permanent sections. Histochemical staining was used when necessary to confirm the cell type. Incisional biopsies were utilized to establish the diagnosis with the exception of retroperitoneal and intra-abdominal sarcomas. In most instances, these were resected without pre-operative tissue diagnosis. No frozen sections were utilized during surgery. All the patients underwent radical resections that included compartmental resections for extremity sarcomas or wide resections for truncal tumors. Sarcomas of the torso were widely resected with 5 cm margins. Small bowel sarcomas were resected with wide margins with the mesentery. Sarcomas of the colon or stomach were resected in standard radical fashions. Retroperitoneal and pelvic sarcomas were resected (without prior tissue diagnosis) as far as the anatomical location allowed. Margins of resections were free of tumor clinically and pathologically. In this study, we used the three grade system. Post-operative radiation therapy was requested on patients with G2 and G3 sarcomas. None of the patients received pre-operative radiation therapy or brachytherapy. Six patients with T2 G3 STS (high grade and over 5 cm in size) received post-operative chemotherapy that consisted of mesna + doxorubicin + ifosfamide + dacarbazine (MAID) followed by radiation therapy.

All patients underwent surgical resections of the primary tumors by one senior surgical oncologist. Those with upper and/or lower extremities sarcomas were managed by compartmental resections. Some of the patients with large sarcomas of the forearms or legs underwent extended radical compartmental resections. An extended radical resection included the resection of more than one compartment and a large area of skin. Despite wide resections margins that were grossly free of tumor, wounds resulting from an extended radical resection were not closed by any tissue, but dressed open until the permanent pathology sections became available (Figure 1). When negative margins of resection were confirmed, the large defects were closed with skin grafts. This method was utilized for several reasons: 1) to save as many normal functioning muscles in the event that the tumor had crossed the facial barrier between the muscular compartments, 2) allow granulation tissue to develop which helps in skin graft take, and 3) skin-grafts allow for easier visual inspection of the area for any evidence of local recurrence.

D. Tumor grade (G) and tumor size (T) The incidence of tumor grade correlated to tumor size is expressed in Table 1. Thirty-three patients (63%) had high-grade tumors, 9 (17%) had intermediate-grade, and 10 (19%) had low-grade.

III. Results The study population consists of fifty-two patients treated between July 1983 and March 2001. All were newly diagnosed with resectable primary STS. Most of the patients had somatic soft tissue sarcoma. Only ten patients had tumors of retroperitoneal or visceral origin. There were 24 men and 28 women. The ages ranged from 12 to 90 years with a median of 58. There were 6 AfricanAmericans and 46 Caucasians. Twenty patients were 60 years of age or older. The duration of follow-up ranged from three months (due to early death) to 22 years with a median of 7 years.

E. Post-operative therapy



Twenty-nine patients received post-operative radiation therapy (RT); 21 had G3 tumors (18 of them were T2), and eight had G2 tumors. Twenty-three of the 52 patients received no adjuvant irradiation; 10 had G1 tumors. The other thirteen did not receive adjuvant irradiation for a variety of reasons. These included: 1) patients refused radiation (n=3), 2) radiation oncologist felt there was no need for radiation (n=4), and 3) radiation therapy was not appropriate due to tumor location or patientâ&#x20AC;&#x2122;s condition (n=6).

A. Cell types The majority of patients had malignant fibrous histiocytoma (MFH) and liposarcoma (13 patients with each diagnosis). Other STS were less frequent [6 leiomyosarcoma, 5 angiosarcoma, 4 adult rhabdomyosarcoma, 3 synovial sarcoma, 2 malignant peripheral nerve sheath tumor (MPNST), i.e., malignant schwannoma]. There was one patient with each of the following diagnoses: Ewing's soft tissue sarcoma [primitive neuro-ectodermal tumor (PNET)], osteogenic soft tissue sarcoma, chondrosarcoma, alveolar soft tissue sarcoma, epitheloid sarcoma, and fibrosarcoma.

F. Survival Thirty-one patients (59.6%) are alive; 28 are disease free, and two are living with disease. Twenty-one patients (40.4%) died; three died from unrelated causes: stroke, heart attack, and metastatic carcinoma of the urinary bladder. Eighteen patients (36.5%) died of the disease, which were mostly high-grade sarcomas (Table 1). Four patients died during the first year of follow-up and seven died during the second year. Survival was correlated to tumor cell type, grade, and size. All patients with angiosarcoma, 10 of 13 with MFH, and three of six of the patients with leiomyosarcoma had G3 tumors. On the other hand, only five of 13 patients with liposarcoma had G3 (Table 2).

B. Anatomical sites of origin Five of the patients had sarcomas that originated in the head and neck area, 8 in the upper extremity, 9 in the torso, 20 in the lower extremity, 6 in the retroperitoneal


Cancer Therapy Vol 6, page 49

Figure 1. Shows large defect in a forearm, status post-resection of rhabdomyosarcoma, dorsal aspect. This wound was dressed without tissue coverage for four days until the final pathology revealed no evidence of tumor at the margins of resection. This wound was then covered by autologous skin graft.

Table 1. The overall survival correlated to tumor grade (G) and tumor size (T)


G1T1 0

G1T2 10

G2T1 4

G2T2 5

G3T1 7

G3T2 26













3(b, c)


Totals 52 31 (59.6%) 21 (40.4%)

n = number of patients (a) = one patient died of stoke (b) = one patient died of heart attack (c) = one patient died of urinary bladder cancer

Ten patients had intra-abdominal sarcomas and their survival was correlated to tumor location, cell type, grade, and size (Table 3). All had T2 tumors (> 5 cm in size) and six of 10 had G3 tumors. Five of these 10 patients (50%) were alive. However, only two of the six patients with G3 sarcoma were living. Nine of the 52 patients developed local recurrences as the first sign of failure. Of these, seven had G3 sarcomas, one with G2, and one had G1 (Table 4). Seven had not received adjuvant radiation therapy and two had received adjuvant radiation therapy postoperatively. Seven of the nine patients underwent resection of their recurrences and four were living. Adjuvant RT was not administered for previously stated reasons. In one instance, the radiation oncologist felt that the resection was more than adequate and the patient (grade 3 MFH of thenar muscles) developed local recurrence 3 years later. He underwent re-resection and received RT.

Sixteen patients developed distant metastases as the first sign of recurrence or treatment failure. Thirteen of the 16 had G3 and three had G2 tumors. The site of metastases include lungs (n = 10), lungs and liver (n = 2), intraperitoneal (n = 1), kidney (n = 1), regional lymph nodes (n = 1), and spleen, ribs, and viscera (n = 1). The time from the initial diagnosis to the development of metastases ranged from five to 72 months. Ten patients underwent resection of their metastases. Thirteen of the 16 patients died of their disease. Survival, after the first event, ranged from one to 9 years. Three are alive: two with no evidence of disease at 6 to 8 years post-resection of their metastases and one alive with disease 16 years postresection of the first metastases to the liver. This patient underwent resection of a second distant metastasis to the lung (Table 5). It was noted that some cell types are extremely aggressive and have higher tendency for local recurrence and systemic metastases. These included angiosarcoma


Elias et al: Management of soft tissue sarcoma and MPNST. There were five patients with angiosarcoma (all G3) and four died of their disease. In addition, we encountered two cases of MPNST and both were grade 3 sarcomas. In one case, the primary site was in the thigh and the patient underwent compartmental resection of the quadriceps muscles. This tumor recurred 3 years later requiring re-resection with part of the femur, which was replaced by allogenic bone graft. This patient is alive and

free of disease 19 years later. The second patient who had MPNST (malignant schwannoma) had an unusual presentation. The tumor began in the pelvis and extended through the greater sciatic notch to the gluteal region in an hourglass formation. This was resected sacrificing the sciatic nerve. This tumor recurred one month later and the patient died within few months.

Table 2. The overall survival correlated to tumor cell type, grade (G), and size (T) Cell type


MFH Liposarcoma Leiomyosarcoma Angiosarcoma Rhabdomyosarcoma Synovial Sarcoma MPNST Others

13 13 6 5 4 3 2 6

G1T2 A/D 4/0 0/1 0/1

G2T1 A/D 2/0 1/0 1/0 0/1 -

G2T2 A/D 0/1 2/1 1/0 0/1

G3T1 A/D 0/2 2/0 1/0 1/1 1/0

G3T2 A/D 5/3 2/1 0/2 0/3 2/1 2/1 1/1 2/1

Totals A/D 7/6 11/2 3/3 1/4 2/2 2/1 1/1 3/3

n = number of patients; A/D = number alive over number dead; MFH = malignant fibrous histiocytoma Others included one of each: PNET (Ewingâ&#x20AC;&#x2122;s soft tissue sarcoma), Osteogenic Sarcoma, Chondrosarcoma, Alveolar Cell Sarcoma, Epitheloid Sarcoma, and Fibrosarcoma.

Table 3. The overall survival of patients with intra-abdominal sarcomas correlated to tumor cell type, grade (G), and size (T) Site of primary Retroperitoneal

# of patients 6

# alive 4

# dead 2









Cell type Liposarcoma Liposarcoma Liposarcoma Liposarcoma Angiosarcoma MFH* Leiomyosarcoma* Leiomyosarcoma Leiomyosarcoma MPNST

G 3 3 1 1 3 3 1 3 2 3

T 2 2 2 2 2 2 2 2 2 2


* means received adjuvant chemo (MAID); MFH = malignant fibrous histiocytoma; MPNST = malignant peripheral nerve sheet tumor (malignant schwannoma); A = alive; D = dead

Table 4. The disease free and overall survival of the patients who developed local recurrence as the first sign of failure correlated to tumor cell type, grade (G), size (T) and site of origin Cell type



Site of origin

MFH MFH Leiomyosarcoma Leiomyosarcoma MPNST MPNST Liposarcoma Liposarcoma Liposarcoma

3 3 1 3 3 3 3 2 3

2 2 2 1 2 2 2 2 2

Hand Neck Viscera Thigh Thigh Pelvis Retroperitoneal Peri-parotid Retroperitoneal

DFS (mo) 36 15 12 120 36 1 48 weeks 48

OS (mo) 60 36 24 156 228 3 60 6 60


DFS = disease-free survival; OS = overall survival; MFH = malignant fibrous histiocytoma; MPNST = malignant peripheral nerve sheet tumor (malignant schwannoma); A = alive; D = dead


Cancer Therapy Vol 6, page 51 Table 5. The disease-free and overall survival of patients who developed distant metastases as the first sign of failure correlated to tumor cell type, grade (G), size (T), site of origin, and site of metastases Cell type



Site of origin

MFH MFH MFH MFH Angiosarcoma Angiosarcoma Angiosarcoma Angiosarcoma Leiomyosarcoma Leiomyosarcoma Leiomyosarcoma Synovial synovial PNET Chondrosarcoma Rhabdomyosarcoma

3 2 3 3 3 3 3 3 2 3 3 3 3 3 2 3

2 2 1 2 2 1 2 2 2 2 2 2 2 2 2 1

forearm thigh leg thigh retroperitoneal neck thigh scalp pelvis groin viscera calf back thigh thigh forearm

DFS (mo) 12 24 12 17 12 5 12 7 48 24 72 36 6 21

OS (mo) 48 24 24 48 12 108 48 12 61 48 48 60 144 108 48 26


Site of metastases


Lungs Lungs Popliteal L N Lungs Lungs Lungs Lungs Lungs Liver & lung Liver & lungs Peritoneal Lungs Lung metastases, resected Right kidney metastasis, resected * Lungs Spleen + ribs + viscera

* received chemotherapy before and after the nephrectomy DFS = disease-free survival; OS = overall survival; A = alive D= dead; LN = lymph nodes; PNET = primitive neuroectodermal tumor (Ewingâ&#x20AC;&#x2122;s sarcoma)

+ radiation therapy versus patients treated by the three modalities (surgery + chemotherapy + radiation therapy). While no statistical differences were noted, the poorest outcome was observed in patients who were treated by surgery only with a median disease-free survival of 42 months (Figure 2), and a median overall survival of 54 months (Figure 3). It was noted that that there was little difference between the disease-free survival and overall survival among all the treatment groups. However, our data suggest that the addition of chemotherapy may prove beneficial, but more data are needed. The results of the survival analysis comparing the different treatment groups are summarized in Table 6.

G. Adjuvant systemic chemotherapy Six patients received adjuvant systemic chemotherapy prior to RT. All had G3 tumors and 5 had tumors over 5cm in size (T2). Three of the six patients had MFH, 1 rhabdomyosarcoma, 1 leiomyosarcoma, and another had alveolar soft tissue sarcoma of the breast (G3 T1) with lung metastases. The chemotherapy administered was MAID, which consisted of mesna 2,500 mg/m2 per day for 4 days, doxorubicin 20mg/m2 per day for 3 days, ifosfamide 2,000 mg/m2 per day for 3 days, and dacarbazine 250mgm/m2 per day for 4 days. These were administered intravenously for a total of 4 courses followed by irradiation in the range of 44 to 50 Gy. After a period of follow-up that ranged from 31 to 72 months, three patients succumbed to their disease and three are alive (two free of disease and one with disease). In addition, one patient with Ewingâ&#x20AC;&#x2122;s soft tissue sarcoma (PNET) of the thigh (G3 T2) developed metastases to the right kidney, received three courses of chemotherapy prior to nephrectomy and two courses after the nephrectomy. Each course consisted of cyclophosphamide, vincristine, doxorubicin, and ifosfamide. This patient is alive free of disease 6 years later. Another patient with alveolar soft tissue sarcoma (G3 T1) of the breast was found to have lung metastases at the time of diagnosis. This patient underwent mastectomy, resection of the lung metastases, post-operative chemotherapy (MAID), and no radiation therapy. This patient is alive free of disease for over 5 years. Life-table analysis was performed to determine if there was difference in the disease-free survival and overall survival rates among patients with G3 T2 sarcoma treated by surgery only versus those who received surgery

IV. Discussion The majority of the patients had somatic STS and only ten had sarcomas that originated from the retroperitoneum or viscera. We excluded sarcomas that arose from the skin or subcutaneous tissues. Dermatofibrosarcoma is a localized disease with a tendency for local recurrence with almost no potential for distant metastases. Kaposiâ&#x20AC;&#x2122;s sarcoma is managed primarily by irradiation. Gastrointestinal stromal tumors and uterine sarcoma constitute different and distinct clinical entities. Desmoids and sarcomas arising in subcutaneous tissue were excluded as these tumors have better survival than either somatic soft tissue sarcomas or visceral and retroperitoneal sarcomas. All 52 patients included in this study had deep sarcomas according to AJCC Cancer Staging Manual (Greene et al, 2001). It has been reported that the two most common STS are MFH and liposarcoma (Fletcher, 1992). In this study, there were


Elias et al: Management of soft tissue sarcoma

Figure 2. Disease-free survival by treatment groups of the 26 patients with large (over 5 cm) high-grade (G3) sarcomas. Note that those treated by surgery alone had the poorest outcome.

Figure 3. Overall survival by treatment groups of the 26 patients with large (over 5 cm) high-grade (G3) sarcomas. Note that those treated by surgery only had the poorest survival.

Table 6. Disease-free and overall cumulative probability of survival at 72 months follow-up Treatment Group Surgery only Surgery + irradiation Surgery + chemotherapy + irradiation

Disease-Free survival .50 .57 .83

Overall Survival .50 .75 .83

Incidental Findings: Three patients developed second malignancies. These included breast, urinary bladder and prostatic cancers. 13 MFHâ&#x20AC;&#x2122;s and 13 liposarcomas that constituted 50% of the study patients. Several methods of establishing the diagnosis have been reported ranging from fine needle aspiration or a large bore needle biopsy to open incisional biopsy. Each method has its advantages and disadvantages. However, accurate diagnosis with grade must be established if neoadjuvant therapy is to be considered. A needle biopsy may not yield sufficient tissue to determine the accurate

diagnosis with the cell type and grade. It may suggest the presence of malignant cells and can be misleading. This is demonstrated by a case of a patient who presented with a large gluteal tumor. The radiologist felt that the tumor had the characteristics of a synovial sarcoma. The needle biopsy was interpreted as soft tissue chondrosarcoma. After the resection, the final diagnosis was changed to MFH. The microscopical examination of the specimen revealed areas mimicking chondrosarcoma, but the 52

Cancer Therapy Vol 6, page 53 majority of the specimen revealed MFH. Therefore, some of STS may consist of a variety of tissues that may lead to misdiagnosis even after an open biopsy. Frozen sections should be avoided because the interpretation by the pathologist can be difficult. The most common primary site of origin in this study was the lower extremity with 37% incidence, which is comparable to the national average. This was followed in frequency by intra-abdominal sarcoma, which included the retro-peritoneal space, viscera and pelvis. This was followed by the torso then upper extremity. It should be noted that primary sarcomas of the head and neck region are rare in the adult population. The technique of dressing the wound without tissue coverage until the pathology report for the permanent sections becomes available may be beneficial. If further resection is not required, the surgical defect can be closed with autologous skin graft. This approach can potentially save function by sparing some uninvolved muscles. The use of a skin graft to cover the surgical defect permits future inspection of the site allowing early detection of local recurrence. This technique could be very beneficial in the management of large STS of forearms and legs. We used the three-tiered system for grading (not the four-tiered system) because of its simplicity and the small study population. Sixty-two percent of the study patients had high-grade tumors and over 75% of the study population had sarcomas over 5 cm in size (T2). These two factors; grade and size, constituted the majority of the cases within the high-risk population. Our data suggests that patients with intermediate and high-grade sarcomas have almost similar survival and they are managed in the same fashion. This may justify the 2-grade (low and high) system proposed to the AJCC (Greene et al, 2001). The status of the margins of resection is another prognostic indicator and correlates to the tumor size and the primary site. In this study, the margins of resection were sufficiently wide grossly and pathologically except for the retroperitoneal, intra-peritoneal, and pelvic sarcomas. In these areas anatomical locations and vital structures restricted resections. The limitation on resection for adequate margins explains the role of site of origin on overall outcome as patients with intra-peritoneal and retroperitoneal sarcomas have a poor prognosis (Jacque et al, 1990; Heslin et al, 1997; Lewis et al, 1998). Furthermore, in this study, all retroperitoneal and intraperitoneal sarcomas were T2 and six of the ten patients had high-grade tumors. The overall survival of the patients with intra-abdominal sarcomas was 50%. The best results were noted in patients with G1 tumors (Table 3). The incidence of local recurrence and distant metastases correlated well to tumor grade. Seven of nine patients with local recurrences had G3 tumors and 13 of 16 patients with distant metastases had G3 sarcomas. One patient with G1 tumor developed local recurrence in the viscera and died of the disease demonstrating that the primary site can be a prognostic factor. On the other hand, nine patients had G2 tumors; four died of their disease, and five are alive. It seems that the tumor size plays a secondary role in the outcome in every grade.

Patients who developed radiation-induced sarcomas have a very poor prognosis for two reasons: the patient cannot receive additional radiation therapy and the tumors are usually high-grade and aggressive with potential risk for local recurrence and distant metastases. The survival remains poor despite radical resections (Thijssens et al, 2005). Two patients developed G3 T2 STS in the neck in the field of previous RT for Hodgkinâ&#x20AC;&#x2122;s disease. One was 28 year-old who developed MFH and the other was 23 year-old who developed angiosarcoma. The patient with MFH developed local recurrence followed by systemic metastases and the patient with angiosarcoma developed systemic metastases as the first sign of failure. Both failed to respond to MAID chemotherapy. However, other systemic chemotherapy should be considered as different cell-types may respond to different agents. Another example of extremely aggressive behavior was noted in one patient with MPNST (G3 T2) of the pelvis that extended through the greater sciatic notch into the gluteal region that required the sacrifice of the lumbar-sacral roots and the sciatic nerve. This tumor recurred in the pelvis in less than 4 weeks. Neoadjuvant chemotherapy should be considered if a preoperative diagnosis can be made in these tumors. Patients with G3 T2 sarcomas have the poorest survival as 12 of 26 (46%) died of systemic metastases, primarily to lungs (Table 1). In addition, certain cell types were recognized as always high-grade and patients with these cell types are at risk of recurrence and metastases. Angiosarcoma and PNST have these tendencies and may be considered for preoperative (neo-adjuvant) chemotherapy if the diagnosis can be established prior to resection. Preoperative RT may not be effective in bulky tumors. RT is most effective when delivered to a very low tumor load. For this reason, it should be administered postoperatively. On the other hand, brachytherapy may play a role in reducing the incidence of local recurrence. Even then, in high-grade sarcomas, post-operative irradiation is necessary regardless of the extent of the margins of resection. An example is one of our patients with high-grade MFH of the thenar muscles of the hand who was denied RT and developed local recurrence 3 years later. Overall, there were more local recurrences in patients who did not receive RT regardless of the tumor cell type. Our results suggest that a short latent period (from diagnosis to first event) could signify poor prognosis. This was true for local recurrences (Table 4), as well as for distant metastases (Table 5). Recurrences that develop within a short time of the resection of the primary or a previous recurrence signifies poor outcome. A longer latent period could indicate a better prognosis. The anatomical locations of some sarcomas strongly suggest that STS do arise from a primitive multipotenital mesenchymal cells that differentiate along different lines. Examples of this include: 1) synovial sarcoma in the muscle of the back or calf away from any synovial membrane or joint, 2) Ewingâ&#x20AC;&#x2122;s sarcoma in the muscles of the thigh, away from any bone in an adult, and 3) chondrosarcoma in the muscles of the back away from any cartilage.


Elias et al: Management of soft tissue sarcoma Frustaci S, Gherlinzoni F, De Paoli A, Bonetti M, Azzarelli A, Comandone A, Olmi P, Buonadonna A, Pignatti G, Barbieri E, Apice G, Zmerly H, Serraino D, Picci P (2001) Adjuvant chemotherapy for adult soft tissue sarcomas of the extremities and girdles: Results of the Italian Randomized Cooperative Trial. J Clin Oncol 19, 1238-1247. Greene FL, Page DL, Fleming ID, Fritz AG, Balch CM, Haller DG, Morrow M (eds.) (2002) Soft Tissue Sarcoma. AJCC Cancer Staging Manual, 6th Edition, (pp. 193-197) New York: Springer-Verlag. Heslin MJ, Lewis JJ, Nadler E, Newman E, Woodruff JM, Casper ES, Leung D, Brennan MF (1997) Prognostic factors associated with long-term survival for retroperitoneal sarcoma: implications for management. J Clin Oncol 15, 2832-2837. Jacque DP, Coit DG, Hajdu SI, Brennan MF (1990) Management of primary and recurrent softJemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ (2007) Cancer statistics, 2007. CA Cancer J Clin 57, 43-66. Lewis JJ, Leung DH, Woodruff JM, Brennan MF (1998) Retroperitoneal soft tissue sarcoma. Analysis of 500 patients treated and followed at a single institution. Ann Surg 228, 355-366. Mills SE (1995) Sometimes we don’t look like our parents Mod Path 8, 347. Rosenberg SA, Tepper J, Glatstein E, Costa J, Young R, Baker A, Brennan MF, Demoss EV, Seipp C, Sindelar WF, Sugarbaker P, Wesley R (1983) Prospective randomized evaluation of adjuvant chemotherapy in adults with soft tissue sarcomas of the extremities. Cancer 52, 424-434. Rossai J (1996) Soft tissues. Ackerman’s Surgical Pathology, 8th Edition, (pp 2021-2133) St. Louis: Mosby. Thijssens KM, van Ginkel RJ, Suurmeijer AJ, Pras E, van der Graaf WT, Hollander M, Hoekstra HJ (2005) Radiationinduced sarcoma: A challenge for the surgeon. Ann Surg Oncol 12, 237-245. Tierney, JF, Stewart, LA, Parmar, MKB (1997) Sarcoma metaanalysis collaboration. Adjuvant chemotherapy for localized resectable soft-tissue sarcoma of adults: meta-analysis of individual data. Lancet 350, 1647-1654.

While local recurrences and distant metastases carry guarded prognosis, surgical resection should be considered (if appropriate) followed by systemic therapy. This approach yielded some long-term survivals. Adjuvant chemotherapy prior to RT was utilized in a small number of patients with high-risk sarcoma. This seems to be beneficial as four of six patients were alive. The suggested benefit is displayed in Figures 2 and 3. However, controversy continues to exist regarding the use of adjuvant chemotherapy. Early reports indicated that the use of single agent doxorubicin showed no benefit (Alvegard et al, 1989). However, later it was reported that the use of poly-chemotherapy resulted in borderline benefits (Rosenberg et al, 1983; Frustaci et al, 2001). Several other reports followed showing the beneficial results of the use of postoperative adjuvant chemotherapy (Tierney et al, 1997; Eilber et al, 2005). The fact remains that, while tumor grade is an important prognostic factor, we must also correlate the cell type to survival. We must keep in mind that different cell types may respond differently to different chemotherapeutic or targeted therapeutic agents. Therefore, trials with chemotherapy and/or targeted therapy, utilizing new agents, should be tested in various cell types rather than restricting the use to doxorubicin and ifosfamide based chemotherapy for all cell types of STS as it was suggested over a decade ago (Elias, 1992). Several new agents have been suggested including gemcitabine, paclitaxel, ecteinascidin, vinorelbine, and imatinib, which showed activity in desmoid tumors and gastrointestinal stromal tumors (Dileo and Demetri, 2005). Adjuvant chemotherapy should be considered in high-risk patients who refuse RT or if the radiation is not recommended because of previous irradiation. Adjuvant chemotherapy could prove to be ideal in patients with visceral or retroperitoneal sarcomas in place of RT that may result in bowel injury.

References Alvegård TA, Sigurdsson H, Mouridsen H, Solheim O, Unsgaard B, Ringborg U, Dahl O, Nordentoft AM, Blomqvist C, Rydholm A, et al (1989) Adjuvant chemotherapy with doxorubicin in high-grade soft tissue sarcoma: A randomized trial of the Scandinavian Sarcoma Group. J Clin Oncol 7, 1504-1513. Dileo P, Demetri GD (2005) Update on new diagnostic and therapeutic approaches for sarcoma. Clinical Advances in Hematology and Oncology 3, 781-791. Eilber FC, Eilber FR, Eckardt J, Rosen G, Riedel E, Maki RG, Brennan MF, Singer S (2004) The impact of chemotherapy on the survival of patients with high-grade primary extremity liposarcoma. Ann Surg 240, 686-695. Elias AD (1992) Future directions in the management of soft tissue sarcoma. Hemat Oncol 10, 53-60. Fletcher CD (1992) Pleomorphic malignant fibrous histiocytoma: Facts or fiction? A critical reappraisal based on 159 tumors diagnosed as pleomorphic sarcoma. Am J Surg Pathol 16, 213-228.

E. George Elias


Cancer Therapy Vol 6, page 55 Cancer Therapy Vol 6, 55-66, 2008

Tumor acidity and malignancy: novel aspects in the design of anti-tumor therapy Review Article

Elisabetta Iessi, Maria Lucia Marino, Francesco Lozupone, Stefano Fais, Angelo De Milito* Department of Drug Research and Evaluation, Section of Drug Resistance and Experimental Therapeutic, Istituto Superiore di Sanità

__________________________________________________________________________________ *Correspondence: Angelo De Milito, PhD, Department of Drug Research and Evaluation, Section of Drug Resistance and Experimental Therapeutic, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome Italy; Phone: +39 06 49902153; Fax: +39 06 49903691; Email: Key words: pH regulators, glycolisis, acidic vesicles, tumor biology, Microvesicles, tumors, Chemoresistance, Cannibalism acidity, hypoxia, cellular metabolism, Cellular mechanisms, cancer therapy Abbreviations: acridine orange, (AO); acute lymphoblastic leukemia, (ALL); ATP binding cassette, (ABC); breast cancer resistance protein, (BCRP); extracellular matrix, (ECM); hypoxia-inducible factor-1, (HIF-1!); intracellular pH, (pHi); lung resistance protein, (LRP); magnetic resonance spectroscopy, (MRS); matrix metalloproteinases, (MMPs); multi-drug resistance associated protein-1, (MRP-1); multi-drug resistance, (MDR); Na+/H + exchanger, (NHE); P-glycoprotein, (P-gp or MDR-1); pH-low insertion peptides, (pHLIP); Proton Pump Inhibitors, (PPI); reactive oxygen species, (ROS); trans-Golgi network, (TGN); Vacuolar H +-ATPase, (VATPase); vascular endothelial growth factor, (VEGF) Received: 9 January 2008; Accepted: 17 January 2008; electronically published: February 2008

Summary The peculiar features of hypoxia and acidity characterizing tumor microenvironment represent key factors in tumor progression, metastasis and response to therapies. Studies aiming at dissecting the metabolic pathways leading to such a hostile microenvironment are still scanty despite the early intuition of the Nobel price Otto Warburg that tumors develop as a “metabolic disease” based on their selective use of glycolysis to generate energy. As a result of the acid production through the glycolysis occurring also in the presence of oxygen, tumors need to extrude protons in order to survive. The mechanisms used to eliminate protons include up-regulation of ion pumps like Vacuolar H+-ATPase (V-ATPase) and Na+/H+ exchanger (NHE) and increased turn over of acidic vesicles. However, such mechanisms are also involved in the generation of the malignant behaviour of tumors which include unresponsiveness or resistance to chemotherapy, metastatic and invasive potential and altered survival/apoptosis regulation. As a consequence, studies aimed at better understanding these mechanisms will help to develop new therapeutic strategies to target cancer. We discuss here recent results showing new and sometimes neglected aspects of tumor biology whose importance needs to be evaluated in the design and application of novel anti-cancer therapies.

acidity, factors that contribute to the progression from benign to malignant growth and that normally are not permissive for growth of normal cells. In order to survive in the unfavourable environment created by them, tumor cells up-regulate several proton extrusion mechanisms (Izumi et al, 2003; Sennoune et al, 2004). Among the cellular pH regulators whose activity is enhanced in many tumors are the V-ATPase (Martinez-Zaguilàn, 1993), the Na+/H+ exchanger (NHE) (Lee and Tannock, 1998) and the carbonic anhydrase (Robertson et al, 2004). As shown by several methods both in vitro and in vivo, the consequence of such an aberrant activity of ion pumps is

I. Introduction Classical model of carcinogenesis are based on a sequence of mutations or epigenetic changes in the expression or activity of oncogenes, protooncogenes, tumor suppressor genes or apoptotic pathways (Nowell, 1976; Fearon and Vogelstein, 1990; Gillies and Gatenby, 2007). Recently, the role of tumor microenvironment in determining tumor malignancy has received renewed interest with the suggestion that environmental conditions may drive the selection of the cancerous phenotype (Gillies and Gatenby, 2004, 2007). Features of tumor microenvironment include hypoxia, low blood supply and 55

Iessi et al: Tumor acidity and malignancy that the extracellular tumor environment has an acidic pH whereas the intracellular (cytosolic) pH is alkaline, therefore resulting in a reversed pH gradient across the plasma membrane. In many tumors, the chronic exposure to acidic pHe has been reported to promote invasiveness (Martinez-Zaguilàn, 1996; Rofstad et al, 2006), metastatic behaviour (Martinez-Zaguilàn et al, 1996) and resistance to cytotoxic agents (Wachsberger et al, 1997; Raghunand et al, 2001). The specific mechanisms tumors use to acquire a malignant phenotype are still largely unknown but recent finding suggest that the abnormal pH control may regulate several biological functions like trafficking of acidic vesicles (Glunde et al, 2003), drug resistance (Martinez-Zaguilàn et al, 1999), activity of proteases to digest the extracellular matrix (Glunde et al, 2003) and ability to phagocyte (Lugini et al, 2003) and/or cannibalize cells (Lugini et al, 2006). This review will attempt to emphasize the importance of pH regulation in many tumor functions correlated to malignancy and to evaluate possible therapeutic strategies based on a new thinking of tumors as a chronic disease.

to survive in hypoxic-anoxic environment through the upregulation of hypoxia-inducible factor 1-! and the adaptation of a glycolytic phenotype with the generation of lactate (Gatenby and Gillies, 2004). However, some in vivo experiments have suggested that the production of lactate via glycolysis is likely not the major mechanism responsible for the development of an acidic environment within solid tumours (Newell et al, 1992; Yamagata et al, 1998; Provent et al, 2007).

1. Hypoxia and acidity It is commonly believed that hypoxia and acidity are a direct result of the chaotic and heterogeneous microvasculature structure of solid tumors (Gillies et al, 1999; Schornack and Gillies, 2003). During carcinogenesis, solid tumors are hyper-proliferative and are separated several hundreds of microns from blood vessels. Blood vessels are the primary mode for delivery of glucose and O2 and for the removal of metabolic H+ and lactate (Schornack and Gillies, 2003). As a result, oxygen and glucose must diffuse from the vessels through layers of tumour cells, where they are metabolized (Gatenby and Gillies, 2004). Oxygen concentrations decreases with distance from a capillary and tumor cells are even more distant from their blood supply. Tumor cells generally are highly proliferative and then require a more robust source of nutrients than normal tissues. Therefore, pre-malignant lesions will inevitably develop hypoxic regions and with low blood supply. Hence, there is a clear requirement for new microvasculature in the growth of tumors. Recent evidence indicates that tumor cells secrete active angiogenesis factors (Knighton et al, 1983; Gillies et al, 1999) and the most important of these factors in tumors is the vascular endothelial growth factor (VEGF). The VEGF mRNA levels dramatically increase within a few hours after exposing cell cultures to hypoxia and return to background when normal oxygen supply is resumed. VEGF is also induced by glucose deprivation (Shweiki et al, 1992). Notably, hypoxia and low blood supply continuously translate into an altered pH gradient between the extracellular environment and the cytoplasm and between the cytoplasm and endo-lysosomal vesicles (Simon et al, 1994; Mahoney et al, 2003; Luciani et al, 2004). There is evidence that hypoxia up-regulates both the expression and activity of carbonic anhydrase in order to enhance the extracellular acidification (Svatova et al, 2004), in turn contributing to tumor progression. Hypoxia and low blood supply seem to be involved in cancer progression, but some reports showed that are not the major mechanisms responsible for the development of an acidic environment within solid tumors.

A. Tumor microenvironment: acidity, hypoxia and cellular metabolism The microenvironment of solid human tumors is characterized by heterogeneity in oxygenation and the establishment of hypoxia is believed to occur very early in the development of a tumor, producing a microenvironment that is hypoxic, acidic, and low in nutrients (Wouters BG, Seminars in Radiation Oncology 2003). Human tumors have long been considered acidic based on microelectrode measurement (Wike-Hooley et al, 1984). Non-invasive measurement of tumor pH in animal tumor models by magnetic resonance spectroscopy (MRS) using 3-aminopropylphosphonate and ZK-150471 and experiments performed in solid tumour xenografts have demonstrated that the pHi of tumour cells is neutralalkaline (7.0-7.4) while the pHe of the same tumours is acidic (6.0-6.9), indicating that solid tumours have an acid-outside pH gradient (Negendank, 1992; Gillies et al, 1994; McCoy et al, 1995; Raghunand et al, 1999a; Ojugo et al, 1999). Hence, tumors contain regions with large, acid-outside pH gradients, while normal tissues generally have alkaline-outside pH gradients. The causes for the acidic pH in tumors may include environmental factors which develop during tumor growth such as (i) deficiencies in tumor perfusion, due to the abnormal vascularization of the tumor mass; (ii) hypoxia and metabolic abnormalities associated with transformation and cell growth, and (iii) an increased capacity for transmembrane pH regulation. These environmental conditions may create a sort of vicious circle that favoring the selection of highly malignant tumor cells contributes to maintain the same hostile conditions. Notably, hypoxia and low blood supply continuously translate into an altered pH gradient between the extracellular environment and the cytoplasm and between the cytoplasm and endolysosomal vesicles (Simon et al, 1994; Mahoney et al, 2003; Svastova et al, 2004). An important determinant of tumor acidity is the anaerobic metabolism which allows selection of cells able

2. Acidity and glycolisis Over 75 years ago Otto Warburg postulated that tumors are acidic due to their marked rate of lactic acid production (Warburg, 1956). He observed that tumour cells maintain a high glycolytic rate even in conditions of adequate oxygen supply (aerobic glycolysis or “Warburg effect”) and depend largely on this metabolic pathway for the generation of energy (i.e. ATP). He attributed this 56

Cancer Therapy Vol 6, page 57 metabolic alteration to mitochondrial ‘‘respiration injury’’ and considered this as the most fundamental metabolic alteration in malignant transformation or ‘‘the origin of cancer cells’’ (Warburg, 1956; Xu et al, 2005). The biochemical and molecular mechanisms underlying the “Warburg effect” are extremely complex and remain to be defined. Among the possible mechanisms, mitochondrial malfunction and hypoxia in the tumor microenvironment are considered two major factors contributing to it (Xu et al, 2005; Pelicano et al, 2006). A hypoxic environment within the tumor mass limits the availability of oxygen for use in mitochondrial respiration and synthesis of ATP and forces the cancer cells to up-regulate the glycolytic pathway as the main route of energy production (Guppi, 2002; Xu et al, 2005). Hypoxia favours a shift of glucose metabolism to less efficient glycolytic pathways in many cancer phenotypes (Gatenby and Gillies, 2004; Gatenby and Gillies, 2007), making glycolysis essential for tumour survival (Bartrons and Caro, 2007). A key regulator of the glycolytic response is HIF-1! (hypoxia-inducible factor-1glycolytic response is HIF), a transcriptional activator that controls the expression of most of hypoxia-regulated genes. HIF-1glycolytic response is HIF is overexpressed in a variety of tumours and its expression appears to correlate with poor prognosis and responses to chemo or radiotherapy (Bartrons and Caro, 2007).

The adaptation to hypoxia through the up-regulation of glycolysis has a significant negative consequence, i.e. the increased acid production causing a significant decrease in extracellular pH. Since exposure to acidic microenvironment results in cell death, extracellular acidosis requires an additional adaptation through resistance to apoptosis and up-regulation of membrane ion channels in order to maintain intracellular pH in the range of normality (Gatenby and Gillies, 2004). Indeed, the unfavourable environment may favour tumor cells able to survive in acidic and hypoxic conditions via selection of cells that are resistant to hypoxia, acid-induced cell toxicity and hypoxia-induced, p53-dependent apoptosis (Kim et al, 1997). However, it is also known that the major cause of acidity in tumors it is not the lactic acid production, as also demonstrated in rat gliomas where sites of proton efflux and sites of glycolysis had a different distribution (Provent et al, 2007).

B. Cellular mechanisms altered in tumors 1. pH regulators Maintenance of the intracellular pH (pHi) is crucial to normal cell function because many cellular processes have a narrow pH optimum. Moreover, evidences have shown that tumor cells exist within an acidic microenvironment, probably due to accumulation and poor removal of metabolic acids such as lactic acid and protons derived from glycolysis (Figure 1).

Figure 1. Schematic representation of novel anti-tumor agents targeting metabolic pathways and pH regulation.


Iessi et al: Tumor acidity and malignancy To survive and proliferate under these acidic conditions, tumor cells must up-regulate the proton extrusion mechanisms that maintain the pHi. The influence of pHi on many cellular functions has been studied with respect to cell growth (Helmlinger et al, 1997), cell motility (Martinez-Zaguilàn, 1998), tumorigenesis (Perona and Serrano, 1988), metastasis (Schlappack et al, 1991), apoptosis (Gottlieb et al, 1995), and drug resistance in cancer cells (Thiebaut et al, 1990; Martinez-Zaguilàn et al, 1999). Four major types of pHi regulatory mechanisms have been identified in tumor cells: Na+/H+ exchangers, bicarbonate transporters, proton-lactate symporters and proton pumps. These transmembrane proteins are ion pumps or ion exchangers and pump protons across the plasma membrane from the cytoplasm to the opposite site of the membrane, the extracellular space or the lumen of various organelles. Among these proton pumps one of the most studied are the V-ATPases. V-ATPases are involved in maintaining a relative neutral pHi, an acidic luminal pH and an acidic pHe, through pumping protons into extracellular environment or within the lumen of some membrane-bound organelles (Nishi and Forgac, 2002). These ATP-dependent proton transporters are ubiquitously expressed (Nishi and Forgac, 2002), not only in vacuolar membranes but also in plasma membranes (MartinezZaguilàn et al, 1993, Nishi and Forgac, 2002) of various specialized eukaryotic cells, in which they play important functions (Vaananen et al, 1990; Nanda et al, 1992; Gluck et al, 1996). Recent evidences suggest that V-ATPases are functionally expressed in plasma membranes of human tumor cells and may have specialized functions in cell growth, differentiation, angiogenesis, invasion and metastasis (Martinez-Zaguilàn et al, 1993, 1998). In tumor cells the extrusion of protons by V-ATPases causes intracellular alkalinization and extracellular acidification which are important mechanism favouring growth, resistance to apoptosis and invasion (Martinez-Zaguilàn et al, 1996, 1999). Such a mechanism contributes to the maintenance of an aberrant pH gradient between the alkaline cytosol and the acidic extracellular environment. The low pH of tumor extracellular microenvironment may influence the increased secretion and activation of proteases. Moreover, low pHe may promote the degradation and remoulding of extracellular matrix (ECM) through the activation of proteolytic enzymes, including matrix metalloproteinases (MMPs), bone morphogenetic protein-1-type metalloproteinases, tissue serine proteases, and adamalysin-related membrane proteases, thus contributing to cancer invasion and metastasis (MartinezZaguilàn et al, 1996; Rofstad et al, 2006). Some data suggest that different use of ion exchanger may help to distinguish tumor cells with different metastatic behaviour (Sennoune et al, 2004). In fact, while breast cancer cells with low metastatic potential preferentially utilize Na+/H+ exchangers and HCO3--based H+-transporting mechanisms, highly metastatic cells preferentially use plasma membrane V-ATPases, suggesting that V-ATPases are important in the acquisition of a more metastatic and invasive phenotype (Sennoune et al, 2004). Furthermore, V-ATPases have been involved in the acquisition of the

multidrug resistance phenotype (Laurencot et al, 1995; Martinez-Zaguilàn et al, 1999; Raghunand et al, 1999b) and treatment with inhibitors of the V-ATPases (most of which are highly toxic) may reverse tumor resistance (Altan et al, 1998; Izumi et al, 2003; Ouar et al, 2003; Luciani et al, 2004; De Milito and Fais, 2005). Specific inhibitors of V-ATPase like Bafilomycins have been reported to induce apoptosis in human cancer cells (Nakashima, 2003). Proton Pump Inhibitors (PPI) have been successfully used for the treatment of peptic diseases, due to their anti-acidic properties. After protonation in the acidic spaces of the stomach, PPI irreversibly bind the proton pump, dramatically inhibiting proton translocation and acidification of the extracellular environment. The specific targets of PPI are H+-ATPases contained within the lumen of gastric parietal cells and, though to a lesser activity, they inhibit the activity of VATPases, thus blocking proton transport across membranes. Recent data obtained by our group (De Milito et al, 2007) and by Lu and collaborators (Lu et al, 2005) strongly demonstrated that inhibition of the V-ATPase has an antineoplastic activity (Fais et al, 2007). In fact, the inhibition of V-ATPase function via knockdown of ATP6L (c subunit gene) expression using siRNA suppresses cancer metastasis by decreased proton extrusion and downregulated protease activity (Lu et al, 2005). Our group has shown that proton pump inhibitors induce cell death in human B cell tumors by a mechanism involving oxidative stress and lysosomal membrane perturbation (De Milito et al, 2007). Interestingly, these two different types of antineoplastic strategies resulted in delayed tumor growth in vivo as shown by i) inhibition of B cell lymphoma growth in PPI-treated SCID mice and ii) retarded tumor growth and suppressed cancer metastasis in nude mice. These data suggest that V-ATPases and its activity regulating the reversed tumor pH gradients may represent a potential target for cancer therapy (Izumi et al, 2003; Fais et al, 2007).

2. Role of acidic vesicles in tumor biology Metastatic behaviour of solid tumours has been associated with increased expression of extracellular lysosomal proteases, such as cathepsin D, L and B, and metalloproteases causing tissue remodelling (Sloane et al, 1981; Danö et al, 1985; Liotta et al, 1991; Fais et al, 2007). There is strong evidence indicating also a pivotal role of lysosomal-like vesicles in the degradation of ECM, cell invasion, and cell migration into ECM of some cancers, including breast cancer (Montcourrier et al, 1994; Montcourrier et al, 1997; Glunde et al, 2003). In breast cancer cell lines acidic pHe caused a significant redistribution of lysosomes from the perinuclear region to the cell periphery (Glunde et al, 2003). In malignant tumor cells, cathepsin B+ vesicles redistribution toward the cell periphery and constitutive secretion of active cathepsin B were observed at acidic pHe (Rozhin et al, 1994). Lysosomes displacement to the cell periphery, detected in response to an acidic pHe, may be a mechanism to facilitate increased secretion of degradative enzymes (Glunde et al, 2003), thus facilitating tumor invasion and metastasis.


Cancer Therapy Vol 6, page 59 Moreover, there is increasing evidence that acidic vesicles expressing typical lysosomal or endosomal markers are involved in phagocytic activity of tumor cells (Lugini et al, 2003). Furthermore, highly metastatic cells were found to express more lysosomal proteins, such as Lamp-1 and -2 on the cell surface (Saitonh et al, 1992). In breast cancers very large acidic heterophagosomes were observed, containing high levels of cathepsin D and sufficiently acidic to allow cathepsins activation and degradation of endocytosed proteins, whose presence is correlated with tumor invasion (Montcourrier et al, 1994). Recent data have also shown that only cells derived from metastatic lesions exert a phagocytic activity (they can engulf, ingest and digest latex beads, yeast and apoptotic cells), suggesting that phagocytic behaviour enables malignant cells to survive in the lack of nutrients supply and may be considered an indicator of high-grade malignancy (Lugini et al, 2003). Altogether this data have shown that the phagocytic activity may represent a new tool in discriminating the level of aggressiveness of human malignant cells and the connection of phagosomal vesicles to the actin cytoskeleton supports the use of new antitumor strategies aimed at inhibiting the tumor phagocytic activity (Lugini et al, 2003).

4 Chemoresistance Acquired resistance to chemotherapeutic drugs, a phenomenon known like multi-drug resistance (MDR), is a major cause of treatment failure in cancer patients (Nielsen and Skovsgaard, 1992; Gottesman and Pastan, 1993). Mechanisms underlying drug-resistance are not yet entirely known but the major hypothesis involves biochemical and microenvironmental factors. In classical multidrug resistance, cells exhibit resistance to a wide range of structurally and functionally unrelated compounds, including anticancer drugs such as vinca alkaloids, anthracyclines, taxoids, and other antimitotics (Dalton, 1994; Raghunand et al, 1999b). The MDR cells over-express a variety of transmembrane drug efflux pumps, belonging to the ATP binding cassette (ABC) transporters family, which include P-glycoprotein (P-gp or MDR-1) (Gottesman and Pastan, 1993), the multi-drug resistance associated protein-1 (MRP-1) (Cole et al, 1992), the lung resistance protein (LRP) (Scheper et al, 1993; Izquierdo et al, 1996) and the breast cancer resistance protein (BCRP) (Doyle et al, 1998). These proteins extrude against concentration gradient drug molecules from the cell to the extracellular environment and this phenomenon cause a significant decrease in intracellular drug retention. Although the increase in the expression and activity of these proteins is directly related to the in vitro generated MDR, the same relationship was not shown in the in vivo resistance of solid tumours to the various cytotoxic drugs, seriously opening severe doubts on the clinical relevance of this phenomenon (Leonard et al, 2003). Another mechanism of resistance may be changes in the pH gradient between the extracellular environment and the cell cytoplasm and/or in the pH gradient between the cell cytoplasm and lysosomal compartments observed in tumors (Simon et al, 1994; Mahoney et al, 2003). Since the mechanisms of entry of drugs into the cell are dependent on both concentration gradients and pH gradients, the reversed pH gradients of tumors may severely affect drugs entry (Raghunand and Gillies, 2000; De Milito and Fais, 2005). It is well known that low pHe reduces the uptake of weakly basic chemotherapeutic drugs and, hence, reduces their cytotoxicity preventing weakly basic drugs to reach their intracellular target (Raghunand et al, 1999b). As mentioned above, an important mechanism for chemoresistance involves acidic vesicles and the altered pH gradient between the cytoplasm and intracellular organelles. Many chemotherapeutic drugs such as the anthracyclines and vinca alkaloids are weak bases. They are membrane permeable in their neutral form and relatively membrane impermeant when protonated and charged. Upon entering any of the acidic compartments of the cell (such as the lysosome, recycling endosomes, transGolgi network (TGN), or secretory vesicles), they will be protonated and sequestered within these compartments. It is known that lysosomal-like vesicles present in MDR cells are more acidic respect to their counterpart in drugsensitive cells (Altan et al, 1998). This suggests that after the entry of drugs into the cells, basic drugs are sequestered by acidic compartments and are secreted from

3. Microvesicles and tumors Exosomes are small microvesicles (50-100 nm) biologically active, originating from acidic vesicle turnover and released by cells upon fusion of multivesicular bodies with the plasma membrane (Keller et al, 2006; van Niel et al, 2006). Microvesicles play important regulatory role in a variety of cellular functions including immunomodulation, differentiation and antigen presentation (Keller et al, 2006; Valenti et al, 2007). Data have shown that exosomes produced by mouse dendritic cells pulsed with tumor peptide are able to mediate the rejection of established tumors (Zitvogel et al, 1998, 2000; Keller et al, 2006). Human malignant tumors can secrete exosomes able of inducing antigen-specific tolerance, Fasmediated T-cell apoptosis (Andreola et al, 2002; Abusamra et al, 2005; Taylor and Gercel-Taylor, 2005) and tumor immune escape through expression of functional apoptotic molecules in different types of human tumors (Huber et al, 2005; Valenti et al, 2007). Moreover, exosomes were also shown to play a role in the control of tumor growth and tumor invasion (Taylor and GercelTaylor, 2005; Koga et al, 2005; Liu et al, 2006). In fact, pretreatment of mice with exosomes derived from murine mammary carcinomas augumented tumor growth by inhibiting the cytolytic activity of NK cells. Trafficking of acidic vesicles is also involved in the chemoresistance independently of P-glycoprotein (P-gp) expression (Dietel et al, 1990; Bobichon et al, 1996; Cleary et al, 1997; Bour-Dill et al, 2000). In fact, the increased acidification of lysosomal-like vesicles may substantially determine the sequestration and/or neutralization of drugs into acidic intracellular compartments and their elimination through increased activity of the secretory pathway (Raghunand et al, 1999c, 2003).


Iessi et al: Tumor acidity and malignancy the cell through the normal pathways of vesicular traffic and secretion. Moreover, the possibility that basic drugs are directly protonated and neutralized by the acidic pHe of tumors has to be considered (De Milito and Fais, 2005). Agents that disrupt or normalize the pH gradient in tumors may reverse MDR and/or directly inhibit tumor growth. For example, sodium bicarbonate treatment causes alkalinization of tumor growth in vivo (Raghunand et al, 1999b). Lysosomotropic agents that induce pH gradient modification and alkalinisation of acidic vesicles may reverse anthracycline resistance in multidrug-resistance cells (Ouar et al, 2003). Our group showed that pretreatment with PPI induced i) reversion of drug-resistance in chemoresistant human melanoma cells and ii) increased sensitivity to cytotoxic drugs in MDR cell lines (Luciani et al, 2004). These data suggest that the peculiar acidic pH of human cancers may represent a target mechanism of new and specific anticancer therapies aiming at “normalizing” the reversed pH gradient, thus depriving tumor cells of a crucial homeostatic mechanism for cell survival and proliferation (Fais et al, 2007).

C. New approaches for cancer therapy Recent studies aimed at investigating the mechanisms behind tumor malignancy will provide insights into the identification of novel targets for the treatment of human tumors (Figure 1). We already know that tumors have an alkaline-acidic outside pH gradient generated by the up-regulated activity of proton transporters that maintain an alkaline pHi and an acidic pHe. In cancer cells also the pH gradient between the cytoplasm (alkaline) and the lumen of intracellular vesicles (very acidic) is altered and its regulation is fully controlled by V-ATPases. The homeostatic regulation of such abnormal pH gradients by V-ATPases may represent a suitable and specific target for novel anticancer strategies (Fais et al, 2007). Furthermore, recent evidence has shown that pHi is critical for the cytotoxicity of anticancer agents and V-ATPase has been implicated in the acquisition of the multidrug resistance phenotype (Laurencot et al, 1995; Martinez-Zaguilàn, 1999a,b). Therefore, understanding the mechanisms regulating pHi and tumor acidity is important for developing new approaches to cancer therapy and V-ATPase may represent a potential target for cancer chemotherapy (Torigoe et al, 2002; Sennoune et al, 2004; Fais, 2007). Because of the importance of the reversed pH gradients in malignant progression of tumor cells, the authors tested the specific effects of PPI on (i) drugresistance in a variety of human tumor cells and (ii) growth of B cell tumors, both in vitro and in vivo. Most chemotherapeutics are weak bases and accumulate either in the acidic tumor microenvironment or in the lumen of acidic intracellular vesicles, representing these two factors the bases for cellular resistance to drugs (Raghunand et al, 1999b; De Milito and Fais, 2005). We observed that physiologic concentrations of PPI significantly increase the pH of acidic intracellular vesicles and the pHe, inducing accumulation of acidic vesicles in the cell. Indeed, pre-treatment with PPI in vitro and in vivo (i) reverted chemoresistance of different tumor cells to cisplatin, 5-fluorouracyle and doxorubicin, and (ii) increased the sensitivity of drug-sensitive cells to anticancer agents. These effects were mediated by the intracellular retention of chemotherapeutic agents, associated with a “normalization” of the pH gradients of the tumor cells (Luciani et al, 2004). In the acidic microenvironment of tumors, by analogy with the gastric compartment, PPI may undergo acidity-induced protonation, followed by transformation in the active form that blocks the H+-ATPase, in turn altering the tumor pH gradients. In fact, we found that PPI effect on tumor cells were directly related to the level of acidity of the culture medium (De Milito et al, 2007). PPI induced selective cytotoxicity in B cell tumors that passed through an early massive reactive oxygen species (ROS) activation and lysosomal membranes perturbation, leading to a caspaseindependent cell death (De Milito et al, 2007). PPI caused also alkalinization of acidic vesicles and acidification of the cytosol. The antineoplastic activity of PPI was observed also in pre-B acute lymphoblastic leukaemia cells obtained from patients with acute lymphoblastic leukemia (ALL), as well as in SCID mice engrafted with B

5 Cannibalism More recently malignancy of tumor cells has been related to another important feature, considered a survival option of malignant tumors that live in condition of nutrient deprivation, such as “cannibalism” (Lugini et al, 2006; Fais, 2007). Cannibalism is recognized as a phenomenon used by unicellular and higher organisms, even at single-cell level (Lugini et al, 2006). Cells with cannibalic behaviour have been identified in malignant tumors up to a century ago and recently have been detected in tumors of different histologies (DeSimone et al, 1980; Fujii et al, 1986; Kojima et al, 1998; Kumar et al, 2001). Some data indicate that cannibalism is a feature associated with malignancy but little is known about the mechanisms underlying this phenomenon. Some reports suggested that cannibal tumor cells contain other cells through which feed themselves (Monteagudo et al, 1997; Caruso et al, 2002; Lugini et al, 2006). A recent report showed that cannibalism, like phagocytosis, is an exclusively property of metastatic malignant cells (Lugini et al, 2006). This report demonstrated also that during cannibalism a live lymphocyte established a contact with melanoma cell and then sink into the tumor cells. This leads to the formation of large vacuoles where the lymphocyte remains alive but progressively undergoes necrosis and degeneration (Lugini et al, 2006). The endolysosomal compartment of cannibal cells is rich in caveolin-1, is more acidic and over-express cathepsin B, a proteolytic enzyme involved in tumor invasion and metastasis. Like in phagocytosis, the actin cytoskeleton have a key role in the cannibalic behaviour of metastatic cells, allowing the formation of the “cannibalistic vacuole” that contains a highly efficient digestive machinery (Lugini et al, 2006). Experimental data have shown that cannibalism is increased in acidic culture condition and then inhibition of tumor acidity may represent a further therapeutic approach against cannibalistic activity (Lugini et al, 2006; Fais, 2007).


Cancer Therapy Vol 6, page 61 cell lymphomas, whose growth was significantly reduced following PPI oral administration (De Milito et al, 2007). It is clear from those data that ROS accumulation is an early event in the PPI-mediated antineoplastic effect and that permeabilization of acidic vesicles is a crucial event in this cascade, that is rapidly followed by the acidification of cytosol, with a possible massive activation of protease and other very dangerous lytic enzymes leading the cells to dead through a sort of autodigestion. However, these results provided the proof of concept that PPI may be considered, not only chemosensitizer agents, but also a new class of antineoplastic drugs. Other pharmacological inhibitors of V-ATPases activity have been used in the past with high level of efficacy in vitro but their potential application in clinical settings is hampered by predicted toxicity on normal cells (De Milito and Fais, 2005). PPI are not toxic to normal cells while they exert their action against tumor cells and this is proven to occur in vivo as well (De Milito et al, 2007). The great potential of PPI as V-ATPases inhibitors is that they need (i) an acidic or at least unbuffered medium to work as anticancer drugs and (ii) protonation to be transformed in the active drug (De Milito and Fais, 2005). This in turn means at least two important things: (i) they are recruited by the acidic compartments and (ii) only in these compartments they are activated in order to work as antiacid drugs. Like the V-ATPases, other cellular pH regulators (NHE, MCT, CA) are important in the maintenance of pH homeostasis in cancer cells (Lee and Tannock, 1998; Izumi et al, 2003; Robertson et al, 2004) and may represent suitable targets for anticancer strategies. For each of them, inhibitors have been identified to date but little is known about their cellular mechanism of action. Among the NHE inhibitors, amiloride and its derivatives have been shown to induce apoptosis in leukemic cells but not in normal cells (Izumi et al, 2003). It has been recently shown by that CA IX may control acidification of the tumoral extracellular pH under hypoxic conditions (Svastova et, 2004) and this process can be perturbed by inhibiting CA IX with selective sulfonamide inhibitors (Winum et al, 2007). Many biological and pharmacological data point to the possible use of the inhibition of tumor associated CA IX in the management of hypoxic tumors, which do not respond or poorly respond to the classical chemo- and radiotherapy. However, further experimentation is needed to fully understand whether this approach will lead to new class of antitumor drugs (Winum et al, 2007). In addition, acridine orange (AO) and pH-low insertion peptides (pHLIP) have been proposed as novel intriguing antitumoral strategies employing tumor acidity as a delivery system. In a model of osteosarcoma, AO selectively accumulates in the tumor tissue due to reversed pH gradients, and following photo-dynamic therapy AO is activated and selectively kills cancer cells (Kusukaki et al, 2007). Recently, interesting nanotechnology data identified peptides able to selectively insert into the membrane of cancer cells only at acidic pH, thus providing a powerful tool for selective delivery of therapeutic agents to the acidic site of the tumors (Andreev et al, 2007).

The ability of cancer cells to phagocyte and cannibalize living cells provides another potential target whose regulation is likely dependent on acidic vesicles trafficking and pH (Fais, 2007). These activities are an exclusive property of cells derived from malignant lesions and are regulated by the actin cytoskeleton. In particular, the ERM proteins, known to link membrane proteins to the actin cytoskeleton, are important in the connection of the phagocytic machinery to the actin cytoskeleton (Lugini et al, 2003, 2006). These novel data support the use of new antitumor strategies aimed at inhibiting the tumor phagocytic/cannibal activity through targeting components of the cannibal framework (Fais, 2007). Another class of anticancer agent for new therapeutic strategies could be the inhibitors of glycolytic pathway, important for ATP generation. These drugs have a broad therapeutic application and are particularly effective against cancer cells with mitochondrial defects or under hypoxic conditions, which are frequently associated with cellular resistance to conventional anticancer drugs and radiation therapy (Xu et al, 2005). The glycolytic inhibitors known include the 2-deoxyglucose, an analogue of glucose and able to bind and suppress hexokinase II (Geschwind et al, 2004) and arsenate compounds that cause arsenolysis in the glyceraldehyde-3-phosphate dehydrogenase reaction, although they seem to have toxic effects. It was recently shown that inhibition of hexokinase II by 2-deoxyglucose causes a depletion of cellular ATP, leading to blockage of cell cycle progression and cell death in vitro, and exhibits antitumor activity in vivo (Maher et al, 2004; Maschek et al, 2004). 3bromopyruvate (3-BrPA), another potent inhibitor of hexokinase II (Ko et al, 2001; Geschwind et al, 2004), seems to effective kill liver cancer cells in animal models when given by local infusion (Geschwind et al, 2002). Recent evidence have showed that inhibition of glycolysis by 3-BrPA led to the activation of an apoptotic cascade (Xu et al, 2005). The ability of 3-BrPA to preferentially kill cancer cells with mitochondrial defects and that live in a hypoxic environment provides a biochemical basis to further develop this class of compounds as novel anticancer agents with potentially promising therapeutic activity and selectivity (Xu et al, 2005).

II. Conclusions Despite the great efforts of the scientific community in finding proper treatments for cancer, human tumors responsive to chemotherapy have not changed in the last three decades (e.g., lymphomas, leukemias and some pediatric tumors) while many others such melanomas and most adenocarcinomas still are unresponsive or poorly responsive to most types of therapies. After the partial failure of some approaches, the issue of resistance or refractoriness to chemotherapeutics has become a key problem in the therapy of tumor patients. Our results and recent published observations indicate a new path to anticancer treatment and important suggestions are emerging from these new data. The mechanisms controlling the abnormal pH gradients in tumors should represent a selective and specific target in setting up new anticancer strategies. To our opinion, V61

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ATPases should be considered as the most important of these targets because of its crucial function in determining the acidification of tumor microenvironment. Moreover, tumor acidity should be considered as a specific delivery system for new antitumor strategies, based on drugs that are specifically recruited within acidic environment and activated in situ, thus hijacking an essential survival factor for tumors. Notably, tumor acidity is also related to cannibalism, an important tumor feature considered a survival option of malignant tumors. The molecular basis for evolution of glycolytic phenotype, extracellular acidosis and altered metabolic pathways have to be better clarified and much additional work will be required to fully understand the complex pathways involved in cancer progression. Thus, studies aimed at investigating the major mechanisms involved in proliferation, tumorigenesis, drug-resistance and tumor progression will provide the knowledge to design new strategies in the treatment of human tumors based on the inhibition of mechanisms that allow them to survive in these unfavourable conditions. Among novel anti-cancer drugs, the authors have focused their attention on PPI, drugs able to normalize the pHi and pHe of human tumors, thus rendering tumor cells sensitive to the action of several cytotoxic drugs to which they are normally refractory. However, recent results have provided the proof that PPI may be considered, not only as chemosensitizer agents, but also a new class of antineoplastic drugs. PPI may have an important effect on tumor homeostasis based on their ability to alkalinize the tumor microenvironment. Based on these results new investigations are needed to better understand the mechanisms of tumor acidification, a new and key target of future strategies in the treatment of human tumors.

Acknowledgements We are grateful to Roberta Terlizzi and Zaira Maroccia for excellent technical assistance

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Angelo De Milito


Iessi et al: Tumor acidity and malignancy


Cancer Therapy Vol 6, page 67 Cancer Therapy Vol 6, 67-72, 2008

Subclavian artery infusion as induction and adjuvant chemotherapy for breast conserving treatment of primary breast cancer Research Article

Karl R. Aigner*, Sabine Gailhofer, Emir Selak Department of Surgical Oncology, Medias Klinikum GmbH & Co KG, Krankenhausstrasse 1, 84489 Burghausen, Germany

__________________________________________________________________________________ *Correspondence: Prof. Dr. Karl R. Aigner, Department of Surgical Oncology, Medias Klinikum GmbH & Co KG, Krankenhausstrasse 1, 84489 Burghausen, Germany; Tel: +49-(0)-8677-91600; Fax: +49-(0)-8677-9160120; E-Mail: Key words: breast cancer, regional chemotherapy, subclavian artery infusion, neoadjuvant chemotherapy, tumor downsizing, local recurrence Abbreviations: intraarterial chemotherapy, (IAC); subclavian artery infusion, (SAI) Received: 4 December 2007; Revised: 16 January 2008 Accepted: 24 January 2008; electronically published: February 2008

Summary In recent years the management of breast cancer experienced a tendency towards breast conserving surgery combined with chemotherapy and irradiation. Surgery combined with chemotherapy for downsizing was applied in order to achieve resectability in large tumors or to keep the surgical intervention minimal. However, achievement of impressive response in terms of downstaging is a longlasting procedure, accompanied by prolonged toxic chemotherapy. In an attempt to achieve increased local drug exposure intraarterial infusion chemotherapy of the internal mammary and the subclavian artery was performed in 53 patients with primary breast cancer. The therapy consisted of six cycles of the three drug combination Mitomycin, Adriamycin and Cis-Platin. 34/53 patients had undergone local tumor excision prior to i. a. chemotherapy (IAC), 19/53 patients received induction (neoadjuvant) IAC and restaging with lumpectomy after three treatment cycles. Complete response was noted in 26 % and overall response was 74 %. A significant shift from T3/T4 stages towards T1/T2 was observed within three treatment cycles. Quality of life was generally good and most patients were able to work between the treatment cycles. Main side-effect was drugstreaming causing skin burns, which initially occurred in about 30 % and in recent years in 4 %. Local recurrence rate was 17 % and median survival was not yet reached within the follow up time of 16 years.

et al, 1999). If systemic chemotherapy would be sufficiently effective in the entire organism, local relapse or distant metastases should not occur - the dose-response relationship is steepâ&#x20AC;? Emil Frei (Frei et al, 1980). If highdose chemotherapy could generate sufficient efficacy (GĂśrich et al, 1995; Merajver et al, 1997; Murakami et al, 2001) in the entire organism, resistant cell clones would no longer be resistant and could not survive. Regional chemotherapy, because of precalculated local drug concentrations, in many cases can generate the required drug exposure at the target site (Aigner, 1994; Sainsbury et al, 1990; Stephens, 1988; Stephens et al, 1980a,b; Wang, 1990).

I. Introduction The treatment strategy of breast cancer has changed remarkably during the past 3 decades. Radical surgery is no longer the treatment of choice. Breast cancer is considered a systemic disease already in early stages and therefore mutilating surgery has been abandoned to a great extent. Breast conserving surgery is aimed at whenever justifiable in most latest treatment protocols. Radical axillary dissection has been replaced by removal of the sentinel lymphnode in the first attempt. Radiotherapy maintained its position in combination therapies for destruction of potential axillary lymphnode metastases or for prevention of local recurrences. More importance is given to multidisciplinary management of disease (Carlson


Aigner et al: Subclavian artery infusion as induction and adjuvant chemotherapy MMC, two times 15 mg ADM and two times 30 mg CDDP. During the first three cycles until local resection of the residual tumor, the first two days selective infusion of the internal mammary artery was performed with 10 mg each of Mitomycin and Adriamycin. Then the cycle was completed with subclavian artery infusion of Adriamycin and Cis-Platin according to the protocol. In case the patient continued to be reluctant to any surgical intervention, angiographic therapy was continued throughout the six cycles. In case the patient agreed with surgical placement of a Jet Port Allround subclavian artery catheter (PfM Cologne), further therapies were performed through this catheter (Figure 1).

II. Material and Methods This study was performed in a series of 53 patients who refused mastectomy. In 19/53 patients, after three cycles of regional induction (neoadjuvant) chemotherapy a local excision of the lesion or the area of the prior lesion, respectively, was performed for pathological evaluation. 34/53 patients had had local tumor excision prior to regional chemotherapy. None of these patients had undersigned informed consent of mastectomy.

III. Treatment All patients initially underwent catheter placement in Seldingerâ&#x20AC;&#x2122;s technique and an arteriogram of the subclavian artery with its branches and the internal mammary artery was obtained. For contrast imaging of the vascular branches of the subclavian artery (Doughty et al, 1996), and as well to avoid infusion of the arm, a brachial pneumatic sphygmomanometer was inflated at least to 30 mm/Hg above the systolic pressure during the intraarterial infusion. In order to trace the target of intraarterial infusion and correct positioning of the catheter, indigocarmin blue was injected prior to each therapy. A total of six cycles of subclavian artery infusion (SAI) in four weeks intervals was performed with a three drug combination of mitomycin, adriamycin and cis-platin (Table 1). The average dose for one cycle was 10 mg

IV. Results Local response in terms of tumor downsizing was the leading parameter. In 34 patients the primary tumor (stages T1-3) had been resected prior to adjuvant arterial infusion. In 19 patients, who underwent regional induction (neoadjuvant) chemotherapy, most evidently there was a remarkable shift from larger tumorsizes (T3, T4) down to lower sizes (T1, T2). In 26 % a complete remission was noted histologically (Figures 2, 3) 9 patients (48 %) had a partial remission, whilst 32 % of pretherapeutic T4 tumors were reduced to a histologically confirmed 11% T4 after

Table 1. Protocol of Subclavian Artery Infusion for Breast Cancer. Six cycles in four weeks intervals Day



Infusion Time


Mitomycin C

0,17 mg/kg

15 min



0,25 mg/kg

15 min



0,25 mg/kg

15 min



0,50 mg/kg

15 min



0,50 mg/kg

15 min

Figure 1. End-to-side implantation of subclavian artery Jet Port catheter


Cancer Therapy Vol 6, page 69 arterial chemotherapy. Vice versa, after arterial infusion of all patients the number of T1 sized tumors had increased from initially 10,5 % to 32 % after intraarterial chemotherapy (Table 2). Local recurrence rate after 16 years was 17 %. Kaplan Meier survival estimate is shown

in Figure 4. After a 16 years follow-up the median survival time has not yet been reached. 9/53 patients (17 %) had a local relapse and 7 patients died from relapse and/or distant metastases.

Figure 3. Complete remission after three cycles of intraarterial chemotherapy.

Figure 2. T4 primary breast cancer before therapy

Table 2. Downsizing of primary tumors after 3 cycles of subclavian artery infusion (SAI). TNM-Stage before SAI (n = 19)

after SAI (n = 19) CR n = 5 (26 %) pT1 n = 6 (32 %)


n = 2 (10,5 %)


n = 9 (47 %)


n = 3 (15,75 %)


n = 2 (10,5 %)


n = 1 (5,25 %)


n = 2 (10,5 %) n = 2 (10,5 %)


n = 6 (32 %) unknown

Figure 4. Kaplan Meier Survival estimate curve after SAI for primary breast cancer


Aigner et al: Subclavian artery infusion as induction and adjuvant chemotherapy Side-effects in general were moderate. There was no substantial haematologic toxicity that required antibiotic prophylaxis or substitution of blood components. Tiredness or fatigue have never been observed and patients usually were able to go to work between the treatment cycles. Hairloss mostly was moderate and normally did not occur. Only two patients had visible hairloss (4 %), one of them alopecia. The only real complication, causing problems, was drugstreaming, which, in the beginning had happened in every third case, but could be reduced later on to 4 % after applying the total amount of drug through a constant flow pump within 15 minutes.

account that also T3/4 stages have been included in the study, and that all patients have been treated with local excision only, but without mastectomy. The survival estimate with seven deaths out of 53 treated patients throughout sixteen years suggests that the rate of distant metastases after i. a. infusion is not higher than has to be expected after systemic chemotherapy. This leaves one question open: Is systemic exposure from i. a. infusion as efficient as systemic exposures from systemic chemotherapy, or are both systemic exposures inefficient? Regional chemotherapy for breast cancer is not at all a new method but all the same it never has had its breakthrough (Eedwinek et al, 1981; Carter et al, 1988; Noghuchi et al, 1988; Stephens, 1989, 1990; Wang et al, 1990; Dycker de et al, 1991; GĂśrich et al, 1993, 1995; Takatsuka, 1994; Chrysos et al, 2001; Fiorentini et al, 2003). Data from various published studies report approximately the same response rates as have been noted in our studies (Koyama et al, 1975; Koyama et al, 1985; Iwasa et al, 1988; Aigner et al, 1991, 1988; Dycker de et al, 1991; Nitz et al, 1993; Koyama, 1994; Morandi et al, 1996; Cakmakli et al, 1997; Mohrmann et al, 1999, 2000). At good quality of life they vary around 70%, a number that hardly can be achieved with other therapies without causing severe side-effects. This should be an argument to include the method into guidelines. The pioneers of intraarterial chemotherapy, Biermann and Klopp, after observing the local effect, called intraarterial chemotherapy, a â&#x20AC;&#x153;chemical irradiationâ&#x20AC;? (Biermann and Klopp, 1951). Indeed it is as effective, and it does not cause tissue fibrosis. But most evidently irradiation is easier to handle, and there are not sufficient clinical studies on intraarterial infusion for breast cancer, enough to justify inclusion of the method into treatment guidelines. One of the main reasons might be the adverse effect encountered in most studies, which was drugstreaming and skin burns after internal mammary artery infusion (Ashaishi, 1994; Doughty et al, 1995; McCarter et al, 1995, 1998), we were confronted with the same complications and finally found out, that although a relatively small total dose was infused into the internal mammary artery, the local concentration that was generated was by far above the tissue tolerance. This complication can only be avoided by dilution of the total amount of drug to be applied in a bit larger volume, in combination with corticosteroids given the same route with a constant flow pump (Fiorentini et al, 2003) and not in a small volume through a hand syringe. In conclusion, intraarterial infusion therapy for primary breast cancer can be an important tool, once the technique of application is standardized, allowing excellent results within a short time in favour of breast conservation and non-disturbed quality of life.

V. Discussion Breast cancer is considered a potentially systemic disease and therefore regional chemotherapy has been ranked as non adequate, treating only part of the area to be treated. The problem arising, however is, that systemic chemotherapy, in case of a bulky primary tumor, can hardly generate drug exposures, required for definite tumor eradication. And if so, dose- intense chemotherapy has to be intensified in such a way that toxicity becomes intolerable. Regional chemotherapy, however, in terms of intraarterial infusion is not an isolated procedure, confined exclusively to one well defined segment of the body-like surgery or irradiation. Intraarterial infusion is a systemic therapy given with its first pass through the area to be treated firsthand, the target, which is the primary tumor in the breast and all its lymphatic drainages. The first pass through the arterial supply causes a concentrationdependent higher tissue uptake of cytotoxics in the area to be treated, but also a systemic drug exposure from the cytotoxics exiting the tumor area through the venous drainage. The relatively small doses applied intraarterially are highly effective due to the drug concentrations achieved in arteries with comparatively moderate blood-flow, like the internal mammary artery. It has been shown in a previous study (Aigner, 1994), that in locoregional therapy of breast cancer there is a steep dose-response relationship (Fiorentini et al, 2003), dependent on the applied technique. Therefore in the neoadjuvant cases, it was important to combine subclavian artery infusion with infusion of the internal mammary artery, the diameter of which is much smaller than the diameter of the subclavian artery and thus many generate a much higher and efficient drug concentration at the tumor site. As a consequence, intraarterial application of drugs is more effective in terms of local control, when compared to systemic chemotherapy, where the same, or even higher doses are given intravenously (Stephens et al, 1980). Regarding the response rates in the study presented herein, it is obvious that within only three cycles of regional chemotherapy a substantial response could be achieved, translating into a 26% complete remission and 74 % overall remission rate and a significant shift from large to small tumor diameters. Moreover, the recurrence rate of 17% over 16 years is remarkably low, taking into

References Aigner KR (1994) Regional chemotherapy for breast cancer - the effect of different techniques of drug administration on tumor response. Reg Cancer Treat 7, 127-131.


Cancer Therapy Vol 6, page 71 Aigner KR, Müller H, Jansa J, Kalden M, Thiem N (1991) Regional chemotherapy for locally advanced breast cancer a phase II study with mitomycin C, fluorouracil/folinic acid. In: Taguchi T, Aigner KR, eds: Mitomycin C in Cancer Chemotherapy Today. Excerpta Medica, Tokyo. pp 17-26. Aigner KR, Walther HJ, Müller H, Jansa J, Thiem N (1988) Interarterial infusion chemotherapy for recurrent breast cancer via an implantable system. Reg Cancer Treat 1, 102107. Ashaishi K (1994) Arterial infusion chemotherapy for advanced breast cancer: clinical and histological response and adverse reactions. In: Taguchi T, Nakamura H: Arterial infusion chemotherapy. Jpn J Cancer and Chemother. Pub. Inc.: pp 223-233 Biermann HR (1951) Intra-arterial-catheterization of viscera in man. Am J Roentg 66, 555-562 Cakmakli S, Ersöz S, Tu! T, Karaayvaz M, Akgül H (1997) Intra-arterial infusion chemotherapy in the treatment of locally advanced breast cancer. Acta Oncologica 36, 489-92. Carlson RW, Favret AM (1999) Multidisciplinary management of locally advanced breast cancer. Breast 5, 303-307. Carter RD, Faddis DM, Krementz ET (1988) Treatment of locally advanced breast cancer with regional intraarterial chemotherapy. Reg Cancer Treat 1, 108-111. Chrysos E, Tsiaoussis J, Alexandra K, Athanasakis H, Tsetis D, Varveris C, Fiorentini G, Lucchi SR, Vassilakis JS, Zoras O (2001) Treatment of unresectable malignant abdominal, pelvic and thoracic tumors using abdominal, pelvic and thoracic stop-flow chemotherapy. Anticancer Res 21, 1-7. Doughty JC, Anderson JH, Wilmott N, McArdle CS (1995) Intraarterial administration of Adriamycin-loaded albumin microspheres for locally advanced breast cancer. Postgrad Med J 71, 47-49. Doughty JC, McCarter DH, Kane E, Reid AW, Cooke TG, McArdle CS (1996) Anatomic basis of intra-arterial chemotherapy for patients with locally advanced breast cancer. Brit J Surg 83, 1128-1230. Dycker de RP, Schumacher T, Neumann RL (1991) Preoperative intraarterial chemotherapy of advanced breast cancer: response rate as a prognostic factor for survical. Reg Cancer Treat 4, 75-78. Dycker de RP, Timmermann J, Schumacher T, Schindler AE (1988) The influence of arterial regional chemotherapy on the local recurrence rate of advanced breast cancer. Reg Cancer Treat 1, 112-116. Eedwinek JM, Fineberg B, Lee J, Ocwieza M (1981) Analysis of failures following local treatment of isolated locoregional recurrence of breast cancer. Int J Radiat Oncol 7, 581-585. Fiorentini G, Tsetis D, Bernardeschi P, Varveris C, Rossi S, Kalogeraki A, Athanasakis E, Dentico P, Kanellos P, Biancalani M, Almarashdah S, Zacharioudakis G, Saridaki Z, Chalkiadakis G, Xynos E, Zoras O (2003) First-line intraarterial chemotherapy (IAC) with Epirubicin and Mitoxantrone in locally advanced breast cancer. Anticancer Research 23, 4339-4346. Frei E, Canellos GP (1980) Dose: A critical factor in cancer chemotherapy. Am J Med 69, 585-594. Görich J, Hasan I, Majdali R, Sittek H, Kunze V, Doma A, Reiser M, Brambs HJ (1995) Previously treated, locally recurrent breast cancer: Treatment with superselective intraarterial chemotherapy. Radiology 197, 199-203. Görich J, Hasan I, Sittek H, Jakschik J, Werner H, Hartlapp HJ, Reiser M (1993) Superselektive intraarterielle Chemotherapie bei Mammakarzinomen. Radiologie 33, 308312. Gurney H, Harnett P, Stuart-Harris R, Kefford R (1995) Continuous infusion of Vincristine, Ifosfamide and

Epirubicin over 6 weeks in treatment-resistant advanced breast cancer. Eur J Cancer 31A, 1773-1777. Iwasa Z, Matsunami N, Saeki Y, Kurooka K, Yamato M, Okuno K, Sagara N, Matsuda T, Yasutomi M (1988) The follow up study of intra-arterial infusion chemotherapy with local vein blocking as a surgical neo-adjuvant treatment for locally advanced breast cancer. Jpn J Surg 18, 131-135. Koyama H (1994) Arterial infusion chemotherapy for breast cancer in: Taguchi T, Nakamura H: Arterial infusion chemotherapy. Jpn J Cancer and Chemother. Pub. Inc.: pp 213-222. Koyama H, Nishizawa Y, Wada T, Kabuto T, Shiba E, Iwanaga T, Terasawa T, Wada A (1985) Intra-arterial infusion chemotherapy as an induction therapy in multidisciplinary treatment for locally advanced breast cancer: a long term follow-up study. Cancer 56, 725-729. Koyama H, Wada T, Takahashi Y Wada A, Terazawa T (1975) Intraarterial infusion chemotherapy as a preoperative treatment of locally advanced breast cancer. Cancer 36, 1603-1612. McCarter DHA, Doughty JC, Cooke TG, McArdle CS, Reid AW (1995) Angiographic embolization of the distal internal mammary artery as an adjunct to regional chemotherapy in inoperable breast carcinoma. JVIR 6, 249-251. McCarter DHA, Doughty JC, Cooke TG, McArdle CS, Reid AW (1998) Selective angiographically delivered regional chemotherapy in patients with locally advanced or recurrent breast cancer: A feasibility study. JVIR 9, 91-96. Merajver SD, Weber BL, Cody R, Zhang D, Strawderman M, Calzone KA, LeClaire V, Levin A, Irani J, Halvie M, August D, Wicha M, Lichter A, Pierce LJ (1997) Breast conservation and prolonged chemotherapy for locally advanced breast cancer: The University of Michigan experience. J Clin Oncol 15, 2873-2881. Mohrmann S et al (2000) Locoregional intraarterial chemotherapy in primary incurable breast cancer recurrences. J Cancer Res Clin Oncol 126. Mohrmann S, Nitz U (1999) Intraarterielle Chemotherapie Einsatz bei ausgedehnt vorbehandelten Lokalrezidiven eines Mammakarzinoms. Gynäkologe 32, 689-694. Morandi C et al (1996) Intra-arterial chemotherapy in locally advanced or recurrent breast neoplasms. Radiol Med 92, 101-104 Murakami M, Kuroda Y, Nishimura S, Sano A, Okamoto Y, Taniguchi T, Nakajima T, Kobashi Y, Matsusue S (2001) Intraarterial infusion chemotherapy and radiotherapy with or without surgery for patients with locally advanced or recurrrent breast cancer. Am J Clin Oncol 24, 185-191. Nitz U, Havenith B, Rost B, Mosny D, Ellerbrok G (1993) Die lokoregionäre intraarterielle Chemotherapie bei primär inkurablen Lokalrezidiven eines Mammakarzinoms. Geburtsh und Frauenheilk 53, 760-767. Noguchi S, Miyauchi K, Nishizawa Y, Koyama H, Terasawa T (1988) Management of inflammatory carcinoma of the breast with combined modality therapy including intraarterial infusion chemotherapy as an induction therapy: long term follow-up results of 28 patients. Cancer 61, 1483-91. Sainsbury JR, Walker VA, Ali H (1990) A dose escalation study of mitoxantrone given intraaterially for breast cancer. Reg Cancer Treat 3, 243-247. Stephens FO (1988) Why use regional chemotherapy? Principles and pharmacokinetics. Reg Cancer Treat 1, 1-7. Stephens FO (1989) Advanced breast cancer: primary intraarterial induction chemotherapy. Reg Cancer Treat 2, 5-8. Stephens FO (1990) Intra-arterial induction chemotherapy in locally advanced stage III breast cancer. Cancer 66, 645650.


Aigner et al: Subclavian artery infusion as induction and adjuvant chemotherapy Stephens FO, Crea P, Harker GJ, Roberts BA, Hambly CK (1980a) Intraarterial chemotherapy as basal treatment in advanced and fungating primary breast cancer. The Lancet 1980, 435-438. Stephens FO, Harker GJS, Crea P (1980b) The intraarterial infusion of chemotherapeutic agents as "basal" treatment of cancer: evidence of increased drug activity in regionally infused tissues. Aust N Z J Surg 50, 597-602. Takatsuka Y (1994) Transcatheter arterial chemo-embolization (TAC-E) for patients with locally advanced breast cancer. In: Taguchi T, Nakamura H: Arterial Infusion Chemotherapy. Jpn. J. Cancer and Chemother. Pub. Inc.: pp 234-240. Wang JS, Ho DM, Liu HC, Chen CM, Lui WY (1990) Infusion chemotherapy in breast cancer: histopathological study of 25 cases. Reg Cancer Treat 47-53.

Karl R. Aigner


Cancer Therapy Vol 6, page 73 Cancer Therapy Vol 6, 73-80, 2008

Treatment of malignant pleural mesothelioma Review Article

David F. Heigener*, Martin Reck, Ulrich Gatzemeier Department of Thoracic Oncology, Krankenhaus Grosshansdorf, Germany

__________________________________________________________________________________ *Correspondence: David F. Heigener, Krankenhaus Großhansdorf, Woehrendamm 80, 22927 Großhansdorf, Germany; Tel: 0049 4102 601 261; Fax: 0049 4102 601 247; E-Mail: Key words: Malignant Mesothelioma, Diagnosis, Therapy Abbreviations: endothelial-growth factor-receptor, (EGFR); International mesothelioma interest group, (IMIG); Malignant Pleural Mesothelioma, (MPM); nuclear magnetic resonance imaging, (NMR); overall survival, (OS); positron-emitting tomography, (PET); Serum mesothelin-related protein, (SMRP); simian-virus 40, (SV-40) Received: 13 January; Revised: 30 January Accepted: 4 February; electronically published: February 2008

Summary Malignant Pleural Mesothelioma is an asbestos-related disease with rising incidence. Diagnosis is often late in the course of the disease because of its insidious onset. Treatment is difficult because of the natural resistance of this tumor-entity. The diagnostic work-up as well as therapeutic options are outlined in this review article.

The use of the radioactive, alpha-emitting contrast medium 232ThO2 (Thorotrast®) is another risk factor. Up to 7.8% of exposed patients given more than 20ml of Thorotrast developed MPM (Andersson et al, 1995). There is also a higher incidence of MPM in survivors of the Hiroshima and Nagasaki nuclear attacks (White and Abratt, 2005). In summary, the most important risk factor for MPM is undoubtedly asbestos which is responsible for approximately 70-90% of cases (Yates et al, 1997). MPM is a devastating disease with a grim prognosis. Median survival ranges from approximately 4 to 18 months depending on the histological subtype (sarcomatous has a worse prognosis compared to epithelial subtype), tumour stage and some patient derived factors (general condition, age, weight loss, pain, platelets, asbestos exposure) (Manegold et al, 2006). In a series of 49 consecutive patients at our institution median progression free survival was 8 months (Figure 1).

I. Introduction Before 1950, Malignant Pleural Mesothelioma (MPM) was a very rare condition. With the rising use of asbestos after World War II it became more frequent. In the early nineteen-sixties the link between asbestosexposure and MPM was first recognized (Wagner and Sleggs, 1960; Thomson, 1963). Asbestos exposure is either occupational or environmental. The latter can be due to air pollution by asbestos-processing facilities or “natural” exposure in the soil (Constantoupolos et al, 1985). Although the use of asbestos was widely abandoned since 1980 in the western countries, the incidence of MPM will continue to rise until approximately 2020 (Peto et al, 1999). This is due to the long latency between asbestos exposure and development of MPM (up to 58 years) (Kroidl et al, 2000). Other risk factors for the development of MPM are the simian-virus 40 (SV-40) and ionizing radiation. SV-40 DNA can be found in approximately 60% of MPM but also in ependymomas, brain and bone tumours (Rizzo et al, 1998). The virus is a potent inhibitor of tumoursuppressor genes and is considered a co-factor in carcinogenesis. Simian virus 40 was a contaminant in polio vaccines administered to 10-30 million people in the United States, mostly children, between 1955 and 1963 (Stratton et al, 2002). However, the role of the virus in the pathogenesis of MPM is yet unproven (MacLachlan 2002; Price and Ware, 2004).

II. Diagnosis of MPM A. Signs and Symptoms The onset of symptoms in MPM is subtle and nonspecific, so diagnosis is often late in the course of disease. The majority of patients present with dyspnea and/ or chest pain (Lee et al, 2000). Fatigue, fever and night sweats are also common.


Heigener et al: Treatment of malignant pleural mesothelioma The first diagnostic step after clinical examination is a chest X-ray. The most frequent finding here is a pleural effusion (Lee et al, 2000).

B. Imaging techniques

Figure 1. Progression free survival of 49 consecutive patients with MPM at our institution in months.

In one series the majority of MPM is located on the right side, in five percent of cases there is a bilateral involvement (Antman, 1981). However in our patients there were no bilateral manifestations and 65% right-sided locations in a series of 49 consecutive patients (unpublished data). The next diagnostic step when MPM is suspected should be a computed tomography or a nuclear magnetic resonance imaging (NMR) of the chest. The former is more available than the latter and of nearly similar accuracy. NMR provides little more information concerning chest wall and diaphragm invasion (Heelan et al, 1999). The addition of positron-emitting tomography (PET) can help distinguish between benign pleural thickening and foci of malignancy and makes mediastinal lymph-node staging more accurate (Wang et al, 2004). However this method is not readily available in many institutions. MPM is characterized by local spread involving the mediastinal lymph nodes. There are many case reports about distant metastases in MPM, however in general they seldom occur. Therefore we do not perform any routine imaging technique (i.e. bone-scintigraphy or NMR of the brain) beyond thoracical imaging. Staging should be done based on the TNM staging system by the International mesothelioma interest group (IMIG): the T-descriptors are designated as T1 to T4 describing the local extend of the tumour. T1 is localized disease on the pleura (T1a: parietal pleura only; T1b: involvement of visceral pleura), T2 describes diaphragmal or direct lung involvement, T3 describes involvement of the endothoracic fascia or mediastinal fat (locally advanced but technically resectable) and T4 the diffuse invasion of the chest wall, peritoneum, spine, peri- or myocard as well as the contralateral pleura (locally advanced, unresectable). The N-descriptors N1 to N3 are identical as the ones used in the staging of lung cancer: N1 describes ipsilateral pulmonary or hilar nodes, N2 ipsilateral or subcarinal mediastinal nodes and N3 contralateral mediastinal or supraclavicular nodes. M0

denotes the absence and M1 the presence of distant metastases. The corresponding stages are shown in Table 1 (Rusch, 1995).

B. Achievement of a tissue diagnosis Evidence of MPM in pleural fluid is found in 3384% of cases (Whitaker 2000). Aspiration-cytology of pleural fluid (if present) is specific but lacks sensitivity. The latter can be improved with the measurement of hyaluronic acid in the pleural fluid which is markedly elevated in MPM compared to other malignant effusions (Welker et al, 2007). Nevertheless, obtaining a histological specimen is often necessary for an exact diagnosis. Image guided, trans -thoracical core-biopsies lack direct visualisation of the tumor and therefore carry a higher risk of false-negative results compared to direct thoracoscopic forceps-biopsy either in local anaesthesia or video-assisted under general anaesthesia (VATS). Immunhistochemistry should be performed for an exact diagnosis. Typical positive markers are calretinin (epithelial subtype), MNF 116 and AE1/ AE3 cytokeratins, vimentin and many more (Mueller 2005).

C. Serum markers Two markers deserve special consideration: Serum mesothelin-related protein (SMRP) and serum osteopontin. SMRP has a sensitivity of 83% and a specifity of 95% in detecting MPM in one study. There are also level-changes parallel to the tumour size, suggesting that it might be a good diagnostic tool for monitoring (Robinson, 2005). The addition of the tumour-marker CA125 did not improve sensitivity (Creaney et al, 2007). Serum osteopontin is significantly elevated in patients with asbestos exposure and MPM compared to patients with asbestos exposure alone (Pass, 2005). It seems to have a lower diagnostic accuracy than SMRP due to its low specifity (Grigoriu, 2007). However, by now -lacking large validation series- both markers are not routinely used in our clinical practise. Table 1. Stages of MPM (Rusch, 1995).


Cancer Therapy Vol 6, page 75

Stage Ia Ib II III IV

C. Role of medical therapy

TNM T1aN0M0 T1bN0M0 T2N0M0 Any T3M0; any N1M0; any N2M0 Any T4, any N3; any M1

1. “Older” Regimens Numerous trials have been conducted in the 1980´s and 1990´s with various cytotoxic agents in the treatment of MPM. Focussing on remission there were some agents with moderate efficacy: the anthracyclines, platinum compounds, alkylating agents, mitomycin C and antimetabolites. However, these results were only based on small case series. No large randomized controlled trials were available and the impact on survival was not clarified (Ryan et al, 1998). There were also some case series with combination therapies but there was no clear evidence for superiority over monotherapies (Ryan et al, 1998).

III. Therapy of MPM A. Role of radiation therapy External beam radiation of the hemithorax as a single treatment of MPM has no effect on survival (Baldini, 2004). However some series suggest that it might have its place in a multimodality approach to improve local control, especially when using modern techniques (see below) (Rusch et al, 2001; Lee et al, 2002; Rice et al, 2007). Another questionable indication for radiotherapy in MPM is the irradiation of surgical wounds and drainagesites, fearing that otherwise the tumour will spread to the subcutaneous layer along the surgical tract. There are three small randomized controlled trials and some case series. One of the controlled trials showed a small benefit on local control (Boutin et al, 1995) and the other two showed no benefit at all (Bydder et al, 2004; O´Rourke et al, 2007). In one case series of 85 consecutive patients treated with different chemotherapy regimens without radiation none developed surgical tract metastasis (Pinto et al, 1995). As a result, the practise is discussed with considerably controversy and we do not recommend this procedure routinely although it is implemented in the guidelines of the British Thoracic Society (British Thoracic Society Standards of Care Committee, 2007). External beam radiation is moderately effective in the reduction of pain due to MPM. In one case series of 189 patients 50% had a benefit in terms of pain control when 36 Gray in 4- Gray fractions were used. However pain recurred in a median of 69 days (de GraafStrukowska et al, 1999). In a review on that topic, pain relief is described for 50 to 70% of patients (Baldini, 2004).

2. Vinca-Alkaloids Two modern vinca-alkaloids show promising activity in phase II-trials in MPM: Vinorelbine and vinflunine. Vinorelbine was tested in a weekly infusion regimen in 29 patients. 24% of patients achieved a partial response and 55% had stable disease. There was also some improvement in quality of life (Steele et al, 2000). However the patient number was small and phase III studies are clearly needed. 67 patients were enrolled in a phase- II study with vinflunine. Response rate was 13.8% and median survival was 10.8 months (Talbot et al, 2007).

3. Gemcitabine There are several Phase-II studies of either gemcitabine alone ore in combination with cisplatin (Kindler et al, 2002). Monotherapy failed to show significant activity in two trials (van Meerbeeck et al, 1999; Kindler et al, 2001). The combination achieved up to 47.6% partial responses (Byrne et al, 1999). However, as with vinorelbine, phase-III data are lacking.

4. Taxanes There is only sparse data on paclitaxel in MPM. One trial showed only 9% remissions with high-dose paclitaxel (Vogelzang et al, 1999). Docetaxel also showed at most mildly efficacy as monotherapy (Vorobiof et al, 2002; Belani et al, 2004). Also in combination with irinotecan, the results are disappointing (Knuutila et al, 2000).

B. Role of surgery There are mainly two surgical approaches to MPM. First there is a quite aggressive option, the extrapleural pleuropneumectomy (EPP) or a solely cytoreductive procedure: decortication and/ or pleurectomy. The latter can sometimes be performed via VATS. A case series describing the trimodality approach of EPP plus consecutive chemoradiation showed a five-year survival rate of 22%. However this was a selected group of patients and a control group was lacking (Sugarbaker and Norberto 1998). In another trial the two surgical procedures were compared, showing a benefit in progression free survival for EPP (319 vs. 197 days, p= 0.019) but only a non significant benefit favouring EPP in overall survival (497 vs. 327 days, p=0.079) (Stewart et al, 2004). In both papers the involvement of mediastinal lymph nodes was a predictor of poor survival. To date a large randomized trial is ongoing to clarify the role of radical surgery in MPM (Treasure et al, 2006).

5. Antifolates Alpha folate receptor protein is overexpressed in MPM-Cells in approximately 70% of cases. This might explain the responsiveness to antifolate drugs (Bueno et al, 2001) as shown in a group of 60 patients receiving high dose methotrexate. There were 37% responses with one complete response (Solheim et al, 1992). In the beginning of this century, a new multi-targeted antifolate, pemetrexed, was evaluated for its efficacy in MPM. A phase II -trial including 64 patients showed an overall response rate of 14.1% for monotherapy with the substance. Interestingly, overall survival (OS) was higher in those patients who were supplemented with folic acid and vitamin B12 (13 months OS vs 8 months in the not supplemented group). Moreover, neutropenia was lower in the supplemented group (4 grade ! neutropenias in the supplemented group vs. 11 in the non-supplemented


Heigener et al: Treatment of malignant pleural mesothelioma group) (Scagliotti et al, 2003). However, because of the small subgroups no definite conclusion could be drawn on the impact of vitamin supplementation. A phase III trial with 456 enrolled patients compared pemetrexed and cisplatin with cisplatin alone (Vogelzang et al, 2003). This was the first trial ever in MPM showing a significant survival advantage for a chemotherapy regimen with a median survival of 12.1 months for the cisplatin/ pemetrexed-arm vs 9.3 months for cisplatin alone (p=0.02). Again, toxicities were significantly reduced after adding folic acid and vitamin B12 to the regimen. The combination of cisplatin and pemetrexed can now be considered standard first line therapy in MPM in those patients, who can tolerate this fairly toxic regimen. Because of the considerably toxicity of cisplatin, it is sometimes replaced by carboplatin for patient comfort in this palliative setting (Figure 2). Another combination with an antifolate drug with some efficacy in a phase II trial are oxaliplatin and ralitrexed. 70 patients were enrolled, the overall response rate was 20% and one year survival was 26% (Fizazi et al, 2003). There is one case series suggesting that pemetrexed also has efficacy in second-line treatment after a platinumcontaining doublet (mostly platinum/ vinorelbine). In patients treated with carboplatin + pemetrexed (n=11) there were 18% responses with a median time to progression of 32 weeks. In patients treated with pemetrexed alone the numbers were 21% and 21 weeks respectively. Median survival was 39 weeks and 42 weeks respectively (Sorensen et al, 2007).

showed high expression of EGFR. However treatment with erlotinib did not result in any objective remission (Garland et al, 2007). The reason might be, that simple overexpression of EGFR is not predictive of response to erlotinib in other cancers. Only EGFR-mutations seem to have some predictive value. The antisense-oligonucleotide ranpirnase blocks the 30S-subunit of the ribosome and interferes with protein synthesis. In a phase-II trial a median survival time of 18.5 months was observed (Mikulski et al, 2002). These results encouraged to perform a phase-III study comparing doxorubicin and ranpirase with doxorubicin alone. The recruitment was finished recently and the results are pending.

D. Multimodality treatment As described above, multimodality treatment approaches seem to be promising in a selected group of MPM patients, those in a good performance status (Sugarbaker and Norberto, 1998). In another case series focussing on prognostic factors in MPM, the data suggest that surgery within a multimodality approach improves survival more than surgery alone (pleurectomy or EPP: 10.3 months median survival; pleurectomy or EPP plus chemotherapy and external beam radiotherapy: 20.1 months median survival). There was no statistical significant difference between pleurectomy and EPP in terms of survival when used as a single-modality (median survival 15.8 vs. 14.3 months) (Flores et al, 2007). In a phase-II trial the combination of hyperthermia (41.8° Celsius body temperature) with chemotherapy (ifosfamide, carboplatin and etoposide) resulted in a response rate of 20% and a two-year survival of 20% (Bakhshandeh et al, 2003). However to date, no phase-III data were present to support this approach.

6. â&#x20AC;&#x153;Biologicalsâ&#x20AC;? Although endothelial-growth factor-receptor (EGFR) is overexpressed in the majority of MPM (Dazzi et al, 1990), the inhibition of the downstream signalling of EGFR by means of erlotinib does not seem to work. In a study with 64 patients with MPM, 75% of tumours


Cancer Therapy Vol 6, page 77

Figure 2. CT-scans before (left) and after (right) four cycles of carboplatin and pemetrexed.

However, these recommendations lack clear evidence which will hopefully come with further clinical trials.

E. Palliative treatment Because of the devastating course of the disease almost all patients need sufficient palliative care. Main symptoms are pain and dyspnoea. Pain can be either neuropathic due to infiltration of intercostal nerves or somatic due to chest-wall involvement. Often there is a combination of both. Somatic pain responds to nonsteroidal analgesic drugs and opiates. Neuropathic pain should be managed with opiates and co-analgesics like antidepressants (i.e. amitryptiline) or anticonvulsants (i.e. carbamazepine, pregabaline; Doyle et al, 2004). Dyspnoea results from pleural effusion as well as entrapment of the lung by the pleural tumor. Pleurodesis either by VATS or pleuroscopy should be done in the former case. However the latter needs symptomatic treatment with opioids (Lee YC, 2002).

References Andersson M, Wallin H, Jรถnsson M, Nielsen LL, Visfeldt J, Vyberg M, Bennett WP, De Benedetti VM, Travis LB, Storm HH (1995) Lung carcinoma and malignant mesothelioma in patients exposed to Thorotrast, incidence, histology and p53 status. Int J Cancer 63, 330-6. Antman KH (1981) Clinical presentation and natural history of benign and malignant Mesothelioma. Semin Oncol 8, 31320. Bakhshandeh A, Bruns I, Traynor A, Robins HI, Eberhardt K, Demedts A, Kaukel E, Koschel G, Gatzemeier U, Kohlmann T, Dalhoff K, Ehlers EM, Gruber Y, Zumschlinge R, Hegewisch-Becker S, Peters SO, Wiedemann GJ (2003) Ifosfamide, carboplatin and etoposide combined with 41.8 degrees C whole body hyperthermia for malignant pleural mesothelioma. Lung Cancer Mar 39, 339-45. Baldini EH (2004) External beam radiation therapy for the treatment of pleural mesothelioma. Thorac Surg Clin 14, 543-8. Belani CP, Adak S, Aisner S, Stella PJ, Levitan N, Johnson DH; Eastern Cooperative Oncology Group (2004) Docetaxel for malignant mesothelioma, phase II study of the Eastern Cooperative Oncology Group. Clin Lung Cancer 6, 43-7. Boutin C, Rey F, Viallat JR (1995) Prevention of malignant seeding after invasive diagnostic procedures in patients with pleural mesothelioma. A randomized trial of local radiotherapy. Chest 108, 754-758. British Thoracic Society Standards of Care Committee: BTS Statement on malignant mesothelioma in the UK, 2007. Thorax, Nov 2007; 62 Suppl 2: ii1-ii19 Bueno R, Appasani K, Mercer H, Lester S, Sugarbaker D (2001) The alpha folate receptor is highly activated in malignant pleural mesothelioma. . Thorac Cardiovasc Surg 121, 22533.

IV. Summary MPM is notoriously resistant to therapy. Moreover the natural course can vary widely from only a few months of survival to several years. Because of the diffuse growth pattern, treatment response is not easy to evaluate and inter-observer differences can be great. All these factors make good clinical trials difficult to perform and many issues remain unanswered. As a practical approach we recommend to establish the diagnosis via VATS in patients with adequate performance status or via pleuroscopy. With the former, pleurectomy/ decortication can be done. The role of radical surgery (EPP) needs to be clarified. Afterwards chemotherapy with platinum and pemetrexed should be made. Radiotherapy is indicated for palliative reasons (i.e. pain control) but not for tumour reduction. In patients with poor performance status monotherapy with pemetrexed is a reasonable option. 77

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Lee YC, Light RW, Musk AW (2000) Management of malignant pleural mesothelioma, a critical review. Curr Opin Pulm Med 6, 267-274. Lee TT, Everett DL, Shu HK, Jahan TM, Roach M 3rd, Speight JL, Cameron RB, Phillips TL, Chan A, Jablons DM (2002) Radical pleurectomy/decortication and intraoperative radiotherapy followed by conformal radiation with or without chemotherapy for malignant pleural mesothelioma. J Thorac Cardiovasc Surg 124, 1183-9. MacLachlan DS (2002) SV40 in human tumors, new documents shed light on the apparent controversy. Anticancer Res 22, 3495-3499. Manegold C, Schirren J, Dienemann H, Wannenmacher M in Schmoll HJ, Höffken, K and Possinger, K (2006) Kompedium Internistische Onkologie; Springer Medizin Verlag, Heidelberg pp.3644-45. Mikulski SM, Costanzi JJ, Vogelzang NJ, McCachren S, Taub RN, Chun H, Mittelman A, Panella T, Puccio C, Fine R, Shogen K (2002) Phase II trial of a single weekly intravenous dose of ranpirnase in patients with unresectable malignant mesothelioma. J Clin Oncol 20, 274-81. Mueller KM in Manegold Ch (2005) Pleuramesotheliom; Springer Heidelberg. pp.43-58. O'Rourke N, Garcia JC, Paul J, Lawless C, McMenemin R, Hill J (2007) A randomised controlled trial of intervention site radiotherapy in malignant pleural mesothelioma. Radiother Oncol 84, 18-22. Payne, R; Gozales, R; Foley, K et al.: The management of pain in Doyle, D.; Hanks, G; Cherny, N and Calman, K (eds): Oxford Textbook of Palliative Medicine. Oxford 2004; pp288-458 Pass HI, Lott D, Lonardo F, Harbut M, Liu Z, Tang N, Carbone M, Webb C, Wali A (2005) Asbestos exposure, pleural mesothelioma, and serum osteopontin levels. N Engl J Med 353, 1564-73. Peto J, Decarli A, La Vecchia C, Levi F, Negri E (1999) The European mesothelioma epidemic. Br J Câncer 79, 666-72. Pinto C, Sperandi F, Marino A, Mutri V, Martoni A (1995) Is prophylactic radiotherapy necessary as prevention of tumor seeding following thoracoscopy in malignant pleural mesothelioma? Lung Cancer 49(suppl. S227) Price B, Ware A (2004) Mesothelioma trends in the United States, an update based on Surveillance, Epidemiology, and End Results Program data for 1973 through 2003. Am J Epidemiol ; 159, 107-12. Rice DC, Stevens CW, Correa AM, Vaporciyan AA, Tsao A, Forster KM, Walsh GL, Swisher SG, Hofstetter WL, Mehran RJ, Roth JA, Liao Z, Smythe WR (2007) Outcomes after extrapleural pneumonectomy and intensity-modulated radiation therapy for malignant pleural mesothelioma. Ann Thorac Surg 84, 1685-92. Rizzo P, Di Resta I, Powers A, Matker CM, Zhang A, Mutti L, Kast WM, Pass H, Carbone M (1998) The detection of simian virus 40 in human tumours by polymerase chain reaction. Monaldi Arch Chest Dis 53, 202-10. Robinson BW, Creaney J, Lake R, Nowak A, Musk AW, de Klerk N, Winzell P, Hellstrom KE, Hellstrom I (2005) Soluble mesothelin-related protein--a blood test for mesothelioma. Lung Cancer 49(Suppl 1) Rusch VW (1995) A proposed new international TNM staging system for malignant pleural mesothelioma. From the International Mesothelioma Interest Group. Chest 108, 11228. Rusch VW, Rosenzweig K, Venkatraman E, Leon L, Raben A, Harrison L, Bains MS, Downey RJ, Ginsberg RJ (2001) A phase II trial of surgical resection and adjuvant high-dose


Cancer Therapy Vol 6, page 79 hemithoracic radiation for malignant pleural mesothelioma. J Thorac Cardiovasc Surg 122, 788-95. Ryan CW, Herndon J, Vogelzang NJ (1998) A review of chemotherapy trials for Malignant Mesothelioma. Chest ; 113(1 Suppl) Scagliotti GV, Shin DM, Kindler HL, Vasconcelles MJ, Keppler U, Manegold C, Burris H, Gatzemeier U, Blatter J, Symanowski JT, Rusthoven JJ (2003) Phase II Study of Pemetrexed With and Without Folic Acid and Vitamin B12 as Front-Line Therapy in Malignant Pleural Mesothelioma. J Clin Oncol 21, 1556-1561. Solheim OP, Saeter G, Finnanger AM, Stenwig AE (1992) Highdose methotrexate in the treatment of malignant mesothelioma of the pleura, a phase II study. Br J Cancer ; 65, 956-60. Sørensen JB, Sundstrøm S, Perell K, Thielsen AK (2007) Pemetrexed as second-line treatment in malignant pleural mesothelioma after platinum-based first-line treatment. J Thorac Oncol 2, 147-52. Steele JP, Shamash J, Evans MT, Gower NH, Tischkowitz MD, Rudd RM (2000) Phase II Study of Vinorelbine in Patients With Malignant Pleural Mesothelioma. J Clin Oncol 18, 3912-3917. Stewart DJ, Martin-Ucar A, Pilling JE, Edwards JG, O'Byrne KJ, Waller DA (2004) The effect of extent of local resection on patterns of disease progression in malignant pleural mesothelioma.Ann Thorac Surg 78, 245-52. Stratton K, Almario DA, McCormick MC, eds. (2002) Immunization safety review, SV40 contamination of polio vaccine and cancer. Immunization Safety Review Committee. Washington, DC, National Academies Press, . Sugarbaker DJ, Norberto JJ (1998) Multimodality management of malignant pleural mesothelioma. Chest113, 61S-65S. Talbot DC, Margery J, Dabouis G, Dark G, Taylor H, Boussemart H, Cadic V, Pinel MC, Rivière A, Ollivier L, Ruffié P (2007) Phase II study of vinflunine in malignant pleural mesothelioma. J Clin Oncol 25, 4751-6. Thomson JG (1963) Exposure to asbestos dust and diffuse pleural mesotheliomas.Br Med J 1, 123. Treasure T, Tan C, Lang-Lazdunski L, Waller D (2006) The MARS trial, mesothelioma and radical surgery. Interact Cardiovasc Thorac Surg 5, 58-9. van Meerbeeck JP, Baas P, Debruyne C, Groen HJ, Manegold C, Ardizzoni A, Gridelli C, van Marck EA, Lentz M, Giaccone G (1999) A Phase II study of gemcitabine in patients with malignant pleural mesothelioma. European Organization for Research and Treatment of Cancer Lung Cancer Cooperative Group. Cancer 85, 2577-82.

Vogelzang NJ, Herndon JE 2nd, Miller A, Strauss G, Clamon G, Stewart FM, Aisner J, Lyss A, Cooper MR, Suzuki Y, Green MR (1999) High-dose paclitaxel plus G-CSF for malignant mesothelioma, CALGB phase II study 9234. Ann Oncol 10, 597-600. Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C, Kaukel E, Ruffie P, Gatzemeier U, Boyer M, Emri S, Manegold C, Niyikiza C, Paoletti P (2003) Phase III Study of Pemetrexed in Combination With Cisplatin Versus Cisplatin Alone in Patients With Malignant Pleural Mesothelioma. J Clin Oncol 21, 2636-44. Vorobiof DA, Rapoport BL, Chasen MR, Abratt RP, Cronje N, Fourie L, McMichael G, Hacking D (2002) Malignant pleural mesothelioma, a phase II trial with docetaxel. Ann Oncol 13, 412-5. Wagner JC, Sleggs CA, Marchand P (1960) Diffuse pleural mesothelioma and asbestos exposure in the North Western Cape Province. Br J Ind Med 17, 260-71. Wang ZJ, Reddy GP, Gotway MB, Higgins CB, Jablons DM, Ramaswamy M, Hawkins RA, Webb WR (2004) Malignant pleural mesothelioma, evaluation with CT, MR imaging, and PET. Radiographics 24, 105-119. Welker L, Müller M, Holz O, Vollmer E, Magnussen H, Jörres RA (2007) Cytological diagnosis of malignant mesothelioma--improvement by additional analysis of hyaluronic acid in pleural effusions. Virchows Arch 450, 455-61. Whitaker D (2000) The cytology of malignant mesothelioma. Cytopathology 11, 139-151. White NW, Abratt RP in Manegold, Ch (2005) Pleuramesotheliom; Springer Heidelberg. p4. Yates DH, Corrin B, Stidolph PN, Browne K (1997) Malignant mesothelioma in south east England, clinicopathological experience of 272 cases. Thorax 52, 507-12.

David F. Heigener


Heigener et al: Treatment of malignant pleural mesothelioma


Cancer Therapy Vol 6, page 81 Cancer Therapy Vol 6, 81-94, 2008

Diagnosis and multimodality management of stage III non-small cell lung cancer Review Article

Kevin Sullivan, Zujun Li, John Rescigno, Michael Fanucchi* St. Vincentâ&#x20AC;&#x2122;s Comprehensive Cancer Center and New York Medical College, New York, NY

__________________________________________________________________________________ *Correspondence: Michael Fanucchi, MD, St. Vincent's Comprehensive Cancer Center, 325 West 15th Street, New York, NY 10011, USA; Tel: (212) 604-6011; Fax: (212) 604-6038; e-mail: Key words: Diagnosis, staging, Treatment, N2 disease, Preoperative chemotherapy, Preoperative chemoradiotherapy, radiotherapy, neoadjuvant therapy, Adjuvant therapy, Superior sulcus tumors, Satellite nodules, Mediastinal invasion, Targeted therapy, Immunotherapy, Prophylactic cranial irradiation Abbreviations: 3-dimensional, (3D); Adjuvant Navelbine International Trialist Association, (ANITA); American Society of Clinical Oncology, (ASCO); Cancer and Leukemia Group B, (CALGB); Cancer Care Ontario, (CCO); central nervous system, (CNS); computed tomography, (CT); disease free survival, (DFS); Eastern Cooperative Oncology Group, (ECOG); Electromagnetic navigation bronchoscopy, (ENB); endobronchial ultrasound-guided transbronchial needle aspiration, (EBUS-TBNA); epidermal growth factor receptor, (EGFR); European Organization for Research and Treatment of Cancer, (EORTC); European Society of Thoracic Surgeons, (ESTS); fine needle aspiration, (FNA); Fluoro-Deoxy-Glucose, (FDG); granulocyte macrophage colony stimulating factor, (GM-CSF); hazard ratio, (HR); hyperfractionated RT, (HART); intensity modulated radiotherapy, (IMRT); International Adjuvant Lung Cancer Trial, (IALT); International Association for the Study of Lung Cancer, (IASLC); International Staging System, (ISS); magnetic resonance imaging, (MRI); median survival, (MS); National Comprehensive Cancer Network, (NCCN); Non-Small Cell lung cancer, (NSCLC); overall survival, (OS); performance status, (PS); Positron emission tomography, (PET); progression free survival, (PFS); Prophylactic cranial irradiation, (PCI); Radiation Therapy Oncology Group, (RTOG); radiation therapy, (RT); response rate, (RR); Southwest Oncology Group, (SWOG); time to progression, (TTP); tumor-node-metastasis, (TNM); video-assisted thoracoscopic surgery, (VATS) Received: 31 December 2007; Revised: 30 January 2008 Accepted: 1 February 2008; electronically published: February 2008

Summary Stage III non-small cell lung cancer (NSCLC) encompasses a heterogeneous patient population with locoregionally advanced disease. The multimodality management of patients with this stage of disease is reviewed, including diagnosis, staging and treatment. Evidenced-based treatment approaches are emphasized including chemotherapy and radiotherapy in the neoadjuvant, adjuvant and definitive setting. The role of surgery in select patients is discussed.

International Association for the Study of Lung Cancer (IASLC) (Rami-Porta et al, 2007), the ISS, which was last revised in 1997, is still in current use. Stage III NSCLC is defined as locoregionally advanced disease, due to involvement of mediastinal lymph nodes, involvement or invasion of extrapulmonary structures, or the presence of a malignant pleural or pericardial effusion, without evidence of distant metastatic disease. Stage III NSCLC is subdivided into Stage IIIA and IIIB disease. Stage IIIA disease is a T1 or T2 tumor with involvement of ipsilateral mediastinal lymph nodes (N2), or a T3 lesion with hilar (N1) or mediastinal (N2) lymph node involvement. Stage IIIA patients are further clinically subdivided into bulky and non-bulky disease. Bulky disease includes lymph nodes which are >2cm in diameter as measured by

I. Introduction In the United States, lung cancer is the leading cause of cancer death in both men and women. An estimated 213,380 new cases of lung cancer were diagnosed in 2007, with 68,280 patients having stage IIIA/B disease (Jemal et al, 2007). Non-Small Cell lung cancer (NSCLC) represents about 80% of all histological types of lung cancer and includes adenocarcinoma (including its subset, bronchioloalveolar carcinoma), squamous cell (epidermoid) carcinoma, and large cell carcinoma (Fraire, 1996; Travis et al, 2004). The International Staging System (ISS) is the most commonly used staging system for NSCLC (Mountain, 1997). Although there is a proposal to modify the TNM staging of lung cancer under consideration by the 81

Sullivan et al: Diagnosis and multimodality management of stage III non-small cell lung cancer computed tomography (CT), or conglomeration of multiple smaller lymph nodes (Robinson et al, 2003). Patients with non-bulky N2 disease may be rendered disease free by surgical resection, whereas patients with bulky disease are generally inoperable due to extent of nodal disease. Stage IIIB disease is based upon the presence of a T4 tumor, or involvement of supraclavicular lymph nodes or contralateral mediastinal or hilar lymph nodes (N3). Stage IIIB disease due to the presence of a malignant pleural or pericardial effusion is managed primarily with chemotherapy alone and will not be discussed here.

mediastinal lymph nodes (Chin et al, 1995). PET scan was compared with CT scan for accuracy in identifying N2 and N3 disease, and the PET scan was found to be more sensitive (81%) versus CT scan (76%) (Kerstine et al, 1998). The most accurate method of clinically staging the mediastinal lymph nodes is integrated PET/CT (Lardinois et al, 2003). Integrated PET/CT provided additional information in 41% of patients beyond that provided by visual correlation of PET and CT. Patients that have a negative PET/CT evaluation of the mediastinal lymph nodes may proceed to surgical resection without preoperative mediastinoscopy (Vansteenkiste 2006). The European Society of Thoracic Surgeons (ESTS) recommends invasive staging despite a negative PET/CT evaluation of mediastinal lymph nodes in cases of central tumors, Fluoro-Deoxy-Glucose (FDG)-avid hilar lymph nodes, low FDG uptake of the primary tumor and lymph nodes greater than or equal to 16 millimeters (mm) on CT scan (De Leyn et al, 2007). Most clinicians recommend pathologic evaluation of FDG-avid mediastinal lymph nodes, particularly for patients with non-bulky disease by CT. Endoscopic ultrasound imaging permits rapid and accurate identification and subsequent needle biopsy of mediastinal lymph nodes. Transesophageal endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) have been useful for lymph node staging of lung cancer (Vilmann et al, 2005). EBUSTBNA was compared with CT and PET for evaluating mediastinal and hilar nodes in patients with lung cancer and had a sensitivity and specificity of 92% and 100% respectively (Yasufuku et al, 2006). Transesophageal ultrasound-guided lymph node biopsy was compared to surgical mediastinoscopy and was shown to be highly sensitive for the detection of metastatic nodes, especially in the paratracheal and subcarinal regions (Larsen et al, 2005). Endoscopic ultrasound with FNA is being increasingly used in lung cancer staging, and may decrease the need for mediastinoscopy. Lung cancer causes brain metastases in 18-64% of patients during the course of their illness (Lassman, 2003). At presentation, 10% of all patients with NSCLC have central nervous system (CNS) involvement, with up to 20% of patients with adenocarcinoma having occult brain metastases (Newman, 1974; White et al, 1981). Brain magnetic resonance imaging (MRI) to evaluate for asymptomatic brain metastases is recommended in those patients with clinical stage III disease (Mayr et al, 1995), as outlined in the National Comprehensive Cancer Network (NCCN) guidelines (NCCN Clinical Practice Guidelines in Oncology, 2008).

II. Diagnosis and staging evaluation The diagnosis of NSCLC requires a tissue biopsy to identify the histopathologic subtype as well as proper staging of the tumor, according to tumor-node-metastasis (TNM) descriptions (Fossella et al, 2003). Tissue specimens may be obtained via bronchial washings, bronchial brushings, fine needle aspiration (FNA) biopsy, core needle biopsy, endobronchial biopsy, or transbronchial biopsy. Electromagnetic navigation bronchoscopy (ENB) can be used to biopsy peripheral lesions with a higher accuracy and lower risk of pneumothorax than CT-guided percutaneous FNA biopsy (Eberhardt et al, 2007). Occasionally, mediastinoscopy, parasternal mediastinotomy, video-assisted thoracoscopic surgery (VATS) or open thoracotomy is required to obtain tissue. CT scanning of the chest and upper abdomen is often used for the initial assessment of disease stage, but there are limitations in the evaluation of hilar and mediastinal lymph node involvement (Patterson et al, 1987). Node positivity is based on the size of the lymph nodes in CT scanning assessment. Because of this, small metastases that do not cause nodal enlargement will be missed. A 16% false negative result in metastases to lymph nodes that were found to be normal in size on CT scanning has been reported, with subsequent pathologic confirmation of N2 or N3 disease (Arita et al, 1995). Chest CT scans have a sensitivity and specificity of 69% and 71%, respectively, for identifying N2 disease (Dillemans et al, 1994). Another study found a sensitivity of 40-65% and specificity of 45-90% for CT scan detection of mediastinal lymph node involvement, depending on the clinical scenario (McLoud et al, 1992). An additional study reported a positive predictive value of 43% and negative predictive value of 92% for chest CT scans identifying mediastinal lymph node involvement (Seely et al, 1993). Currently, mediastinoscopy is the gold standard for the pre-operative evaluation of mediastinal lymph nodes, and is appropriate to confirm node positivity in those with a positive CT scan. Positron emission tomography (PET) scans detect tumor physiology rather than tumor anatomy, and may therefore be more sensitive than CT scans for clinical staging (Kerr et al, 1992). PET imaging scans have been useful in providing more accurate clinical staging and for evaluating the extent of disease. PET scans have been found to be 78% sensitive and 81% specific, with a negative predictive value of 89% when used to stage the

III. Treatment The treatment plan depends on whether the patient was diagnosed with stage III disease before or after surgical resection. Patients with clinical stage I or II NSCLC that undergo surgical resection, may then be reclassified as stage III based on metastases in mediastinal lymph nodes in the final pathology specimen. These patients are recommended to receive adjuvant 82

Cancer Therapy Vol 6, page 83 chemotherapy to reduce the risk of recurrence (see â&#x20AC;&#x153;Adjuvant Therapy in Resected Patientsâ&#x20AC;? discussed below). Patients who are confirmed to have mediastinal lymph node involvement prior to resection are recommended to have either preoperative therapy or definitive chemotherapy and radiation therapy (RT) without surgical resection.

randomized study of 60 patients with clinical stage IIIA NSCLC comparing perioperative chemotherapy (with cisplatin, cyclophosphamide and etoposide) vs. surgery alone (Roth et al, 1994). The perioperative chemotherapy group received 3 cycles of chemotherapy followed by surgical resection. Three additional cycles of postoperative chemotherapy were given to those patients who responded to the preoperative treatment. The patients in the chemotherapy group had a median survival of 64 months compared with 11 months in the surgery alone group (p<0.008). Two and three year survival rates in the perioperative chemotherapy group were 60% and 56% vs. 25% and 15% respectively in the surgery along group. Rosell and colleagues studied 60 patients with stage IIIA NSCLC randomly assigned to receive surgery alone vs. 3 cycles of chemotherapy with cisplatin, ifosfamide and mitomycin followed by surgery (Rosell et al, 1994). All patients had postoperative mediastinal RT. Median survival was 26 months in the chemotherapy group vs. 8 months in the surgery alone group (p<0.001). In the Spanish Lung Cancer Group Trial 9901, Garrido and colleagues reported survival results for 129 patients with stage IIIA (N2) and IIIB (T4 N0-1) NSCLC receiving induction chemotherapy with three cycles of cisplatin, gemcitabine and docetaxel followed by surgery in responding patients (Garrido et al, 2007). The patients had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0-1. In this study 50.7% were stage IIIA and 49.3% were stage IIIB. Seventy percent of the patients underwent surgery, with 68.9% of the patients undergoing complete resection. Median overall survival (OS) was 15.9 months, 3-year and 5-year survival rate was 36.8% and 21.1% respectively, with no significant survival differences between stage IIIA and IIIB patients. Median survival time was 48.5 months for 62 completely resected patients, 12.9 months for 13 incompletely resected patients and 16.8 months for 15 nonresected patients (p=0.005). Survival rates at 3 and 5 years were 60.1% and 41.4% for completely resected patients, 23.1% and 11.5% for incompletely resected patients, and 31.1% and 0% for nonresected patients.

A. Clinically evident but potentially resectable N2 disease For patients with non-bulky mediastinal lymph node involvement and T1-3 primary tumors, a combined modality treatment approach is recommended for management. Treatment options include preoperative induction therapy with chemotherapy or chemoradiotherapy; or definitive treatment with chemotherapy and radiotherapy given in a sequential fashion or concurrently. As per the NCCN guidelines, induction chemotherapy with or without RT could be considered for patients with T1-2 N2 disease. Definitive concurrent chemoradiotherapy could also be considered for this type of lesion, and is recommended for patients with T3 N2 disease. Additionally, the NCCN recommends the consideration of surgical resection if there has been an excellent response to the induction/definitive treatment (NCCN Clinical Practice Guidelines in Oncology, 2008).

1. Preoperative chemotherapy preoperative chemoradiotherapy


It is unknown whether neoadjuvant chemoradiotherapy is superior to neoadjuvant chemotherapy. This issue is being addressed by the Swiss Group for Clinical Cancer Research conducting a phase III randomized trial of preoperative cisplatin and docetaxel chemotherapy versus chemoradiotherapy with the same agents followed by surgical resection in patients with stage IIIA NSCLC. Radiation Therapy Oncology Group (RTOG) trial 0412 and Southwest Oncology Group (SWOG) trial S0332 is a phase III randomized trial in favorable prognosis patients with stage IIIA NSCLC comparing preoperative chemotherapy (with cisplatin and docetaxel) to preoperative concurrent chemoradiotherapy (50.4 Gy thoracic RT with the same chemotherapy agents). Both arms receive postoperative chemotherapy with docetaxel. This study closed prematurely due to slow accrual. Several small phase II and III studies substantiate the benefit of preoperative chemotherapy. Skarin and colleagues reported a median survival of 32 months with 1-year survival of 75% in 41 patients receiving neoadjuvant cyclophosphamide, adriamycin, and cisplatin chemotherapy followed by RT and subsequent resection (Skarin et al, 1989). Pass and colleagues reported a prospective, randomized trial in 27 patients with stage IIIA (N2) NSCLC comparing preoperative etoposide-platinum chemotherapy followed by surgery and postoperative chemotherapy with surgery followed by postoperative mediastinal RT (Pass et al, 1992). A trend toward increased survival was reported for the preoperative chemotherapy group (28.7 months vs. 15.6 months, p=0.095). Roth and colleagues performed a prospective,

2. Surgical resection vs. following neoadjuvant therapy


The role of surgical resection, in comparison to RT, following induction therapy in patients with stage IIIA disease is unclear. Two large randomized phase III trials have not found a survival benefit for surgery in comparison to RT following chemotherapy or for surgery following concurrent chemoradiotherapy. Chemoradiotherapy in this setting may be sufficient. The European Organization for Research and Treatment of Cancer (EORTC) study 08941 examined the issue of radiotherapy versus surgical resection following induction chemotherapy in patients with clinical stage IIIA-N2 NSCLC (van Meerbeeck et al, 2007). In this study, 579 patients received induction chemotherapy with three cycles of platinum-based chemotherapy. Following chemotherapy, 332 patients with at least stable disease were randomized to receive definitive RT [60-62.5 Gray (Gy) to the primary tumor and involved mediastinum with 83

Sullivan et al: Diagnosis and multimodality management of stage III non-small cell lung cancer 40-46 Gy to the uninvolved mediastinum] vs. surgical resection. Surgical resection did not improve OS or progression free survival (PFS) compared with RT. The Intergroup 0139 trial evaluated 429 patients with stage IIIA NSCLC that were treated initially with concurrent RT (45 Gy) plus chemotherapy with cisplatin (50 mg/m2 on days 1, 8, 29, and 35) plus etoposide (50 mg/m2 on days 1 to 5, and 29 to 33); 396 patients with at least stable disease were then randomized to surgical resection or a continuation of RT (to 61 Gy) (Albain et al, 2005). Two additional cycles of chemotherapy were given to both groups. A planned interim analysis, reported in abstract form, revealed that surgery was associated with a significant increase in five-year PFS (22 vs. 11 percent), but only a trend toward better OS (27 vs. 20 percent, p=0.10) due to increased post-operative deaths in patients in the surgery arm who required pneumonectomy. Over one-fourth of the patients who required pneumonectomy (n=54) died within 30 days post-operatively, but the survival curves crossed at approximately 18 months follow-up, and the hazard ratio for death was 0.87 for those randomized to surgery (p=0.24). Local relapse was significantly less in patients who underwent surgery (10% vs. 22%, p=0.002). Five-year survival for patients â&#x20AC;&#x153;downstagedâ&#x20AC;? to node negative was 41% as compared to 24% for those with residual nodal disease. Approximately 13%

suffered isolated brain metastases and the overall distant metastases rate was 40%. Table 1 summarizes the OS data comparing surgery with RT. These data suggest that surgery does not have an established role in the treatment of patients with clinical N2 disease. However, surgery may have a role after preoperative chemoradiation in patients who do not require pneumonectomy, particularly if there has been a response to the preoperative therapy. Additionally, there may be a role for surgery in patients with small volume N2 disease who receive preoperative chemotherapy or preoperative chemoradiotherapy. There is not a consensus from the NCCN in this regard (NCCN Clinical Practice Guidelines in Oncology, 2008). Re-staging should be considered following neoadjuvant treatment, but it is unclear if complete elimination of N2 disease is a requirement to benefit from surgery.

B. Unresectable Stage III NSCLC In patients with unresectable stage III NSCLC due to T4 primary tumors, bulky N2 disease, or N3 disease, chemotherapy and RT have been combined in various algorithms in an effort to treat both locoregional and micrometastatic disease. Table 2 summarizes the median and 5-year survival rates comparing RT alone, sequential chemoradiotherapy and concurrent chemoradiotherapy in select studies.

Table 1. OS at 5 years comparing surgery with radiotherapy after induction therapy. Study EORTC 08941 Intergroup 0139

Randomized patients 332 396

Surgical resection 15.7% 27%


P value


14% 20%

0.60 0.10

van Meerbeeck et al, 2007 Albain et al, 2005

Table 2. Outcomes of sequential and concurrent chemoradiotherapy compared with radiotherapy alone. *NA = Not Available.


Randomized patients

Median survival (mos) 10

5-year survival rate (%) 6

Sequential Chemoradiotherapy






Sequential Chemoradiotherapy








2 (3-year)


16 (3-year)


2 (3-year)


10 (3-year)

Treatment Radiotherapy

CALGB 8433








Cakir et al


Radiotherapy Sequential Chemoradiotherapy Radiotherapy Concurrent Chemoradiotherapy Radiotherapy Concurrent Chemoradiotherapy


P value






Reference Dillman et al, 1996 Sause et al, 2000 Le Chevalier et al, 1994 SchaakeKoning et al, 1992 Cakir, 2004 Dillman et al, 1996 Sause et al, 2000 Le Chevalier et al, 1994 SchaakeKoning et al, 1992

Cancer Therapy Vol 6, page 85 progression at nodes not clinically involved at the time of diagnosis. In this regard, potential improvements in the ability of radiation to eradicate known locoregional sites of disease through dose-escalation, and perhaps looking forward, improved chemosensitization and more effective systemic treatment for occult distant disease, will force a re-evaluation of the role of elective nodal irradiation. 3-D conformal therapy techniques allow the development of complex multiple field radiotherapy plans that decrease the amount of normal tissue exposed to high doses. The feasibility of delivering much higher doses of radiotherapy with modern techniques has been demonstrated in phase I and II studies. A dose escalation study reported by Haymen and colleagues involved 104 patients, most of whom had stage III disease (Hayman et al, 2001). The dose escalation schema depended on the amount of normal lung in the radiation field based on the 3-D conformal therapy plan. For patients with the greatest quintile of normal lung exposed to radiation, a maximum tolerated dose of 65.1 Gy at 2.1 Gy per day was reached; however, for the other four groups, continued escalation beyond 75.6 Gy and up to more than 100 Gy in the two quintiles with the lowest normal lung exposure was associated with acceptable toxicity. The RTOG conducted a phase I/II dose-escalation trial that incorporated 3D conformal therapy in 179 patients (Bradley et al, 2005). Concurrent chemotherapy was not given. Dose escalation was based on the volume of lung exposed to 20 Gy, which is near the tolerance dose of normal lung and has been shown to correlate with pneumonitis risk. Radiation was delivered at 2.15 Gy per daily fraction. Doses of 77.4 Gy were found to be tolerable, and in patients with low-volume disease, doses of 83.8 Gy were feasible. Local failure at two years was 38%. However, there have been no randomized phase III trials demonstrating that dose escalation improves survival. Better delineation of the target volume can be achieved with FDG-PET, and modern RT treatment planning systems can accommodate target definition using fused PET and CT images. The target volume when PET is used has been shown to change in a significant proportion of patients as compared with CT planning alone. The RT target volume can decrease (due to the ability of PET to differentiate atelectatic lung from tumor) or increase (due to FDG uptake at mediastinal lymph nodes that were not positive by CT size criteria alone) (Lavrenkov et al, 2005; Nestle et al, 2006). In the increasingly common situation today when elective nodal irradiation is avoided, more accurate definition of involved sites of disease with PET decreases the likelihood that tumor bearing nodes will not be encompassed in the target volume. The use of techniques that account for mobility of the tumor with respiration take on greater importance when 3D conformal treatment planning is utilized. By accounting for tumor motion on an individualized basis, smaller margins can be utilized, thereby decreasing exposure to normal lung tissue. One approach to this problem is the use of respiratory gating or breath hold technique. Gating the treatment with the respiratory cycle

1. Radiotherapy techniques and definitive radiotherapy Definitive RT is a treatment option for patients with stage III NSCLC who are not candidates for combined modality therapy due to poor PS or medical conditions. The RTOG 73-01 was designed to assess optimal dose of radiotherapy for patients with locally advanced disease (Perez et al, 1987). Local control and 2 year survival were better with 60 Gy in 6 weeks compared with lower doses. RTOG 83-11 tested whether improved results could be obtained with higher RT doses and twice-daily fractionation (hyperfractioned accelerated RT-HART), a technique that could potentially spare normal tissues from late toxicity (Cox et al, 1990). In this study, 840 patients were treated with 1.2 Gy twice daily with 4-6 hours between fractions to total doses ranging from 60-79.2 Gy. The best results were obtained with HART of 69.6 Gy at 1.2 Gy twice daily over 6! weeks. In patients with N2 disease, weight loss of 5% or less, and good performance status, 3 year survival of 20% was achieved with HART as compared with 7% survival for similar patients treated on earlier RTOG trials of standard fractionation. Saunders and colleagues conducted a phase III randomized trial comparing 60 Gy in 6 weeks and 54 Gy given at 1.5 Gy three times daily (six hour interfraction interval), seven days per week (continuous hyperfractionated accelerated radiotherapy-CHART) (Saunders et al, 1999). Three year survival was 20% with CHART vs. 13% with standard RT, at the expense of more moderate and severe acute dysphagia. This trial validated the concept of accelerated hyperfractionation as safe and appeared to be more effective than standard fractionation when RT is used as single modality therapy, however this was the only trial that showed improved results and has not been confirmed. Efforts have also been made to increase the efficacy of RT through dose-escalation, which is made possible by decreasing the volume of normal tissue in the radiation field. The use of 3-dimensional (3D)-conformal techniques, which are now standard, has made possible a decrease in normal tissues receiving high doses. It has also raised the question of whether reductions in radiation target volume by avoidance of elective nodal irradiation may be appropriate. Since most locoregional failures occur at the sites of initial gross disease, exclusion of elective nodal irradiation (so-called involved-field radiotherapy) has become increasingly utilized. A recent study by Rosenszweig and colleagues found only 6.1 % isolated failure at nodes that were outside the high dose radiation volume in 524 patients treated with 3D conformal radiotherapy (Rosenzweig et al, 2007). This is, however, at odds with surgical data suggesting high rates of occult involvement of mediastinal nodes even with small primary tumors. A therapeutic benefit from lower dose incidental radiation of nearby nodal stations when multiple-field 3D dose-escalated conformal techniques are used in conjunction with chemotherapy may effectively treat subclinical nodal disease. However, competing risks of progression at distant metastatic sites and death from both local recurrence and distant metastases are likely to be the primary reasons few patients are reported to have 85

Sullivan et al: Diagnosis and multimodality management of stage III non-small cell lung cancer or treating with breath hold can help to reduce the planning target volume or avoid marginal miss. Another method incorporates so-called 4D imaging. Use of rapid spiral CT scanning and acquisition of multiple images during breathing allows for better definition of the target volume, so that changes in the shape and location of the tumor during the breathing cycle can be taken into account in radiation delivery. With this technique temporal changes in tumor position and anatomy are incorporated into the treatment planning process. Radiotherapy delivery that adjusts in real-time to changes in tumor and normal anatomy holds further promise to decrease the necessary tumor margin and exposure to uninvolved lung. Image guided radiotherapy may also improve the therapeutic ratio. Accurate patient set-up with the use of radiopaque markers placed in the tumor by ENB or VATS, or use of daily CT scan imaging can essentially eliminate any additional margin that might otherwise be needed for daily patient set-up variability. Adaptive radiotherapy can also be employed, in which the radiation treatment plan is modified to account for dimunition in the size of the primary tumor or mediastinal lymph nodes during the course of treatment. Use of intensity modulated radiotherapy (IMRT) is also being studied. With this technique, the intensity of the beam is varied across the two dimensions of each field, and delivery is accomplished using multiple fields at different angles. The advantage of IMRT is that high dose gradients between target and normal tissue can be generated. The primary disadvantage is that a greater volume of normal tissue gets low doses. Since the normal lung has low tolerance to even small doses, this technique has not gained general acceptance in the treatment of locally advanced non-small cell carcinoma. There are additional unsolved technological problems of moving targets and the necessity of using time-consuming respiratory gating.

other two arms of the study. The HART arm, although better, was not statistically superior in survival compared with the standard RT arm. Two year survival was 32 % with induction chemotherapy, 24 % for HART, and 19 % for standard RT. Three year survival was 17 %, 14 %, and 11 %, respectively. Five year survival was 8%, 6%, and 5%, respectively. A French randomized trial of 353 patients with stage III disease compared 3 cycles of induction chemotherapy with vinblastine-cisplatin-cyclophosphamide and 65 Gy thoracic RT followed by 3 more cycles of the same chemotherapy vs. RT alone to the same dose (Le Chevalier et al, 1994). Three year survival was improved with the use of induction and consolidation chemotherapy (12 % vs. 4 %, p=0.02), as was 5 year survival (6% vs. 3 %, p<0.02). This is the only study that employed systematic post-treatment bronchoscopy to evaluate locoregional control. Only 16% of patients were locally controlled at one year post-treatment, with no difference between arms, suggesting that the survival benefit afforded by chemotherapy was mainly due to its impact on occult distant metastases. A study from the EORTC reported a 3 year survival of 16 % for patients receiving chemoradiotherapy with daily concurrent cisplatin, 13 % with weekly concurrent cisplatin, and only 2 % for RT alone (p=0.009 for comparison of daily concurrent cisplatin vs. RT alone) (Schaake-Koning et al, 1992). A more recent study of 176 patients revealed improvement in 3 year survival from 2% to 10% in patients who received daily cisplatin on weeks 2 and 6 of RT (total dose 64 Gy) (Cakir, 2004). All of these trials used standard radiation doses of 55 to 60 Gy and were performed prior to the modern era of 3D conformal therapy, which allows safe delivery of higher doses. Nonetheless, these trials, summarized in Table 2, established the use of induction chemotherapy followed by RT, or concurrent chemoradiotherapy, as superior to RT alone.

2. Sequential and conurrent chemotherapy and RT vs. RT alone

3. Concurrent chemotherapy and RT vs. sequential chemotherapy and RT

Clinical trials have established the superiority of sequential therapy compared with RT alone. In Cancer and Leukemia Group B (CALGB) trial 8433, cisplatinvinblastine for two cycles followed by thoracic radiotherapy to a dose of 60 Gy in 6 weeks was compared with the same radiotherapy alone in 155 randomized patients (Dillman et al, 1996). Induction chemotherapy improved median survival (13.8 months vs. 9.7 months, p=0.007), 3 year survival (23 % vs. 10 %, p=0.01) and 7 year survival (13 % vs. 6 %, p=0.01). This was the first study to demonstrate a survival benefit with the use of induction chemotherapy followed by radiotherapy for patients with good PS (ECOG 0-1) and weight loss of less than 5%. These results were confirmed in an intergroup study of 452 patients with stage III NSCLC (also with ECOG PS 0-1 and weight loss of less than 5%) randomized to the positive arm of the CALGB trial (induction vinblastine-cisplatin followed by RT) vs. the best arm of RTOG 83-11 (HART alone to 69.6 Gy) vs. standard fractionation RT of 60 Gy in six weeks (Sause et al, 2000). OS was statistically superior for the patients receiving induction chemotherapy followed by RT vs. the

Concurrent chemoradiotherapy has become the standard treatment for most unresectable patients with stage III disease based on randomized trials that have established the superiority of this approach compared with sequential therapy in patients with good PS (Table 3). To illustrate this, a Japanese study randomized 320 patients with unresectable stage III NSCLC to cisplatin, vindesine, and mitomycin concurrent with split course RT (56 Gy total dose, 10 day break) versus the same chemotherapy followed by continuous course RT to the same total dose (Furuse et al, 1999). Over 90% of the patients had an ECOG PS of 0-1. Concurrent therapy was associated with significantly better response rate (RR) of 84 vs. 66 percent, median survival (MS) of 17 vs. 13 months, and two- and five-year survival of 35 vs. 17 percent and 16 vs. 9 percent. Patients who received concurrent treatment had more myelosuppression, however there was no difference in esophagitis rates, perhaps because the concurrent radiation was given split course. Zatloukal and colleagues performed a randomized phase II trial involving 102 86

Cancer Therapy Vol 6, page 87 patients to compare concurrent or sequential cisplatin and vinorelbine with RT to a dose of 60 Gy (Zatloukal et al, 2004). Improved median survival of 16.6 months vs. 12.9 months was found with concurrent administration (p=0.02). RTOG 9410 is the largest trial assessing the value of concurrent vs. sequential therapy (Curran et al, 2003). In this trial, 610 patients with unresected stage III disease were randomized to three arms: the positive arm of the CALBG trial reported by Dillman and colleagues (induction cisplatin-vinblastine for two cycles followed by RT to 63Gy) vs. the same chemotherapy given concurrently vs. a third arm of oral etoposide and weekly cisplatin given concurrently with 69.6 Gy hyperfractionated RT (HART). Four-year survival was significantly improved with concurrent cisplatinvinblastine and standard fractionated RT vs. sequential therapy and standard fractionated RT (21% vs. 12%). The rates of acute grade 3-4 non-hematologic toxicities were higher with concurrent than sequential therapy, but late toxicities were similar (1-3% with esophagitis and 11-13% with pneumonitis). Table 4 summarizes the acute toxicities in this trial comparing concurrent vs. sequential therapy. Huber and colleagues compared concurrent chemoradiotherapy (with weekly paclitaxel) with RT alone following induction chemotherapy with carboplatin and paclitaxel in 300 patients with stage III NSCLC (Huber et al, 2006). Median time to progression (TTP) favored the chemoradiotherapy group (11.5 months vs. 6.3 months), although there was not a survival advantage observed in this trial. For patients with a compromised PS, the greater acute toxicities with concurrent therapy make sequential therapy a better choice.

4. Induction chemotherapy prior to definitive chemoradiotherapy The CALGB examined the issue of adding platinumbased induction chemotherapy prior to concurrent chemoradiotherapy in a phase III trial of 366 patients (Vokes et al, 2007). Immediate concurrent chemoradiotherapy with carboplatin AUC of 2 and paclitaxel 50 mg/m2 given weekly during 66 Gy of chest RT was compared with induction chemotherapy with two cycles of carboplatin AUC 6 and paclitaxel 200 mg/m2 administered every 21 days followed by identical chemoradiotherapy. The addition of induction chemotherapy did not provide a survival benefit over concurrent therapy alone and was associated with an increased toxicity. This is the only prospective study comparing induction chemotherapy prior to concurrent chemoradiotherapy with concurrent chemoradiotherapy alone. However, the survival times were inferior to previously reported values for patients with stage III disease treated with concomitant chemoradiotherapy. The only other study addressing the role of induction chemotherapy was by Huang and colleagues who published a retrospective outcome analysis of 265 patients treated with definitive chemoradiotherapy for unresectable, locally advanced NSCLC, of whom 127 patients received induction chemotherapy (Huang et al, 2006). The induction chemotherapy group had an improved median OS of 1.9 years vs. 1.4 years for the group that did not receive induction chemotherapy. Five year survival rate significantly favored the induction chemotherapy group (25% vs. 12%; p<0.001). A subgroup analysis revealed that the survival benefit was isolated to those with adenocarcinoma or large cell carcinoma, but not squamous cell carcinoma. Thus, there may be a role for induction chemotherapy prior to chemoradiotherapy, but this is as yet undefined.

Table 3. Outcomes of "equential vs. concurrent chemoradiotherapy.

Study West Japan Lung Cancer Group RTOG 9410

Randomized patients

Chemoradiotherapy treatment Concurrent

Median survival (mos)

Survival rate (%)


16 (5-year)





9 (5-year)

Concurrent Sequential

17 14.6

21 (4-year) 12 (4-year)

P value



Furuse et al, 1999


Curran et al, 2003

Table 4. Grade 3 or greater acute toxicities comparing sequential chemoradiotherapy with concurrent chemoradiotherapy in RTOG 9410 (Curran et al, 2003). Toxicity (grade) Esophagitis (3-4) Pneumonitis (3-5) Neutropenia (4-5) Thrombocytopenia (4-5) Any Grade 5

Sequential chemoradiotherapy (%) 4 7 56 2 3


Concurrent chemoradiotherapy (%) 25-47 3-4 48-58 4-8 2-3

Sullivan et al: Diagnosis and multimodality management of stage III non-small cell lung cancer versus observation in 1867 patients of whom 39% had stage III disease (Arriagada et al, 2004). With a median duration of follow-up of 56 months, the chemotherapy group had a significantly improved disease free survival (DFS) of 39.4% vs. 34.3% and OS of 44.5% vs. 40.4% compared with the observation group. The Adjuvant Navelbine International Trialist Association (ANITA) looked at adjuvant chemotherapy with cisplatin and vinorelbine versus observation in 799 patients with stage IB, II, or IIIA NSCLC, of whom 39% had stage IIIA disease (Douillard et al, 2006). RT was optional and was administered to 24% of patients in the chemotherapy group and 33% in the observation group. The median survival was significantly increased with chemotherapy, at a median follow-up of 76 months (66 vs. 44 months with observation). The absolute OS benefit with adjuvant chemotherapy was 8.6 percent at five years and 8.4 percent at seven years. The survival benefit was limited to patients with pathologic stage II or IIIA disease. Pathologic N2 disease was present in 27% of patients. Five year survival for the N2 subset was 40% (95% CI 3049%) with chemotherapy vs. 19% (95% CI 11-27%) without chemotherapy. Of patients with N2 disease randomized to chemotherapy and who also received RT (n=73) 5 year survival was 47% as compared to 34% in those patients who did not receive RT (n=152). Of patients with N2 disease randomized to observation who received RT (n=128), 5 year survival was 21% as compared to 17% in those patients who did not receive any adjuvant therapy. Adjuvant chemoradiotherapy has not been shown to be beneficial over surgery alone in a randomized study of patients with completely resected stage II and stage IIIA NSCLC, suggesting a possible detriment from the RT component (Keller et al, 2000). However, if numerous mediastinal lymph nodes are involved, patients may benefit from adjuvant RT to the mediastinum. In a population-based cohort study, Lally and colleagues found in a subset analysis that post-operative RT was associated with a significant improved survival for patients with N2 disease (Lally et al, 2006). For patients with N0 or N1 disease, post-operative RT was associated with a significant decrease in survival. An American intergroup trial and an EORTC trial are presently underway to reevaluate the role of radiotherapy for patients with N2 disease using conformal techniques. The NCCN recommends adjuvant chemotherapy, for patients with stage IIIA disease after resection, with a cisplatin-based doublet but there is disagreement among panel members regarding the role of RT (NCCN Clinical Practice Guidelines in Oncology, 2008). A recent Cancer Care Ontario (CCO) and ASCO guideline recommends adjuvant cisplatin-based chemotherapy for patients with stage IIIA disease (Pisters et al, 2007). Due to the lack of prospective, randomized clinical trial data evaluating its efficacy, adjuvant RT was not recommended for patients with stage IIIA disease (Pisters et al, 2007).

5. Consolidation or maintenance chemotherapy following chemoradiotherapy Consolidation chemotherapy was incorporated in a treatment regimen for patients with unresectable stage IIIA/B disease by the SWOG, but recent studies do not support a survival benefit. Two phase II nonrandomized SWOG studies suggested that treating patients with three cycles of docetaxel consolidation therapy, following completion of definitive treatment with cisplatin and etoposide with concurrent thoracic RT, might improve survival (Albain et al, 2002; Gandara et al, 2003). Despite a lack of randomized studies, consolidation docetaxel has been used by 50% of surveyed oncologists (Green, 2007). Preliminary results from a study by Carter and colleagues, found a worse survival with the use of carboplatin, paclitaxel, and radiotherapy followed by consolidative weekly taxol vs. no consolidation therapy, primarily due to toxicity associated with consolidative therapy (Carter et al, 2006). Recently, the Hoosier Oncology Group randomized 203 patients who did not progress on cisplatin-etoposide and radiotherapy to 3 cycles of consolidation docetaxel vs. no consolidation. There was no difference between arms in survival (3 year survival 28%), and docetaxel consolidation was associated with increased toxicity (Bedano et al, 2007; Hanna et al, 2007). Kelly and colleagues evaluated 243 patients in 2007 with Stage III NSCLC treated with chemoradiotherapy with cisplatin and etoposide followed by docetaxel consolidation therapy, who were then randomized to receive Gefitinib maintenance therapy or placebo. Gefitinib maintenance therapy was associated with an inferior survival compared with placebo.

C. Adjuvant therapy in resected patients Patients who are thought to have stage I or II disease preoperatively, who undergo surgical resection, may be discovered to have stage III disease in the final pathology specimen when mediastinal lymph node involvement is discovered. In one study, 13% of patients with clinical stage IA disease, who had a pre-operative high resolution CT scan without enlargement of mediastinal lymph nodes, were reclassified as stage IIIA disease after resection (Yoshino et al, 2006). Patients with stage III NSCLC that have been completely resected are at high risk of local recurrence and the development of distant metastatic disease. Improved survival with adjuvant chemotherapy with platinum-based drug regimens was confirmed in a meta-analysis that was presented at the American Society of Clinical Oncology (ASCO) annual meeting in June 2006 (Pignon et al, 2006) This meta-analysis pooled data from 5 large clinical trials, all of which used platinumbased chemotherapy and included 4584 patients. Three of the included trials allowed the use of thoracic RT at the discretion of the treating physician. Adjuvant chemotherapy was associated with an absolute increase in survival of 4.2 percent (hazard ratio [HR] 0.89, 95% CI 0.82-0.96) at a median follow-up of 5.1 years. The data did not sort out the best agent to combine with cisplatin, and differing doses of cisplatin were used, depending on the trial. The International Adjuvant Lung Cancer Trial (IALT) examined cisplatin-based adjuvant chemotherapy

D. Special situations/considerations 1. Superior sulcus tumors Patients with superior sulcus (Pancoast) tumors with hilar lymph node involvement (T3, N1) are uncommon. 88

Cancer Therapy Vol 6, page 89 Improved outcomes with chemoradiotherapy in the treatment of non-Pancoast, locally advanced, stage III NSCLC has led to its use in patients with superior sulcus tumors. Comparison to historical controls indicate that these patients should be treated with concurrent chemoradiotherapy, followed by surgical resection if there has not been progression of disease, and this is the approach recommended by the NCCN (NCCN Clinical Practice Guidelines in Oncology, 2008). A cooperative intergroup study evaluated concurrent chemotherapy (2 cycles of cisplatin and etoposide) with thoracic RT (45Gy in 25 fractions) followed by resection 3-5 weeks later (in those without progression of disease) and two additional cycles of postoperative chemotherapy in 111 patients with pathologically proven T3-4, N0-1 NSCLC presenting in the superior sulcus (Rusch et al, 2001, 2007). The percent of patients with N1 disease was not provided in the report, but 28% of patients had T4 tumors. Mature results of the study revealed a five year survival of 44% for all patients with no difference between T3 and T4 tumors. Japan Clinical Oncology Group Trial 9806 was a phase II study examining the efficacy and safety of preoperative chemoradiotherapy with two cycles of cisplatin, mitomycin, vindesine and 45Gy RT to the primary tumor and ipsilateral supraclavicular nodes in 76 patients with superior sulcus NSCLC (Kunitoh et al, 2008). Seventy four percent of the patients had T3 tumors and 26% had T4 disease. All of the T4 cases involved the spine. Induction therapy was completed in 95% of the patients and 76% underwent surgical resection. Complete resection was accomplished in 68% of the patients and 12 patients had a pathologic complete response. DFS and OS at 5 years was 45% and 56% respectively.

of tumors that respond to the neoadjuvant treatment (Albain et al, 1995). The NCCN is in agreement with this approach (NCCN Clinical Practice Guidelines in Oncology, 2008). The Spanish Lung Cancer Group Trial 9901 (described above) included 34 patients with tumor infiltration of the great vessels, 2 patients with tracheal infiltration, 6 patients with invasion of the carina, 2 patients with infiltration of the esophagus, 4 patients with invasion of the heart and 22 patients with mediastinal invasion (Garrido et al, 2007). The patients received induction chemotherapy followed by surgery in responding patients. The survival was similar to that of stage IIIA (N2) patients (median survival of 15.6 and 16.8 months for patients with stage IIIA and IIIB disease, respectively).

4. Targeted therapy The tyrosine kinase signaling pathway, including the epidermal growth factor receptor (EGFR) is known to be altered in NSCLC. EGFR is overexpressed in a variety of tumors including NSCLC. Erlotinib, a small molecule EGFR inhibitor, is approved for use as second-line treatment of metastatic NSCLC (Shepherd et al, 2005). Additionally, the anti-vascular endothelial growth factor inhibitor bevacizumab has improved survival in first-line treatment of metastatic NSCLC when combined with carboplatin and paclitaxel (Sandler et al, 2006). The optimal use of targeted therapies in the neoadjuvant, adjuvant or definitive treatment of stage III NSCLC remains to be defined.

5. Immunotherapy A goal of cancer research has been to incite an immune response against cancer cells. Tumor antigens can stimulate an immune response and the use of tumor vaccines may have a role in resectable and unresectable NSCLC in conjuction with conventional therapies. Tumor vaccines have been used to target known tumor-specific antigens and, in an autologous fashion, to target unique antigens derived from a patientâ&#x20AC;&#x2122;s own tumor. Dendritic cells are antigen-presenting cells under investigation in tumor vaccine development and have been shown to have therapeutic potential (Hirschowitz et al, 2004, Ishikawa et al, 2005). Encouraging results in NSCLC patients immunized with an autologous tumor cell vaccine expressing granulocyte macrophage colony stimulating factor (GM-CSF) support the rationale for further investigation (Nemunaitis et al, 2004). Muc 1 is a cell surface glycoprotein overexpressed in NSCLC and has been administered as a liposomal vaccine to stimulate an immune response. A phase II trial in patients with advanced stage NSCLC showed a trend toward improved survival with use of the vaccine compared with best supportive care (Butts et al, 2005). A phase III trial is underway. Further investigation of immunotherapy in NSCLC is warranted.

2. Satellite nodules Patients with T4N0-1 lesions, based upon the presence of satellite nodules within the same lobe as the primary tumor, may be candidates for surgical resection as the primary method of treatment. To illustrate this, Port and colleagues conducted a retrospective review of 53 patients with resected lung cancer containing intralobar satellite lesions and reported a 5-year OS rate of 47% for all patients in the study and an OS of 58% for those with N0 disease (Port et al, 2007). Treatment modalities other than surgery were not reported in this study. Osaki and colleagues conducted a retrospective review of 76 patients with surgically resected T4 NSCLC lesions, 36 of whom had satellite nodules. A 5-year OS rate of 26.7% was reported for the 36 patients, fifteen of whom received adjuvant chemotherapy and/or RT (Osaki et al, 2003). For resectable T4, N0-1 tumors due to satellite lesions, the NCCN recommends surgical resection followed by adjuvant chemotherapy (NCCN Clinical Practice Guidelines in Oncology, 2008).

3. Mediastinal invasion Patients with T4 tumors due to invasion of the mediastinum are generally unresectable and the standard treatment is chemoradiotherapy. Anecdotal experience suggests that neoadjuvant treatment with chemotherapy or chemoradiotherapy may be beneficial, enabling resection

6. Prophylactic cranial irradiation (PCI) Patients with stage III NSCLC are at high risk of developing CNS metastases, suggesting a role for PCI. A SWOG retrospective study found that 26% of patients 89

Sullivan et al: Diagnosis and multimodality management of stage III non-small cell lung cancer 0139 (RTOG 9309). (Abst 7014). J Clin Oncol 23, (Supp No. 16S). Arita T, Kuramitsu T, Kawamura M, et al (1995) Bronchogenic carcinoma: incidence of metastases to normal sized lymph nodes. Thorax 50, 1267-1269. Arriagada R, Bergman B, Dunant A, Le Chevalier T, Pignon JP, Vansteenkiste J; International Adjuvant Lung Cancer Trial Collaborative Group. (2004) Cisplatin-based adjuvant chemotherapy in patients with completely resected nonsmall-cell lung cancer. N Engl J Med 350, 351-60. Bedano PM, Neubauer M, Ansari R, Govindan R, Einhorn L, Bruetman D, White A, Breen T, Juliar B, Hanna N (2007) Phase III study of cisplatin (P) plus etoposide (E) with concurrent chest radiation (XRT) followed by docetaxel (D) vs. observation in patients (pts) with stage III non-small cell lung cancer (NSCLC): An interim toxicity analysis of consolidation therapy (Abst 7043). J Clin Oncol 24, (Supp No. 18S). 24, 374s. Bradley J, Graham MV, Winter K, Purdy JA, Komaki R, Roa WH, Ryu JK, Bosch W, Emami B. (2005) Toxicity and outcome results of RTOG 9311: a phase I-II dose-escalation study using three-dimensional conformal radiotherapy in patients with inoperable non-small-cell lung carcinoma. Int J Radiat Oncol Biol Phys 61, 318-328. Butts C, Murray N, Maksymiuk A, Goss G, Marshall E, Soulières D, Cormier Y, Ellis P, Price A, Sawhney R, Davis M, Mansi J, Smith C, Vergidis D, Ellis P, MacNeil M, Palmer M (2005) Randomized Phase IIB Trial of BLP25 Liposome Vaccine in Stage IIIB and IV Non-Small-Cell Lung Cancer. J Clin Oncol 23, 6674-81. Cakir S, Egehan I (2004) A randomised clinical trial of radiotherapy plus cisplatin versus radiotherapy alone in stage III non-small cell lung cancer. Lung Cancer 43, 309-316. Carter D, Keller A, Tolley R, Johnson DB, Hathorn J, Mundis RJ, O'Rourke MA, legbodu D, Asmar L (2006) A randomized phase III trial of combined paclitaxel, carboplatin and radiation therapy followed by either weekly paclitaxel or observation in patients with stage III non small cell lung cancer (Abst 7076). J Clin Oncol 22, (Supp No. 14S). 22, 635s. Chin R, Ward R, Keyes JW, Choplin RH, Reed JC, Wallenhaupt S, Hudspeth AS, Haponik EF (1995) Mediastinal staging of non-small-cell llung cancer with positron emission tomography. Am J Respir Crit Care Med 152, 2090-2096. Cox JD, Azarnia N, Byhardt RW, Shin KH, Emami B, Pajak TF (1990) A randomized phase I/II trial of hyperfractionated radiation therapy with total doses of 60.0 Gy to 79.2 Gy: possible survival benefit with greater than or equal to 69.6 Gy in favorable patients with Radiation Therapy Oncology Group stage III non-small-cell lung carcinoma: report of Radiation Therapy Oncology Group 83-11. J Clin Oncol 8, 1543-1555. Curran WJ, Scott CB, Langer CJ, Komaki R, Lee JS, Hauser S, Movsas B, Wasserman T, Sause W, Cox JD (2003) Longterm benefit is observed in a phase III comparison of sequential vs. concurrent chemo-radiation for patients with unresected stage III non small cell lung cancer: RTOG 9410 (Abst 2499). Proc Am Soc Clin Oncol 22, 621. De Leyn P, Lardinois D, Van Schil PE, Rami-Porta R, Passlick B, Zielinski M, Waller DA, Lerut T, Weder W (2007) ESTS Guidelines for Preoperative Lymph Node Staging for NonSmall Cell Lung Cancer. Eur J Cardiothoracic Surg. 32, 18. Dillemans B, Deneffe G, Verschakelen J, Decramer M (1994) Value of computed tomography and mediastinoscopy in preoperative evaluation of mediastinal nodes in non small cell lung cancer. A study of 569 patients. Eur J Cardiothorac Surg 8, 37-42.

with stage III NSCLC subsequently develop brain metastases and were more common in patients under the age of 60 and with non-squamous histologies (Gaspar et al, 2005). A German randomized trial with 112 patients examined the role of PCI following a trimodality treatment protocol of chemotherapy, chemoradiotherapy and surgery in patients with operable stage IIIA NSCLC (PĂśttgen et al, 2007). PCI significantly reduced the probability of brain metastases as the first site of failure (7.8% vs. 34.7% at 5 years, p=0.02) and reduced the overall brain relapse rate (9.1% vs. 27.2% at 5 years, p=0.04). There was no difference in 5-year OS (18%). Neurocognitive late effects in 11 long-term survivors were not significantly different between those patients treated with or without PCI. The RTOG is presently conducting a study of patients with stage III non-small cell carcinoma who do not have progressive disease to evaluate the potential benefit of PCI. Patients will be randomized to 30Gy in 15 fractions vs. observation after definitive local therapy. The primary endpoint is survival and secondary endpoints are the rate of CNS metastasis, quality of life, and neurocognitive effects.

IV. Conclusions Patients with stage III NSCLC encompass a heterogeneous group whose optimal management approach is dependent on multiple factors and remains to be defined for some patients. A combined modality approach with concurrent chemoradiotherapy with a platinum-based regimen has become the preferred treatment for the majority of patients with stage III disease detected clinically. The role of surgery in patients with pre-operatively detected but non-bulky mediastinal lymph node involvement and T1-3 primary tumors who respond to chemotherapy or chemoradiotherapy remains unclear, but seems reasonable in those patients with down-staged nodal disease after induction therapy who do not require pneumonectomy. Adjuvant platinum-based chemotherapy should be offered to those patients found to have stage III disease at the time of surgery. There is vast room for improving outcomes for patients with this disease. A multidisciplinary approach to managing this diverse group of patients is recommended.

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Sullivan et al: Diagnosis and multimodality management of stage III non-small cell lung cancer


Cancer Therapy Vol 6, page 95 Cancer Therapy Vol 6, 95-102, 2008

Targeted agents in Non Small Cell Lung Cancer Review Article

Elizabeth Blanchard Caritas St Elizabethâ&#x20AC;&#x2122;s Medical Center, Division of Hematology/Oncology and Tufts University School of Medicine

__________________________________________________________________________________ *Correspondence: Elizabeth Blanchard, MD, Caritas St Elizabethâ&#x20AC;&#x2122;s Medical Center, Division of Hematology/Oncology, Assistant Professor of Medicine, Tufts University School of Medicine, 736 Cambridge Street, Boston, Massachusetts 02135, USA; e-mail: Key words: Angiogenesis, angiogenesis inhibitors, epidermal growth factor receptor, Clinical performance, tyrosine kinase inhibitors, Predictors of response Abbreviations: chronic myelogenous leukemia, (CML); epidermal growth factor receptor, (EGFR); Non small cell lung cancer, (NSCLC); overall response rate, (ORR); time to progression, (TTP); tyrosine kinase inhibitors, (TKIs); vascular endothelial growth factor receptor, (VEGFR); vascular endothelial growth factor, (VEGF) Received: 9 July 2007; Revised: 27 October 2007 Accepted: 7 November 2007; electronically published: February 2008

Summary Non small cell lung cancer (NSCLC) is a common and often devastating cancer. For patients with disease that cannot be managed surgically, particularly those with metastatic disease, options have traditionally been limited to chemotherapy, which provides a modest survival benefit. Recently, there have been several new agents that appear to improve survival without introducing unmanageable toxicity. Bevacizumab is an anti-angiogenesis agent that has been shown to improve survival in selected patients with metastatic NSCLC when used with a traditional chemotherapy doublet. The small molecule tyrosine kinase inhibitors (TKIs) have been fairly extensively studied over the past several years in several different settings and erlotinib has emerged as a beneficial drug in second line treatment of metastatic NSCLC. Much work remains, however. Clinical characteristics and tumor specific markers are currently under study in an effort to understand which patients might derive the most benefit from these agents. Research efforts are focusing on tumor biology and genetics to better characterize tumors, which will lead to better targeted agents and more effective, less toxic therapy for this often difficult disease. This review discusses the evidence for the use of targeted agents in non small cell lung cancer in various settings as well as the molecular basis for the use of such therapies and the challenges faced in determining the optimal targeted therapy for individual patients and tumor types.

with resulting treatment regimens that are more effective and, often, less toxic. This overview will examine the mechanisms and data behind the use of targeted therapy in NSCLC including small molecule tyrosine kinase inhibitors (TKIs) as well as the use of the vascular endothelial growth factor (VEGF) inhibitor bevacizumab in first line therapy for non small cell lung cancer with standard chemotherapy.

I. Introduction Lung cancer is the leading cause of cancer related mortality worldwide with approximately 1.3 million deaths per year (World Health Organization). Non small cell lung cancer (NSCLC) is the most common type of lung cancer diagnosed and includes the histologies of adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Often, lung cancer is diagnosed at an advanced stage when treatment options have traditionally been limited and life expectancy poor. Chemotherapy has been the mainstay of treatment for metastatic lung cancer, while chemotherapy and radiation are often employed for locally advanced lung cancer not amenable to surgical resection. Chemotherapy can offer a modest survival advantage in patients of good performance status with metastatic disease, though the therapy itself has many side effects (Non-small Cell Lung Cancer Collaborative Group, 1995). The past several years have increasingly seen the use of targeted agents in advanced non small cell lung cancer

II. Angiogenesis Angiogenesis is a complicated and multifaceted process that plays an integral and crucial role in tumor growth of many cell types, including non-small-cell lung cancer. It is this process of new blood vessel growth, which in turn facilitates passage of oxygen, nutrients, growth factors and hormones allowing tumor cells to survive and disseminate (Folkman 1989; Fidler and Ellis, 1994; Hicklin and Ellis, 2005). Indeed, vascularization of a tumor marks the ability of the tumor to grow and 95

Blanchard et al: Targeted agents in Non Small Cell Lung Cancer metastasize (Fidler and Ellis, 1994). Increased markers of angiogenesis such as tumor expression of VEGF and vascular endothelial growth factor receptor (VEGFR) as well as tumor microvessel density are associated with diminished survival. In one series of patients with lung cancer, patients whose tumors expressed VEGF and VEGFR had shortened survival times compared with patients whose tumors did not express either VEGF or VEGFR (Seto et al, 2006). Another series (Fontanini et al, 1997) looked at tumor microvessel density in patients with NSCLC and found that the highest levels of microvessel density were associated with diminished survival and that progressively increasing microvessel counts correlated with increasingly poor survival. Microvessel counts were noted in this study to be an independent poor prognostic sign in multivariate analyses as well.

cisplatin/gemcitabine, bevacizumab 15mg/kg plus cisplatin/gemcitabine and cisplatin/gemcitabine plus placebo. Notably, bevacizumab was continued after chemotherapy until disease progression in the experimental arm and crossover to bevacizumab was not allowed for patients in the placebo arm. Progression free survival was significantly improved in the group randomized to cisplatin/gemcitabine plus bevacizumab 7.5mg/kg with a hazard ratio of 0.75 (95% CI 0.62, 0.91; p value of 0.0026). Interestingly, PFS was also increased in the group receiving bevacizumab 15mg/kg compared with placebo in similar proportions to the 7.5mg/kg dose. Median survival was 6.7 months in the group receiving bevacizumab 7.5mg/kg plus chemotherapy, 6.5 months in the group receiving bevacizumab 15mg/kg plus chemotherapy and 6.1 months in the group receiving chemotherapy alone. Overall survival data are awaited. It does raise the question, however, as to what the optimal dose of bevacizumab is in this setting.

III. Use of angiogenesis inhibitors Observations such as these have led to the use of angiogenesis inhibitors for clinical use. Though many are under investigation, bevacizumab is the most commonly used in clinical practice. Bevacizumab is a recombinant IgG antibody of approximately 149 kilodaltons with human and murine components that binds to VEGF and inhibits its contact with VEGF receptors, thus neutralizing the function of VEGF, including its role in angiogenesis (Package insert, bevacizumab; Margolin et al, 2001). In a phase II study of patients with advanced lung cancer, the addition of bevacizumab to a standard chemotherapy regimen of carboplatin and paclitaxel was associated with increased time to progression and better response rates compared with carboplatin and paclitaxel alone (Johnson et al, 2004). Of note, six patients on the arm containing bevacizumab had major bleeding, including four fatal events. Bleeding episodes were found to be associated with squamous histology, central tumors located near major blood vessels and cavitary lesions. Minor mucocutaneous bleeding was also more common in the arm that included bevacizumab. These results led to the large randomized phase III trial of similar design, though patients with squamous histology, clinically significant hemoptysis or brain metastases were excluded from the trial (Sandler et al, 2006). This pivotal study showed increased response rates and increased time to progression with the addition of bevacizumab to a standard chemotherapy regimen of paclitaxel and carboplatin as well as improved survival in the group treated with all three agents. Notably, though, there was more clinically significant bleeding and more treatment related deaths in the bevacizumab containing arm. This has now become the first line standard of care in patients who have stage IV NSCLC with a good performance status, non-squamous histology, no risk factors for major bleeding and no history of hemoptysis. Recently, preliminary results from the AVAiL trial were presented at the American Society of Clinical Oncology meeting in June 2007 (Manegold et al, 2007). This multinational and multicenter trial enrolled more than 1000 patients with advanced NSCLC (stage IIIb or IV) who had not been treated previously. There were three trial arms: bevacizumab 7.5mg/kg plus

IV. Targeting the epidermal growth factor receptor The mutation or alteration of tyrosine kinases has been discovered to play an integral role in the pathogenesis of a wide variety of cancer types. For example, in chronic myelogenous leukemia (CML), the fusion of Bcr and Abl to form the abnormally activated intracellular tyrosine kinase is the cause of CML and also a target for its treatment (Smith et al, 2003). In breast cancer, over expression of the HER-2/neu receptor, which is a transmembrane tyrosine kinase in the same family as the epidermal growth factor receptor (EGFR), is associated with a poor prognosis and is also a target for therapy (Aguilar et al, 1999). In NSCLC, mutations in the EGFR are well described (Sequist et al, 2007) and therapeutic agents that target the EGFR have been shown to improve survival (Shepherd et al, 2005). The EGFR is a transmembrane receptor with an extracellular portion that is involved in ligand binding and an intracellular portion that contains the tyrosine kinase function, which is composed of two segments: one that binds ATP and magnesium and an activation segment. When the extracellular domain is bound by its ligand, the activation segment becomes phosphorylated, which changes the conformation of the intracellular portion resulting in an increase in enzymatic activity and various signaling processes are initiated. (Krause and Van Etten 2005) Small molecule TK inhibitors, such as gefitinib and erlotinib antagonize this process. Gefitinib competes for the ATP binding site on EGFR and once bound, it effectively blocks the normal epidermal growth factor induced activity of phosphorylation and thereby shuts down further signaling (Wakeling et al, 2002). Similarly erlotinib is also a competitive inhibitor of ATP on EGFR and inhibits subsequent phosphorylation. It has been shown to both decrease cell proliferation and induce apoptosis in tumor cell lines in vitro (Moyer et al, 1997). After exposure to erlotinib, an increase of the percentage of cells in the cell cycle phase G1 was noted, with a decrease in the percentage of cells in other phases of the cell cycle including mitosis, implying an arrest in G1. It is 96

Cancer Therapy Vol 6, page 97 speculated that the loss of EGFR signaling (blocked by erlotinib) is responsible for the inability of the cells to progress through the cell cycle and thus proliferate. Further evidence from the same in vitro study also demonstrated increased apoptosis in tumor cell lines exposed to erlotinib that was similar to the degree of apoptosis induced by exposure to cisplatin in the same tumor cell lines. This implies that TKIs have both cytostatic as well as more direct cytotoxic effects on tumor cells.

V. Clinical performance tyrosine kinase inhibitors


improvement rate in both groups, though symptom improvement was correlated with response to therapy. Diarrhea and skin toxicities were most common, though no cases of interstitial lung disease were observed. Based on this data, a phase III trial looking at overall survival was launched. The ISEL (Thatcher et al, 2005) (Iressa Survival Evaluation in Lung Cancer) trial enrolled 1692 patients from multiple centers worldwide and randomized them in a 2:1 fashion to gefitinib (at 250mg daily) or placebo with best supportive care. Patients with locally advanced or metastatic NSCLC treated with at least one prior chemotherapy regimen and found to be either refractory or intolerant were eligible. Survival was the primary end point. Median survival among all patients enrolled was 5.6 months for the gefitinib arm and 5.1 months for the placebo arm, a difference that was not statistically significant. Patients who were of Asian origin or who had never smoked did have an improvement in overall survival in a planned subset analysis, though the subgroup of patients with adenocarcinoma histology did not show an overall survival benefit when treated with gefitinib when compared with placebo. As a result, the use of gefitinib has now been restricted to patients who were previously treated with gefitinib and are responding to the medication. Newer data may broaden its use, however. Results from the INTEREST trial were recently presented at the 12th World Conference on Lung Cancer. This multi-center, multi-national trial included more than 1400 patients with previously treated locally advanced or metastatic NSCLC. All patients had a platinum incorporated into their prior regimen and approximately 20% of patients enrolled were of Asian origin. After enrollment, patients were randomized to either docetaxel or gefitinib. Adverse events were the expected toxicities associated with both of these agents including an increased incidence of rash and diarrhea in the gefitinib group and increased neutropenia, fatigue, nausea and alopecia in the docetaxel group. Gefitinib was found to be non-inferior to docetaxel with a median overall survival of 7.6 months compared with 8 months in the docetaxel group, but was associated with improved symptoms and quality of life in two out of three models used. Of note, subgroup analyses of patients with EGFR mutations, high EGFR gene copy number or EGFR expression showed no difference in survival irrespective of their treatment group. Erlotinib has also shown benefit in patients with advanced NSCLC who have failed chemotherapy (Table 1) and is often used as second or third line therapy. The BR.21 study (Shepherd et al, 2005) was a randomized, double blind, placebo controlled phase III trial which enrolled 731 previously treated patients with NSCLC. More than 90% had been treated with a platinum in prior regimens and more than half had been treated with two prior chemotherapy regimens. Patients were randomized in a 2:1 ratio, with two patients randomized to erlotinib for every one patient randomized to the placebo arm. The response rate (CR and PR) was 8.9% in the erlotinib group compared with <1% in the placebo group. Overall survival was also better in the erlotinib group with a median survival of 6.7 months in the erlotinib group compared


Several dose finding phase I trials demonstrated that the small molecule TKI gefitinib was safe and well tolerated, which led to the phase II trials of Iressa Dose Evaluation in Advanced Lung Cancer trials (IDEAL-1 and IDEAL-2). IDEAL-1 (Fukuoka et al, 2003) was a non-US, multicenter phase II trial that included patients with nonsmall-cell lung cancer that was metastatic or locally advanced following progression on one or more chemotherapy regimens, one of which had to have included a platinum. More than 200 patients were randomized in a double blind fashion to gefitinib 250mg or 500mg. Of note, about half of the patients were of Japanese origin. Response rates and disease related quality of life were measured. Response rates did not differ between doses (18.4% and 19% in the 250mg and 500mg groups respectively) nor did disease control rate (54.4% and 51.4%). Disease control rate referred to the best response of CR, PR or SD that persisted for 4 weeks or longer. Symptom improvement was noted and was similar between the dose groups with increasing symptom improvement rates with increasing responses. The main toxicities were the expected adverse effects of skin reactions, diarrhea and elevated transaminases. In addition, pulmonary interstitial type illness was seen in two patients. IDEAL-2 (Kris et al, 2003) was a similar phase II trial with multi-center participation in the US. There are some important differences in the patient populations of the IDEAL 1 and 2 trials. IDEAL-2 included patients with stage IIIb or Stage IV NSCLC if they had progressed or had unacceptable toxicity after two or more prior chemotherapy regimens containing a platinum agent and docetaxel. Enrollment in this trial came entirely from centers based in the United States. In addition, patients were only eligible if they had symptomatic and measurable disease. Approximately 20% were ECOG performance status of 2, the remainder were 0-1. In the IDEAL-1 trial, approximately 40% of patients had failed two prior treatment regimens, while all had failed one. About 65-70% of patients were symptomatic at study entry. Less than 15% of patients were performance status 2, the remainder were 0-1. As part of the study design, half of patients were Japanese. In the IDEAL-2 trial more than two hundred patients were enrolled and randomized to either 250mg or 500mg of gefitinib. There were no complete responses and a partial response rate of 12% and 9% for the 250mg and 500mg doses of gefitinib respectively. In addition, there was a 25% overall increase in the symptomatic 97

Blanchard et al: Targeted agents in Non Small Cell Lung Cancer

Table 1. Selected Phase III trials involving erlotinib and gefitinib Trial TRIBUTE TALENT BR.21

Type Phase III Phase III Phase III


Phase III


Phase III

Patients Untreated stage IIIB/IV NSCLC Untreated stage IIIB/IV NSCLC Previously treated patients with stage IIIB/IV NSCLC Previously treated patients with stage IIIB/IV NSCLC Previously treated patients with locally advanced or metastatic NSCLC

Treatment Carboplatin paclitaxel vs. carboplatin/paclitaxel/erlotinib Cisplatin/gemcitabine vs. cisplatin/gemcitabine/erlotinib Erlotinib vs. placebo

Results No difference in RR, TTP or MS No difference in RR, TTP, OS, QoL Increased RR, PFS and OS in erlotinib group

Gefitinib vs. placebo

No difference in median survival between the two groups Similar median survival, but less toxicity in gefitinib group

Gefitinib vs. docetaxel

RR=response rate, TTP=time to progression, MS=median survival, OS=overall survival, QoL=quality of life

with 4.7 months in the placebo group (adjusted hazard ratio of 0.70; 95% CI: 0.58-0.85, p<0.001). In addition, the median time to symptom worsening was longer for patients taking erlotinib including the cancer related symptoms of cough, dyspnea and pain. Several studies have investigated the value of adding tyrosine kinase inhibitors with chemotherapy. INTACT-1 (Giaccone et al, 2004) and INTACT-2 (Herbst et al, 2004) trials were phase III trials of patients with advanced NSCLC who were chemotherapy na誰ve and treated with either chemotherapy or chemotherapy plus gefitinib. In INTACT-1, more than one thousand patients were enrolled and randomized to a standard chemotherapy doublet of cisplatin/gemcitabine or the same doublet plus gefitinib at a dose of either 250mg or 500mg. There was no significant difference in response rates, time to progression or survival between the groups. INTACT-2 was a similar large phase III trial that randomized patients with advanced NSCLC to carboplatin/paclitaxel versus carboplatin/paclitaxel plus gefitinib. The results were also similar: no significant difference in response rates, time to progression or overall survival. The TRIBUTE study (Herbst et al, 2005) randomized previously untreated patients to standard chemotherapy with carboplatin and paclitaxel vs. the same chemotherapy plus concurrent erlotinib. More than one thousand patients were enrolled. Response rates, time to progression and overall survival were not statistically different between the two arms. Similarly, adding erlotinib to another common doublet used in advanced non-small-cell lung cancer, cisplatin and gemcitabine, did not yield any added benefit to chemotherapy alone in terms of response rate, time to progression or survival as reported in the TALENT study (Gatzemeier et al, 2007). It does not appear therefore that adding tyrosine kinase inhibitors to standard chemotherapy schedules offers any benefit. In one of the few large intergroup trials in stage III NSCLC, the investigators of SWOG 0023 looked at maintenance gefitinib after treatment with

cisplatin/etoposide and concurrent radiation, followed by consolidation with docetaxel (Kelly et al, 2007). After consolidation, patients were randomized to gefitinib or placebo. The primary endpoint was overall survival. In patients who were randomized, progression free survival was statistically similar, but survival was greater in the placebo arm with median survival of 23 months in the gefitinib arm and 35 months in the placebo arm (P value of 0.01, HR 0.63, 95% CI 0.44-0.91). The main cause of death was overwhelmingly cancer related, and toxicity was not felt by the investigators to be a significant cause of diminished survival in the gefitinib arm. The use of maintenance gefitinib in stage III disease should be limited to clinical trials.

VI. Predictors of response to tyrosine kinase inhibitors Several clinical characteristics have consistently predicted for response to the small molecule TKIs including a history of never smoking, Asian origin, female gender and adenocarcinoma histology (Kris et al, 2003; Shepherd et al, 2005; West et al, 2006). Molecular correlates have also been studied extensively. Retrospective data, particularly (though not exclusively) out of Asia, supports the notion that the presence of an EGFR mutation is associated with improved survival when those patients are treated with a tyrosine kinase inhibitor (gefitinib or erlotinib), (Cortes-Funes et al, 2005; Han et al, 2005) but prospective phase III trials have not been able to confirm this. In the several phase III clinical trials that have evaluated the use of TKIs in advanced non small cell lung cancer, most of them have also included molecular correlates as part of the study design. From the IDEAL-1 (Fukuoka et al, 2003) and IDEAL-2 (Kris et al, 2003) trials, samples from 155 patients out of 425 enrolled were available for analysis of EGFR mutational status and presence of EGFR amplification. There were 666 tumor samples available from the more than two thousand


Cancer Therapy Vol 6, page 99 participants in the INTACT trials that were subjected to analysis for EGFR mutation status and amplification. Between 59-86% of samples were subsequently successfully analyzed (Bell et al, 2005). EGFR mutations were found in 18% of the analyzed samples from the IDEAL trials and 10% from the INTACT trials. Consistent with other studies of mutations in EGFR, most of the mutations were in frame deletions found in exon 19, followed by missense mutations in the L858R in exon 21 followed by a small percentage of novel variants. Similar to other series, in both the IDEAL and INTACT trials mutations were found more frequently in patients with known clinical predictors of response to therapy including adenocarcinoma histology, women, non smokers and Asian patients. In contrast, the presence of EGFR gene amplification was not associated with any of the previously described characteristics associated with clinical response. In the IDEAL series, the overall response rate to gefitinib was higher in the group of patients whose tumors were found to have an EGFR mutation (46% compared with 10%, p=.005) (Bell et al, 2005). In addition, patients whose tumors contained mutations had a longer median time to progression (TTP) compared with patients whose tumors did not contain EGFR mutations, but there was no difference in overall survival between the groups. The numbers of patients with gene amplifications was very small, limiting the comparison, but there was no statistically significant difference in response to treatment between patients with high gene copy numbers and lower copy numbers. In the INTACT trial series, neither mutational status nor the presence of gene amplification seemed to impact response rates to chemotherapy plus gefitinib, as they were not statistically different in patients with or without the mutation or gene amplification. The addition of gefitinib did not affect overall survival in patients who were mutation positive (hazard ratio 1.77; 95% CI, 0.5-6.23). Similarly, several trials have looked at erlotinib and molecular correlates that may predict for response. The BR.21 (Shepherd et al, 2005) trial was a phase III study in patients with previously treated NSCLC who progressed after chemotherapy and were randomized to either erlotinib or placebo. Three different aspects of EGFR were investigated using tumor samples from this trial: expression of EGFR by the tumor, EGFR gene amplification and EGFR mutations (Tsao et al, 2005). Expression of EGFR protein was established by immunohistochemistry and considered positive if 10 percent of tumor cells had staining of any intensity. Mutational analysis was carried out first with amplification by PCR, followed by sequence analysis. EGFR gene copy number was determined by FISH, with FISH positive tumors corresponding to high degree of amplification. In multivariate analysis, expression of EGFR was associated with increased responsiveness to erlotinib, but neither expression of EGFR, EGFR gene amplification nor the presence of an EGFR mutation predicted for improved survival in patients treated with erlotinib. Of the 1079 patients enrolled in the TRIBUTE (Eberhard et al, 2005) study, tumor specimens from 274 of

these patients were satisfactory and available for DNA sequencing. EGFR mutations, when detected, were confirmed with independent PCR and sequencing. EGFR mutations were found in 12.2% of the tumor samples available and the presence of a mutation was associated with improved overall response rate (ORR) in patients who had an EGFR mutation and were treated with erlotinib (53% vs. 18% p<.01). Patients with EGFR mutations who were treated with CP plus erlotinib had a numerically increased time to progression compared with patients with EGFR mutations treated with CP alone (12.5 months vs. 6.6 months) but the difference was not statistically significant (p=.092, 95% CI for HR 0.2-1.2). Overall survival was not different in patients who were mutation positive regardless of if they received chemotherapy alone or in combination with erlotinib. The outcome parameters and analysis were limited by the small number of EGFR mutation positive patients (29 total). Interestingly, patients with EGFR mutations had improved ORR, TTP and survival independent of their treatment groups compared with patients whose tumors did not have EGFR mutations. One major challenge in attempts to determine molecular correlates to clinical responses to targeted therapy is the analysis process itself. Investigators are limited by small amounts of tissue as fine needle aspirates are increasingly used for diagnosis, the fixatives used to preserve the tissue and the limitations of the analytic methods (Sequist et al, 2007). One prime example is the mutation analysis done in the BR.21 study that found new mutations in 53% of patients who were mutation positive (Tsao et al, 2005) which is indicative of the presence of artifacts. This led the investigators of that trial to reevaluate their mutation analysis (Tsao et al, 2006) in which they included only mutations already described in exon 19 and in exon 21. In the new analysis, the results were similar: patients whose tumors had mutations in those two â&#x20AC;&#x153;classicâ&#x20AC;? areas had better response rates than patients without such mutations, but there was no difference in overall survival (HR for death 0.52 with 95% CI 0.21-1.31, P=0.16). Investigation into new targeted agents continues and there are several currently in clinical trials. AZD2171 is an oral antiangiogenesis drug that inhibits VEGFR tyrosine kinases and has been looked at in multiple phase I and II trials in several different solid tumors and has shown some efficacy in NSCLC (Goss et al 2007). A phase II/III trial is now accruing using this agent plus a carboplatin/paclitaxel doublet and comparing it with the chemotherapy doublet plus placebo. Sunitinib, a multitargeted tyrosine kinase inhibitor best known for its use in renal cell cancer has shown promise in NSCLC in phase II trials (Brahmer et al 2007) and is now being studied in combination with erlotinib in a phase III study now accruing. ZD6474 is an oral tyrosine kinase inhibitor that targets both VEGF and the epidermal growth factor, now being investigated in several phase III trials after showing superior progression free survival versus gefitinib (Natale et al 2006). Active clinical trials can be accessed at There is tremendous potential in newer targeted agents, though challenges still lay ahead. One major


Blanchard et al: Targeted agents in Non Small Cell Lung Cancer Giaccone G, Herbst RS, Manegold C, Scagliotti G, Rosell R, Miller V, Natale RB, Schiller JH, Von Pawel J, Pluzanska A, Gatzemeier U, Grous J, Ochs JS, Averbuch SD, Wolf MK, Rennie P, Fandi A, Johnson DH (2004) Gefitinib in combination with gemcitabine and cisplatin in advanced nonsmall-cell lung cancer: A phase III trial-INTACT-1. J Clin Oncol 22, 777-784. Goss GD, Laurie S, Shepherd F, Leighl N, Chen E, Gauthier I, Reaume N, Feld R, Powers J, Seymour L (2007) IND 175: Phase I study of daily oral AZD2171, a vascular endothelial growth factor receptor inhibitor (VEGFRI), in combination with a gemcitabine and cisplatin (G/C) in patients with advanced non-small cell lung cancer (ANSCLC): a study of the NCIC clinical trials group (Abstr 7649). J Clin Oncol 25, (Supp No. 18S). Han SW, Kim TY, Hwang PG, Jeong S, Kim J, Choi IS, Oh DY, Kim JH, Kim DW, Chung DH, Im SA, Kim YT, Lee JS, Heo DS, Bang YJ, Kim NK (2005) Predictive and Prognostic Impact of Epidermal Growth Factor Receptor Mutation in Non–Small-Cell Lung Cancer Patients Treated With Gefitinib. J Clin Oncol 23, 2493-2501. Herbst RS, Giaccone G, Schiller JH, Natale RB, Miller V, Manegold C, Scagliotti G, Rosell R, Oliff I, Reeves JA, Wolf MK, Krebs AD, Averbuch SD, Ochs JS, Grous J, Fandi A, Johnson DH (2004) Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: A phase III trial-INTACT-2. J Clin Oncol 22, 785-794. Herbst RS, Prager D, Hermann R, Fehrenbacher L, Johnson BE, Sandler A, Kris MG, Tran HT, Klein P, Li X, Ramies D, Johnson DH, Miller VA; TRIBUTE Investigator Group (2005) TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 23, 5892-5899. Hicklin DJ, Ellis LM (2005) Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 23, 1011-1027. Johnson DH, Fehrenbacher L, Novotny WF, Herbst RS, Nemunaitis JJ, Jablons DM, Langer CJ, DeVore RF, Gaudreault J, Damico LA, Holmgren E, Kabbinavar F (2004) Randomized Phase II Trial Comparing Bevacizumab Plus Carboplatin and Paclitaxel With Carboplatin and Paclitaxel Alone in Previously Untreated Locally Advanced or Metastatic Non-Small-Cell Lung Cancer. J Clin Oncol 22, 2184-2191. Kelly K, Chansky K, Gaspar LE, Jett JR, Ung Y, Albain KS, Crowley JJ, Gandara DR (2007) Updated analysis of SWOG 0023: A randomized phase III trial of gefitinib versus placebo maintenance after definitive chemoradiation followed by docetaxel in patients with locally advanced stage III non-small cell lung cancer (Abstr 7513). J Clin Oncol 25, (Supp No. 18S). Krause DS, Van Etten RA (2005) Tyrosine kinases as targets for cancer therapy. New Engl J Med 353, 172-187. Kris MG, Natale RB, Herbst RS, Lynch TJ Jr, Prager D, Belani CP, Schiller JH, Kelly K, Spiridonidis H, Sandler A, Albain KS, Cella D, Wolf MK, Averbuch SD, Ochs JJ, Kay AC (2003) Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer. JAMA 290, 21492158 Manegold C, von Pawel J, Zatloukal P, Ramlau R, Gorbounova V, Hirsh V, Leighl N, Mezger J, Archer V, Reck M, the BO17704 study group (2007) A randomized, double-blind, phase III study of bevacizumab in combination with cisplatin and gemcitabine in chemo-naïve patients with advanced or recurrent non-squamous NSCLC (Abstr LBA7514). J Clin Oncol 25, (Supp No. 18S).

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Elizabeth Blanchard


Blanchard et al: Targeted agents in Non Small Cell Lung Cancer


Cancer Therapy Vol 6, page 103 Cancer Therapy Vol 6, 103-116, 2008

The molecular bases of cannabinoid action in cancer Review Article

Stefano Fogli*, Maria Cristina Breschi Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, 56126-Pisa, Italy

__________________________________________________________________________________ *Correspondence: Stefano Fogli, PharmD, PhD, Assistant Professor of Pharmacology, Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa; Via Bonanno, 6; Pisa, PI 56126, Italy; Phone: ++39-050-2219511; Fax: ++39050-2219609; E-mail: Key words: Cannabinoids, cancer, apoptosis, angiogenesis, inflammation Abbreviations: 2-arachidonoylglycerol, (2-AG); apoptotic protease-activating factor-1, (APAF-1); arachidonoyl-serotonin, (AA-5-HT); cannabinoid type 1, (CB1); cannabinoid type 2, (CB2); carcinoma-associated fibroblasts, (CAFs); cyclin-dependent kinase, (cdk); cyclooxygenase, (COX-2); extracellular signal-regulated kinase, (ERK); fatty acid amide hydrolase, (FAAH); fibroblast growth factors, (FGFs); inducible form of nitric oxide synthase, (iNOS); interleukin, (IL); mitogen-activated protein kinase, (MAPK); nerve growth factor, (NGF); nuclear factor-!B, (NF-!B); proHeparin-binding EGF-like growth factor, (proHB-EGF); prostaglandin E2, (PGE2); protein kinase A, (PKA); retinoblastoma protein, (pRb); stromal cell-derived factor 1, (SDF-1); tetrahydrocannabinol, (THC); TNF-" converting enzyme, (TACE); transient receptor potential vanilloid type 1 receptor, (TRPV1); tumor necrosis factor, (TNF); vascular endothelial growth factor, (VEGF) Received: 15 December 2007; Revised: 17 December 2007 Accepted: 31 December 2007; electronically published: February 2008

Summary In the last two decades, research in the cannabinoid field has generated a rich body of knowledge revealing a role of the endocannabinoid system in the regulation of several processes involved in cancer development and progression. Cannabimimetic drugs have been shown to inhibit tumor growth by modulating pivotal regulatory circuits in the genetically altered malignant cells and in their supporting coconspirators in the stromal compartment. Nonetheless, the complexity of the integrative actions of these pleiotropic molecules in tumor cells, mesenchymal cells and cells of the immune system makes difficult to understand the precise mechanism of action responsible of their effects. In this review, we highlight the molecular mechanisms of cannabinoid action in cancer by focusing on key cellular and molecular processes that collectively dictate the molecular signature of the basic machinery of cancer.

understand the molecular bases of cannabinoid action should be encouraged. This issue appears to be particularly relevant if considering that both mitogenic and antimitogenic potentials of cannabinoid-based treatments have been observed, with the type of biological response depending on a number of extrinsic and intrinsic factors, such as the specific tumor phenotype (e.g, receptor status and dependence on growth signaling), the pharmacological profile of cannabinoid compounds (e.g, receptor subtype selectivity and agonist or antagonist properties) and the mechanism involved in their action (e.g, receptordependent or receptor-independent effects). A great number of derivatives of different origin, including endogenous lipids (endocannabinoids) as well as plant- and synthetic-derived compounds with cannabinoidlike activity, bind specific receptor subtypes to induce cannabimimetic responses on target tissues. Three endocannabinoid receptors have been characterized; these are cannabinoid type 1 (CB1) and type 2 (CB2) receptors and transient receptor potential vanilloid type 1 receptor

I. Introduction The investigation of the therapeutic effects of cannabinoids on some cancer-related disorders has provided evidence of their effectiveness in the alleviation of several symptoms observed in cancer patients, such as chemotherapy-induced nausea and vomiting, appetite loss and neuropathic pain (Hall et al, 2005). Beyond the palliative effects induced by these compounds, recent advances in the knowledge of the biological role of endocannabinoids and novel insights into the molecular signaling of cannabinoid receptors, support the participation of the endocannabinoid system in the regulation of key processes involved in the development of cancer, including cell growth and apoptosis (Bifulco and Di Marzo, 2002; Guzman et al, 2002), angiogenesis (Pisanti et al, 2007), inflammation and immune cell function (Klein, 2005). However, despite the potential therapeutic application of these findings, caution is required when envisaging new options in cancer therapy and more intense research efforts aimed to better 103

Fogli and Breschi: Cannabinoids and cancer (TRPV1) ion channels (for review see Pacher et al, 2006). Information regarding novel non-CB1/CB2 receptors has been also recently provided; however, the molecular identity and function of these receptors remain substantially unclear (Brown, 2007). CB1 and CB2 are seven-transmembrane G-protein-coupled receptors (GPCR) that are coupled to Gi/Go heterotrimeric proteins and adenylyl cyclase, but other second messengers and signaling components are also involved in their activity (Bisogno et al, 2005; Demuth and Molleman, 2006). In this review article, we will summarize information on the molecular mechanisms of cannabinoid action currently available by focusing on the cross-talk between the CB1/CB2 signaling network and different target molecules in tumor cells and in the surrounding tissue that serve as protagonists and active collaborators in the neoplastic cell agenda. Such a viewpoint may be helpful in understanding the value of cannabinoid receptors as novel promising targets for therapeutic interventions aimed to disrupt cancer machinery.

highlighting the relevance of considering tumors as complex tissues in which different actors contribute to their phenotypic signature (Figure 1). An illustrative example of this concept comes from evidence demonstrating that chronic inflammation is a pathophysiologic condition able to deregulate cellular homeostasis and drive carcinogenesis, particularly in epithelial-derived malignancies (Hussain et al, 2003; Angelo and Kurzrock, 2007); in this view, inflammatory cells attracted to sites of neoplasia may promote (rather than eliminate) cancer cells. As a matter of fact, the protective effect observed in cancer patients following the administration of the selective COX-2 inhibitor, celecoxib (Altorki et al, 2003), indicates that molecules interfering with inflammatory signaling pathways either in the tumor or in adjacent nontumor tissues which cooperate in the multiple events that characterize tumor growth, can effectively elicit antitumor effects. Concerning the molecular mechanisms underlying the complex interplay between inflammation and tumor development, it has been recognized that tumor-infiltrating lymphocytes such macrophages, T cells, B cells, natural killer cells, neutrophils and granulocytes, release a large amounts of inflammatory cytokines into the microenvironment. These cytokines may enhance the tumorigenic process by upregulating important mediators of angiogenesis, including vascular endothelial growth factor (VEGF) and interleukin (IL)-8, and/or may serve as autocrine and paracrine growth factors for several types of cancers. Of note, VEGF, in turn, increases vascularity at the site of inflammation causing the reaction to be more severe (Angelo and Kurzrock, 2007).

II. The heterotypic cell biology view of cancer Cancer death is caused by a number of molecular and biochemical processes which ultimately result in tumorcell metastatic spreading (Hanahan and Weinberg, 2000). Several lines of evidence suggest that besides genetic and epigenetic signatures of a specific cancer cell type, the interactions between malignant cells and cells in the host microenvironment, appear to be critical in the selection and expansion of the tumor mass (for review see Liotta and Kohn, 2001; Orimo and Weinberg, 2006), thus

Figure 1. Overview of the multistep process involved in tumor progression. Cancer cell genotype deriving from genetic defects in regulatory circuits governing normal cell homeostasis drives the clonal expansion of different cell variants. Cytokine and enzyme exchange between cells populating the stromal compartment surrounding the tumor (i.e, immune, inflammatory and vascular cells) and neoplastic cells stimulates migration of both cell types towards each other which ultimately results in invasion and metastasis.


Cancer Therapy Vol 6, page 105 The ability to induce and sustain angiogenesis is a fundamental step during tumor development and metastatic progression (Mehlen and Puisieux, 2006). The angiogenesis-initiating signals are exemplified by VEGF and fibroblast growth factors (FGFs) which interact with transmembrane tyrosine kinase receptors displayed by endothelial cells (Folkman, 2002). Of note, it has been recently demonstrated that host inflammatory responses induced by FGF-2 are associated with FGF-2-induced tumor progression (Tsunoda et al, 2007). Finally, FGF-2 is known for its mitogenic effects on human cancer cells and it is also a potent stimulator of cell survival (Vandermoere et al, 2007). These notions highlight how a complete understanding of the complex interplay between different elements which play a pivotal role into the tumor-host interface circuit, might represent a rational basis for selecting innovative strategies in cancer therapy.

(Caffarel et al, 2006), prostate (Sarfaraz et al, 2006), colorectal (Ligresti et al, 2003), glioma (Massi et al, 2004), melanoma (Blazquez et al, 2006) and leukemia (McKallip et al, 2006), and with the notion that the endocannabinoids, 2-arachidonoylglycerol (2-AG; Nithipatikom et al, 2004) and anandamide (AEA; Ligresti et al, 2003), have been demonstrated to play a role as endogenous inhibitors of cancer cell proliferation. In contrast with the abovementioned results, some published data seem instead to support a mitogenic role for endocannabinoids. For example, the extent of CB2 expression was directly related with tumor malignancy in biopsies from human astrocytomas (Sanchez et al, 2001), and real-time quantitative PCR analyses in human breast tumor samples demonstrated a correlation between CB2 expression and several prognostic markers such as histological grade of tumors, estrogen/progesterone receptor status, and expression of the HER2/neu oncogene (Caffarel et al, 2006). Despite the causative role of cannabinoid receptor expression cannot be establised by association studies, it should be noted that CB1 receptor antagonist SR141716A, when administered alone, did not enhance, but instead slightly inhibited, the growth of rat thyroid transformed cells both in vitro and in tumor xenografts induced in vivo (Bifulco et al, 2004). In line with this notion, CB1 antagonism by SR141716A induced a cytostatic effect on human breast cancer cell growth that was more pronounced in highly invasive metastatic cells than in less-invasive cells (Sarnataro et al, 2006). The antiproliferative effect of SR141716A was characterized by a G1/S-phase cell cycle arrest with a concomitant induction of the cyclin-dependent kinase inhibitor p27KIP1 (Sarnataro et al, 2006). Of note, such a mechanism has been also demonstrated for trastuzumab (Lane et al, 2000), a FDA-approved monoclonal antibody for the treatment of patients with metastatic breast cancer, whose tumors overexpress the HER2/neu protein (Bernard-Marty et al, 2006). In summary, although their action may substantially depend on the cell type and pathophysiologic context, it is conceivable that endocannabinoids, through their receptors, may act as autocrine and/or paracrine growth factor-like molecules by promoting positive feedback loops which confer to cancer cells a self-sufficiency from other cells within the tissue, a mechanism shared by most types of human cancers. This hypothesis seems to be in agreement with evidence demonstrating that nanomolar concentrations of cannabinoids may elicit a receptormediated tumor cell proliferation (Hart et al, 2004), a mechanism most probably linked to the stimulation of mitogen-activated protein kinase (MAPK) (Bouaboula et al, 1995; Galve-Roperh et al, 2002) and phosphatidylinositol 3-kinase and Akt (PI3K/Akt) pathways (Sanchez et al, 2003; Fernandez-Ruiz et al, 2007) (Figure 2). Otherwise, overstimulation of these receptors induced by micromolar concentration of cannabimimetic drugs or increased amount of endocannabinoids (Nithipatikom et al, 2004) as well as receptor antagonism (Sarnataro et al, 2006), may promote disarray in the homeostatic regulation of receptor function and signaling that finally results in dismantling of the

III. The role of the endocannabinoid system in the regulation of neoplastic cell circuits A. Cannabinoid receptors in human cancer Several lines of evidence suggest that specific cannabinoid receptor subtypes play a determining role in hematological and solid malignancies. The differential expression of these receptors between normal and tumor tissues may therefore be of interest to better understand the role of the endocannabinoid system in the maintenance of the neoplastic cell phenotype. For example, it has been recently demonstrated that expression levels of both CB1 and CB2 were significantly higher in human prostate cancer cells than in prostate epithelial cells (Sarfaraz et al, 2005), and in human hepatocellular carcinoma samples, comparing to their counterpart in normal liver (Xu et al, 2006). While examination of a number of human leukemia and lymphoma cell lines revealed that they express CB2, but not CB1 (McKallip et al, 2002), results from other groups demonstrated a specific expression of CB1 in human lymphoma cells but not in normal human B lymphocytes (Ek et al, 2002; Islam et al, 2003; Flygare et al, 2005; Gustafsson et al, 2006). Furthermore, interesting and potentially very important findings considering the induction of CB1 gene expression via CB2 in human malignant T lymphocytes indicate the presence of a crosstalk between these two receptors, at the transcriptional level (Borner et al, 2007). Regarding the pathogenetic significance of cannabinoid receptor overexpression in cancer development, Xu and co-workers demonstrated in 2006 that disease-free survival was significantly better in hepatocellular carcinoma patients with both high CB1 and CB2 expression levels compared with those with low CB1 and CB2 expression levels, thus suggesting that endocannabinoid system might be part of the anti-growth factor signaling circuit. This concept seems to be in line with most of the in vitro studies finding that ligandinduced activation of cannabinoid receptors reduces cell proliferation in different types of tumors including breast 105

Fogli and Breschi: Cannabinoids and cancer cancer cell network. As an illustrative example, the ability of both the CB1 agonist AEA (De Petrocellis et al, 1998) and the CB1 antagonist SR141716A (Sarnataro et al, 2006) to inhibit the proliferation of human breast cancer MCF-7 cells, has been demonstrated.

ligand-activated growth factor receptors, such as those observed in the SOS-Ras-Raf-MAPK mitogenic cascade (Medema et al, 1993). Furthermore, cross-talking connections with other pathways enable extracellular signals to elicit multiple cell biological effects, as demonstrated by the interaction of the Ras protein with the survival-promoting PI3K/Akt (Downward et al, 1998). Therefore, it is generally assumed that activation of these signaling pathways leads to cell protection from death stimuli by acting either independently or in a coordinate manner (Figure 2). The endocannabinoid system has been demonstrated to be involved in the modulation of critical components of the downstream cytoplasmic circuitry, including PI3K/Akt (Gomez del Pulgar et al, 2000), extracellular signal-regulated kinase (ERK) (Bouaboula et al, 1995), c-Jun N-terminal kinase and MAPK cascades (Rueda et al, 2000). Of note, it appears that the time of cannabinoid exposure can affect the type of response observed. For example, CB1-mediated short-term ERK activation and PI3K stimulation protect glial cells from ceramide-induced apoptosis (Galve-Roperh et al, 2002), whereas sustained ERK activation promotes apoptosis (Galve-Roperh et al, 2000). Furthermore, inhibition of PI3K/Akt and ERK signaling pathways and activation of apoptosis in tumor cells, seems to be a common feature of cannabinoid action after sustained treatment with cannabinoid receptor ligands (Ellert-Miklaszewska et al, 2005; Jia et al, 2006), thus suggesting that prolonged exposure to these compounds may disrupt the interplay between mitogenic and antiapoptotic signaling molecules in tumor cells, in such a way that favors inhibition of cell proliferation and/or induction of cell death (Figure 2).

B. Interference with growth signaling pathways Many tumor cells acquire the ability to synthesize autocrine growth factors to which they are responsive, thus reducing their dependence from exogenous growth stimulation. Therefore, blocking the growth factor action on their cognate receptors represents a powerful means to arrest cancer cell proliferation. It has been reported that the endocannabinoid AEA, through CB1-like receptors, can exert a cytostatic effect on human breast cancer MCF7 and EFM-19 cells by downregulating the levels of the receptor of prolactin, a hormone which operates as an autocrine signal, in this cell type (De Petrocellis et al, 1998) (Figure 2). Likewise, autonomous production of nerve growth factor (NGF) by epithelial cancer cells, including those isolated from breast (Dolle et al, 2004) and prostate tumors (Djakiew et al, 2000) has an important role in malignant progression. AEA and 2-AG promoted a CB1-mediated inhibition of the NGF-induced proliferation of MCF-7 cells by suppressing the levels of the high affinity trk NGF receptors (Melck et al, 2000) (Figure 2). Biological responses to cannabinoids critically depend on several factors, including drug concentration at the site of action and specific cell type examined. For instance, submicromolar concentrations of AEA and arvanil (CB1 agonist), potently inhibit the prolactininduced proliferation of prostate cancer cells, while inhibition of basal cell proliferation was observed only at high doses (i.e, #5 ÂľM) (Melck et al, 2000). Moreover, a concentration-dependent biphasic stimulant/inhibitory effect of the marijuana derivative !9-tetrahydrocannabinol (THC) on NGF production by prostate cancer cells has been also observed (Velasco et al, 2001). In contrast to the antiproliferative properties of cannabinoids mediated by negative regulation of growth factor signaling, evidence has been provided on the ability of several ligands such as THC, AEA, and the synthetic cannabinoids HU-210 and Win55,212-2, to rapidly induce epidermal growth factor (EGF) receptor and HER2/neu signal transactivation and proliferation in different cancer cell lines (Hart et al, 2004) (Figure 2). Of note, cannabinoid-promoted mitogenic action by transactivation processes has been mostly observed in cancer cell lines where the EGF signaling pathway plays a pivotal role in determining the neoplastic phenotype (Hart et al, 2004; Zhao et al, 2005). From the molecular viewpoint, signal cross-communication between cannabinoid and EGF receptors was mediated by shedding of proAmphiregulin (proAR) and/or proHeparin-binding EGF-like growth factor (proHB-EGF) by tumor necrosis factor (TNF)-" converting enzyme (TACE) (Hart et al, 2004) (Figure 2). Beyond the autocrine growth factor production, another mechanism of self-sufficiency acquisition in growth signals comes from abnormalities in the cytoplasmic components that receive signals emitted by

C. Cell cycle and apoptosis At variance with normal tissues, malignant tumor cells can be hypothesized as being resistant to the antiproliferative signals. Much of the signaling pathways that enable normal cells to respond to antigrowth signals are associated with the mitotic cell cycle, specifically the components regulating the transit of the cell through the G1 phase. At the molecular level, antiproliferative signals target the retinoblastoma protein (pRb) that, in the hypophosphorylated state, negatively regulates cell proliferation by altering the function of E2F, a transcription factor essential for progression from G1 into S phase (Khidr and Chen, 2006) (Figure 2). Of note, the antiproliferative action of cannabinoids on melanoma cells is caused, at least in part, by cannabinoid receptormediated cell cycle arrest at the G1/S transition through inhibition of the prosurvival protein Akt and hypophosphorylation of the pRb (Blazquez et al, 2006). Furthermore, on human prostate cancer cells, inhibition of cell growth and induction of apoptosis by WIN-55,212-2 has been related to cannabinoid receptor-mediated cell cycle arrest in the G0/G1 phase, downregulation of cyclins D, E and cyclin-dependent kinases (cdk)-2, -4, and -6, decrease in protein expression of pRb and downregulation of E2F (Sarfaraz et al, 2006) (Figure 2). Apoptosis is a potent mechanism that limits the expansion of tumor cells by triggering their suicide, while defects in apoptosis underpin both tumorigenesis and 106

Cancer Therapy Vol 6, page 107 metastasis (Mehlen and Puisieux, 2006). Two independent pathways lead to the execution of programmed cell death: (i) the extrinsic apoptotic pathway, mediated by deathinducing signaling complex formation (e.g, FAS ligand/Fas receptor) and caspase-8 activation, and (ii) the intrinsic mitochondria-dependent pathway, characterized by the release of cytochrome c and synthesis of the ternary complex cytochrome c/procaspase-9/apoptotic proteaseactivating factor-1 (APAF-1). Both pathways then converge to activate the executioner enzyme, caspase-3, which ultimately leads to apoptotic cell dismantling (Fesik, 2005) (Figure 2). Evidence has been obtained that stimulation of cannabinoid receptors by their ligands induces cell death by interfering with relevant signaling nodes of the apoptotic cascade. Exposure to the nonpsychoactive cannabinoid, cannabidiol, leads to CB2-dependent cell killing by activation of caspase-8 and caspase-9, which

precedes the activation of caspase-3 and the appearance of apoptosis in leukemia cells (McKallip et al, 2006). Despite these findings suggest that cannabinoids may affect both intrinsic and extrinsic mechanisms of apoptotic cell death, the intrinsic pathway seems to play a more critical role in the antitumor action of cannabinoids. Indeed, apoptosis after treatment of androgen-dependent and -independent prostate cancer cells with WIN-55,212-2 results from the cannabinoid receptor-mediated increase in Bax/Bcl-2 ratio, (Sarfaraz et al, 2006), and THC-induced apoptosis in Jurkat leukemia T cells takes place via translocation of the proapoptotic Bcl-2 family member, Bad, to mitochondria (Jia et al, 2006). Of note, data from Lombard and colleagues (2005) also suggest that the extrinsic pathway may facilitate apoptosis via cross-talk with the intrinsic pathway, since tumor cells deficient in FADD or caspase-8 were partially resistant to THC-induced apoptosis (Figure 2).

Figure 2. Central growth factor signaling pathways in tumor cells involved in the molecular mechanisms of cannabinoid action. Ligand-induced activation or mutation of receptor tyrosine kinases (RTKs), promote the formation of protein complexes containing autophosphorylated growth factor receptors with adaptor proteins, including growth factor receptor bound-2 (GRB2) and exchange factors such as Son-of-sevenless (SOS) which, in turn, activate Ras. In its GTP-bound form, Ras stimulates the mitogen-activated protein kinase cascade (Raf, MEK, MAPK) resulting in the activation of different factors that regulate cell cycle progression (e.g, activation of the MAPK pathway induces cyclin D expression, retinoblastoma protein phosphorylation and E2F activation). This cascade is also linked via a variety of cross-talking connections with other pathways. For example, the direct interaction of the Ras protein with the PI3 kinase (PI3K) enables growth signals to concurrently evoke survival signals by the signaling kinase AKT, which provides strong antiapoptotic signals through its negative regulation of Bad. The apoptotic cell death is also regulated by ceramide generation via neutral sphingomyelinase (SMase) activation and enhanced synthesis de novo (see text for further details). FADD, Fas-associated death domain protein; pRb, retinoblastoma protein, i-E2F, inactive form of E2F; a-E2F, active form of E2F. Bcl2, Bax, and Bad are Bcl2 family members of apoptotic regulatory proteins. Solid and dashed lines indicate activation and inhibition of signaling pathways, respectively.


Fogli and Breschi: Cannabinoids and cancer CB1 receptor is coupled to the generation of ceramide, a sphingosine-based lipid second messenger molecule critically involved in the regulation of apoptosis. Particularly, short-term ceramide generation involves sphingomyelin hydrolysis via sphingomyelinase, while long-term ceramide generation might occur via serine palmitoyltransferase induction and enhanced ceramide synthesis de novo (Guzman et al, 2001) (Figure 2). Experiments carried out using different C6 glioma cell subclones showed that sustained, but not acute, ceramide generation is responsible for cannabinoid-induced apoptosis (Galve-Roperh et al, 2000), indicating that de novo synthesis of ceramide may have a major role in the apoptotic response induced by these compounds. In line with this proposal, up-regulation of the stress-regulated protein p8, an essential mediator of cannabinoid inducedapoptosis, is dependent on de novo-synthesized ceramide (Carracedo et al, 2006). Receptor-ligand binding appears to take a significant part in the proapoptotic effect induced by these molecules. For instance, local administration of the selective CB2 agonist, JWH-133, induces a considerable regression of malignant tumors generated by inoculation of C6 glioma cells to RAG-2-deficient mice, a strain of genetically altered mice lacking B- and T-lymphocytes. Of note, in vitro experiments on the same cell line underline that selective activation of CB2, promotes apoptosis via enhanced ceramide synthesis de novo (Sanchez et al, 2001). Nevertheless, evidences obtained in cancer cells which express cannabinoid receptors underscore the irrelevance of receptor status in determining the proapoptotic response to cannabinoids (Sanchez et al, 1998; Ruiz et al, 1999; Fogli et al, 2006).

independent mechanism, regardless the type of response induced (Hinz et al, 2004; Gardner et al, 2003). Since endocannabinoids are also substrates of COX-2 (Patrignani et al, 2005), the possible interplay between COX-2 and the endocannabinoid system might be mediated by intermediate metabolites involved in specific pathways regulating cell multiplication. In line with this notion, AEA can be metabolized to prostaglandinethanolamide by COX-2 in colorectal carcinoma cells (Kozak et al, 2002), and AEA-induced cytotoxicity in this type of neoplasia has been observed only in those cell lines overexpressing COX-2 (Patsos et al, 2005). Furthermore, selective inhibition of COX-2 enzyme activity by NS398 significantly reduced the antiproliferative effect of AEA, while blocking the enzyme responsible for the inactivation of AEA (i.e, fatty acid amide hydrolase) in combination with AEA treatment enhanced the cell death, as compared to AEA alone (Patsos et al, 2005). These findings suggest that upregulation of COX-2 may yield to a large amount of anandamide by-products (i.e, prostaglandinethanolamides) which may eventually account, at least in part, for the AEA-induced cell death. With regard for the molecular mechanism responsible for the COX-2 modulation by cannabinoids, it has been shown that cAMP is able to activate protein kinase A (PKA) and other intracellular mechanisms that are implicated in the COX-2 basal or induced expression (Tang et al, 2001). Therefore, a cannabinoid receptormediated decrease in cAMP (i.e, the most frequently observed cannabinoid response) may contribute to the negative modulation of this enzyme with the consequent inhibition of cell proliferation (Figure 2). However, under particular circumstances yielding to a cannabinoidmediated increase in cAMP production (Glass and Felder, 1997; Tang et al, 2001), a mitogenic effect might also occur.

D. Cyclooxygenases The increased expression of the inducible isoform of cyclooxygenase (COX-2) was firstly demonstrated in human colon carcinoma (Eberhart et al, 1994) and afterwards, in other neoplastic cells (Chan et al, 1999). Cannabinoid regulation of arachidonate metabolism and prostaglandin production may represent a mechanism by which these molecules modulate proliferative events in cancer cells. Despite the ability of cannabinoids to increase the formation of prostaglandins in tumor cells has been demonstrated, the consequence of such an effect is still unclear. For example, AEA and its metabolically stable analogue, met-AEA, cause apoptotic death of neuroglioma cells by a mechanism involving de novo expression of COX-2 (Hinz et al, 2004). Of note, since elevations of COX-2 mRNA and prostaglandin E2 (PGE2) levels were observed at the time of maximum apoptosis and the pro-apoptotic action of met-AEA was mimicked by PGE2 (Hinz et al, 2004), it is conceivable that increased levels of this metabolite may play a role in the antitumor action of endocannabinoids. In contrast with these observations, met-AEA increases murine lung cancer growth by inducing COX-2 expression and elevation of PGE2 levels at the site of the tumor (Gardner et al, 2003), indicating that the type of response to prostaglandins may be tumor-specific. Of note, modulation of COX-2 expression by endocannabinoids occurs via a receptor-

E. Modulation meatabolism



Beside targeting cannabinoid receptors directly, the use of â&#x20AC;&#x153;indirectâ&#x20AC;? agonists, i.e, compounds that act by inhibiting the degradation of endocannabinoids has been proposed as promising therapeutic strategy against cancer (Bifulco et al, 2004; Nithipatikom et al, 2005). In vitro findings from Ligresti and colleagues demonstrated in 2003 that differentiation of the colorectal cancer cell line, CaCo-2, into noninvasive cells results in increased fatty acid amide hydrolase (FAAH) expression, decrease in endocannabinoid levels and no responsiveness to cannabinoids. Modulation of the endogenous levels of endocannabinoids by the selective FAAH inhibitor, arachidonoyl-serotonin (AA-5-HT), inhibited CaCo-2 cell proliferation and such an effect was antagonized by the CB1 antagonist SR141716A (Ligresti et al, 2003). These data were also confirmed in vivo, where pharmacological enhancement of endocannabinoid levels (through inhibition of endocannabinoid hydrolysis) reduced the growth of tumor xenografts induced by the subcutaneous injection of rat thyroid transformed cells (Bifulco et al,


Cancer Therapy Vol 6, page 109 2004) as well as the development of precancerous lesions in the mouse colon (Izzo et al, 2007).

antitumor activity of cannabinoids have been performed by using immune-deficient mice, it has been demonstrated that intraperitoneal administration of THC to immunecompetent mice leads to an accelerated growth of tumor implants in murine lung cancer models (Zhu et al, 2000). Such a mechanism has been related to the CB2-dependent inhibition of the ability of antigen-presenting cells and T cells to generate alloreactivity (Zhu et al, 2000). It should be noted that immunosuppressive effects of cannabinoids seem to be evident mainly in tumors that express low levels of cannabinoid receptors and are resistant to cannabinoid-induced cytotoxicity (McKallip et al, 2005), thus indicating that the balance between tumor progression and regression might critically depend on the intrinsic capacity of a specific type of tumor to respond to cannabinoid treatment. The demonstrated ability of the immune system to affect the pathogenesis and development of cancer should be also taking into consideration to better understand the mechanism of cannabinoid action. For example, many tumor types are characterized by the presence of host leukocytes in tumor zones and in peritumoral regions, a process caused by tumor-derived chemokines, such as MCP-1 and RANTES, which are functional in inducing cell migration. Leukocytes then respond to the tumor by secreting IL-1$, IL-6 and TNF-" which, in turn, induces proliferation of the tumor cells and up-regulation of the VEGF gene (Angelo and Kurzrock, 2007) (Figure 3). These proinflammatory cytokines also promote upregulation of COX-2 in stromal cells which, in turn, may contribute to cancer development by producing adhesion molecules and activating neoangiogenesis, particularly through the production of prostanoids (Cianchi et al, 2004). PGE2 has been implicated in carcinogenesis (Watanabe et al, 1999; Sheng et al, 2001) and thromboxane A2 has been reported to activate angiogenesis and tumor progression (Daniel et al, 1999), suggesting that pathophysiologic changes involved in chronic inflammation may facilitate cancer development and progression. Although cannabinoid-based drugs have been proposed as anti-inflammatory therapeutics (Klein, 2005), to the best of our knowledge, no substantial evidence has been provided on the relationship between the endocannabinoid system and the regulation of COX-2 signaling in adjacent nontumour tissues as well as on the biological relevance of such an effect. With regard for the type of response induced by cannabinoids, in terms of COX-2-stimulating or -inhibiting effect, contrasting evidences have been reported in several inflamation models. For example, the synthetic derivatives HU-210 and WIN-55,212-2 were able to inhibit COX-2 upregulation and PGE2 release in an animal model of arthritis (Mbvundula et al, 2006), while cannabidiol, a nonpsychoactive analog of THC, showed opposite effects in the rat paw edema (Costa et al, 2004). Furthermore, nanomolar concentrations of endogenous or synthetic cannabinoids induce COX-2 in mast cells derived from sensitized guinea-pigs (Vannacci et al, 2004), whereas micromolar doses of CB1 and CB2 synthetic agonists have been shown to inhibit COX-2 expression in bovine chondrocytes (Mbvundula et al, 2005).

IV. Relevance of the antiangiogenic, anti-inflammatory and immunoregulatory functions of cannabinoids in tumor progression A. Angiogenesis Angiogenesis, i.e, the process of blood vessel formation in adults, is essential for supplying nutrients to the tumor (Folkman, 2002) and much effort has been directed towards the development of antiangiogenic agents that disrupt this process. The prototype compound of this drug class is bevacizumab, an antibody against VEGF that has been licensed for the first-line treatment of metastatic colorectal cancer, in combination with 5-fluorouracilbased chemotherapy (Ellis, 2005). Vascular endothelial cells express functional CB1 (Liu et al, 2000) and CB2 (Blazquez et al, 2003) receptors and the anticancer effect by cannabinoids has been associated with cannabinoid receptor-mediated deregulation of tumor angiogenesis in a variety of cancer models, in vitro and in vivo (Pisanti et al, 2007). Concerning the molecular mechanism accounting for the antiangiogenic properties of cannabinoids, met-AEA significantly inhibited the expression of the VEGF and its receptor, flt-1/VEGFR-1, in rat thyroid carcinoma implanted in athymic mice, and these effects were antagonized by the selective CB1 antagonist, SR141716A (Portella et al, 2003). On the other hand, selective activation of the CB2 receptor by JWH-133 has been shown to be responsible for the antiangiogenic action of this compound in malignant glioma, which included the direct inhibition of vascular endothelial cell migration and survival, as well as the decrease in the expression of the pro-angiogenic factors, VEGF and angiopoietin-2 (Blazquez et al, 2003). Finally, local administration of the mixed CB1/CB2 agonist, WIN-55,212-2, or the selective CB2 agonist, JWH-133, induced a considerable growth inhibition of malignant tumors generated by inoculation of epidermal tumor cells into athymic mice (Casanova et al, 2003). Of note, such an effect was associated to the decrease in EGF receptor mRNA levels as well as receptor activation and in the expression of VEGF, placenta growth factor, and angiopoietin-2 (Casanova et al, 2003), suggesting that inhibition of angiogenesis plays a significant role in the antitumor action of cannabinoids (Figure 3).

B. Inflammation and immune system regulation It is widely recognized that natural and endogenous cannabinoid derivatives affect most of the components of the immune response system and regulate cytokine signaling (Klein, 2005; Massi et al, 2006). Cannabinoids may be immunosuppressive with the consequent inhibition of host antitumor immunity (Srivastava et al, 1998), a particular aspect of cannabinoid action that may represent a serious drawback in their eventual use as antitumor drugs. While most of the studies investigating the in vivo


Fogli and Breschi: Cannabinoids and cancer

Figure 3. Cannabinoid action and the heterotypic cell biology view of cancer. Inflammatory cytokines, chemokines and lymphocytes represent critical elements of autocrine and paracrine signaling networks involved in tumor growth and metastasis. Tumor-derived chemokines, such as MCP-1 and RANTES, induce leukocyte cell migration into the tumor from the circulation, thus establishing the population of tumor-associated macrophages (TAMs). TAMs respond to the tumor by secreting various inflammatory cytokines including IL-1$, IL-6, and tumor necrosis factor-(TNF)-" which, in turn, promote proliferation of tumor cells, up-regulation of the VEGF gene, induction of COX-2 and prostaglandin E2 (PGE 2). VEGF and PGE2 released from tumor cells can then act on endothelial cells causing angiogenesis. TAM may also favor tumor escape from immune surveillance by producing immunotoxin molecules that induce dendritic cell inactivation. Stromal fibroblast fractions, named carcinoma-associated fibroblasts (CAFs), secrete elevated levels of stromal cell-derived factor 1 (SDF-1, also called CXCL12). This cytokine enhances CXCR4 receptor-mediated tumor progression trough recruitment of circulating endothelial progenitor cells (EPCs) into the tumor mass, angiogenesis stimulation, direct stimulation of tumor cells and homing of metastatic cells to specific organs. CB: a cannabinoid ligand.

Such discrepancies may be ascribed to a number of factors including the nature of the agonists, the drug concentration employed, the evidence or not of a receptor-mediated mechanism and the eventual receptor subtype involved (Croxford et al, 2005; Bifulco et al, 2006). The cross-talk between nitric oxide (NO) and COX systems in the surrounding tissue has been recognized as one of the biological events that perpetuate the consequence of oncogenic stimuli (Hussain et al, 2003). The nuclear factor-!B (NF-!B), a transcription factor that modulates the expression of several genes involved in apoptosis and in cytokine production (Gloire et al, 2006), represents a critical point of the potential synergism between COX-2 and the inducible form of nitric oxide synthase (iNOS). In several cell lines, particularly immune cells, NF-!B is a potent inducer of iNOS and COX-2 (Mollace et al, 2005) and the NO-derived peroxynitrite is able to induce NF-kB production which, in turn,

perpetuates iNOS and COX-2 activities. Furthermore, NF!B activates VEGF and IL-8 gene expression in tumor cells and inhibitors of NF-!B decrease the levels of VEGF produced in leukemia and glioma (Angelo and Kurzrock, 2007), thus suggesting that NF-!B targeting may elicit antitumor effects. It has been reported that THC inhibits NF-!B/Rel activation and iNOS expression in murine macrophages (Jeon et al, 1996), and activates NF-!Bdependent apoptosis in dendritic cells, thymocytes and splenocytes (Do et al, 2004). WIN55212-2 is able to reduce the release of IL-8 from the human epithelial cells by inhibiting the activation of NF-!B and such an effect may be mediated through the CB2 receptors (Ihenetu et al, 2003; Mormina et al, 2006). Since the deregulation of the COX-2/iNOS balance over a prolonged period of time may favor tumorigenesis (Hussain et al, 2003), these findings suggest that an interference with such a faulty


Cancer Therapy Vol 6, page 111 mechanism by cannabinoids may be taken into account as a part of their antitumor action. Finally, it has been demonstrated that carcinomaassociated fibroblasts (CAFs), i.e, stromal fibroblast fractions extracted from invasive human breast carcinomas, release elevated levels of stromal cell-derived factor 1 (SDF-1, also called CXCL12), which play a pivotal role in tumor growth and angiogenesis (Orimo and Weinberg, 2006). Such a chemokine is expressed in typical metastatic sites of breast cancer and acts on CXCR4-expressing breast tumor cells to direct their motility. Mostly important, CB2 activation yields to the inhibition of the CXCL12/CXCR4-induced chemotaxis and transendothelial migration of leukemia cells (Ghosh et al, 2006), thus highlighting the potential of cannabinoids to disrupt a mechanism that has been demonstrated to be relevant in directing metastatic organ preference (Muller et al, 2001) (Figure 3).

glioma cells they inhibit Akt (Gomez del Pulgar et al, 2002b).

VI. Conclusive remarks Beside the well-regognized palliative effect of cannabinoid derivatives observed in cancer patients, a great deal of preclinical data indicate that pharmacological interventions tailored on the endocannabinoid system can significantly affect a variety of signaling molecules in the neoplastic cell circuit that collaborate to the multistep process of tumorigenesis. Furthermore, the interference with cross-talk signals between tumor cells and critical elements in tissues surrounding the tumor mass may be also part of the molecular mechanism of action of these compounds. Nevertheless, much effort should be dedicated to better define the cellular and molecular context that might have the potential to improve the ability to predict the likelihood of cannabinoid response. With regard for this, investigations in the cannabinoid research field should be always performed by using specific cannabinoid receptor antagonists, in order to delineate the precise mechanism of action of these compounds in terms of CB1- and CB2-mediated effects (Table 1) or non-CBmediated effects. Furthermore, strategies by which tumor cells can be profiled for their genomic background may be helpful in studying the molecular signature that matches the mechanism of action of cannabinoids. One of the most intriguing and clinically relevant aspects in the cannabinoid action regards the relationship between ligand concentration at the target site and the type of biological response obtained. In fact, micromolar levels of cannabinoid agonists promote a receptor-mediated decrease in tumor cell proliferation, while in the nanomolar range of concentrations, that seems more likely to reflect a therapeutically relevant situation (Grotenhermen, 2004), these compounds may also produce the opposite effect. This notion together with the fact that sustained, but not acute, exposure to cannabinoids is responsible for apoptosis induction, suggest that transdermal delivery should be preferred to inhalation or oral administration. To date, while intratumoral (Sanchez et al, 2001; Casanova et al, 2003) or peritumoral (Blazquez et al, 2006) injection of these compounds has been performed in several in vivo studies and very recently, THC delivery by intracranial route has been used in patients with glioblastoma multiforme (Guzman et al, 2006), systemic administration of cannabinoids have not been adequately investigated in the preclinical setting. Development of less lipophilic cannabinoids should be also encouraged in order to allow their parental use and to reduce receptor-independent effects, a mechanism observed when using micromolar concentrations of these compounds. Finally, in vitro investigations of combination schedules that allow obtaining increased sensitivity and/or decreased resistance to the chemotherapeutic agents routinely used in the management of cancer patients may provide information that would facilitate the design of randomized, add-on studies aimed to evaluate the clinical benefit of cannabinoid-based anticancer therapies.

V. Selective action towards tumor cells Fighting cancer by selective targeting of tumor cells is one of the most promising strategies to improve the therapeutic index of anticancer treatments. Therefore, when envisaging the potential clinical use of novel anticancer strategies, the comparison of drug action towards tumor cells with respect to their normal counterpart, represents a critical point that should be carefully examined. Several reports indicate that cannabinoid action is selective for tumor versus nontumor cells (Bifulco et al, 2001; McAllister et al, 2005; Blazquez et al, 2006) and the presence of cannabinoid receptors and their expression levels are some of the fundamental elements which may provide a possible explanation for the selective effect of cannabinoid ligands. For instance, treatment of the human prostate cancer LNCaP cells with WIN-55,212-2 at 1-10 ÂľM for 24 and 48 h significantly decreased cell viability, while similar doses had no effect on the prostate epithelial PrEC cells (Sarfaraz et al, 2005). Of note in this study, expression levels of both CB1 and CB2 receptors were significantly higher in cancer cells than in normal cells (Sarfaraz et al, 2005). In addition, ligand-induced activation of CB2 receptors reduces human breast cancer cell proliferation, while the proliferation pattern of normal human mammary epithelial cells which do not express significantly this receptor subtype, was much less affected (Sarfaraz et al, 2005). A number of data support the hypothesis that cannabinoid receptors may regulate cell survival pathways differently in tumor and nontumor cells that express cannabinoid receptors, a further mechanism that could account for the selectivity of cannabinoid action. Indeed, despite the nontumorigenic cell line of human melanocytes, Hermes 2b, expressed CB1 receptors at levels comparable to the tumorigenic A375 cell line, the antiproliferative effect of cannabinoids was selective for tumor cells and was related to inhibition of the prosurvival protein Akt (Blazquez et al, 2006). Interestingly, in primary astrocytes (Gomez Del Pulgar et al, 2002a) and oligodendrocytes (Molina-Holgado et al, 2002) transfected with the CB1 cDNA, cannabinoids activate Akt, while in 111

Fogli and Breschi: Cannabinoids and cancer Table 1. Type of response obtained after treatment with cannabinoids and specific receptor subtype involved. Biological response Antiproliferative action

Tumor type Breast, prostate

Receptor subtype CB1

Melanoma Colon


References De Petrocellis et al, 1998; Melck et al, 2000 Blazquez et al, 2006 Ligresti et al, 2003

Apoptosis induction

Prostate Skin Glioma Leukemia


Sarfaraz et al, 2006 Casanova et al, 2003 Sanchez et al, 2001 McKallip et al, 2006

Mitogenic action

Astrocytoma, lung


Hart et al., 2004

Antiangiogenic effect

Thyroid Glioma Skin


Portella et al, 2003 Blazquez et al, 2003 Casanova et al, 2003

Inhibition chemotaxis



Ghosh et al, 2006


*Without using specific antagonists.

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Stefano Fogli


Fogli and Breschi: Cannabinoids and cancer


Cancer Therapy Vol 6, page 117 Cancer Therapy Vol 6, 117-130, 2008

General review of polysaccharopeptides (PSP) from C. versicolor: Pharmacological and clinical studies Review Article

King-Fai Cheng, Ping-Chung Leung* Institute of Chinese Medicine, The Chinese University of Hong Kong

__________________________________________________________________________________ *Correspondence: Prof. Ping-Chung LEUNG, DsocS(Hon), D.Sc, M.S, Director, Centre for Clinical Trials on Chinese Medicine 5/F, The CUHK Hong Kong Jockey Club School of Public Health Building, Prince of Wales Hospital, Shatin, NT, Hong Kong SAR; Tel: (852) 2632 2723/ 2252 8872; Fax: (852) 2686 8463/ 2632 5441; E-mail: Key words: Coriolus Versicolor (Yunzhi); polysaccharopeptide (PSP); immunomodulation; anti-tumor Abbreviations: biological response modifiers (BRM); Coriolus versicolor (CV); cyclophosphamide (CPA); human immunodeficiency virus (HIV); Interleukine-2 (IL-2); intraperitoneal (i.p.); National Center for Complementary and Alternative Medicine (NCCAM); Natural Killer Cells (NK cells); Office of Dietary Supplements (ODS); polysaccharopeptide (PSP); polysaccharopeptide Krestin (PSK) Received: 20 December 2007; Revised: 30 January 2008 Accepted: 5 February 2008; electronically published: February 2008

Summary In China, C. versicolor is named Yun Zhi (meaning “cloud-like mushroom”). C. versicolor is mainly used as an adjuvant in the treatment of cancer. The active principle derived from C. versicolor belongs to a new class of elements called biological response modifiers (BRM) which are defined as agents capable of stimulating the immune system and thereby, they express various therapeutic effects. The best know commercial polysaccharopeptide preparations of C. versicolor are polysaccharopeptide Krestin (PSK) and polysaccharopeptide (PSP). One of the most important functions of PSP and PSK is their immunomodulatory and anti-cancer actions. The present paper reviewed and summarized the pharmacological and clinical properties, as well as its background of Coriolus versicolor or PSP.

According to the record of Ben Cao Gang Mu (!"#$) written during the Ming Dynasty (1368-1644), there were over 120 strain of C. versicolor (Hyde and Adams, 1960). Recent literatures report that more than 270 medicinal fungi are used in traditional Chinese medicine for their preventive and/or curative effects (Ding, 1987; Ying et al, 1987). In the clinical practice of traditional Chinese medicine, C. versicolor is recommended for various types of cancers, chronic hepatitis, and infections of the upper respiratory, urinary, and digestive tracts (Jong and Yang, 1999; Li, 2003). In Asia, C. versicolor extract is available as a health supplement and can be purchased without a prescription. In both China and Japan, health authorities regard C. versicolor extract as a valuable adjuvant for combination chemotherapy or radiotherapy in the treatment of various cancers (Mizuno, 1999; Yan et al, 2000; Chu et al, 2002). The present paper reviews and summarizes the pharmacological and clinical properties, as well as its background of Coriolus versicolor or PSP.

I. Introduction Mushrooms have an established history of use in traditional oriental therapies. In Asian cultures, mushrooms are combined with herbal mixtures to treat cancer. The mushroom Coriolus Versicolor (C. versicolor) is a macrofungi belonging to the Basidiomycetes class, which encompasses about 20,000 and 25,000 known species (Gregory and Hirst, 1957; Hyde and Adams, 1960). In China, C. versicolor is named Yun Zhi (meaning “cloud-like mushroom”). Researches have found that this mushroom has antimicrobial, antiviral and anti-tumor properties (Jong and Birmingham, 1993; Ulrike et al, 2005). Nowadays C. versicolor is mainly used as an adjuvant in the treatment of cancer (Tsang et al, 2003; Hattori et al, 2004). It has been demonstrated that extracts obtained from this mushroom are likely to show stimulatory effects on the immune system and to inhibit the growth of cancer cells. Because of these properties, Yun Zhi is called a biological response modifier (BRM) (Leung et al, 2006).


Cheng and Leung: General review of polysaccharopeptides (PSP) from C. versicolor PSP and PSK are light or dark brown powders that are soluble and stable in hot water. They are chemically similar and posse similar physiological activity profiles, PSK and PSP differ mainly in the presence of fucose in PSK and rhamnose and arabinose in PSP (Table 1).

II. Composition of C. versicolor The composition of the polysaccharopeptide is depending on the source of the material and the method of extraction used. For example, polysaccharopeptide Krestin (PSK) is obtained from the extraction of C. versicolor (CM-101) strains, while polysaccharopeptide (PSP) obtained from the extraction of C. versicolor (Cov-1) strains (Chu et al, 2002; Zhou et al, 2007). The active principle derived from C. versicolor belongs to a new class of elements called BRM. BRM are defined as agents capable of stimulating the immune system and thereby, they express various therapeutic effects (Leung et al, 2006). Polysaccharides linked to a small protein (or peptide) are at the base of this immunomodulatory activity. This “polysaccharidepeptide” is termed polysaccharopeptide, or PSP in its abbreviated form. The strain used for PSP production is called Cov-1 and was obtained through careful selection of over 80 wild strains collected from various areas in China.

IV. Safety studies The toxicological experiments conducted with a variety of animals (dogs, monkey and guinea pigs) had shown negative results for acute, genetic, reproductive and chronic toxicity.

A. Acute toxicity The LD50 value of PSP is 26-300.36mg/kg for mice administered by intraperitoneous injection. The highest daily tolerant dose was over 18-20g/kg for mice (Jin, 1999; Ze et al, 2003). According to the “Procedures for Toxicological Assessment on Food Safety Acute Toxicity Test (GB15193.3-94)”, PSP is considered to be non-toxic (Procedures for Toxicological Assessment on Food Safety Acute Toxicity Test GB15193.3-94).

III. The difference between PSP and PSK

B. Long-term toxicity

The best know commercial polysaccharopeptide preparations of C. versicolor are PSK and PSP. Both products are obtained from extraction of C. versicolor mycelia (Figure 1). PSK is a Japanese product, while PSP is Chinese product which was first isolated in 1986 (Yang and Van, 1986). Both products have similar physiological activities but are structurally different. PSK is produced from CM-101 strain of C. versicolor, the extraction is done by salting out with ammonium sulfate from the hot water extract. PSP is produced from Cov-1 strain of C. versicolor (Figure 2), the extraction is done by using alcoholic precipitation from the hot water extract.

Subchronic and chronic toxicity studies were done by Jian et al who continuously administrated 4 oral doses (0, 1.5, 3.0, and 6.0 mg/kg) to 80 rats for 62 days. The results showed no toxic symptoms or death. Neither were there any obvious toxic changes in blood and serum biochemistry (Jian, 1999). Rats and monkeys were administrated with PSP by oral at a dose of higher than 200 and 100 times of human dose separately daily for 6 months, no abnormal changes in development, hematology, blood chemistry, and electrocardiography were observed (Zou et al, 2003).

Figure 1. Trametes (Coriolus) versicolor (


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Figure 2. Typical partial structures of polysaccharide portions of the polysaccharide peptide (PSP) of COV-1 strain of Coriolus versicolor (Zhou and Yang, 1999).

Table 1. The differences of PSP and PSK

Source Produced in Appeared on the market in Extract method

Physicochemical properties

Chemical composition

Biological properties


PSP Mycelia of Coriolus versicolor Cov-1 strain China 1987 Recovered by alcoholic precipitation from hot water extract Brown in color; soluble in water; insoluble in organic solvents; stable to heat; mean MW=100kDa (1!3) !-glucan branched at 4’ and 6’ positions

PSK Mycelia of Coriolus versicolor CM-101 strain Japan 1977 Recovered from hot water extracts of the biomass by salting out with ammonium sulfate Brown in color; soluble in water; insoluble in organic solvents; stable to heat; mean MW=100kDa 18%--38% w/w protein (1!3) !-glucan branched at 4’ and 6’ positions rhamnose, arabinose fucose In vitro and in vivo In vitro and in vivo immunorestorative and antiimmunorestorative and antitumor activities tumor activities Sakagami and Takeda, 1993; Ng et al, 1999; Kidd, 2000; Cui and Yusuf, 2003

Accumulating evidence suggested that the polysaccharopeptides were nontoxic even when administered at several times the therapeutically effective dosage and over extended periods. Extended use of PSP at 100-fold the normal clinical dose had not induced acute and chronic toxicity in animals (Ng et al, 1997).

The results of chromosomal aberration tests and cytogenetic lesions in mice showed that the number of chromosomes had not changed in PSP treated groups even at the high dose rate 126 mg/kg.

D. Reproductive test The possible effects on male and female reproductive physiology and embryonic development were also examined. Results from these studies suggested that PSP could not cause sperm aberration at a dosage 100 times higher that the usual clinical dose (Qian et al, 1993). The lack of deleterious effects on ovarian follicular development, ovulation, pregnancy and embryo development in mice was also demonstrated (Ng et al, 1997). Polysaccharopeptides appear to be safe during pregnancy. No adverse effects of PSP had been observed in female reproductive and embryonic development in mice (Ng et al, 1997).

C. Genetic toxicity (Zhong et al, 1999) Mutagenicity of PSP was assessed with the Ames test and with the chromosomal aberration test of bone marrow cells in mice. It was concluded that PSP showed no evidence of mutagenic or cytogenetic activity. Cytotoxicity tests of PSP with V79 Chinese hamster cells in vitro also showed no toxic effects against the V79 cell line. In vivo micronucleus tests to assess the cytogenotoxicity on mammalian somatic cells, the results indicated that PSP showed no evidence of mutagenic potential.


Cheng and Leung: General review of polysaccharopeptides (PSP) from C. versicolor Researches demonstrated that PSP can antagonize the immunosuppression caused by such chemotherapeutic agents (Qian et al, 1997; Li, 1999). The administration of PSP (at a dosage of 2 grams/kg day) on cyclophosphamide-induced immunosupressed rats demonstrated that the mushroom extract was effective in restoring their immune system. It did so by stimulating lymphocytes proliferation, NK cell functions, and the growth of spleen and thymus where lymphocytes mature and transit (Qian et al, 1997).

V. Pharmacological actions A. Immunomodulatory effects The immunological activities of PSP and PSK have been extensively investigated both in vitro and in vivo. PSP strengthening the immunological functions was studied. Its anti-tumor effect seemed to be mediated more through immunomodulatory regulation rather than by direct cytotoxicity as was the case for most anticancer drugs currently used. Numerous reports had demonstrated the ability of PSK and PSP to active cellular and humoral components of the host immune system. In addition, these polysaccharides had been shown to inhibit the growth of tumor cell lines and to have in vivo anti-tumor activity (Tzianabos, 2000). The effect of PSP on the phagocytic functions had been tested in normal ICR mice. It was determined that the carbon clearance rate of the groups given oral doses of 0.5-1.5g PSP/kg or intraperitoneal (i.p.) injections of 100, 200, 400mg/kg was similar to that of groups treated with acanthopanax (300mg/kg). Regardless of the route of administration, PSP increased the carbon clearance rate in mice and it suggested that PSP could increase the phagocytic function of normal animals (Yang et al, 1993). It was reported that PSP in different concentrations promoted the proliferation of T-lymphocytes both in human peripheral blood and mouse splenocytes (Li, 1999). PSP augmented T-helper cell (CD4+) activation, and also increased the ratio of CD4+/T suppressor (CD8+) production (Li, 1999). It appeared that the basic mechanisms for PSP to inhibit tumor cells included the activation of 1) macrophages, 2) natural killer cells and 3) T-helper cells (CD4+) which induce T-killer cells, antibody production, and interleukins (Li, 1999). Studies were conducted in vitro with numerous cell lines to investigate the immunomodulatory effects of PSP. It was recognized that Interleukine-2 (IL-2) plays a critical role in immune defense against tumors, because it is a potent inducer of activation of NK cells. In a study designed to evaluate the anti-tumor potential of IL-2 and PSP, it was demonstrated that the combination of the two agents had the most dramatic anti-tumor effect (Mao et al, 1996). Moreover, PSP could effectively stimulate the generation of Interferon-" (IFN-") and markedly improve the production of IFN-# (Yang et al, 1999). A study demonstrated that PSP did not exert a direct cytotoxic effect on tumor cell lines but rather stimulated macrophages, thus strengthening the hypothesis that C. versicolor anti-tumor effects were mediated by an immunomodulatory mechanism (Liu et al, 1999). In vivo studies had revealed that PSP usually had no significant immunological effects on a normal host, but could restore a depressed immunological responsiveness as seen in cancer or with chemotherapy (Chu et al, 2002). PSP can also counteract the depressive effect of cyclophosphamide (CPA) on the white blood cell count and interleukin-2 production. CPA has become the leading drug in the clinical treatment of cancer, particularly for lymphomas, leukemia and solid tumors. This drug is cytotoxic, kills rapidly dividing neoplasic and normal cells, but has deleterious effect on the immune system.

B. Anti-tumor effects in vivo Many studies have been done since 1980â&#x20AC;&#x2122;s to demonstrate the effectiveness of PSP, and its counterpart PSK (Table 2). The anti-tumor actions are predominantly considered to be host-mediated. The preliminary determination of antitumor activity is usually assessed by a bioassay system normally using Sarcoma 180 in mice and B16 melanomabearing mice through transplantable animal tumor (Yang et al, 2007). Several hundreds of polysaccharides or polysaccharide-protein complexes have been screened for their anti-tumor activity, and three of them, namely schizophyllan, lentinan and protein-bound polysaccharides (PSK and PSP), have been used clinically (Kobayashi et al, 1993). PSK has been successful in postoperative treatment of respectable cancer in humans, increasing survival rates (Ito et al, 2004). Protein-bound polysaccharide (PSK) prepared from the cultured mycelia of Coriolus versicolor in Japan demonstrated a significant anti-tumor effect against allogeneic tumors such as Sarcoma 180 and Ehrlich carcinoma of experimental animals by both intraperitoneal and oral administration (Taniguchi et al, 1984; Kobayashi et al, 1993). Researches indicated that PSK and PSP can suppress pulmonary metastasis from induced sarcomas, induced prostate cancer, lymphatic metastasis of mouse leukemia P388, and that both could prolong the survival period in spontaneous metastasis model (Xu, 1999). Polysaccharopeptides from C. versicolor influence cancer metastasis in a number of ways: 1) by suppression of intravasation through the inhibition of tumor cells infiltration, 2) by suppression of tumor cell attachment to endothelial cells through the inhibition of tumor cellinduced platelet aggregation, 3) by suppression of tumor cell migration after extravasation through the inhibition of tumor cell mobility, and 4) by suppression of tumor growth after extravasation through the inhabitation of angiogenesis, the modulation of cytokine production and the augmentation of effector cell function (Liu et al, 2004). In addition to anti-cancer effect, animal study also demonstrated that PSP had central analgesic effect (Yin et al, 2002). In vitro antitumour study, the C. versicolor extract was also found to tumour-selectively and dosedependently inhibit the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway (Lau et al, 2004; Zhou et al, 2007). However, both immune property and anticancer potency of polysaccharopeptide-Coriolus versicolor were affected by the fermentation duration of the fungi (Lee et al, 2006).


Cancer Therapy Vol 6, page 121 Table 2. Some animal anti-tumor studies.

1 2

Animal Mice

Test article PSP

Administration By oral

Nude mice


















Dosage 1-2g/kg/day

Duration Results Reference 15-20 days Inhibited growth of Zeng et al, 1993 human lung adenocarcinoma by 50-70% 50mg/kg/day 3 weeks Inhibited the growth Wang et al, 1993 of Lewis lung cancer by nearly 45% 2 weeks Decreased the Dong et al, 1996 incidence of tumor growth, tumor size in control group about 35 times bigger than the PSP group 150mg/kg 3 weeks Prolonged the survival Fujii et al, 1995 twice a week period of mammary tumor-bearing rats 2, 5 g/kg 4 weeks Tumor inhibition rate Zeng et al, 1999 daily of 77 and 63% in treated groups 5-20mg/kg 3-15 days Exerted inhibitory Wei et al, 1996 effects on experimental and clinical tumors 1.2g/day 21 days Oral PSP did not Kanoh et al, prevent CTX2-induced 1994 cytopenia in rats

Coriolus versicolor polysaccharides Cyclophosphamide

When PSK, obtained from cultured mycelia of Coriolus versicolor, was administered in mice, in vivo tumor-induced angiogenesis was suppressed (Mao et al, 2001). However, some animal experiments could not demonstrate a specific effect of polysaccharopeptides extracted from the mushroom C. versicolor. For example, for a long-term control of brain tumors, intraperitoneal injection of 2mg of PSP with radiotherapy did not increase radiation efficacy (Mao et al, 1996). Another study investigated the effect of low-dose administration of IL-2 or PSP alone in a herpes virus type-2 transformed murine tumor in mice. The results indicated that IL-2 or PSP alone could slow down tumor progression, but the combination of the two modalities had no synergistic effects on tumor growth (Liu, 2001). These results warranted further investigation to determine if PSP could be effectively used for all types of tumors, or cancers, in which immunosuppression was a prominent feature. In general, it seemed that oral administration of PSP reduced the incidence of tumor or prolonged the survival period, following chemical carcinogen-induced, radiationinduced and spontaneously developed animal cancer models. Coriolus versicolor (CV) regulated cytokine production and possessed both anti-tumor and immunopotentiating activities (Ho and Lau, 2004). The

main mechanism might be an anti-teratogenic effect attributed to free radical trapping and prevention of chromosome injury, coupled to an immunomodulating effect linked to the modulation of cytokines production and effect cell function. Table 3 summaries the possible mechanisms of anticancer effects of PSP (Hobbs, 2004).

C. Antimicrobial effects Besides its documented anti-cancer effects, polysaccharopeptides from C. versicolor could be useful for other conditions as well. PSK has been demonstrated to have antiviral activities against ectromelia virus (Taniguchi et al, 1984) and cytomegalovirus infections (90) and human immunodeficiency virus (HIV) (Zou and Zhu, 2003). In China, C. versicolor is also considered useful in the treatment of hepatitis. Preliminary studies had indicated that PSP was effective in protecting liver from hepatotoxins in laboratory animals (Song and Liang, 2000; Zou et al, 2003). As with antiviral action, the multivalent manner in which PSP acts to protect liver cells and detoxification mechanisms, demonstrates the potential usefulness of C. versicolor extracts to prevent damages from reactive compounds which could be carcinogenic agents (Jong and Yang, 1999; Yeung, 1999).


Cheng and Leung: General review of polysaccharopeptides (PSP) from C. versicolor Table 3. Mechanisms of anticancer effects of extracts of C. versicolor. • Inhibition of DNA of tumour cells • Enhancement of cytokine production • Antitumour activity in wide range of animal systems • Tumour cell killing effect • Inhibition of carcinogen-induced cancers in rats • Antioxidant effects in tumour-bearing rats • Induction of apoptosis • Antiproliferative effect on many cancer cell lines • Anti-invasion effects • Angiogenesis effects • Tumouricidal and cytotxicity effects • Antimetastic activity • Immunoprotective effects during radiation and chemotherapy

have an impact on the survival rate of different types of cancer. PSK used in Japanese trials significantly extended the survival rate at five years for stomach, colorectal, esophagus, nasopharynx, breast and lung cancers. Study also showed that PSP has effect of cancer prevention and development (Zhou et al, 2001). The following were the clinical trial results selected from research papers conducted in Japan (Table 4, 5).

D. Anti-nociceptive effects In an animal study, two different mice pain models were used to investigate the anti-nociceptive effects of PSP. The results demonstrated that PSP could induce hyperalgesia. PSP-induced hyperalgesia was related the activation of peritoneal macrophages and mast cells and, hence, increased the release of inflammatory mediators (Chan and Yeung, 2006).

A. Breast cancer

VI. Clinical studies

As early as 1970, breast cancer patients received long term combination chemotherapy along with C. versicolor extract immunotherapy. Addition of the extract to the regimen significantly extended the survival rate. In a large trial done in 1995 in Japan, the survival rate at 10 years was 81% for the PSK plus chemotherapy group which the rate fell to 64% for the group that used chemotherapy alone. The study concluded that immuno-chemotherapy with mushroom extract could improve the prognosis of patients with resectable breast cancer with vascular invasion. Mild and well tolerated side effects such as leukopenia and nausea were observed in 5 out 227 patients (Iino et al, 1995).

The therapeutic use of PSP or PSK as an adjuvant therapy in cancer treatment has been substantiated by numerous clinical trials. Table 6 summarizes the methodological parameters and the results of some these trials. As an adjuvant in the treatment of many types of cancers, Yun Zhi (PSP) was subjected to Phase I, II and III clinical trials in Shanghai, China. The results showed that addition of PSP to radiotherapeutic or chemotherapeutic protocols can greatly improve the quality of life of cancer patients and reduce chemotherapy-induced side effects (Zhong et al, 2001), because PSP alleviated weakness, anorexia, vomiting, dryness of throat, spontaneous sweat and pain symptoms. In addition to symptoms improvement, polysaccharopeptides from Yun Zhi also Table 4. Randomized controlled trials for breast cancer. Patients 914 cases Standard or Radical mastectomy


227 cases operable breast cancer with v+ and/or n+ involvement 134 cases Typed as HLAA, HLA-B and HLA-C

Treatment 1.Patients(ER+tumours) chemo +/- tamoxifen 2.Patients (ER- tumour) Chemo +/- PSK Chemo (n=77) Chemo +LMS (n=76) Chemo +, PSK (n=74)

Operable with v+ and/or nv+

Previously randomised into two groups(ref 23): 1. Chemo 2. Chemo +PSK Each group stratified by HLA type B40+ or B40-.


Outcome Longer overall survival for patients in Stage IIA T2N1 cancer ER- and node-negative treated with chemo + PSK compared with other ERsubgroups without PSK. Risk ratio lower in the chemo+ PSK group. Overall and diseasefree survival rates not significant for all groups.

Refs Toi et al, 1992

Years for chemo + PSK group: HLA-B40+ : 100%; HLA-B40- : 76% and 55%, respectively. Significant difference at p =0.05.

Yokoe et al, 1997

Iino et al, 1995; Yokoe et al, 1997

Cancer Therapy Vol 6, page 123 Table 5. Selected randomized controlled trials for colorectal cancer. Patients 55 cases 56 controls

Multicentre 221 cases 227 controls Total 207 134 cases 67 controls 6 withdrew

Stage Advance (II/III)

Primary (II/III)

Total 205 137 cases 68 controls

Primary (II/III)

Colon cancer with lymph node metastasis Total 441 220 cases 221 controls

Dukes A:7% B:45.5% C:47.3%

202 primary colon cancer patients 5-FU + PSK:99 5-FU alone:103

Primary colon cancer

Treatment 1. Surgery + placebo 2. Surgery + PSK (3gm/day for 2 months; 2g/day for 24 months; 1 gm/day thereafter) 1.Chemo 2.Chemo+ PSK (3g/day for 3 years) 1. Chemo 2.Chemo+ PSK (3g per day for >2 yrs)

All patients received Mitomycin-C postsurgery. 1. Chemo 2. Chemo + PSK (3g/ day) Both treatments for 2 yrs All patients received chemo after surgery for 3-4 weeks, then 10 courses of treatment. 1. PSK 4 weeks then 4 weeks chemo. 2. 4 weeks rest then 4 weeks chemo. 99 underwent adjuvant treatment with PSK and 5-FU (PSK group), 103 were treated with 5FU alone (5-FU group).

Outcome 8-yrs survival rate significant in the PSK group( p<0.05); Disease-free interval (p<0.05)

Refs Torisu et al, 1990

Disease-free interval and survival significantly better for PSK in the colon group (p<0.05 in both) Overall survival rate higher in the PSK group but not significant (p=0.21). 3-year disease-free survival rate significantly higher in the PSK group (p=0.02). Stage III. Patients 3-year overall and disease-free survival rates in the PSK significant (p=0.02; p =0.01) 5-year overall survival rate significantly higher in the PSK group (p<0.016, p <0.056 respectively). Stage III patients: Overall and 5-year disease-free survival in PSK group (p <0.003; p<0.002). Seven- year survival rate Significantly higher in the PSK group (p=0.019). Overall survival or disease-free rates not significant.

Mitomi et al, 1992

The presence of diffuse nuclear accumulation-type !-catenin activation identifies patients with colon cancer who respond better to immunotherapy with polysaccharide K.

Yamashita et al, 2007

Ohwada et al, 2003

Ohwada et al, 2004

Ito et al, 2004

5-FU = 5-fluorouracil

patients in three clinical trials. The aim of the metaanalysis was to evaluate the effect of PSK as adjuvant immunochemotherapy for patients with curatively resectal colorectal cancer. The results suggested that adjuvant immunochemotherapy with PSK can improve both survival and disease-free survival of patients with curatively resectal colorectal cancer.

B. Colorectal cancer C. versicolor extract was assessed for its potential anticancer activity in patients with advanced colorectal cancer (stages III and IV) (Torisu et al, 1990). PSK was given at 3 grams/day for two months after surgery, followed by 2 grams/day until the end of the second year and 1 gram/day thereafter. This study found that the leukocyte activity of the PSK group was remarkably enhanced. Notably, polymorphonuclear leukocytes from PSK-treated patients showed enhancement in their activities, such as random and/or chemotactic locomotion, and phagocytic activity, when compared with those of the control group. The survival rate of the PSK group reached 40%, a net improvement over the 25% rate registered for the placebo group. From this study, it was concluded that PSK could be useful as a maintenance therapy for patients after their curative surgical operations for colorectal cancer. Sakamoto and colleagues performed in 2006 a metaanalysis of colorectal cancer, which involved 1094

C. Gastric and esophageal cancer Stomach cancer is a major cause of mortality in Japan and China and, for that reason, has been the object of many clinical trials with polysaccharopeptides from C. versicolor. Trials done in the 1970s and 80s had evidenced a better survival rate at two or three years. More recent clinical studies, done in the 1980s and 90s, established that PSK could improve the survival rate at five years and beyond in stomach cancer patients, including some patients with advanced Stage III and IV cancer with metastasis (Kidd, 2000).


Cheng and Leung: General review of polysaccharopeptides (PSP) from C. versicolor Table 6. Assessment of methodological parameters on clinical trials of PSP or PSK. Reference

Mitomi et al, 1993

Jadad Study design Score (max = 5) 2 Randomized control, followup study

Nakazato et al, 3 1989

Ichihashi et al, 3 1987

Sample size

Inclusion/Exclusion criteria

Patients younger than 75 years of age with stage III or IV colorectal cancer and consented form obtained were included Patients with WBC<4000/mm3, PLT<100,000/mm3, TP<6.0g/dl, Alb<3.0g/dl, A/G<1, SGOT/SGPT>100U, urine protein (+), Crea>1.5mg/dl were excluded Randomized 262 gastric Patients with age<75 control, follow- cancer patients years, PPD(+), diagnose up trial with radical confirmed by pathology surgery were included. Patients who underwent any radiotherapy, chemotherapy, or immunotherapy or having multiple cancers, or any abnormal hematological findings were excluded Randomized 168 patients with Inclusions included: age control follow- stomach cancer <75 years, without up study multiple cancers, normal hematological findings and diagnosis confirmed by post-operation pathology

Guo, 2000


Double-blind placebocontrolled randomized study

Ze et al, 2003


Randomized controlled trial

Ebihara and 2 Minamishima, 1984

Randomized controlled trial

448 colorectal cancer patients treated with chemotherapy

34 patients with Completed conventional non-small cell treatment for advanced lung cancer NSCLC

Treatment schedule


227/221 patients were randomly assigned to control (chemotherapy for 6 months) and treatment (chemotherapy 6 months plus PSK 3g/day for 3 years)

The disease-free survival and the survival of the PSK group were better than those of the control group (Disease-free survival: p=0.0214, Survival: p=0.0272)

129/124 were randomly assigned to control (chemotherapy) and treatment (chemotherapy plus PSK 3g/day for 4 weeks)

The disease-free survival curves and overall survival curves of PSK treatment group were significant (p=0.018 and p=0.045) better than those of control group.

49/47/28 patients were randomly assigned to receive chemotherapy (CQ), PSK (3g/50kg) plus CQ for 13 months and no CQ and PSK treatment (as control). Patients received 28-day administration of PSP

Overall 7 year survival rate in PSK group higher than chemotherapy group but no significant difference.

After 28-day treatment, there was a significant improvement in blood leukocyte and neutrophil counts, serum IgG and IgM, and percent of body fat among the PSP, but not the control, patients (p<0.05) 201 patients with Age < 75 year and 137/68 patients The three-year, stage II or III historical confirmed treated with 3g PSK disease-free survival colorectal cancer colorectal cancer. Exclude: plus 300mg tegafur rate was 80.6% in underwent any /uracil or 300mg PSK group (p=0.02) radiotherapy, tegafur/uracil alone compared with 68.7% chemotherapy or for 2 years in the control group. immunotherapy. 25 advanced Advanced malignant Treatment group Quality of life and malignant cancer cancer patients without orally taken PSP 5g symptoms were patients immunotherapy each time 3 times a improved in PSPday for 2 months treated group plus symptoms compared with control treatment control group (p<0.01 Control group only and p< 0.05) gave symptoms control treatment


Cancer Therapy Vol 6, page 125 In a randomized clinical trial including 579 patients with gastric cancer receiving chemotherapy, C. versicolor extract was administrated orally as an adjuvant to surgery in a combination therapy to part of the group, at a daily dose of 3 grams for 1 year. Results from this study showed a significant increase in the 5-year survival rate for the PSK-treated group when compared with the other groups (Niimoto et al, 1988). In an additional study involving more than 260 patients who underwent surgery for stomach cancer at 46 hospitals in Japan, those who received PSK along with chemotherapy experienced a higher 5-year disease-free rate and a better 5-year survival rate than subjects who underwent chemotherapy alone (73% vs. 60%). The two regimens had slight toxic effects, consisting of nausea, leucopenia, and liver function impairment but no characteristic toxic effects were linked to PSK administration (Nakazato et al, 1994). Trials with PSP indicated that C. versicolor extract has the potential to alleviate the side effects normally associated with chemotherapy (lassitude, inappetence, spontaneous perspiration, etc.) in patients with stomach cancer classified as stage I to IV (Zhang et al, 1999). Moreover, an increase in the immunological functions and a concomitant decrease of the adverse hematological side effects of chemotherapeutical drugs was demonstrated for stomach cancer patients following the administration of PSP (1 gram three times a day for 8 weeks) (Wu et al, 1999). Results from a prospective multi-centre study including 158 esophageal cancer patients, indicated that those who received PSK (3 grams/day for three months after surgery) had a significantly better survival rate at five years (55% and 58%) compared with those without PSK supplementation (26% and 31%) (Ogoshi et al, 1995a). Another study reported the results of 174 patients who underwent esophagectomy and were then assigned to receive radiotherapy or chemotherapy with or without PSK. There was a tendency for longer survival on PSK, but statistical significance was not reached. However, regression analysis indicated that C. versicolor extract might have a beneficial effect on esophageal carcinoma when combined with radiotherapy and chemotherapy (Ogoshi et al, 1995b). Better survival rates were also achieved with PSP. A hundred patients with esophageal carcinoma were randomly divided into two groups: one group was treated with radiotherapy alone while other one received radiotherapy plus PSP (3 grams daily for a total dose of 190 grams during the period of radiation time). The results demonstrated that patients treated with radiotherapy plus PSP had higher one, three and five survival rates (67%, 38%, and 19% respectively) versus the control group (47%, 21% and 14%) (Yao, 1999). Addition of PSP to the regimen improved the relief of major symptoms commonly associated with esophageal cancer, such as change of weight, alteration in the hemogram profile and in immunological functions. The relief of these symptoms was quantified to reach 61% in the PSP-treated group, while it was 31% in the control group (Wu and Wang, 1999).

A Meta-analysis study was performed to evaluate the effect of immunochemotherapy on survival in patients with curative resections of gastric cancer. The metaanalysis included 8,009 patients from eight randomized controlled trials after central randomization. The results suggest that adjuvant immunochemotherapy with PSK improves the survival of patients after curative gastric cancer resection (Oba et al, 2007).

D. Leukemia A study with PSK as an adjuvant to chemotherapy done in the early 1980â&#x20AC;&#x2122;s (with 28 patients) found that remission and survival were significantly prolonged for patients who received PSK plus chemotherapy over those who received chemotherapy alone (Nagao et al, 1981). In another multi-center trial including 67 patients in remission of acute non-lymphocytic leukemia (ANLL) in Japan, patients who received a maintenance chemotherapy plus immunotherapy with PSK tended to have longer survival over the group that received chemotherapy alone but without significance. However, analysis of the data of patients who had maintained complete remission for more than 270 days revealed that immunotherapy had a suggestive beneficial effect (p=0.105), prolonging the 50% remission period by 418 days (885 vs. 467 days). It was concluded that PSK may help in the treatment of adult ANLL when used for maintenance therapy in combination with chemotherapy, especially in patients with a good prognosis.

E. Lung cancer A clinical trial conducted in Japan was done with patients having different lung cancers at stages I-III; a group was selected to receive 3 grams of PSK daily after cessation of radiation therapy. PSK was given in repeating cycles of two weeks on and two weeks off. After 5 years, 27% of the patients treated with PSK were alive compared to 7% for those not given the mushroom extract (Hayakawa et al, 1993). The study also demonstrated that patients with Stage III disease who received PSK had a better prognosis than those with stages I and II without PSK. A study investigated the effects of PSK as an accessory treatment for lung cancer (Ke et al, 1999), 30 patients were administered the mushroom extract while they received chemotherapy. The symptoms (side effects) improvement for PSP-treated patients was over 87%, while it was 47% for the control group. Thirty-four patients, with no significant difference in their baseline demographic, clinical or tumor characteristics, or previous treatment regimes (p>0.05), were recruited into each of the PSP and control arms. After 28-day treatment, there was a significant improvement in blood leukocyte and neutrophil counts, serum IgG and IgM, and percent of body fat among the PSP, but not the control patients (p<0.05) (Tsang et al, 2003).

VII. Discussion and Conclusion Since 1970s numerous mushroom fungi have been increasingly used as a source of medicinal compounds and


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therapeutic adjutants or health food supplements. PSP has been previously shown to have immuno-stimulatory, antitumour and analgesic effects (Zhong et al, 2001) in animal models. When used as an adjunct in cancer chemotherapy in clinical trials carried out in China, PSP improved the quality of life in the patients by improving appetite and alleviating symptoms associated with cancer chemotherapy. Cancer chemotherapy and radiotherapy are standard treatments of cancer in the past 30 years, often associated with side effects such as immuno-suppression, poor appetite, and vomiting, which seriously affected the therapy. Studies in Japan and China have suggested mushroom proteoglycans may provide a possible solution, with Polysaccharide Krestin and PSP being systemically studied as adjunct to cancer chemotherapy (Kidd, 2000). Nowadays the biological activities of PSP and PSK have received much attention in biomedical sciences. One of the most important functions of PSP and PSK is their immunomodulation and anti-cancer actions. Various experimental evidences demonstrated that the anti-tumor action of mushroom polysaccharides is due to the enhancement and potentiation of cell-mediated immune system through the regulation of immunomodulatory cytokines and activation of the complement system and Natural Killer Cells (NK cells) (Ohwada et al, 2006). However the mechanism of anti-tumor actions of PSP and PSK from most fungi is still not clear. It is accepted that anti-tumor polysaccharides enhance various immune responses, and act as biological response modifiers (Ohwada et al, 2006). PSK/PSP are nonspecific immunopotentiators and exert immunomodulatory actions by promoting the proliferation of T-lymphocytes, the activation of macrophages, natural killer cells, and Thelper cells, thereby inducing the production of antibody and interleukins (Mao et al, 1996; Li, 1999). PSK also has favorable effect on the activation of leucocyte chemotactic locomotion and phagocytic activity (Torisu et al, 1990). However further studies on the mechanisms behind anticancer, immunostimulatory, and biological response modifying effects of PSK or PSP are needed.

Acknowledgments This project was made possible by Grant Number 1 P50 AT002779-01 from the National Center for Complementary and Alternative Medicine (NCCAM) and the Office of Dietary Supplements (ODS). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCCAM, ODS or the National Institute of Health.

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From left to right: King-Fai Cheng, Ping-Chung Leung


Cheng and Leung: General review of polysaccharopeptides (PSP) from C. versicolor


Cancer Therapy Vol 6, page 131 Cancer Therapy Vol 6, 131-136, 2008

Therapeutic modalities for intraocular lymphoma Review Article

Sapna Gangaputra, Robert Nussenblatt, Grace Levy-Clarke* National Eye Institute, National Institutes of Health

__________________________________________________________________________________ *Correspondence: Grace Levy Clarke, MD, Uveitis Specialist, St. Luke’s Cataract and Laser Institute, 9400 9th St N (MLK), St. Petersburg, Fl 33702, USA; Tel: 727 328 7700; Fax:727 321 2301; E-mail: Key words: Immunotoxin HA22 – B, T cell murine models, Clinical presentation, Diagnosis, High dose MTX, Local Chemotherapy, Radiotherapy (RT), Rituximab, Treatment options, Trofosfamide, Ifosfamide Abbreviations: blood-brain barrier, (BBB); central nervous system, (CNS); fluorescein angiography, (FA); Non Hodgkin’s Lymphoma, (NHL); primary central nervous system lymphoma, (PCNSL); Primary intraocular lymphoma, (PIOL); Radiotherapy, (RT); retinal pigment epithelium, (RPE) Received: 1 July 2007; Revised: 31 August 2007 Accepted: 18 February 2008; electronically published: February 2008

Summary PIOL is a rare tumor that primarily affects intraocular tissues. It is a subset of PCNSL. It usually presents as unremitting uveitis that is refractory to corticosteroid therapy. A work-up is warranted in every patient presenting with a picture clinically compatible with PIOL. The diagnostic algorithm followed at NEI outlines the critical steps to rule out a lymphoma. This paper also reviews the current therapeutic modalities available for the treatment of PIOL. The treatment algorithm developed at NEI, offers a comprehensive treatment plan based on the clinical presentation of the PIOL. A consortium of physicians including neurologists, oncologists and ophthalmologists working together will achieve improved tumor remission and survival. More studies are needed to better understand the pathophysiology of PIOL and develop treatment targeting the lymphoma cells at a molecular level.

and overall patient survival. Prompt and accurate diagnosis together with improving treatment plans can lead to better visual and systemic outcomes (Choi et al, 2006).

I. Introduction Primary intraocular lymphoma (PIOL) is a form of non-Hodgkin’s lymphoma that involves primarily intraocular structures. It was first described in 1951 by Cooper and Riker as “reticulum cell sarcoma” (Cooper and Riker, 1951). PIOL refers to a type of primary central nervous system lymphoma (PCNSL), which presents in the eye with or without CNS involvement. PIOL is a rare malignancy, but the exact incidence is unknown. The frequency of this rare condition has increased over the past years in immunosuppressed as well as immunocompetent patients. It has been shown that chronic antigenic stimulation may result in the development of PIOL (Gunduz et al, 2006). Histologically, PIOL is usually a diffuse large-cell B-cell lymphoma. Intraocular lymphoma of T-cell type is rare, and most cases represent a primary cutaneous T-cell lymphoma, most commonly Mycosis Fungoides or secondary manifestations of a systemic Tcell lymphoma in conjunction with systemic leukemia, such as Adult T cell leukemia (Levy-Clarke et al, 2002; Coupland et al, 2005). PIOL presents a diagnostic and therapeutic challenge as it is a rare and lethal malignancy. It often masquerades as uveitis, and delay in prompt and accurate diagnosis can affect treatment, ocular outcome

II. Clinical presentation The most common presentation of Non Hodgkin’s Lymphoma (NHL) of the central nervous system (CNS) is an older patient with chronic uveitis that is not responsive to corticosteroids. These patients may complain of painless loss of vision with floaters. Patients may also rarely complain of red eye, photophobia, or ocular pain, or even be clinically silent (Gill and Jampol, 2001). The vitreous cavity usually contains a cellular infiltrate composed of neoplastic and reactive inflammatory cells by cytology. Clinical exam reveals vitreous cells and vitreous haze. The infiltrate often forms clumps and sheets in the vitreous and may obscure visualization of the retina. Retinal findings commonly seen in patients with PIOL include scattered punctate, deep retinal lesions and yellowish subretinal infiltrates that may enlarge and coalesce (Buggage et al, 2001). Atypical findings on ophthalmic examination may include the presence of keratic precipitates, and mild cellular reaction in the


Gangaputra et al: Therapeutic modalities for intraocular lymphoma anterior chamber. Noninvasive ancillary tests such as fluorescein angiography (FA) can play an important role in the early diagnosis of PIOL. Fluorescein angiography is a technique for examining the blood circulation of the choroid and retina. A bolus of Fluorescein is injected intravenously and after a few minutes is visible in the retinal vessels, when visualized using blue light. Serial photographs of the filling and emptying of dye from the retinal and choroidal circulation are used to study the vascular tree and retinal perfusion. The most common angiographic characteristics include disturbances at the level of the retinal pigment epithelium (RPE), such as granularity, blockage and late staining. These changes correlate to histopathologic findings of lymphoma cells between the RPE and Bruchâ&#x20AC;&#x2122;s membrane. Perivascular staining or leakage and cystoid macular edema are not frequent findings (Velez et al, 2002).

be done (Levy-Clarke et al, 2001). When a definite diagnosis is obtained, a neuro-oncologist should participate in the treatment and follow up of the patient (Levy-Clarke et al, 2005).

IV. Treatment options At National Eye Institute, we have formulated a treatment algorithm based on disease presentation (LevyClarke et al, 2005). At initial presentation, systemic chemotherapy is offered for PIOL without parenchymal or leptomeningeal involvement. Adjunct local chemotherapy is typically added when there is high tumor burden in the eye or systemic therapy is contraindicated. If there is PIOL with intraparenchymal or leptomeningeal involvement, then systemic and intrathecal chemotherapy should be offered with management by oncologist and ophthalmologist. When there is refractory or recurrent intraocular disease, then additional systemic chemotherapy with adjunctive intravitreal methotrexate maybe administered, or radiotherapy can be used. If intravitreal methotrexate is used, then a baseline electroretinogram to assess pre-treatment retinal function and to assess for subsequent treatment toxicity is recommended. In addition, rescue therapy for limbal stem cells with topical leucovorin should be attempted. Topical leucovorin should be formulated by the local pharmacist and administered as 0.003% eyedrops, four times a day or as needed. A diagnosis of keratoconjunctivitis sicca is treated as a relative contraindication for intravitreal MTX due to the high incidence of corneal epitheliopathy (Smith et al, 2002). A thorough corneal evaluation with Schirmer testing is recommended prior to institution of this therapy. Monitoring for recurrence of disease during disease free intervals should include neuroimaging and cytoanalysis of the CSF. Vitreous specimen for cytopathology and cytokine and molecular analyses should be included when clinically indicated.

III. Diagnosis Cytological examination of vitreous is the best test to exclude lymphoma in patients with persistent idiopathic uveitis. Chorioretinal biopsies increase the chances of diagnosing or excluding a PIOL. Biopsies also allow for exact subtyping of the malignant lymphoma (Levy-Clarke et al, 2001; Coupland et al, 2003; Gonzales and Chan, 2007). Recommendations to maximize utility of cytologic evaluation for PIOL include discontinuation of systemic corticosteroids before vitrectomy, obtaining cells from the subretinal pigment epithelium space when possible, and to process the cells immediately for cytologic evaluation. Corticosteroids have a cytotoxic effect on lymphoma cells and consequently will decrease the availability of malignant cells at the time of biopsy. Immunohistochemistry and flow cytometric immunophenotyping may help to support the diagnosis (Zaldivar et al, 2004). Judicial handling of the vitreal biopsy and evaluation of the slides by an experienced cytopathologist are also key factors in the diagnostic process (Karma et al, 2007). Molecular analysis using microdissection and PCR in the examination of vitreous specimens in patients with PIOL have shown diagnostic utility. Clonal rearrangements of the immunoglobin heavy chain or the T-cell receptor genes and are well reported adjunctive studies utilized in the assessment of PIOL patients (Shen et al, 1998; Chan, 2003; Merle-BĂŠral et al, 2004; Baehring et al, 2005; Coupland et al, 2005). Chan and colleagues have published extensively on the unique molecular patterns of PIOL compared with other systemic lymphomas (Wallace et al, 2006). At the National Eye Institute we follow a diagnostic algorithm. Every patient suspicious for PIOL undergoes neuroradiologic imaging and CSF examination. No further ocular diagnostic tests are required in patients with positive CSF findings by cytology and/or flow cytometry. In patients with a negative CSF study, a vitrectomy or vitreous tap should be performed in the eye with more severe vitritis or worse visual acuity. This sample should be sent for immediate cytolopathologic analysis. Adjunctive studies may include immunophenotyping/ flow cytometry, cytokine analysis, microdissection and PCR (Chan and Wallace, 2004). If indicated, chorioretinal biopsy or subretinal aspirate may

V. High dose MTX Advances in PCNSL treatment has shown evidence that high-dose methotrexate-based chemotherapy regimens improve survival compared to those treated with radiotherapy alone. However, there is no optimal treatment and therapy can be associated with long-term neurotoxicity. Gavrilovic and colleagues reported on their cohort of PCNSL who were treated with high dose MTX chemotherapy (Gavrilovic et al, 2006). Whole brain radiotherapy was used when necessary. The 10 year follow up period showed remission in almost half of the enrolled patients with just chemotherapy. They observed that the rate of death due to neurotoxicity was equivalent to the rate of death due to progression of the tumor. Batchellor and colleagues studied the effects of high dose methotrexate in patients with concurrent PIOL and PCNSL. They had satisfactory rates of remission. Refractory cases were treated with radiation. The concentration of methotrexate in the vitreous and aqueous four hours post infusion, was high enough to be cytotoxic to the lymphoma cells (Batchelor et al, 2003). In another study, patients with recurrent or refractory lymphoma were treated with methotrexate and hematopoietic stem 132

Cancer Therapy Vol 6, page 133 cell rescue. They showed improved survival with the regimen (Soussain et al, 2001).

VIII. Newer therapies A. Trofosfamide and Ifosfamide Trofosfamide and Ifosfamide are alkylating oxazaphosphorine derivatives. They are prodrugs which require hydroxylation at the cyclic carbon-4 position by hepatic cytochrome P-450 isoenzymes. IFO and TRO penetrate through the blood-brain barrier (BBB) due to their lipid solubility, small molecular size, and minimal binding to plasma and tissue proteins. Jahnke and colleagues published in 2004 their experience using oral trofosfamide. Two patients, who were not good candidates for radiation therapy or systemic chemotherapy, were seen to have complete remission on this regimen (Jahnke et al, 2004). One patient carried a diagnosis of PIOL while the other was diagnosed with oculocerebral lymphoma. The same investigators later evaluated the efficacy and aqueous penetration of intravenous ifosfamide and oral trofosfamide on 10 patients with PIOL. All patients responded and showed evidence of aqueous penetration of the active metabolite (Jahnke et al, 2005).

VI. Local chemotherapy In an attempt to provide more focused drug delivery, Smith and colleagues report in 2002 treating patients with intravitreal methotrexate according to a standard induction-consolidation-maintenance regimen. The induction phase consisted of twice weekly intravitreal MTX injections for one month. A dose of 400µg in 0.1ml was injected at the level of pars plana using a 30G needle. After induction, weekly consolidation doses were given for a month. The maintenance phase involved monthly MTX injections for a year. They achieved a 100% remission, defined as clinical absence of tumor cells and resolution of retinal and optic nerve infiltration. The side effects commonly observed were cataract, corneal epitheliopathy, and maculopathy. All patients were also on systemic chemotherapy at the same time (Smith et al, 2002). Intrathecal MTX and Ara-C delivered by means of an Ommaya reservoir was also tried. Patients showed complete remission and good visual outcome (Valluri et al, 1995; Mason and Fischer, 2003). Rabbit eye studies showed that methotrexate when delivered intravitreally, remained at therapeutic levels for about 48 hours in the vitreous. It was proven to be safe and efficacious when given with a single injection of fluorouracil and dexamethasone (Velez et al, 2001). Based on their results, the optimal dosing schedule for intravitreal methotrexate is every 48-72 hours. There have been other case reports showing success in inducing complete remission in patients with PIOL, using intravitreal MTX as an adjunct to systemic chemotherapy (Fishburne et al, 1997; de Smet et al, 1999; Velez et al, 2002).

B. Rituximab It is a monoclonal antibody against the B cell specific CD20 antigen. Though proven useful to treat CNS B Lymphomas, the CSF levels of rituximab after infusion are not sufficiently high and it does not appear to cross the blood brain barrier (Harjunpää et al, 2001). Investigations of the pharmacokinetics following intravitreal administration showed drug levels in aqueous and vitreous chambers with a half life of 4.7 days (Kim et al, 2006). An investigation was conducted to look at the efficacy of intrathecal monotherapy using a Phase I dose escalation study, enrolling patients with recurrent CNS non Hodgkin's lymphoma. The results suggested that an intrathecal dose of 10-25 mg would be an effective dose to use in patients with PCNSL (Rubenstein et al, 2007). A recent case series found rituximab to be histologically non toxic to rabbit and human eyes, at the dosage of 1.0mg/01ml. The effectiveness of the drug to induce regression of the lymphoma was not confirmed and more studies are needed to prove its efficacy (Kitzmann et al, 2007).

VII. Radiotherapy (RT) Studies published in 1980s indicated that RT was the first choice of treatment as lymphoma cells are highly sensitive to radiation. Complications as a result of radiotherapy were common. The most frequently reported was cataract. Dry eye was also relatively common, while serious complications of radiation retinopathy and optic atrophy were less frequent. However, it should be noted that these complications occur late and survival of the patients may compromise reporting (Hoffman et al, 2003). Ocular external beam radiotherapy is proven to be highly effective in controlling PIOL and is associated with tolerable ocular complications. One reported regimen in a retrospective, interventional case series used 35-40 Gy given in 15 fractions to both globes to minimize normal tissue toxicity (Berenbom et al, 2006). A multiinstitutional survey in Japan summarized the experience of 17 institutions performing radiotherapy for PIOL. The conclusive recommendation was to deliver 40 Gy of ocular Radiotherapy, in conjunction with prophylactic cranial irradiation at doses 30 Gy and high-dose MTX to control PIOL (Isobe et al, 2006).

IX. Immunotoxin HA22 - B and T cell murine models Researchers at NEI developed a murine model of PIOL by direct inoculation of adult mouse vitreous with immunogenic variants of mouse T cell lymphomas. Although this model was not a B-cell lymphoma, there were many features of human PIOL, such as clinical presentation, histologic findings, and cytokine expression pattern (Chan et al, 2005). To mimic the human PIOL, a B cell murine model was developed by intravitreally injecting human B-cell lymphoma cells into severe combined immunodeficient mice. Results showed that the murine model resembled human PIOL closely. Pathologic examination revealed that the tumor cells initially colonized on the retinal surface, later infiltrated through the retinal layers and eventually penetrated through the retinal pigment epithelium into the 133

Gangaputra et al: Therapeutic modalities for intraocular lymphoma Chan CC (2003) Molecular pathology of primary intraocular lymphoma. Trans Am Ophthalmol Soc 101, 275-92. Chan CC, Fischette M, Shen D, Mahesh SP, Nussenblatt RB, Hochman J (2005) Murine model of primary intraocular lymphoma. Invest Ophthalmol Vis Sci 46, 415-9. Chan CC, Wallace DJ (2004) Intraocular lymphoma, update on diagnosis and management. Cancer Control 11, 285-95. Choi JY, Kafkala C, Foster CS (2006) Primary intraocular lymphoma, A review. Semin Ophthalmol 21, 125-33. Cooper EL, Riker JL (1951) Malignant lymphoma of the uveal tract. Am J Ophthalmol 34, 1153-8. Coupland SE, Anastassiou G, Bornfeld N, Hummel M, Stein H (2005) Primary intraocular lymphoma of T-cell type, report of a case and review of the literature. Graefes Arch Clin Exp Ophthalmol 243, 189-97. Coupland SE, Bechrakis NE, Anastassiou G, Foerster AM, Heiligenhaus A, Pleyer U, Hummel M, Stein H (2003) Evaluation of vitrectomy specimens and chorioretinal biopsies in the diagnosis of primary intraocular lymphoma in patients with Masquerade syndrome. Graefes Arch Clin Exp Ophthalmol 241, 860-70. Coupland SE, Hummel M, Muller HH, Stein H (2005) Molecular analysis of immunoglobulin genes in primary intraocular lymphoma. Invest Ophthalmol Vis Sci 46, 3507-14. de Smet MD, Vancs VS, Kohler D, Solomon D, Chan CC (1999) Intravitreal chemotherapy for the treatment of recurrent intraocular lymphoma. Br J Ophthalmol 83, 448-51. Deangelis LM, Iwamoto FM (2006) An update on therapy of primary central nervous system lymphoma. Hematology Am Soc Hematol Educ Program 311-6 Fishburne BC, Wilson DJ, Rosenbaum JT, Neuwelt EA (1997) Intravitreal methotrexate as an adjunctive treatment of intraocular lymphoma. Arch Ophthalmol 115, 1152-6. Gavrilovic IT, Hormigo A, Yahalom J, DeAngelis LM, Abrey LE (2006) Long-term follow-up of high-dose methotrexatebased therapy with and without whole brain irradiation for newly diagnosed primary CNS lymphoma. J Clin Oncol 24, 4570-4. Gill MK, Jampol LM (2001) Variations in the presentation of primary intraocular lymphoma, case reports and a review. Surv Ophthalmol 45, 463-71. Gonzales JA, Chan CC (2007) Biopsy techniques and yields in diagnosing primary intraocular lymphoma. Int Ophthalmol Gunduz K, Pulido JS, McCannel CA, O'Neill BP (2006) Ocular manifestations and treatment of central nervous system lymphomas. Neurosurg Focus 21, E9. Harjunp채채 A, Wiklund T, Collan J, Janes R, Rosenberg J, Lee D, Grillo-L처pez A, Meri S (2001) Complement activation in circulation and central nervous system after rituximab (antiCD20) treatment of B-cell lymphoma. Leuk Lymphoma 42, 731-8. Hoffman PM, McKelvie P, Hall AJ, Stawell RJ, Santamaria JD (2003) Intraocular lymphoma, a series of 14 patients with clinicopathological features and treatment outcomes. Eye 17, 513-21. Isobe K, Ejima Y, Tokumaru S, Shikama N, Suzuki G, Takemoto M, Tsuchida E, Nomura M, Shibamoto Y, Hayabuchi N (2006) Treatment of primary intraocular lymphoma with radiation therapy, a multi-institutional survey in Japan. Leuk Lymphoma 47, 1800-5. Jahnke K, Bechrakis NE, Coupland SE, Schmittel A, Foerster MH, Fischer L, Thiel E, Korfel A (2004) Treatment of primary intraocular lymphoma with oral trofosfamide, report of two cases and review of the literature. Graefes Arch Clin Exp Ophthalmol 242, 771-6. Jahnke K, Wagner T, Bechrakis NE, Willerding G, Coupland SE, Fischer L, Thiel E, Korfel A (2005) Pharmacokinetics and

choroid. Several putative molecular markers for human PIOL were expressed in vivo in this model. Tumor metastasis into the central nervous system was also observed. Having developed a closely resembling model, the next step was to use the model to test therapeutic strategies. A recently developed immunotoxin combining a monoclonal antibody against CD22 and a Pseudomonas exotoxin (BL22) has been successfully used in a phase I clinical trial for treating hairy cell leukemia. Immunotoxin HA22 is a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity. The therapeutic effectiveness of immunotoxin HA22 was tested by injecting the drug intravitreally into the B cell murine model. A single intravitreal injection of immunotoxin HA22 resulted in complete regression of the tumor (Li et al, 2006). PIOL is a difficult disease to diagnose and treat and the best therapeutic options have not yet been formalized. Earlier publications centered on radiotherapy, but approaches over the last 2 decades have focused on systemic and intrathecal chemotherapy. Within the new realm, high dose MTX still remains the first option for many oncologists. Our experience at NEI has shown that as a first line therapy, it is very effective at treating PIOL (a subset of PCNSL) especially in patients with large subretinal tumor bulk. Patients with recurrent disease have been treated with local intravitreal MTX and have shown good initial response but we have seen a high rate of recurrence, with the need for multiple courses of therapy. For patients with refractory disease, radiotherapy still remains a good therapeutic option. Radiation is also effective at treating leptomeningeal involvement with associated optic nerve infiltration, which is an ophthalmologic emergency. The patient can have rapidly progressive vision loss and leading to blindness. In conclusion, PIOL continues to be a diagnostic and therapeutic challenge. The International CNS and Ocular Lymphoma workshop group (Nussenblatt et al, 2006) recommend new approaches to diagnose PIOL with better accuracy and sensitivity. In this regard, an animal model for the rare disease will help in understanding the biology of the disease. Other methods for disease detection should be explored so that accurate diagnosis, staging and treatment regimens can be formulated. In order for such studies to have greatest impact, national and international collaboration should be coordinated.

References Baehring JM, Androudi S, Longtine JJ, Betensky RA, Sklar J, Foster CS, Hochberg FH (2005) Analysis of clonal immunoglobulin heavy chain rearrangements in ocular lymphoma. Cancer 104, 591-7. Batchelor TT, Kolak G, Ciordia R, Foster CS, Henson JW (2003) High-dose methotrexate for intraocular lymphoma. Clin Cancer Res 9, 711-5. Berenbom A, Davila RM, Lin HS, Harbour JW (2006) Treatment outcomes for primary intraocular lymphoma, implications for external beam radiotherapy. Eye. Buggage RR, Chan CC, Nussenblatt RB (2001) Ocular manifestations of central nervous system lymphoma. Curr Opin Oncol 13, 137-42.


Cancer Therapy Vol 6, page 135 efficacy of ifosfamide or trofosfamide in patients with intraocular lymphoma. Ann Oncol 16, 1974-8. Karma A, von Willebrand EO, Tommila PV, Paetau AE, Oskala PS, Immonen IJ (2007) Primary Intraocular Lymphoma Improving the Diagnostic Procedure. Ophthalmology Kim H, Csaky KG, Chan CC, Bungay PM, Lutz RJ, Dedrick RL, Yuan P, Rosenberg J, Grillo-Lopez AJ, Wilson WH, Robinson MR (2006) The pharmacokinetics of rituximab following an intravitreal injection. Exp Eye Res 82, 760-6. Kitzmann AS, Pulido JS, Mohney BG, et al. Intraocular use of rituximab. Eye 2007 Levy-Clarke GA, Buggage RR, Shen D, Vaughn LO, Chan CC, Davis JL (2002) Human T-cell lymphotropic virus type-1 associated t-cell leukemia/lymphoma masquerading as necrotizing retinal vasculitis. Ophthalmology 109, 1717-22. Levy-Clarke GA, Byrnes GA, Buggage RR, Shen DF, Filie AC, Caruso RC, Nussenblatt RB, Chan CC (2001) Primary intraocular lymphoma diagnosed by fine needle aspiration biopsy of a subretinal lesion. Retina 21, 281-4. Levy-Clarke GA, Chan CC, Nussenblatt RB (2005) Diagnosis and management of primary intraocular lymphoma. Hematol Oncol Clin North Am 19, 739-49, viii. Li Z, Mahesh SP, Shen de F, Liu B, Siu WO, Hwang FS, Wang QC, Chan CC, Pastan I, Nussenblatt RB (2006) Eradication of tumor colonization and invasion by a B cell-specific immunotoxin in a murine model for human primary intraocular lymphoma. Cancer Res 66, 10586-93. Mason JO, Fischer DH (2003) Intrathecal chemotherapy for recurrent central nervous system intraocular lymphoma. Ophthalmology 110, 1241-4. Merle-BĂŠral H, Davi F, Cassoux N, Baudet S, Colin C, Gourdet T, Bodaghi B, LeHoang P (2004) Biological diagnosis of primary intraocular lymphoma. Br J Haematol 124, 469-73. Nussenblatt RB, Chan CC, Wilson WH, Hochman J, Gottesman M (2006) International Central Nervous System and Ocular Lymphoma Workshop, recommendations for the future. Ocul Immunol Inflamm 14, 139-44. Rubenstein JL, Fridlyand J, Abrey L, Shen A, Karch J, Wang E, Issa S, Damon L, Prados M, McDermott M, O'Brien J, Haqq C, Shuman M (2007) Phase I study of intraventricular administration of rituximab in patients with recurrent CNS and intraocular lymphoma. J Clin Oncol 25, 1350-6. Shen DF, Zhuang Z, LeHoang P, BĂśni R, Zheng S, Nussenblatt RB, Chan CC (1998) Utility of microdissection and polymerase chain reaction for the detection of immunoglobulin gene rearrangement and translocation in primary intraocular lymphoma. Ophthalmology 105, 16649. Smith JR, Rosenbaum JT, Wilson DJ, Doolittle ND, Siegal T, Neuwelt EA, Pe'er J (2002) Role of intravitreal methotrexate in the management of primary central nervous system

lymphoma with ocular involvement. Ophthalmology 109, 1709-16. Soussain C, Suzan F, Hoang-Xuan K, Cassoux N, Levy V, Azar N, Belanger C, Achour E, Ribrag V, Gerber S, Delattre JY, Leblond V (2001) Results of intensive chemotherapy followed by hematopoietic stem-cell rescue in 22 patients with refractory or recurrent primary CNS lymphoma or intraocular lymphoma. J Clin Oncol 19, 742-9. Valluri S, Moorthy RS, Khan A, Rao NA (1995) Combination treatment of intraocular lymphoma. Retina 15, 125-9. Velez G, Boldt HC, Whitcup SM, Nussenblatt RB, Robinson MR (2002) Local methotrexate and dexamethasone phosphate for the treatment of recurrent primary intraocular lymphoma. Ophthalmic Surg Lasers 33, 329-33. Velez G, Chan CC, Csaky KG (2002) Fluorescein angiographic findings in primary intraocular lymphoma. Retina 22, 37-43. Velez G, Yuan P, Sung C, Tansey G, Reed GF, Chan CC, Nussenblatt RB, Robinson MR (2001) Pharmacokinetics and toxicity of intravitreal chemotherapy for primary intraocular lymphoma. Arch Ophthalmol 119, 1518-24. Wallace DJ, Shen D, Reed GF, Miyanaga M, Mochizuki M, Sen HN, Dahr SS, Buggage RR, Nussenblatt RB, Chan CC (2006) Detection of the bcl-2 t(14;18) translocation and proto-oncogene expression in primary intraocular lymphoma. Invest Ophthalmol Vis Sci 47, 2750-6. Zaldivar RA, Martin DF, Holden JT, Grossniklaus HE (2004) Primary intraocular lymphoma, clinical, cytologic, and flow cytometric analysis. Ophthalmology 111, 1762-7.

Grace Levy-Clarke


Gangaputra et al: Therapeutic modalities for intraocular lymphoma


Cancer Therapy Vol 6, page 137 Cancer Therapy Vol 6, 137-148, 2008

DNA methylation in cancer: techniques and preliminary evidence of hypermethylation in canine lymphoma Research Article

Jeffrey N. Bryan1, ,#, Kristen H. Taylor2, Carolyn J. Henry1,3, Kimberly A. Selting1, Farah Rahmatpanah2, Michael R. Lewis1,4, Charles W. Caldwell2,5 1

Department of Veterinary Medicine and Surgery, Department of Pathology and Anatomical Sciences, 3 Department of Internal Medicine, Division of Hematology/Oncology, University of Missouri-Columbia 4 Research Service, Harry S. Truman Memorial Veteransâ&#x20AC;&#x2122; Hospital, Columbia, MO 5 Ellis Fischel Cancer Center, University of Missouri-Columbia. 2

__________________________________________________________________________________ *Correspondence: Jeffrey N. Bryan, Washington State University, PO Box 646610, Pullman, WA 99164-6610, USA; Phone: (509) 335-0711; Fax: (509) 335-0880; E-mail: # Dr. Bryanâ&#x20AC;&#x2122;s current address is: Department of Veterinary Clinical Sciences, Washington State University, PO Box 646610, Pullman, WA 99164-6610. Key words: DLC1, Dogs, Lymphoma, DNA hypermethylation, Epigenetic Abbreviations: basic local alignment search tool, (BLAST); chronic lymphocytic leukemia, (CLL); combined bisulfite restriction analysis, (COBRA); Differential methylation hybridization, (DMH); diffuse large B-cell lymphomas, (DLBCL); DNA methyltransferases, (DNMT); histone deacetylases, (HDAC); insulin-like growth factor-1, (IGF-1); interleukin-6, (IL-6); Methylation specific PCR, (MSP); immunoprecipitation, (IP); O6-methylguanine-DNA methyltransferase, (MGMT); polymerase chain reaction, (PCR); Quantitative MSP, (Q-MSP); restriction landmark genomic scanning technique, (RLGS); Rho-GTPase Activating Protein, (RhoGAP); sterile alpha motif homology 2, (SAM2); steroidogenic acute regulatory protein, (START); vascular endothelial growth factor, (VEGF) Received: 6 February 2008; electronically published: June 2008

This work was funded in part by the National Library of Medicine Biomedical and Health Informatics Research training grant LM07089. Presented in the Theilen Tribute Symposium at UC Davis 31 st May- 1st June 2008.

Summary Epigetic changes in cancer, including DNA methylation and histone modification, are now recognized to be integrally involved in generation and maintenance of the neoplastic phenotype. The addition of a methyl group to cytosines occurs normally in genomic, low-density CpG dinucleotides, and is normally blocked in high density accumulations of CpG dinucleotides in control regions of genes, termed CpG islands. Global hypomethylation of cytosines and local hypermethylation of CpG islands are hallmarks of the neoplastic epigenome. Hypomethylation can result in overexpression of growth factors or oncogenes, alterations in DNA repair enzymes, and loss of genomic stability. Hypermethylation of CpG islands can silence tumor suppressor genes, promoting cell cycling. In concert, alterations in methylation may be the primary players in pluripotent cell expansion that develops into cancer. In this paper we review the current molecular procedures to elucidate the methylation status of DNA on a genomewide basis and for specific gene determination for veterinary cancer researchers. We also report the results of a pilot study in dogs of the orthologue of a recognized human tumor-suppressor gene, DLC1. The functional canine sequence orthologous to human DLC1 was identified using web-based tools. Methylation specific PCR (MSP) and combined bisulfite restriction analysis (COBRA) were performed. MSP demonstrated methylated DNA to be present in six of 13 canine NHL samples and two of three canine chronic lymphocytic leukemia (CLL) samples. COBRA identified methylation in nine of 13 NHL and two of three CLL samples. This study provides compelling pilot data that hypermethylation occurs in canine NHL. This change in neoplastic cells warrants further detailed investigation as a marker for diagnosis and classification of disease or as a therapeutic target. 137

Bryan et al: DNA Methylation review using S-adenosyl methionine as the methyl donor. Maintenance methylation, occurring shortly after DNA replication, serves the purpose of conserving patterns of methylation that silence transposons and maintain monoallellic expression of genes imprinted during development (Rollins et al, 2006). De novo methylation allows the dynamic modulation of gene expression through methylation of CpG dinucleotides in control regions of genes, including the promoter, first exon, and first intron. Waves of methylation and demethylation are critical events in normal embryogenesis and in utero development (Siegfried et al, 1999; Herman and Baylin, 2003). Methylation patterns are mitotically heritable for generations (Jones and Baylin, 2007). Across the genome, CpG dinucleotides are statistically relatively scarce. This is likely because methylcytosine that undergoes spontaneous deamination becomes thymine, and the change is inefficiently corrected. As a result, most genomic CpG dinucleotides are low density and are methylated (Rollins et al, 2006). Regions with higher density of CpG dinucleotides are referred to as CpG islands. These are functional units defined by a mathematical construct rather than well-defined physical boundaries. CpG islands are regions of DNA spanning at least 500 bases with a G + C content > 55% and an observed/expected ratio of CpG > 0.6 (Takai and Jones, 2002). In most cases, CpG islands are associated with the promoter and first exon regions of genes, and are largely unmethylated (Takai and Jones, 2002; Rollins et al, 2006). When these regions are predominantly methylated, as in imprinted genes or parasitic DNA sequences, histone modifications occur, chromatin compaction occurs, transcription factors no longer bind, and transcription is silenced (Figure 2) (Herman and Baylin, 2003).

I. Introduction A. Epigenetic changes modifying gene expression In 1948, Rollin Hotchkiss reported the presence of a fifth base in a DNA chromatogram (Hotchkiss, 1949). Subsequently identified as 5-methylcytosine, a modification of the standard base, its role in eukaryotic cells was largely unexplored for decades. In 1993, Holliday postulated that DNA methylation contributes to the neoplastic phenotype (Holliday, 1993). Subsequent research is identifying the mechanisms of Dr. Hotchkissâ&#x20AC;&#x2122; contribution at an increasing rate. Epigenetic changes are covalent modifications of DNA bases or histones that alter the tertiary structure of chromatin, the binding of transcription factors, and, ultimately, the expression patterns of genes within a cell. In human cell lines and primary tumors, abnormal DNA methylation has now been demonstrated to contribute to cancer of the breast, colon, stomach, kidney, prostate, skin, hemolymphatic organs, and other tissues (Cui et al, 2003; Kim et al, 2004; Leu et al, 2004; Liu et al, 2004; Murai et al, 2005; Schulz and Hatina, 2006; Shamay et al, 2006). The presence of aberrant methylation patterns in neoplastic tissues offers the promise of novel markers for diagnosis and prognosis, untapped therapeutic targets, and may hold the key to stem cell carcinogenesis. In normal cells, methylcytosine mediates transcriptional silencing of transposons, imprinted silencing of somatic genes, inactivation of one copy of the X chromosome of females, and the discrimination of self from invading DNA of pathogens (Singal and Ginder, 1999; Rollins et al, 2006) Most commonly, cytosine in a C followed by G (CpG) dinucleotide is methylated by enzymes called DNA methyltransferases (DNMT) (Figure 1). These enzymes are responsible for either maintenance (DNMT1) or de novo methylation (DNMT3a, DNMT3b), Uracil







N Deoxyribose

N Deoxyribose







N Deoxyribose

S-Adenosyl Methionine





N Deoxyribose



Figure 1. Cytosine becomes 5-methylcytosine by an electrophilic methyl substitution at the 5 position catalyzed by DNA methyltransferases. A deamination event results in transformation of cytosine to uracil and methylcytosine to thymine as illustrated.


Cancer Therapy Vol 6, page 139 Often this change is associated with modifications of histone tails as well. The order of events in silencing has not been entirely elucidated. Open, transcriptional chromatin typically contains histone H3 with acetylated tails, phosphorylation of Serine 10, and a methyl group added to lysine-4, as well as acetylation of lysine 8 of histone H4 (Herman and Baylin, 2003). Condensed, transcriptionally silenced chromatin, however, generally lacks acetylation and methylation of lysine-4 on histone H3, and demonstrates methylation of histone H3 lysine-9. The presence of DNA hypermethylation can attract methylcytosine binding proteins, which recruit histone deacetylases (HDAC) and histone methyltransferases (Herman and Baylin, 2003). Some studies have demonstrated that histone modification events may occur in the absence of promoter methylation as well (Bachman et al, 2003; Leu et al, 2004). Modifications of histone tails have been dubbed the â&#x20AC;&#x153;histone codeâ&#x20AC;? (Mellor, 2006).

to suggest that epigenetic changes, particularly global hypomethylation, are the earliest events in carcinogenesis. Recent literature advances the hypothesis that carcinogens injure many cells rather than mutate a few (Jaffe, 2003; Feinberg et al, 2006). A novel paradigm has been proposed that suggests that epigenetic disruption of progenitor cells precedes gatekeeper mutations, followed by a period of epigenetic and genetic plasticity that allows promotion and progression in the more classic sense (Feinberg et al, 2006). Epigenetic changes are clearly more than coincidental occurrences in cancer and represent targets of prevention and therapy. Methylation is a post-replicative event. As such, the standard polymerase chain reaction (PCR) adds cytosine, not methylcytosine to the growing DNA strand. To preserve information about methylcytosine bases, several techniques have been employed.

1. Restriction scanning

B. Laboratory techniques to evaluate methylation



Large-scale information about methylation of CpG dinucleotides can be determined by the use of methylation-sensitive restriction enzyme digestion with a restriction landmark genomic scanning technique (RLGS) (Park et al, 2005; Ando and Hayashizaki, 2006). In this procedure, genomic DNA is digested, but methylated targets will not be cut. Analysis of the pattern of fragments reveals the overall degree of methylation present at CpGcontaining restriction sites in the sample.

In human cancers, results of recent studies have begun to elucidate the contribution of DNA methylation to the neoplastic phenotype. Early studies identified a global loss of methylation in cancer cells. This was shown to lead to hypomethylation of Ras oncogenes, among others, that could result in overexpression of growth factors participating in neoplastic transformation (Feinberg and Vogelstein, 1983). Global genomic hypomethylation appears to contribute to genomic instability and the acceleration of the accumulation of genetic abnormalities that characterize cancer cells (Herman and Baylin, 2003). Concurrently, cancer cells exhibit hypermethylation of CpG islands in promoter regions, leading to the silencing of tumor suppressor genes (Herman and Baylin, 2003). The DNA methylation changes typical of cancer cells are illustrated in Figure 2. Accumulating evidence has begun

2. Bisulfite conversion To understand the precise pattern of cytosine methylation, DNA may be treated with bisulfite to convert unmethylated cytosine to uracil. This, in turn, becomes thymine in subsequent PCR amplification of the bisulfite converted DNA. When compared to the original sequence,

Normal gene

Hypomethylated , normally imprinted gene

Hypermethylated gene

Figure 2. Cytosine methylation patterns of DNA in normal cells, cells expressing normally imprinted alleles due to hypomethylation, and cells with silencing of tumor suppressor genes due to hypermethylation. Circles represent CpG dinucleotides along the DNA (double lines) with black representing the methylated condition and white the unmethylated condition. Boxes represent the promoter/first exon regions of the gene. The region containing more densely spaced CpG dinucleotides represents a CpG island. The grey arrows represent the activity of transcription.


Bryan et al: DNA Methylation review the presence of a thymine, instead of a cytosine, prior to a guanosine indicates the presence of unmethylated cytosine in the original DNA strand. Preservation of a cytosine is evidence that methylcytosine was present originally (Clark et al, 1994).

dinucleotides in each forward and reverse primer. Two sets of primers are designed: One, for methylated sequences, that retains the CpG complementarity; a second, for unmethylated sequences, that is complementary to a TpG sequence. The presence of a band using the methylated primers is evidence for methylation in the original sequence (Figure 3) (Cottrell and Laird, 2003). This technique is sensitive, but does not convey information about the density of methylation.

3. COBRA This assay uses bisulfite converted DNA as the template for PCR. A region of interest of a gene, usually in the promoter region, is amplified with primers designed to avoid CpG dinucleotides. Thus, the DNA will be amplified whether the CpG island is hypermethylated or not. Between these primers will have been identified restriction enzyme sites that contain CpG dinucleotides. When treated with methylation sensitive enzymes such as Hpy99I, HpyCH4IV, TaqaI, and BstUI, sequences that originally contained methylcytosine will be cut, whereas those with unmethylated cytosine will have been converted to thymidine and not be recognized (Figure 3). Some idea of the density of methylation can be gleaned by including multiple cut-sites within the sequence to be studied. Greater numbers of fragments positively correlate with a greater degree of methylation (Fraga and Esteller, 2002).

5. Quantitative MSP (Q-MSP) Similar to MSP, this is performed using a thermocycler with real-time detection capability. Using the TaqMan technology, a probe containing a fluorophore is designed complementary to the PCR amplicon. The relative intensity of fluorescence between the methylated and unmethylated primers allows quantification of the degree of methylation in the sample (Cottrell and Laird, 2003).

6. Bisulfite sequencing by cloning Fragments of bisulfite treated DNA amplified for COBRA or MSP analysis are cloned by phage into bacteria. The resulting colonies are harvested and submitted for sequencing with universal primers. The resulting sequence is compared to the original sequence to determine the degree of methylation in that region of the gene (Clark et al, 1994).

4. MSP Using bisulfite converted DNA, PCR is performed with primers designed to include at least two CpG

Methylation Specific PCR Normal Patient 1


Neoplastic U



2 3


Combined Bisulfite Restriction Analysis Normal




Figure 3. The top panel represents the gel results for MSP. Unless imprinted, normal tissue will rarely yield a methylated band (column M) and will display an unmethylated band (column U). Neoplastic cells that contain a hypermethylated gene of interest will yield a methylated band, and possibly unmethylated bands of varying size as well. The combination may be due to heterogeneity of the tumor, heterozygosity within tumor cells, or a mixed tumor/normal cell population. This can occur with COBRA as well (lower panel). Normal samples will not demonstrate digestion with the restriction enzyme, whereas the hypermethylated gene will be digested, resulting in two smaller bands. An unmethylated band may be seen for the aforementioned reasons.


Cancer Therapy Vol 6, page 141 in many other human and canine cancers. As a potential diagnostic screening test, the loss of imprinting of the IGF2 gene was examined in colon cancer. (Cui et al, 2003). The investigators identified an odds ratio for loss of imprinting in peripheral blood lymphocytes of 21.7 for patients with colorectal carcinoma. Such screening techniques could lead to much less invasive, and potentially more cancer-specific, tests for markers of risk for disease, of early disease, or the presence of residual disease. Promoter hypermethylation has been demonstrated to silence many tumor suppressor genes in human cancers and cancer cell lines (Herman and Baylin, 2003). The gene RASSF1A is frequently silenced in head and neck carcinoma (Dong et al, 2003). In one study the silencing of the gene was inversely associated with the presence of human papillomavirus infection, a common predisposing infection for these carcinomas (Dong et al, 2003). Such differentiation among etiologies could inform therapeutic decisions for clinical patients. In vitro studies have demonstrated that the gene could be demethylated and reexpressed by treatment with 5-aza-2â&#x20AC;&#x2122;-deoxycytidine, making methylated RASSF1A a potential therapy target (Dong et al, 2003). The presence of methylated RASSF1A has also been evaluated as a diagnostic test in urine samples of humans with urinary transitional cell carcinoma (Chan et al, 2003). The test was more sensitive than cytology for detecting low-grade and early cases. This demonstrates the potential utility of DNA methylation as a method of specific primary diagnostic screening. This would also yield a marker to be followed throughout therapy to determine efficacy and detect early relapse. Occasionally, aberrant promoter methylation may result in a beneficial change for cells. The gene coding O6methylguanine-DNA methyltransferase (MGMT) is an important DNA repair enzyme that can lead to chemotherapy resistance by removing potentially lethal DNA adducts that form after chemotherapy exposure. In human diffuse large B-cell lymphomas (DLBCL) lack of expression of MGMT due to DNA methylation has been shown to be positively prognostic for chemotherapy response and outcome (Al-Kuraya et al, 2006). The same has been shown to be true in glioblastoma, in which methylated MGMT can be used to predict a positive response to temozolamide chemotherapy (Hegi et al, 2005; Donson et al, 2007). Discovery of more such markers in cancer could be used to categorize patients into useful therapeutic subgroups. Genomic hypomethylation has been identified in canine lymphoproliferative disease (Pelham et al, 2003). Using RLGS, investigators demonstrated differences in methylation between normal and neoplastic lymphoid tissue (Pelham et al, 2003). Further, they demonstrated that the normal pattern of methylation was preserved in peripheral lymphocytes of dogs with lymphoma (Pelham et al, 2003). This was the first paper to demonstrate the similarity of methylation change between human and canine neoplastic disease. This underscores the potential for companion animal cancer to be instrumental as a clinical model for diagnosis and therapy of similar

7. Differential methylation hybridization microarray (DMH) Genomic DNA from a tumor is digested using restriction enzymes to 200 base-pair fragments, leaving CpG islands relatively intact. Methylation-sensitive restriction enzymes are then used to digest the fragments that are CpG rich. Cut sites that are methylated are ignored by the restriction enzyme, leaving those sequences intact. The resulting fragments, both methylated and unmethylated are tagged with a fluorophore different from that used to tag normal genomic DNA from a similar tissue. The two tagged samples are co-hybridized on a microarray chip containing sequences from regions of interest that span the cut sites of the restriction enzyme used. Those experimental sequences that remain intact will hybridize to the chip, and those that have been cut will not. The resulting fluorophore intensity correlates to the degree of methylation in the tumor sample relative to the normal tissue. This is a discovery tool for candidate methylated sequences. Validation is required by other described techniques. No canine chip exists (Yan et al, 2000).

8. CpG island and promoter tiling arrays Microarray platforms have been developed that give coverage at a resolution of 50 to 100bp covering all CpG islands or promoter regions of genes. Chromatin immunoprecipitation (IP) is performed using an antimethylcytosine antibody to concentrate fragmented genomic DNA that is rich in methylcytosine. This is then fluorescent labeled and competitively hybridized on the chip with the input, complete genomic DNA labeled with a different color. Islands can be viewed as a whole, looking for increased intensity of IP signal to genomic to suggest hypermethylation (Bock and Lengauer, 2008).

9. High-throughput bisulfite sequencing Current research is utilizing high-throughput sequencing methods to bisulfite sequence PCR product to evaluate the methylome. A limitation of this technique is the conversion of much of the genome to a nearly three base, rather than a four base, system. This can make alignment with the complete sequenced genome a bioinformatics challenge, as statistical uniqueness declines significantly (Taylor et al, 2007a).

C. Hypermethylation in cancer Loss of imprinting or normal methylation of a gene promoter can lead to overexpression of that geneâ&#x20AC;&#x2122;s product, contributing to the neoplastic phenotype. An example of this is the overexpression of the NOTCH ligand, JAG2, in malignant human plasma cells. Houde and others demonstrated that hypomethylation of the promoter region of this gene in malignant cells, compared to normal cells, resulted in higher levels of expression of the JAG2 protein (Houde et al, 2004). This induced the secretion of interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and insulin-like growth factor-1 (IGF-1). Secretion of IL-6 could be blocked by inhibition of the NOTCH pathway, confirming the mechanism (Houde et al, 2004). It is likely that such alterations exist


Bryan et al: DNA Methylation review diseases in humans. Recently, promoter hypermethylation was reported by Chuammitri and others in abstract form at the Genes, Dogs, and Cancer: Fourth Annual Canine Cancer Conference, 2006-Chicago, IL. This group identified promoter hypermethylation in the E-cadherin gene in canine mammary tumors, and in the TIMP3 and DAPK1 genes in a small number of canine lymphomas. No methylation was demonstrated in the RASSF1A gene in canine lymphomas. To date, no peer-reviewed literature exists that documents the presence of promoter hypermethylation in dogs. Expression of the tumor suppressor gene DLC1 has been shown to be silenced in multiple cancers, most recently in NHL. Originally identified as a deletion of chromosome 8p21.3-22 in human hepatocellular carcinoma, lack of expression was observed later in the absence such of a deletion (Yuan et al, 1998; Wong et al, 2003). Cells from non-small cell lung cancer, neuroectodermal cancer, breast, colon, prostate, and gastric cancer have shown lack of DLC1 mRNA when the gene promoter region was hypermethylated (Kim et al, 2003; Herman and Baylin, 2003; Wong et al, 2003; Yuan et al, 2003, 2004; Pang et al, 2005). Shi and colleagues examined human NHL cell lines and patient samples for hypermethylation of CpG islands using a CpG island microarray (Shi et al, 2006). The DLC1 gene was found to be hypermethylated in all six NHL cell lines examined, and, in every case, expression was silenced. In several lines, expression could be upregulated by treatment with a combination of a demethylating agent and a histone deacetylase inhibitor. Seventy-five NHL patient samples were examined for methylation of several candidate genes, including DLC1. Of these, 87% demonstrated hypermethylation of DLC1. Overall, expression of mRNA for this gene was significantly downregulated in tumor tissue compared to normal tissue.

suggested to be appropriate, naturally occurring clinical models of human NHL (Greenlee et al, 1990; Hansen and Khanna, 2004). The canine orthologue of the human gene DLC1 was identified and supported in silico using multiple prediction methods. This pilot study used DNA harvested from normal canine lymph nodes and lymph node aspirate samples from dogs with lymphoma stored in saline at -80oC to develop MSP and COBRA assays and evaluate the diseased samples for preliminary evidence of hypermethylation of a candidate gene. The overall hypothesis of this study was that hypermethylation occurs in canine lymphoma cells, as it does in human nonHodgkin’s lymphoma.

II. Materials and Methods A. In silico methods The NCBI reference number for the human DLC1 isoform 1 gene, NM_182643, was used to search a canine genome database ( for the canine ortholog. The 5‘ region of the gene was examined using the MethPrimer ( index1/html) CpG island analysis tool to identify candidate CpG islands (Li and Dahiya, 2002). This region was also examined using the Promoterscan transcription factor binding analysis tool to identify a promoter region within the sequence (Prestridge, 1995). The putative promoter region of the gene was then confirmed to match the human gene promoter region using the UCSC Genome Browser ( A predicted mRNA sequence was constructed by concatenating the canine orthologs of the human exons of DLC1 identified using a basic local alignment search tool (BLAST). The resulting sequence was examined using the InterProScan ( web-tool to identify protein functional groups (Zdobnov and Apweiler, 2001). The same procedure was performed using the N-Scan predicted mRNA sequence.

B. In vivo methods 1. Sample collection and preparation

D. Pilot evaluation of methylation of DLC1 in canine NHL

Lymph node aspirates were performed on clinical patients with multicentric, node-based lymphoma using 22ga. needles and preserved in Hank’s balanced salt solution at -80ºC until analysis. Peripheral blood mononuclear cells were isolated over a ficoll-hypaque (Sigma-Aldrich, St. Louis, MO) gradient and preserved at -80ºC until analysis. DNA was extracted using the Qiagen DNeasy Tissue (Qiagen USA, Valencia, CA) kit, and then bisulfite treated using the Zymo Research EZ DNA Methylation Gold (Zymo Research Corporation, Orange, CA) kit. Bisulfite treatment converts unmethylated cytosine to uracil, which becomes thymidine in subsequent polymerase chain reactions. Methylated cytosine is protected from conversion. Bisulfite treated DNA from normal canine lymph nodes was treated with SssI (New England Biolabs, Ipswich, MA), a DNA methyltransferase, and SAMe to methylate all CpG dinucleotides in the sequence and serve as a positive control.

The DLC1 gene product functions as a tumor suppressor gene (Yuan et al, 1998). The protein is a RhoGTPase Activating Protein (RhoGAP) that counteracts the feed forward signaling of RhoA and Cdc42 among other RAS signaling proteins (Wong et al, 2003). Specifically, the RhoGAP protein causes catalysis of GTP to GDP when bound to the Rho proteins, causing them to become inactive (Wong et al, 2003). Loss of this function results in unconstrained growth signaling from the surface of the cell to the nucleus, changes in cell mobility, and signaling between the cell and its extracellular environment (Yuan et al, 1998; Sahai and Marshall, 2002; Wong et al, 2003). Such changes could confer significant growth advantages, contributing to the initiation, promotion, or progression of cancer, as well as metastasis. The loss of function of DLC1 has been demonstrated to be a significant contributor to many human cancers as described above. However, this gene and its protein have not yet been characterized in the dog. The purpose of the present study was to screen clinical lymphoma samples for the presence of hypermethylation. Companion dogs with NHL have been

2. Methylation specific PCR The MethPrimer website was used to construct primers to amplify a 183bp region in the proximal predicted first intron, located between bases 39,535,120-39,535,307 of chromosome 16 (Table 1). Using bisulfite treated normal canine DNA, the conditions for MSP were optimized. The methylated primer set was used to amplify the region with an annealing temperature of 62°C for 30s, an extension temperature of 72°C for 30s, and a melting temperature of 95°C for 15s, repeating for 32 cycles. The


Cancer Therapy Vol 6, page 143 unmethylated primer set was used to amplify the region with an annealing temperature of 62°C for 60s, an extension temperature of 72°C for 60s, and a melting temperature of 95°C for 15s, repeating for 32 cycles. The PCR products were run on a 1.5% agarose gel with ethidium bromide. The negative control was bisulfite converted DNA from normal lymph node, and the positive control was DNA from a normal lymph node methylated in vitro using SssI, then bisulfite converted.

III. Results Searching the canine genome using the reference number NM_182643 for DLC1 yielded a highly significant similarity with a canine sequence in chromosome 16 (score 1328, Expect Value 0.0). This region is part of a predicted RhoGAP protein. The sequence of the canine 5‘ region corresponding to the human DLC1 promoter was retrieved (Figure 1). This sequence yielded two CpG islands in the MethPrimer analysis (Figure 1). These islands are large, 1,187 and 557 bp, respectively, with a gap of 223bp between them. The PromoterScan analysis of the reverse strand yielded an extremely high promoter score of 260.20 with a minimum promoter cutoff of 53.00 from bases 39,535,766 to 39,536,015. This identified promoter region corresponds highly to the reported human gene promoter region (Yuan et al, 1998). The promoter analysis identified 16 Sp-1 binding sites in close proximity with proper orientation to gene transcription. The transcription factor binding sites identified by Promoterscan in dogs and humans are listed in Table 1. Analysis of the sequence downstream of the predicted promoter region using the InterProScan webtool, revealed the presence of code for all the major functional units of the human DLC1 protein. These include the Rho-GAP active site, a lipid binding steroidogenic acute regulatory protein (START) site, and a sterile alpha motif homology 2 (SAM2) unit. The algorithm N-Scan predicts the location of a gene in the region that this examination predicts the DLC1 gene of dogs with all functional subunits included (van Baren and Brent, 2006).

3. COBRA The MethPrimer website was used to construct primer pairs for COBRA to amplify a 284bp region in the proximal predicted first intron, located between bases 39,534,204-39,534,492 of chromosome 16 (Table 1). These primers do not contain CpG dinucleotides, and will amplify DNA whether or not methylation is present in the gene. The conditions for PCR were an annealing temperature of 58°C for 60s, an extension temperature of 72°C for 60s, and a melting temperature of 95°C for 15s, repeating for 32 cycles. The product size of these primers is 284 bp and contains two BstUI (New England Biolabs, Ipswich, MA) cutsites that recognize the sequence CGCG and yield fragments of 27 bp, 38 bp, and 219 bp. For BstUI, 10!L of PCR product was added to 2.5!L of Buffer 2, 1!L of BstUI, and 11.5!L of HyPure water, and incubated at 60ºC for 4h. Controls were identical to MSP. The PCR products were run on a 1.5% agarose gel with ethidium bromide.

4. Statistical analysis DNA sequences were considered similar in BLAST search if the score was > 900 and the Expect Value was < 0.0001. Promoter score cutoff was set at a minimum of 53 on the Promoterscan software to identify a promoter region (Prestridge, 1995). For the CpG island determination, parameters were set to identify a sequence with a minimum length of 100 bases, a GC percentage > 50%, and a CpG observed/expected ratio of 0.6 (Li and Dahiya, 2002).

Figure 4. This is a composite of gels showing the results from the first 13 lymph node aspirate samples and CLL samples evaluated. M denotes methylated primers; U denotes unmethylated primers; UCX is unmethylated control; MCX is the SssI-treated methylated control; lymphoma samples are numbered one through 13. CLL samples are one through three. Interpretation of each lane is presented in Table 2.


Bryan et al: DNA Methylation review

Table 1. Transcription factors identified by Promoterscan in orthologous dog and human promoter regions of DLC1. Dog Transcription Factor Similar Factors Sp1 JCV_repeated_seq T-Ag GCF Differing Factors AP-2 NGFI-C KROX24 UCE.2 MRE_CS6 JunB-US2


Human Transcription Factor


16 2 2 2

Sp1 JCV_repeated_seq T-Ag GCF

8 1 1 1

2 1 1 0 0 0


0 0 0 1 1 1

Figure 5. This gel shows lymphoma samples one through 13. The symbol “–“ denotes the negative methylation control and “+” the SssI -treated methylation control. Digestion of the PCR product with BstUI could yield bands of 219bp, 38bp, and 27bp from the original 284bp product. Interpretation of each lane is presented in Table 2.

Table 2. Results of MSPCR for thirteen canine lymph node aspiration samples. The symbol “+” denotes amplification with primers for methylated or unmethylated DNA. – denotes failure to amplify for the specified primer. Three patients showed no amplification with either primer set. Immunophenotype is denoted by B for B-cell, T for T-cell of N for not determined. Dog Unmethylated Methylated No Amplification COBRA CpG I 2 Immunophenotype

Normal + -

1 + -

2 +

3 + +

4 + +

5 + -

6 + +


+ B

+ B

+ N

+ B

+ N

+ N


8 + +


Examination of the CpG island containing the promoter region by MSP demonstrated the presence of bands from methylated primers in six of 13 NHL samples and two of three CLL samples (Table 1, Figure 4). Nine



10 + +

11 + -


+ T

+ + N



13 + -

+ N

+ T

of 13 NHL samples and two of three CLL samples demonstrated bands from unmethylated primers. Three NHL samples did not amplify with either primer set. Examination of the second CpG island by COBRA 144

Cancer Therapy Vol 6, page 145 demonstrated methylation in nine of 13 samples with the same three samples failing to amplify (Table 1, Figure 5). Immunophenotype was determined for six of the dogs with four B-cell and two T-cell lymphomas. Of these, two of four B-cell samples showed methylation by MSP and one did not amplify. No T-cell sample showed methylation by MSP, but both showed methylation in the 3â&#x20AC;&#x2122; CpG island by COBRA. Immunophenotype was not determined for CLL. No normal sample was positive for methylation. The DNA of at least three individual normal dogs was used in the development of the assay.

COBRA assay, which examined the CpG island located further downstream. It is significant that this methylation was identified using samples collected by fine needle aspiration. This underscores the ease of analysis and potential diagnostic utility of epigenetic changes that can be assayed by aspiration. Three canine NHL samples did not amplify. All three had lower DNA concentration and appeared at least partially degraded on agarose gel analysis. The lack of amplification could also be a result of deletion. Loss of the region of canine chromosome 16 containing this gene location has been reported in one case of T-cell lymphoma in a female Cocker spaniel (Thomas et al, 2003). Further study will be necessary to elucidate the frequency of such an event. Five of the six NHL and both methylated CLL samples also had a positive unmethylated band. The significance of this ummethylated band is not entirely clear at this time. It is certain that the cell population from which DNA was extracted was heterogeneous in composition, with neoplastic and normal cells mixed. The samples were lymph node aspirates, so would have contained more than one cell type. It is also possible that the neoplastic populations were heterogeneous in nature. Finally, the neoplastic cells could be individually heterozygous for methylation. Simultaneous demonstration of methylated and unmethylated markers is seen in human tumors as well (Kovalchuk et al, 2004; Shi et al, 2006; Taylor et al, 2007b) Whatever the source of the unmethylated DNA, the presence of hypermethylation of an important tumor suppressor gene in naturally occurring canine cancer represents the first report of its kind and is a significant find. It is significant that the observed methylation was present only in neoplastic tissue samples. As for the human DLC1 gene, methylation of the canine gene at this location may serve as a useful marker of the neoplastic phenotype (Wang et al, 2007). A methylation assay may identify early or even pre-neoplastic lesions that could be used to monitor nodes for minimal residual disease, or detect early relapse. To have a cancer-specific test that is robust and could be performed on lymph node aspiration samples rapidly and inexpensively would facilitate efficient diagnosis of canine lymphoma. Such a diagnostic test could also serve as a pre-clinical evaluation of similar technology in humans. Finally, as the clinical use of demethylating therapy is better defined, such a test may serve as a prognostic marker of likely response to therapy, even if expression of the specific gene identified is not modified.

IV. Discussion Epigenetic mechanisms have been shown to play an important role in many human cancers, including NHL. Whether these changes are causative or the result of neoplastic transformation has not been clearly established (Herman and Baylin, 2003). Environmental influences have been identified which modify the methylation pattern of DNA in laboratory animals (Ho et al, 2006). Such changes may cause or promote human cancers, however the epidemiology remains controversial (Welshons et al, 2003). Companion animals tend to share the lives and environmental exposures of their human counterparts (Hansen and Khanna, 2004). This represents an untapped resource of information on the interplay of environment and disease. Developing evidence that demonstrates similar epigenetic alterations in human and canine disease is critical to the development of this naturally occurring model of cancer. The sequence for the canine RhoGAP gene identified using the NCBI site is clearly orthologous to the human DLC1 gene. The high score and low Expect value make it a statistical impossibility that this gene is anything but the DLC1 ortholog. This gene lies on the long arm of canine chromosome 16, a 64Mbp chromosome. Through eons of recombination and fragmentation, this chromosome in the dog contains portions of the genes of human chromosome 8, along with genes located on human chromosomes 4 and 7. The sequence of the predicted promoter region lies within a CpG island, similar to the arrangement of the human gene, with a second, smaller CpG island present that is not seen in the human sequence. The promoter score is extremely high for a dog sequence using the webbased tool. This is the highest score observed by the authors for a canine gene. It is much higher than the score for the human promoter region of DLC1, 99.09. The UCSC Genome Browser clearly shows good alignment of this predicted region with the promoter, first exon, and first intron of the human DLC1 gene. The presence of numerous Sp-1 transcription factor binding sites in the promoter region also suggests that methylation could play a role in control of the gene. This transcription factor has been demonstrated in humans to be inhibited from binding by the presence of methylation (Mancini et al, 1999). The similarity between the predicted canine promoter region and the human tumor suppressor gene makes the identified area the most likely 5â&#x20AC;&#x2122; region of this canine ortholog. Methylation of the DLC1 CpG islands was evident in the majority of the canine NHL and CLL when assayed by MSP and COBRA. The proportion was higher in the

Acknowledgements This work was presented in part at the 22nd Annual Meeting of the Veterinary Cancer Society.

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Cancer Therapy Vol 6, page 149 Cancer Therapy Vol 6, 149-162, 2008

Estrogen receptor ! mediates the protective effects of estrogen in colon cancer Review Article

Maria Marino*, Paola Galluzzo Department of Biology, University Roma Tre, Viale G. Marconi 446, I-00146 Roma, Italy.

__________________________________________________________________________________ *Correspondence: Maria Marino, Department of Biology, University Roma Tre, Viale G. Marconi, 446, I-00146 Roma, Italy; Fax: +39-06-55176321; E-mail: Key words: 17!-Estradiol, Estrogen receptor !, Estrogen receptor palmitoylation, Colon cancer cell, Apoptosis, Membrane association of estrogen receptors, Estrogen receptor signal transduction Abbreviations: 2-Bromopalmitate, (2Br); activating factor-1, (AP-1); colorectal cancer, (CRC); cyclic AMP, (cAMP); diarylpropionitrile, (DPN); DNA binding domain, (DBD); estrogen receptor, (ER); estrogen response element, (ERE); inflammatory bowel disease, (IBD); ligand binding domain, (LBD); mitogen activated protein kinase, (MAPK); nitric oxide, (NO); palmitoyl acyl transferase, (PAT); phosphatidyl inositol 3 kinase, (PI3K), phospholipase C, (PLC); poly(ADP-ribose) polymerase, (PARP); protein kinase C, (PKCs); stimulating factor-1 (Sp-1); wild-type, (WT) Received: 4 January 2008; Revised: 22 February 2008 Accepted: 26 February 2008; electronically published: March 2008

Summary Epidemiological studies indicate that colorectal cancer is more common in men than in women, the difference being more striking amongst pre-menopausal women and age-matched men. These differences suggest that the sex steroid hormone estrogens could elicit protective effects against this disease. Estrogen receptors (ER" and ER!) are differently expressed in colon. In particular, ER" is minimally expressed in normal colon mucosa and colon cancer cells, whereas ER! is the predominant subtype expressed in human colon. Thus, the protective effects of estrogen on colon cancer should be mediated by specific signal transduction pathways activated by ER!. ER! is significantly decreased in colonic tumors compared with normal mucosa. The loss of ER! leads to hyperproliferation, loss of differentiation, and decreased apoptosis in the epithelium of the colon, suggesting a pivotal role for ER! in the organization and architectural maintenance of the colon, and its potential role in the regulation of colon tumor growth. The mechanisms underlying estrogen effects against colon cancer are starting to be elucidated. Here, the epidemiological and experimental evidence as well as the molecular mechanisms sustaining the ER! role as a tumor-suppressor in colon cancer will be reviewed.

hypothesized to reflect full length ER! (Enmark et al, 1997). The following year, Ogawa and co-workers reported the cloning of an additional ER!, consisting of 530 amino acids, which is now considered to represent the full length ER! (Ogawa et al, 1998a). A few months later, Moore and co-workers also identified the same 530 amino acid sequence as the full length ER!, as well as various other isoforms (Moore et al, 1998). It would not be an overstatement to say that this discovery electrified the field of estrogen biology, causing a re-examination of all our understanding and assumptions about the mechanism of estrogen action. Early studies, naturally, focused on cloning ER! from additional species (Mosselman et al, 1996; Tremblay et al, 1997), describing ER! ligand specificity (Kuiper et al, 1997; Kuiper et al, 1998b; Harris et al, 2002) and tissue distribution (Couse et al, 1997;

I. Introduction Estrogens, in particular 17!-estradiol (E2) the most potent and dominant estrogen in humans, elicit a myriad of biological responses directed towards profoundly changing female physiology. Since the early 1960s it has been accepted that these actions are receptor mediated. When an estrogen receptor (ER) was cloned in 1986 (Green et al, 1986a,b) and its ablation in mice had the typical E2-deficiency phenotype in many organs (Lubahn et al, 1993), it seemed a plausible conclusion that only one ER existed. It was, thus, an enormous surprise when the second ER, ER!, was cloned from a prostate cDNA library (Kuiper et al, 1996). At first, a human ER! with 477 amino acids was reported (Mosselman et al, 1996). A few months later, Enmark and co-workers reported the identification of an ER! with 485 amino acids, and it was


Marino and Galluzzo: ER! and colon cancer Shughrue et al, 1997; Kuiper et al, 1998a; Shughure et al, 1998; Harris, 2007). Presently, the research is focused on the comprehension of the role played by the receptor isoform in the E2 effects (Imanov et al, 2005; Koehler et al, 2005; Harris, 2007). Indeed, the discovery of ER! changed the concept of estrogen signaling, opening a new chapter in E2 effects and in the design of estrogenic pharmaceuticals. This widened the role of E2 in human physiology (Ascenzi et al, 2006; Deroo and Korach, 2006; Hewitt et al, 2000), which at the present is not only limited to reproductive female functions, but has been also found in the modulation of the growth of different tissues, bone integrity, cardiovascular apparatus, immune system, and nervous system physiology as well as in the regulation of male physiology (Ascenzi et al, 2006). Given this widespread role of E2 in human physiology, it is not surprising that estrogens are also implicated in the development or progression of diseases, which include various types of cancer (breast, ovarian, and endometrial), osteoporosis, neurodegenerative and cardiovascular diseases, insulin resistance, lupus erythematosus, endometriosis, and obesity (Ascenzi et al, 2006; Deroo and Korach, 2006). However, recent epidemiological, clinical, and experimental evidence shows that E2 also confers protection against cell proliferation and malignant transformation (e.g., prostate and colon cancer) (Horvath et al, 2001; Konstantinopoulos et al, 2003; Bardin et al, 2004a; Acconcia et al, 2005; Koehler et al, 2005; Caiazza et al, 2007; Galluzzo et al, 2007). The role of each ER isoform and the E2-dependent signal transduction mechanisms have recently started to be clarified. ER" seems to mediate proliferative effects of E2 (Castoria et al, 1999, 2001; Lobenhofer et al, 2000; Marino et al, 2002; Fernando and Wimalasena, 2004); on the contrary, recent evidence (Acconcia et al, 2005; Marino et al, 2006a; Caiazza et al, 2007; Galluzzo et al, 2007) indicates that ER! directs the anti-proliferative effects of E2. This hypothesis needs to be confirmed, but could make sense in view of the location of ER! on chromosome 14q (Enmark et al, 1997). A loss of 14q has been detected by comparative genomic hybridization in some breast cancers (Burki et al, 2000; Loveday et al, 2000). Interestingly, in

ovarian cancer, two potential tumor-suppressor gene loci have been mapped to 14q (Bandera et al, 1997). 14q deletions are also observed in colon carcinoma (Young et al, 1993) and prostate cancer (Kasahara et al, 2002). These overall findings suggest a potential tumor-suppressive function for ER!. In this review the epidemiological and experimental evidence as well as the molecular mechanism(s) supporting the role of ER! as a tumor-suppressor will be discussed with particular attention to colon cancer.

II. ER! structure Human ER!, like ER", is a modular protein sharing common regions, named A/B, C, D, E/F, with all the members of the nuclear receptor super-family. These regions participate in the formation of structurally independent but functionally interacting domains: the Nterminal transactivation domain, the DNA binding domain (DBD), the dimerization domain(s), the nuclear localization sequence, and the ligand binding domain (LBD) (Figure 1) (Ribeiro et al, 1995; Mosselman et al, 1996; Kumar and Thompson, 1999; Nillson et al, 2001; Claessens and Gewirth, 2004; Kumar et al, 2004; Ascenzi et al, 2006). The N-terminal domain (A/B region) is involved in both inter-molecular and intra-molecular interactions as well as in the activation of gene transcription. The DBD (C region) allows ERs to dimerize and to bind to the specific estrogen responsive element (ERE) sequence on DNA through its two â&#x20AC;&#x153;zinc fingerâ&#x20AC;? structures. The hinge domain (D region) has a role in receptor dimerization and in binding to chaperone heat-shock proteins (Hsp). The LBD (E/F region, C-terminal) comprises the E2-binding domain and acts, synergistically with the N-terminal domain in the regulation of gene transcription (Mosselman et al, 1996; Nilsson et al, 2001; Claessens and Gewirth, 2004; Kumar et al, 2004). The two regions present in ERs that contribute to transcriptional activity are called activation functions (AFs). AF-1 is located in the Nterminal region and could be activated even in a ligandindependent manner depending on the phosphorylation

Figure 1. A schematic structural comparison of human ER" and ER! functional domains. Receptor domains are illustrated with different colored boxes, and the approximate size of each domain is indicated. The A/B domain contains the ligand-independent transcriptional-activation function AF-1, the C domain represents the DNA-binding-domain (DBD), the D domain corresponds to the hinge region, and the E domain contains the hormone-binding domain (LBD) and the hormone-dependent transcriptional-activation function AF-2. The number inside each box of ER! refers to the percentage of amino acid identity. For details, see text.


Cancer Therapy Vol 6, page 151 status of ERs (Bunone et al, 1996; Weigel and Zhang, 1998; Tremblay et al, 1999). AF-2 is present at the Cterminus and shows a ligand-dependent activation (Wrenn and Katzenellenbogen, 1993; Weihua et al, 2003; Galluzzo and Marino, 2006). Besides full-length ERs, numerous mRNA splice variants exist for both ERs and these have been found in a number of different normal and diseased tissues frequently co-expressed with their wild-type counterparts. The exact function and potential role of ER! and ER! splice variants in physiology and human disease remain to be elucidated (see Ascenzi et al, 2006; Herynk and Fuqua, 2004), but several experiments indicate that ER! isoforms can differentially modulate estrogen signaling and, as a consequence, impact upon target gene regulation. At least five splice variant isoforms of the ER! gene product (ER!1-ER!5) have been described (Moore et al, 1998; Wong et al, 2005) (Figure 2): the 530-amino acid human ER! isoform is currently regarded as the wild-type ER!;

ER!2 (also called ER!cx) (Ogawa et al, 1998b) is identical to the ER! long form, except that 26 unique amino acid residues replaces the C-terminal of LBD. Additionally, two truncated isoforms have been identified and named ER!4 and ER!5. Several additional ER! isoforms have been reported, although full-length sequences have not been determined (Ascenzi et al, 2006; Matthews and Gustaffson, 2003; Heldring et al, 2007).

III. ER mechanism of action The mechanisms of ER! and ER! action are complex pathways that involve two distinct types of signaling which lead to protein kinase activation (rapid non-genomic mechanism) and direct or indirect transcription of target genes (genomic mechanism) (Figure 3). All these pathways synergize to determine the over all effects of E2.

Figure 2: ER! splicing variants. The striped fill patterns of the 3â&#x20AC;&#x2122; end of hER!2 (also named hER!cx), hER!4, and hER!5 represent the differing C-terminal regions of these isoforms. For details, see text.

Figure 3: Schematic model illustrating the localization at and maintenance to the plasma membrane of ER!. In the first panel (a) is depicted the classical interaction of the activated receptor with ERE on DNA. In panels b and c are representations of the indirect effects of ERs on transcription interactions. This occurs through protein-protein interactions with the Sp1 (b), AP-1 (c). The panel d represents the E2-non-genomic mechanism. For details, see text. AP-1, activating factor-1; Sp-1, stimulating factor-1. For details, see text.


Marino and Galluzzo: ER! and colon cancer promoter resulted in attenuation of promoter responsiveness to E2 (Marino et al, 2002, 2003). Unlike ER", E2-bound ER! represses cyclin D1 expression (Acconcia et al, 2005) and blocks ER"-E2-mediated induction when both receptor isoforms are present (Matthew and Gustafsson, 2003). Consequently, these differences in transcriptional activity between the ER" and ER! may account for the major differences in their tissuespecific biological actions. This complexity is further enhanced by the presence of different ER! splice variants, through the ability of the ER isoforms to form homodimers and hetero-dimers, and by their capacity to interact with different co-regulators (Bardin et al, 2004a; Marino et al, 2006b). ER!1 is essential for ER!-induced transcription initiation at ERE, whereas the other ER! isoforms have no innate transcriptional activity but play an enhancement role when dimerized with ER!1. ER!1 is the only full-functioning ER!, which preferentially heterodimerize with other ER! isoforms, particularly ER!4 and ER!5 forming “variable dimer partners” under the stimulation of estrogens (Leung et al, 2006). Furthermore, ER!2 (ER!cx), which is unable to bind ligands or coactivators and has no transcriptional activity in reporter assays, shows preferential hetero-dimerization with ER" rather than with ER!. ER!2 inhibits ER" DNA binding and has a dominant-negative effect on ligand-dependent ER! reporter gene activity (Ogawa et al, 1998b). These data suggest that the ER! isoforms could differentially modulate E2 action (Ascenzi et al, 2006) and make elucidating the physiological role of this receptor more difficult.

A. ER! genomic mechanism In the “classical” mechanism of action, estrogens diffuse into the cell and bind to the ERs. The nuclear ER"or ER!-E2 complex directly binds DNA through the ERE sequences or indirectly through protein-protein interactions with activator protein-1 (AP-1) or stimulating protein (Sp-1), resulting in recruitment of coregulatory proteins (coactivators or corepressors) to the promoter, increased or decreased mRNA levels, protein production, and physiological responses (Ascenzi et al, 2006; Deroo and Korach, 2006). A large subset of coregulatory proteins (e.g., steroid receptor coactivator-1, 2, and 3) help to recruit histone acetyltransferases and methyltransferases which, in turn, possess chromatin-remodeling ability and tether activated receptors to the basal transcriptional machinery (Smith and O’Malley, 2004). Both ER" and ER! are capable of regulating gene transcription through this classical mechanism involving ERE, but ER! seems to be a weaker transactivator (Cowley and Parker, 1999). AF-1 activity of ER! is weak compared with that of ER" on ERE, whereas their AF-2 activities are similar (Cowley and Parker, 1999). In turn, when both AF-1 and AF-2 functions are active in a particular cell and/or on a particular promoter, the activity of ER" greatly exceeds that of ER!, whereas ER" and ER! activities are similar when only AF-2 is required (McInerney et al, 1998; Cowley and Parker, 1999; Ascenzi et al, 2006). It has been postulated that differences in the ER! and ER! activities are due to differences in the ability of the receptors to interact with coregulatory proteins, because of the low amino acid identity in A/B domain of ERs (Figure 1) (Smith and O’Malley, 2004; Ascenzi et al, 2006). Another category of gene promoters, lacking any ERE-like sequences, requires a second DNA-binding transcription factor (e.g. Sp-1 and AP-1) to mediate ER association with the DNA (O’Lone et al, 2004). Although ER" and ER! have similar effects on ERE-mediated gene transcription, the receptors show opposite effects on promoters containing AP-1 (Paech et al, 1997). E2 activates AP-1-mediated gene transcription when bound to ER" but inhibits promoter activity when bound to ER! (Paech et al, 1997). The converse is true for anti-estrogens, such as tamoxifen, raloxifene, and ICI 164384, which are AP-1 transcriptional suppressors via ER" and activators via ER! (Paech et al, 1997; Weyant, et al, 2001). Similar to AP-1, E2 binding to ER" induces transcriptional activation when associated with Sp-1 in GC-rich regions. However, E2 binding to ER! does not result in the formation of a transcriptionally active complex at a promoter containing Sp-1 elements (Saville et al, 2000). As an example ER" and ER!, in the presence of E2, oppose each other’s function in the regulation of the cyclin D1 promoter (Liu et al, 2002). There is considerable evidence that cyclin D1, important for the progression of cells through the G1 phase of the cell cycle, is a welldefined target for ER"-E2 action in mammary carcinoma cells (Altucci et al, 1996; Foster and Wimalasena, 1996; Prall et al, 1997), although no detectable “perfect” or ERE-like sequence in the cyclin D1 gene promoter has been reported (Herber et al, 1994). Deletion of AP-1 and Sp-1 responsive element motifs in the cyclin D1 gene

B. ER! non-genomic mechanism The “classical” genomic mechanism of ER" and ER! action typically occurs over the course of hours. In contrast, E2 can act more quickly (within seconds or minutes) via “non-genomic” mechanisms that require the presence of ER" at the plasma membrane and resulting in such cellular responses as increased levels of Ca2+ or NO, and activation of kinases, such as phospholipase C (PLC)/protein kinase C (PKCs); Ras/Raf/mitogen activated protein kinase (MAPK), phosphatidyl inositol 3 kinase (PI3K)/AKT, c-src/MAPK, and cyclic AMP (cAMP)/ protein kinase A (PKA) (Castoria et al, 2001; Levin, 2005; Marino et al, 2006b and literature cited therein). Only limited information is available on the role played by the ER!-E2 complex in the activation of rapid non-genomic mechanisms, whereas a considerable volume of evidence (Ansonoff and Etgen, 1998; Improta-Brears et al, 1999; Kahlert et al, 2000; Marino et al, 2001, 2005, 2006b; Chambliss and Shaul, 2002; Dos Santos et al, 2002; Chambliss et al, 2005; Kim and Bender, 2005; Kupzig et al, 2005) points to ER" as the primary endogenous mediator of rapid E2-induced signalling, which contributes to the proliferative effects of the hormone. Data from cell culture, gene expression, and knockout mice indicate that E2-activated ER! can function as a dominant negative suppressor of the proliferative activity of ER" (Couse and Korach, 1999; Paruthiyil et al, 2004; Strom et al, 2004; Weihua et al, 152

Cancer Therapy Vol 6, page 153 2003). These studies support a functional antagonism between ER" and ER! with respect to the E2-induced cell proliferation, but do not clarify either the putative role of ER! in E2-induced apoptosis or the signal transduction pathways involved. The ability of the ER!-E2 complex to activate rapid non-genomic mechanisms has been reported, however these data are limited and conflicting. A sub-population of ER! transfected into Chinese Hamster ovary cells is capable of stimulating IP3 production, ERK/MAPK activation, and c-JNK phosphorylation (Razandi et al, 1999). Geraldes and coworkers reported that E2 reduces ERK activity through ER! stimulation in porcine smooth muscle cells (Geraldes et al, 2003). In ER!-transfected HeLa cell, we have recently reported ER! rapidly induces a persistent membrane-initiated activation of p38/MAPK which in turn is involved in caspase-3 activation and cleavage of poly(ADP-ribose) polymerase (PARP), driving cells to the apoptosis. Consequently, besides its role as a negative modulator of ER" activities, our findings indicate that ER! directs the anti-proliferative effects of E2 sustaining the tumor suppressor functions of ER! (Acconcia et al, 2005). Several labs have shown that kinase signaling cascades promote gene expression through the phosphorylation of coactivators. In particular, p38/MAPK phosphorylates and potentiates the SRC-2 coactivator (Frigo et al, 2006). How this result impacts on E2-induced protective effects on colon cancer growth is still unknown. However, these data support the idea that E2-induced rapid signals synergize with genomic events to maintain the pleiotropic hormone effects.

expression of the differentiation markers (Wada-Hiraike et al, 2006b). As a whole, the loss of ER! leads to hyperproliferation, loss of differentiation, and decreased apoptosis in the cells of the colonic epithelium suggesting a pivotal role for ER! in the organization and architectural maintenance of the epithelial barrier (Wada-Hiraike et al, 2006a).

V. ER! and colon cancer Epidemiological studies have ascertained that colorectal cancer (CRC) is the second to fourth most common fatal malignancy in industrialized countries (Potter, 1999; Slattery et al, 2001). The prognosis for patients with this disease is heavily dependent on the stage of the malignancy and on time of diagnosis and almost all patients will require surgical intervention. Therefore early diagnosis and prevention is extremely important. CRC is thought to develop as a consequence of aberrant crypt proliferation or progression of benign hyperplasia to benign adenoma and then in most cases to adenocarcinoma (Vogelstein et al, 1988; Wada-Hiraike et al, 2006). CRC is a common malignancy in both sexes (DeCosse et al, 1993). However, several sex-related differences in incidence, molecular characteristics and response to chemotherapy have been reported. It has therefore been suggested that exposure to E2 and/or estrogenic compounds may underlie these differences. CRC is more common in men than women, the difference being more striking amongst premenopausal women and age-matched men (DeCosse et al, 1993; Wong et al, 2005). In the early 1970s, a transient decrease in colon cancer incidence occurred among women aged 35-44 years old, but not among men (McMichael and Potter, 1980). This observation correlated with a peak in fertility and the use of high dose oral contraceptives during the preceding decade. The authors concluded that either high fertility or exposure to exogenous steroid hormones protected women from colon cancer. Based on metaanalysis of 18 epidemiological studies, the use of hormone replacement therapy by postmenopausal women was associated with a 20% decrease in colon cancer risk (Bhat et al, 2003; Guo et al, 2004). Other studies also demonstrated that women with a history of current or past hormone replacement therapy had a significantly decreased risk of colon cancer and showed that there are gender differences regarding cancer location and type within the colorectum (Cho et al, 2007). These findings have led many investigators to search for the ER isoform involved in E2 protection against colorectal cancer. Since ER" is reported to be minimally expressed in normal colon mucosa and colon cancer cells (Campbell-Thompson et al, 2001; Waliszewski et al, 1997), the effects of E2 on colon cancer susceptibility could be mediated by ER! (Bardin et al, 2004a). ER!1, ER!2 and ER!5 have been demonstrated in normal colorectal mucosa and at much higher levels than ER" (Foley et al, 2000; Campbell-Thompson et al, 2001). As described above, only the full-length ER! protein, equivalent to the ER!1 isoform, can activate ERE in reporter assays (Peng et al, 2003; Wong et al, 2005). On the other hand, the presence of different ER!-isoforms

IV. ER! and colon differentiation Although, mouse physiology is clearly different from human, knockout experiments targeting ER! genes in mice have been useful in understanding the role played by ER" and ER! in human physiology. These are valuable experimental models which provide a basic insight into the normal functions of genes during development and maturity. Indeed, targeted disruption of ER! in mice (Kuiper et al, 1996) has revealed roles for ER! in many tissues and organs, including the ovary, uterus, mammary gland, ventral prostate, salivary gland, immune system, and the central nervous system (Nilsson et al, 2001; Forster et al, 2002; Rossouw et al, 2002; Shim et al, 2003; Imanov et al, 2004). In particular, several studies have revealed that ER! is the predominant ER expressed in colonic tissues (Campbell-Thompson et al, 2001; Konstantinopoulos et al, 2003), its expression being selectively lost in human malignant colon tissue (Foley et al, 2000; Issa et al, 1994; Wada-Hiraike et al, 2006a). In a recent study, to better understand the physiological role of ER! in colonic tissue, Wada-Hiraike and coworkers compared in 2006 morphology, proliferation, and differentiation of colonic epithelium in ER!-/- mice and wild-type (WT) littermates. BrdUrd labeling revealed that the number of proliferating cells was higher in ER!-/- mice than in WT and that the migration of labeled cells toward the luminal surface was faster in ER!/mice than in WT littermates. Additionally, in the absence of ER!, there was a decrease in apoptosis and in the 153

Marino and Galluzzo: ER! and colon cancer (Galluzzo et al, 2007). Upon E2 stimulation, ER" undergoes de-palmitoylation that is paralleled by an increased association of the receptor with caveolin-1 and to p38. The physical association ER"-caveolin-1 and p38 increases the abundance of ER" at the plasma membrane, impairing its association to other signaling proteins which characterize E2-induced ER!-mediated cell survival and proliferation (e.g., Src, ERK/MAPK and PI3K/AKT) (Galluzzo et al, 2007). On the other hand, the E2-induced ER" association with p38 impacts greatly on DLD-1 colon cancer cells. Since p38 activation is required for the establishment of a downstream pro-apoptotic cascade involving caspase-3 and PARP cleavage. E2-induced p38/MAPK is also fundamental both for the rapid increase of ER" mRNA translation and for the slow ER" gene transcription (Caiazza et al, 2007). The mRNAs encoding hormone receptors are commonly regulated by their own hormones to create auto-regulatory feedback loops. Moreover, different hormones, including steroid hormones, regulate concentrations of various gene products primarily by altering mRNA translation and stability (Ing, 2005). The final consequence is an increased level of ER" in DLD-1 cells which, in the presence of E2, will further increase the hormone protective effect against tumor growth (Figure 4). The signalling cascade initiated through ER! at the plasma membrane seems to be mainly involved in the protective effects of E2 against colon cancer. In fact, the treatment of these cells with the palmitoylation inhibitor 2Bromopalmitate (2Br) completely removed ER! from the plasma membrane impairing p38 activation. This condition prevents the activation of the pro-apoptotic cascade without interfering with ER! transcriptional activity, which is still able to promote ERE-dependent gene transcription. Experimental studies with nitric oxide (NO) support the hypothesis that E2-induced rapid signaling through ER! is involved in the protective effects of E2 in colon cancer. NO is a diatomic molecule whose presence, induced by several hormones, E2 included, is important for gastrointestinal motility. NO mainly acts through Snitrosylation of Cys residues in target proteins modulating their activity (Beckman and Koppenol, 1996; Jaffrey et al, 2001; Stamler et al, 2001; Nathan, 2004; Marino et al, 2006a). Modulation of ER activity by NO has been demonstrated. NO is able to link to ERâ&#x20AC;&#x2122;s zinc finger impairing the transcriptional activity of the receptors without interfering with rapid signal pathways. Snitrosylation seems to selectively modulate the biological activity of ERs, shifting the receptor from its role as a transcription factor toward rapid functions. For instance, in DLD-1 colon cancer cells, in the occurrence of NO concentration in micromolar range, normally present during peristalsis, transcriptional activity of ER! is inhibited, but ER! maintains its capability to mediate the pro-apoptotic effects of E2 inducing caspase-3 activation and PARP cleavage. When over produced (e.g., during inflammatory processes) NO worsens the effects. Although the ER!-dependent phosphorylation of p38/MAPK is still present, NO inhibits the caspase-3

could differentially modulate E2 action as previous reported (see section II.B. ER! genomic mechanism). Using semi-quantitative RT-PCR, Campbell-Thompson and colleagues showed 2001 that ER! is the predominant ER subtype in the human colon, and that decreased ER!1 (ER!wt) and ER!2 (ER!cx) mRNA levels are associated with colonic tumorigenesis in women. In accordance, Foley and co-workers in 2000 and Konstantinopoulos and co-workers in 2003 showed that ER! expression was significantly lower in colon cancer cells than in normal colonic epithelium, and that there was a progressive decline in ER! expression, which paralleled the loss of malignant colon cell de-differentiation. A mouse model bearing germ line mutations in the Apc (adenomatosis polyposis coli) gene developed multiple intestinal tumors. In this model, the prevention of Apc-associated tumor formation by estrogen correlated with an increase in ER! expression and a decrease in ER" expression (Weyant et al, 2001; Bardin et al, 2004a). In addition to these experimental models, isolated colon cancer cells have also been found to express primarily ER! (Fiorelli et al, 1999; Qiu et al, 2002), where E2 stimulation (10-1000 nmol/L) consistently induced apoptosis in a dose-dependent manner (Qiu et al, 2002; Guo et al, 2004; Acconcia et al, 2005; Caiazza et al, 2007; Galluzzo et al, 2007). Altogether, these results strongly suggest that the ER":ER! ratio is a possible determinant of the susceptibility of colon to E2-induced carcinogenesis, sustaining the theory that binding of E2 to ER" induces a cancer promoting response, whereas binding to ER! exert a protective action (Weyant et al, 2001).

A. ER! mechanism of action in colon cancer The first mechanism for the anti-proliferative action of ER! was proposed by Paruthiyil and co-workers in 2004 and Strom and co-workers in 2004. They showed that introducing ER! into breast cancer cell lines (MCF-7 and T47D), which also expresses ER", caused an inhibition of proliferation in vitro and prevented tumor formation in a mouse xenograft model in response to E2. ER! inhibited proliferation by repressing components of the cell cycle which are associated with proliferation, such as c-myc, cyclin D1, and cyclin A gene transcription, and by increasing the expression of Cdk inhibitor p21Cip1 and p27Kip1, which leads to a G2 cell cycle arrest. These findings suggested a possible role for ER! as tumor suppressor in breast cancer, impairing ER"-mediated proliferative effects of E2 (Paruthiyil et al, 2004; Strom et al, 2004). But in colonic mucosa and colon cancer cells only ER! is expressed (Campbell-Thompson et al, 2001; Waliszewski et al, 1997), so the protective effects of estrogen on this tissue should be mediated by specific ER!-activated signal transduction pathways. To test this hypothesis, we used DLD-1 colon cancer cells in which only the ER"1 isoform is present. In these cells ER" undergoes palmitoyl acyl transferase (PAT)dependent S-palmitoylation which allows to a small ER" pool to localize at the plasma membrane and associate with caveolin-1 and the p38 member of MAPK family 154

Cancer Therapy Vol 6, page 155 catalytic activity by nitrosylation of the enzymeâ&#x20AC;&#x2122;s cysteine residues (Marino et al, 2006a). In addition to its role as negative modulator of ER" activity as elsewhere reported these novel findings indicate that ER! is able to mediate specific rapid signaling cascades mainly involved in the protective effect of E2 in colon cancer.

peripheral (Bennet et al, 2003) nervous systems, and the immune system (Shim et al, 2003; Koehler et al, 2005). In breast tissues, which express both ER" and ER!, several studies have indicated an increase in ER"/ER! mRNA and protein ratios in cancer as compared with benign tumors and normal tissues. In immunochemical analyses, Roger and colleagues found in a higher percentage of ER!-positive cells in normal mammary glands than carcinoma in situ. In contrast, an increase in ER" protein expression was noted during tumor progression. Moreover, ER! was inversely correlated with Ki67 expression, a cell proliferation marker. Zhao and colleagues also concluded in 2003 that decreased ER! mRNA expression could be associated with breast tumorigenesis and that DNA methylation is an important mechanism for ER! gene silencing in breast cancer.

VI. ER! and other cancers In addition to its presence in colonic tissue, ER! is expressed at high levels in other E2-target tissues, such as prostate (Weihua et al, 2001), salivary glands (Valimaa et al, 2004), testis (Makinen et al, 2001), ovary (Cheng et al, 2002), vascular endothelium (Lindner et al, 1998), smooth muscle (Barchiesi et al, 2004), certain neurons in the central (Shughrue et al, 2000; Mitra et al, 2003) and

Figure 4: Model representing the various modes through which estrogen receptors (ER) can modulate cell functions. Under steady state condition ER! is palmitoylated (black triangle) and localized at the plasma membrane associated to caveolin-1 (cav1) and p38. Upon E2 stimulation ER! undergoes de-palmitoylation, this increases ER! association to cav1, p38, and membrane. This association impairs the ER! re-allocation at the plasma membrane and its association with other signaling proteins. However, ER! association to p38 increases the kinase activity triggering cell functions. ERK, extracellular regulated kinase; MNAR, modulator of nongenomic activity of ER. For details, see text.


Marino and Galluzzo: ER! and colon cancer In 1998 Pujol and coworkers reported an increase in the ER"/ER! mRNA ratio in ovarian carcinomas as compared with normal ovaries and cysts, suggesting that the overexpression of ER" mRNA with respect to ER! mRNA could be considered a marker of ovarian carcinogenesis. Thus the balance between ER" and ER! expression levels seem to be essential for maintaining normal growth function. As the ER! level decreases, this results in the uncontrolled cellular proliferation that leads to a metastatic state (Rutherford et al, 2000). Thus, decreasing levels of ER! expression seem to be a common denominator between breast and ovarian carcinogenesis. The change in the ER"/ER! ratio in prostate cancer seems to be correlated with malignancy, where ER! mRNA level was decreased in most of the tumor samples as compared with normal prostate (Latil et al, 2001), suggesting that ER" and ER! expression status could be used to identify advanced prostate tumor patients. Most studies have shown decreased ER! expression in cancer as compared with benign tumors or normal tissues, whereas ER" expression persists. The loss of ER! expression in cancer cells could reflect tumor cell dedifferentiation but may also represent a critical stage in E2-dependent tumor progression. Consequently, the modulation of the expression of target genes by ER" or ER! sustains that ER! has a differential effect on proliferation as compared with ER". ER! exerts a protective effect and thus constitutes a new target for hormone therapy, based on ligand specific activation (Bardin et al, 2004a,b).

membrane-initiated signaling pathways. Different splicing variants of the ER isoforms may also be important in modulating the cellular response (Leung et al, 2006). Since ER! is the dominant ER isoform in normal colonic mucosa, ER! could represent a target in preventing the malignant transformation of colonic epithelial cells. Results obtained from cellular or animal models, in which ER! was exogenously expressed, show that this receptor is definitely an interesting target for cancer therapy. Strategies that are able to restore or to increase ER! expression or activity, in addition to the selective ER! agonists under development, could definitely be of great interest (Lazennec, 2006). For instance, Schering AG (Berlin, Germany) described two ER isoform selective ligands, which are modified estradiol derivatives (i.e. estradiol 16"-lactone and 8!-vinyl estradiol, for ER" and ER! respectively) (Hillisch et al, 2004). A number of additional subtype-selective ligands have also been described (Koelher et al, 2005): the ER!selective agonists diarylpropionitrile (DPN) (Meyers et al, 2001); indole (Kato et al, 2003), benzoxazole (Barlaam et al, 2002), indenoquinoline (Veeneman et al, 2001), cyclopentachromene (Dodge et al, 2003), cyclopentaindene (Parker et al, 2004), and WAY-358 and ERB-041 (Malamas et al, 2004; Manas et al, 2004). At present, extremely encouraging data for ER!specific agonist action have been reported, for instance in treatment of rodent models of rheumatoid arthritis and endometriosis or atherosclerosis, hypertension, cardiac dysfunction or stroke, and inflammatory bowel disease (IBD). In particular, Harris and co-workers (Harris et al, 2003) have developed and characterized the biological profile of a highly selective ER! agonist, ERB-041. This compound is inactive on traditional target tissues such as the uterus and mammary gland and does not affect bone wasting, inhibit ovulation, or prevent ovariectomy-induced weight gain in clinically predictive rat models. In addition, ERB-041 is not uterotrophic and mammotrophic. However, ERB-041 has a dramatic beneficial effect in the HLA-B27 transgenic rat model of inflammatory bowel disease and the Lewis rat adjuvant-induced arthritis model. Daily oral doses as low as 1 mg/kg reverse the chronic diarrhea of HLA-B27 transgenic rats and dramatically improve histological disease scores in the colon. Thus, other ER!-selective ligands may be therapeutically effective in the treatment of inflammatory bowel disease and/or arthritis (Gustafsson, 2006). The future will be extremely exciting, when results from clinical trials testing the clinical utility of ER! targeted drugs begin to follow. Finally, diet, lifestyle, and other non-genetic factors, such as gut flora, are thought to have a strong impact on CRC risk (Wada-Hiraike et al, 2006a). Diet is known to influence the development of CRC, with high consumption of fruits and vegetables conferring a protective effect. These food categories contain a variety of phytoestrogens capable of modulating ER" and ER! activity (Weyant et al, 2001). Phytoestrogens are believed to play a role as oncoprotective agents and to lower the risk of CRC in rodent models and it has been reported that lignans, plant precursors of the phytoestrogens enterolactone and enterodiol, or lignan-rich food can

VII. Conclusions Epidemiological, clinical, and experimental evidence reported in this review shows that E2 confers protection against colon cell proliferation and malignant transformation, reducing the incidence of colon adenoma and carcinoma by about 20% (Guo et al, 2004). The molecular mechanisms underlying these effects are starting to be clarified. Among others (e.g. epigenetic mechanisms) (Qiu et al, 2002), the specific tissue distribution and the intracellular-generated signals of ERs have opened new avenues for understanding the protective effects of E2 (Acconcia et al. 2005; Weihua et al, 2003; Marino et al, 2006a; Caiazza et al, 2007; Galluzzo et al, 2007). ER! expression in this tissue, especially in the basal region of the colonic crypts, suggests that it has an important role in the growth and regeneration of normal colonic mucosa (Xie et al, 2004). Therefore, an important variable which regulates E2 action on colon tissue is expression and function of ERs (Fiorelli et al, 1999). Numerous clinical and in vitro studies suggest that imbalanced ER"/ER! expression and selective loss of ER! protein is a common feature and could be a critical step in estrogen-dependent tumor progression. ER! seems to play a key role in the mitogenic action of estrogens by providing protection against ER"-induced hyperproliferation and the stimulation of apoptosis. A number of molecular mechanisms could explain the differential roles of ER" and ER!, including differences in ligand affinity and transactivation, distinct cofactor interactions and putative hetero-dimerization, and the activation of distinct 156

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inhibit colon cancer development in animal models (Adlercreutz, 2002; Wada-Hiraike et al, 2006a). Furthermore, some nutritional flavonoids (i.e., quercetin, naringenin, and daidzein) that bind to ER! with higher affinity than ER", act as E2-mimetic in the presence of ER! rapidly activating p38/MAPK (Totta et al, 2004, 2005). Because of their potential to transactivate ER! more than ER", phytoestrogens may be of benefit in tissues in which ER!-mediating signaling plays a significant role (Koheler et al, 2005; Ascenzi et al, 2006; Moutsatsou, 2007). In conclusion, the discovery of ER! has deeply changed our understanding about the biological effects of E2 and the mechanism of ER action. This offers a new prospective for the development of ER! selective drug s for the treatment of diverse clinical conditions.

Acknowledgements The authors wish to thank past and present members of their laboratories who contributed to the ideas presented here through data and discussions. The editorial assistance of Peter DeMuro is also acknowledged. This work was supported by grants from the Ministry of University and Research of Italy (PRIN-COFIN 2006 to M.M.).

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Prediction of very short survival in patients with brain metastases from non-small cell lung cancer Research Article

Carsten Nieder1,2,*, Reinhard Thamm3, Sabrina T. Astner3, Michael Molls3 1

Radiation Oncology Unit, Nordlandssykehuset HF, 8092 Bodø, Norway Institute of Clinical Medicine, Faculty of Medicine, University of Tromsø, Tromsø, Norway 3 Department of Radiation Oncology, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Str. 22, 81675 Munich, Germany 2

__________________________________________________________________________________ *Correspondence: Carsten Nieder, M.D. Ph.D., Radiation Oncology Unit, Medical Department – Oncology, Nordlandssykehuset HF, 8092 Bodø, Norway; Tel: +47 755 78449; Fax: +47 755 34975; e-mail Key words: radiotherapy, brain metastases, lung cancer, whole brain radiotherapy, prognosis Abbreviations: non-small cell lung cancer, (NSCLC); recursive partitioning analysis, (RPA); whole brain radiotherapy, (WBRT) Received: 17 January 2008; Revised: 22 February 2008 Accepted: 28 February 2008; electronically published: March 2008

Summary Current prognostic models are not accurate enough to identify brain metastases patients with non-small cell lung cancer who have very short survival, i.e. <2 months, and therefore are unlikely to derive major benefit from the addition of whole brain radiotherapy (WBRT) to steroid treatment and general supportive measures. Our aim was to develop a more reliable model. A retrospective analysis of patients with squamous cell or adenocarcinoma histology treated with immediate WBRT to 30 Gy was performed. The patients were randomly divided into two groups, i.e. a test and a validation group. The groups included 37 patients each. After analysis of factors predictive for short survival, a risk score based on 5 statistically significant parameters was developed (age, gender, primary tumor histology, extracranial metastases and number of brain metastases). The score performed well with regard to prediction of survival >2 months, but remained unsatisfactory (low positive predictive value) with regard to prediction of survival <2 months even after various optimisation attempts. In conclusion, the prognostic factors for overall survival that form the basis for the recursive partitioning analysis (RPA) classes (age, extracranial metastases, primary tumor control, performance status) do not predict for very short survival. However, we were not able to develop another model that performed accurate enough for clinical decision making.

brain metastases (Gaspar et al, 1997; Nieder et al, 2000; Kepka et al, 2005), including those with primary NSCLC (Rodrigus et al, 2000; Gülbas et al, 2006). However, survival in the most unfavourable class III (defined by Karnofsky performance status, KPS, <70%) is quite variable, with 40-50% of patients dying within 2 months, but 15-20% surviving for more than 6 months (Gaspar et al, 1997; Nieder et al, 2000). Therefore, we aimed at developing a model that is based on readily available clinical parameters and predicts a survival time <2 months in patients with brain metastases from NSCLC better than RPA class and other previous scores.

I. Introduction Whole brain radiotherapy (WBRT) continues to represent an important palliative treatment option for patients with brain metastases from non-small cell lung cancer (NSCLC). The fact that a certain percentage of these patients have very short survival times after WBRT suggests that accurate survival prediction models might help to avoid overtreatment (Lock et al, 2004). Even in brain metastases trials with restrictive inclusion criteria, more than 10% of the patients died within 8 weeks (Mehta et al, 2003; Andrews et al, 2004). It has been reported that survival after corticosteroids alone is in the order of 6-8 weeks (overview in (Khuntia et al, 2006)). In groups with such short survival time, the role of added WBRT is questionable. Prognostic scores such as the recursive partitioning analysis (RPA) classes can support decision making and treatment recommendations in patients with

II. Materials and methods We used a database at the Klinikum rechts der Isar, Munich, Germany, which includes patients with brain metastases treated with 10x3 Gy WBRT and identified all patients with


Nieder et al: Brain metastases from lung cancer primary NSCLC (squamous cell and adenocarcinoma histology). Then, we randomly generated two equally-sized groups (37 patients each) for development and validation of a prognostic score. Patient assignment to these groups was based on year of treatment, where the patients treated in the last year were assigned to the first group, the patients treated in the year before to the second group, and so on in the same alternating fashion (in order to avoid a major bias in treatment period). All patients had received immediate WBRT plus steroids after the diagnosis of brain metastases. No other simultaneous treatments or surgical resection were provided. Treatment decisions were made by a multidisciplinary tumor board. Patients with limited brain disease and favourable prognostic factors were treated during the same time period with either surgery or radiosurgery, but were excluded from this analysis to avoid treatment bias. Chemotherapy after WBRT was given as indicated for extracranial disease. Prognostic factors for overall survival and factors predicting a survival <2 months (60 days) were evaluated by univariate tests in the test group. RPA class, initial KPS, age, presence of extracranial metastases, status of the primary tumor, number of brain metastases, histology, interval from first NSCLC diagnosis to development of brain metastases, gender and dexamethasone dose at the start of WBRT were evaluated. These factors were recorded in all patients. Information on T- and Nstage, tumor grade, tumor markers or other haematology or blood chemistry values was not available. Survival was calculated from the first day of WBRT. Typically, WBRT started within 2 weeks from diagnosis. None of the patients were initially treated with chemotherapy for their brain metastases. We used the KaplanMeier method to generate actuarial survival curves. These were compared with the log rank test to obtain univariate prognostic factors (all performed with the SPSS software). A p-value <0.05 was considered statistically significant. A multivariate analysis of prognostic factors was not performed, as the test group included only 37 patients.

metastases, controlled primary tumor, age less than 65 years) that survived for less than 2 months. In addition, 35% of the patients in class III survived for 3-6 months. Thus, the authors attempted to develop a score based on the 5 significant predictive factors identified in this group. We assigned one point each for male gender, adenocarcinoma, multiple brain metastases, presence of extracranial metastases and age >65 years. When applying this score to the test group, 8 patients were assigned 1 point, 13 patients 2 points, 7 patients 3 points, 6 patients 4 points and 3 patients 5 points. Out of 30 patients with survival >2 months, 29 had 4 or less points while one had 5 points. Out of 7 patients with survival <2 months, 2 had 5 points while 5 had 2-3 points. It was not possible to modify the score in a way that would improve its performance. No single parameter or combination of parameters allowed for reliable prediction of survival <2 months. When looking at the validation group of 37 patients, 9 survived for less than 2 months. These 9 patients scored 1-4 points. Out of 28 patients with survival >2 months, 27 had 4 or less points while one had 5 points. These results confirmed the high specificity (96%) and negative predictive value (75%), however the positive predictive value of 5 score points was 0%. Looking at all 74 patients, there was no pattern identifying the 16 patients that died within 2 months reliably. Whatever prediction model one attempts to use, the risk of withholding WBRT because of suspected poor survival remains higher than 20%. In the present data set, survival <2 months was not observed in patients with initial KPS 90-100%.

III. Results

IV. Discussion

Table 1 shows the patients’ characteristics for the test group. Statistically significant univariate prognostic factors for overall survival included lower RPA class, younger age, higher KPS, absence of extracranial metastases, controlled primary tumor and solitary brain metastasis. However, other factors were important with regard to prediction of survival <2 months, e.g., gender and histology (Table 1). RPA class did not predict survival <2 months reliably. There were, e.g., 2 patients with RPA class I (KPS at least 70%, no extracranial

Outside of prospective clinical trials that include selected patients only, a considerable number of patients with brain metastases from NSCLC die within 2 months, e.g., 22% in this series. The authors tried to develop and validate a clinical risk score that could help to avoid overtreatment in such patients, if one assumes that patients dying within 8 weeks can be managed adequately by best supportive care focused on corticosteroid treatment.

Table 1. Patients’ characteristics in the test group, n=37. Parameter Median age, range Median KPS, range¤ Median time interval*¤ Median dose**¤ % with controlled primary¤ % without extracranial metastases % with solitary brain met. % males % squamous cell cancer

Survival <2 months, n=7 64 yrs., 54-69 80%, 60-80 6 mo., 0-15 12 mg, 8-32 43 43 29 100 29

Survival >2 months, n=30 56 yrs., 44-77 80%, 60-100 7 mo., 0-35 12 mg, 0-32 31 60 46 81 53

KPS: Karnofsky performance status, ¤ not predictive for short survival, i.e. p>0.05, * from lung cancer diagnosis to brain metastases, ** dexamethasone per day at the start of radiotherapy


Cancer Therapy Vol 6, page 165

metastases from non-small-cell lung cancer. Jpn J Clin Oncol 36, 193-196. Jacot W, Quantin X, Boher JM, Andre F, Moreau L, Gainet M, Depierre A, Quoix E, Chevalier TL, Pujol JL; Association d'Enseignement et de Recherche des Internes en Oncologie (2001) Brain metastases at the time of presentation ogf nonsmall cell lung cancer: a multi-centric AERIO analysis of prognostic factors. Br J Cancer 84, 903-909. Kepka L, Cieslak E, Bujko K, Fijuth J, Wierzchowski M (2005) Results of the whole-brain radiotherapy for patients with brain metastases from lung cancer: the RTOG RPA intraclasses analysis. Acta Oncol 44, 389-398. Khuntia D, Brown P, Li J, Mehta MP (2006) Whole-brain radiotherapy in the management of brain metastasis. J Clin Oncol 24, 1295-1304. Lock M, Chow E, Pond GR, Do V, Danjoux C, Dinniwell R, Lea J, Bezjak A (2004) Prognostic factors in brain metastases: can we determine patients who do not benefit from wholebrain radiotherapy? Clin Oncol (R Coll Radiol) 16, 332338. Mehta MP, Rodrigus P, Terhaard CH, Rao A, Suh J, Roa W, Souhami L, Bezjak A, Leibenhaut M, Komaki R, Schultz C, Timmerman R, Curran W, Smith J, Phan SC, Miller RA, Renschler MF (2003) Survival and neurologic outcomes in a randomized trial of motexafin gadolinium and whole-brain radiation therapy in brain metastases. J Clin Oncol 21, 25292536. Nieder C, Nestle U, Motaref B, Walter K, Niewald M, Schnabel K (2000) Prognostic factors in brain metastases: should patients be selected for aggressive treatment according to recursive partitioning analysis (RPA) classes? Int J Radiat Oncol Biol Phys 46, 297-302. Rades D, Schild SE, Lohynska R, Veninga T, Stalpers LJ, Dunst J (2007) Two radiation regimens and prognostic factors for brain metastases in nonsmall cell lung cancer patients. Cancer 110, 1077-1082. Rodrigus P, de Brouwer P, Raaymakers E (2000) Brain metastases and non-small cell lung cancer. Prognostic factors and correlation with survival after irradiation. Lung Cancer 32, 129-136.

The analysis was limited to a narrowly defined group of patients, i.e. those with squamous cell or adenocarcinoma NSCLC, treated with a well defined WBRT regimen, to avoid confounding factors. It was first examined whether RPA class predicts for survival <2 months in the present patient cohort. As this was not the case, attempts to identify other factors were made. From 5 statistically significant parameters, a final score was developed. The majority of these 5 parameters were previously described by other groups, e.g., age and extracranial metastases (Rades et al, 2007) or male gender (Jacot et al, 2001). However, previous analyses were not focussed on short survival. KPS showed a significant impact on overall survival in the present data set, but was not predictive for survival <2 months. This might be explained by the fact that none of our patients had KPS <60%. Usually, patients with very low KPS were not treated with WBRT at our institution. Among those with KPS 90 or 100, all survived for more than 2 months. Eventually, despite of score optimisation attempts, an unacceptable number of patients were not correctly predicted. Lock and colleagues previously evaluated the same endpoint in a larger group of patients, but with different primary tumors (Lock et al, 2004). They found ECOG performance status and number of metastatic sites as important parameters. However, their model classified only 68% of patients correctly, while 55% would have been incorrectly predicted to die early. Thus, all attempts to predict survival <2 months have failed so far. One of the reasons might be that early death in patients with brain metastases might result from different events such as failure to control the intracranial disease, progressive extracranial disease, infectious complications, thromboembolic events etc. Different factors might predict the likelihood of these underlying causes of death, possibly including certain blood count and chemistry abnormalities that were not available for retrospective analysis (leukocyte count, haemoglobin, c-reactive protein etc.). Thus, continuous research is necessary to identify those patients that safely can be managed and palliated without WBRT.

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Cancer Therapy Vol 6, page 167 Cancer Therapy Vol 6, 167-176, 2008

Veterinary radiation oncology: technology, imaging, intervention and future applications Review Article

Ira K. Gordon, Michael S. Kent* Department of Surgical and Radiological Sciences, UC Davis School of Veterinary Medicine, Davis, CA 95616

__________________________________________________________________________________ *Correspondence: Michael S. Kent, Assistant Professor, Department of Surgical and Radiological Sciences, 2112 Tupper Hall, 1 Shields Ave, Davis, CA 95616, USA; Tel: 530-752-1393; Fax: 530-752-9620; E-mail: Key words: Radiation therapy, Oncology, Cancer, Tumor, Veterinary Abbreviations: image guided radiation therapy (IGRT); cone beam computed tomography (CBCT); clinical tumor volume (CTV); computed tomography (CT); functional magnetic resonance imaging (fMRI); Gray (Gy); gross tumor volume (GTV); intensity modulated radiation therapy (IMRT); kilovoltage (kV); magnetic resonance imaging (MRI); magnetic resonance spectroscopic imaging (MRSI); multileaf collimators (MLC); planning target volume (PTV); single-positron emission computed tomography (SPECT); stereotactic radiosurgery (SRS); ultrasound (U/S) Received: 1 February 2008; electronically published: June 2008

Presented at the Theilen Tribute Symposium at UC Davis 31st May- 1st June 2008.

Summary Radiation therapy has become an important modality in treating the veterinary cancer patient. Recent advances in technology such as advanced imaging, electron therapy, custom blocking, computerized treatment planning and advanced treatment techniques such as 3D conformal therapy and intensity modulated radiotherapy have advanced the field. On the near horizon are particle therapy, stereotactic radiosurgery and functional imaging. All these advances allow better targeting and treatment of tumors with the possibility of decreased side effects. This paper reviews the current state of the art of veterinary radiation oncology and looks toward the near future to see where the specialty is going.

versus tumor tissues can be accentuated with dose fractionation. Keeping these principles in mind, most of the recent advances made in radiation therapy have been to spare normal tissue by better targeting of the tumor with external beam radiotherapy techniques and equipment. This allows greater doses of radiation to be delivered to the tumor without increasing the side effects of irradiation. As these are technology driven, they can be expensive to implement but the benefits to the patient can be great.

I. Introduction In simplest terms, the primary goal of the oncologist is to kill cancer cells while sparing normal cells and tissues. In medical oncology, this is usually accomplished by the use of agents that are given systemically but have preferential toxicity to neoplastic cells. Only occasionally is chemotherapy given by a route that targets the desired site (intratumoral, intrathecal, intracavitary). In surgical oncology, the goal is to surgically remove the tumor with the dose of surgery limited by the resulting functional or cosmetic defects. In contrast, the radiation oncologist primarily accomplishes this fundamental goal by maximizing radiation dose to a defined tumor target while minimizing the dose received by surrounding normal tissues. This can be accomplished through the use of various radiation techniques including brachytherapy, external beam radiotherapy, and systemic targeted radiotherapy. In addition, differences in radiation response of normal

II. History and progress in radiation therapy Radiation oncology is a medical science that has existed for just over 100 years. The discovery of X-ray photons is considered the birth of radiation oncology which occurred in Wilhelm Roentgenâ&#x20AC;&#x2122;s lab on November 8, 1895 (Smith et al, 2006). Within months, the first cancer patients were treated with radiotherapy. Shortly thereafter, radioactivity and radium were discovered by


Gordon and Kent: Veterinary radiation oncology Henri Becquerel and Pierre and Marie Curie (Hall and Giaccia, 2006). By 1902, over 100 different conditions were listed that could be treated with X-rays. Brachytherapy was first described and performed in 1904. Many of the early radiation researchers suffered serious injuries or death due to a lack of understanding of radiation biology and dose measurement. The first major advance in radiation technology was the invention of the Coolidge tube in 1912-13 which delivered a more reliable beam in terms of beam energy and penetration and was the precursor to orthovoltage radiotherapy machines (Bernier et al, 2004). In the 1920s and 1930s, in addition to continued improvement of radiation delivery technology, the major advances were the development of dose measurement techniques, central axis treatment planning, and understanding of the benefits of dose fractionation. In 1955, the first patient was treated using the first linear accelerator at Stanford (Hall and Giaccia, 2006). Over the next 30-40 years, significant improvements were made to radiation therapy equipment with higher energy and multiple energy machines being developed. Machines were also made that produce other types of radiation including electrons, neutrons, and protons. Additionally, advances in computer technology allowed for faster and more complex treatment planning software. Despite these advances, the basic process of radiation planning and delivery remained relatively unchanged for most of the 20th century. In the past 10-15 years, while equipment and technology have continued to improve, the most significant clinical improvements in radiation treatments have come from fundamental changes to the basic approach to radiation planning, setup, and treatment delivery. The development and advances of novel imaging techniques, intensity modulated radiation therapy (IMRT), adaptive and image guided radiation therapy (IGRT), and stereotactic radiosurgery (SRS) represent new frontiers for advancement toward the cure of diseases that were previously considered untreatable or uncurable.

deliver a variety of energies and many of the newer machines allow treatment with either photons or electrons (see below for a more detailed explanation). They also allow the use of smaller field sizes, allow for a more homogenous dose to be delivered to deep-seated tumors and can take advantage of newer treatment techniques. The use of advanced treatment planning software allows dosimetric calculations and three-dimensional treatment planning to deliver accurate doses to deep-seated tumors. Systemic radiotherapy using radionucleotides and brachytherapy are also used in treating veterinary patients but are not covered in this review.

IV. Electron therapy Linear accelerators deliver radiation through photons and in newer machines either photons or electrons. Photons provide better deep penetration and often a more homogenous dose distribution in tissue than electrons but there are times that this might pose a problem (Figure 1). If the tumor is located over critical normal structures that you do not want to irradiate such as the spinal cord, intestines or lungs, electrons may be more advantageous. Electrons deliver their dose through a certain depth and then fall off very quickly. By setting the energy of the electrons you can control the depth of penetration. A rule of thumb is penetration in cm is about 1/2 of the energy of electron selected, while the useful beam penetrates to a maximum of about 1/3 of the energy of the electron you choose in cm. For example, treating with 6 MeV electrons corresponds to about 2 cm of tissue effectively treated and using 20 MeV electrons results in approximately 6 cm of tissue being effectively treated. Many of the tumors that we treat in veterinary medicine are located in the skin or subcutaneous tissues allowing electrons to be used effectively.

V. How radiation is dosed The SI unit of absorbed dose used in radiation oncology is the Gray (Gy). The older unit is the rad. 1 Gy is equal to 100 cGy or 100 rads. When considering the total dose to be given, several things need to be taken into account; Specifically: The radiosensitivity of the tumor, the dose delivered in each fraction, the time between fractions, the total number of fractions, the goal of therapy (palliative vs. definitive) and often most importantly, the tolerance of the surrounding normal tissues.

III. Machines used to deliver radiation therapy There are three different types of machines used to deliver external beam radiation therapy in veterinary radiation oncology (McEntee, 2004). Orthovoltage machines, cobalt-60 machines and linear accelerators are used for teletherapy. Orthovoltage machines are no longer widely used and have the disadvantages of increased skin reactions, no computerized treatment planning systems, high bone absorption of dose and the lack of isocentric machines. Cobalt-60 machines are still in use in veterinary medicine but also have some disadvantages compared to linear accelerators, which include increased penumbra at the field edge (leading to larger treatment fields), lower energy (leading to increased skin dosing), lower dose rate, and less penetration of dose. Linear accelerators are becoming more widely available in veterinary radiation oncology and are the standard of care in treating humans. They are able to

VI. Target therapy




It is useful to define several â&#x20AC;&#x153;tumor volumesâ&#x20AC;? when prescribing a radiation dose to a given area. The simplest target to define is the gross tumor volume (GTV). This volume incorporates all palpably or visually abnormal tissue that is evident on physical examination or routine diagnostic imaging studies. In animals that present for radiotherapy after marginal surgical resection of the primary mass, there is no GTV.


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Figure 1. CT image of a dog’s thoracic cavity with an incompletely resected soft tissue sarcoma showing the dose distribution for electrons.

The clinical tumor volume (CTV) represents the gross tumor volume plus all regions that are suspected to contain microscopic disease. In some cases, this may be several centimeters around the gross disease in all directions. In other instances, it may represent the area defined by surgical hemoclips with a several centimeter additional margin. When a tumor is contained by a bony structure or is not felt to contain significant microscopic extension, the CTV may not be much larger than the GTV. A major challenge with delineating the CTV exists as it is unknown how far microscopic disease extends. This decision is therefore based on the biologic behavior of the tumor and the clinical situation of the particular patient and has the risk of missing disease. More commonly, a radiation oncologist will try to overestimate the extent of disease to prevent “geographic miss”. The consequence of these overestimates is increasing the volume of normal tissue that full dose is prescribed to. With advances in physiologic and functional imaging such as PET/CT and fMRI, one goal is to better define the CTV rather than simply adding a margin to the GTV. The planning target volume (PTV) is the CTV plus an additional margin to account for uncertainties associated with radiation delivery (Figure 2) (Purdy, 2004). The PTV is the volume that you ultimately plan to treat with your treatment setup or treatment planning system. The extent of this additional margin is completely

dependant upon your ability to minimize setup uncertainty and patient positioning relative to the radiation field and commonly range from at least 3-5 millimeters up to 1-2 centimeters. These uncertainties include tolerances for the radiation equipment as well as setup inaccuracies and both interfraction and intrafraction patient motion (Purdy, 2004). Each of the following parameters can introduce at least 1-2 mm of uncertainty about the precision of the radiation field relative to the patient although not all are relevant for every patient: 1) Collimator position – The size the collimator is set for versus the actual size of the radiation field 2) Collimator drift- Movement in the collimator during treatment or when the gantry is at different positions 3) Isocenter uncertainty- Movement of the isocenter when the gantry is at different positions 4) Light field uncertainty- Deviation between the radiation field and the light field displayed on the patient 5) Portal imaging uncertainty- The edges and field center as defined by portal image (whether film or digital) 6) Diagnostic imaging uncertainty- This can be from limitations of spatial accuracy with MRI or constraints based on the slice thickness of your diagnostic CT scan 7) Image registration uncertainty- If you merge 2 diagnostic studies such as an MRI and CT scan, there is uncertainty in the precision of that match.


Gordon and Kent: Veterinary radiation oncology

Figure 2. CT image of a cat with an injection site sarcoma showing the GTV (yellow line) CTV (red line) and the PTV (green shading).

8) Positioning and movement uncertainties before and during each treatment 9) Movement of fiducial markers or skin marks relative to the tumor target 10) Penumbra of field- The penumbra of the field is the region at the field edge where dose falloff occurs. This depends on the type and energy of radiation you are using and can be greater than 10 millimeters for Co-60 ! rays or for electron beams. Penumbras for linear accelerators are much smaller. If this is taken into account with your treatment planning system, it may not need to be considered twice by adding a margin into your PTV. However, if not or if you are planning a treatment by hand, the uncertainty of the penumbra region should be incorporated into the PTV. Once again, the tradeoff between a small and large PTV margin is the risk of geographic miss versus increased normal tissue radiation exposure. In stereotactic radiosurgery, which is discussed later, the use of external fiduciary markers with precise positioning devices and lasers minimize setup uncertainty while finely collimated cones reduce penumbra and collimator uncertainties so that a tight PTV can be drawn around the CTV to even further minimize dose to normal tissue.

can be used. Custom blocks can be made for shaping both electron and photon beams (Figure 3). Blocking can be used with both hand and computer plans. A custom electron block can be made in less than 10 minutes. When the patient has finished their treatment course, the block can then be melted and the alloy reused.

VIII. Patient reproducibility



It is essential not to decrease the PTV beyond patient positioning and movement limitations or the tumor may not receive the planned dose. Using a 3-D conformal or IMRT plan that targets the tumor precisely on a patient that cannot be accurately positioned will not deliver the desired dose and does not make sense. Fortunately, imaging techniques to check patient positioning have also improved which allows checking the accuracy of patient positioning on each treatment if needed. A major question that must be answered is how precisely can a patient be setup each day. All of the precision and detail that goes into advanced treatment planning is useless if the patient is unable to be positioned precisely, reliably and reproducibly. The importance of this cannot be overstated, especially as technology allows for finer tumor targeting it can often be just millimeters separating a full dose from a region far below therapeutic doses. Many devices have been developed to help with this over the last few years. These include vacuum bags, bite blocks, headframes, masks and calibrated treatment couches (Figure 4) (Kippenes et al, 2000; Lester et al, 2001; Green et al, 2003). Port films, which are radiographs taken using the treatment machine to check patient positioning are key in determining the accuracy of positioning (Rohrer Bley et al, 2003). They are particularly important for deep seated tumors such as nasal tumors and brain tumors where you cannot directly see the

VII. Custom blocking While the standard collimator on a cobalt-60 machine or linear accelerator can make only squares or rectangles, planned radiation fields are often more complex in shape. There are several different ways that the beam can be shaped to conform to the planned field. The simplest but crudest way is to have pre-cast blocks which can be hand positioned onto plastic trays fitted into the machine. For increased accuracy, either a multileaf collimator, which most commonly have between 80 and 120 leaves, or a custom made block made from a low temperature melting lead alloy to shield part of the field 170

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Figure 3. Images of a (a) photon block and a (b) electron block.

Figure 4. Image of a dog set-up for a radiotherapy treatment using both a vacuum bag and a head mask.

area you are treating. They should be done at least weekly and with particularly difficult fields should even be done on a daily basis for quality assurance (McEntee, 2008). Taking two orthogonal port films allows accurate positioning of a patient to within several millimeters, ensuring that the plan can be accurately carried out regardless of how many beams are used or at what angle they enter. Traditionally, port films have been made using specialized cassettes and film and processed like a regular

piece of radiograph film. Newer techniques such as digital on board portal imaging equipment allow imaging of the patient right before treatment and image registration to compare actual patient positioning with what was planned (Figure 5) (McEntee, 2006, 2008). One limitation of this technique is that imaging with the high energy megavoltage (MV) beam of a linear accelerator yields poor soft tissue contrast. One solution has been to equip a linear accelerator with an on-board kilovoltage (kV) imaging system for diagnostic quality radiographs. 171

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Figure 5. A digitally reconstructed radiograph created from the initial CT scan of a dog with a solitary plasma cell tumor of the maxilla used for treatment planning (a) and a port film taken using an electronic portal imaging device (b).

On board cone beam computed tomography (CBCT) imaging is a technique of producing volumetric CT images at the time of treatment and is now becoming available on newer linear accelerators for even more precise positioning. The use of real-time imaging to adjust patient positioning immediately before and even during treatment is called IGRT (Xing et al, 2006). The power of this technology may reach the point that as the patient or tumor shape changes during the course of treatment, adaptive treatment planning can adjust for positioning changes during a single treatment and shape changes within the patient and/or target over the course of radiotherapy as well. With the increased precision of radiation equipment, the importance of 4-D treatment planning and positioning is now being realized. The â&#x20AC;&#x153;4th dimensionâ&#x20AC;? this refers to is subtle movement over time during and between radiation treatments (intrafraction and interfraction motion). Respiratory motion can result in several centimeters of movement of a tumor target and critical normal tissues including heart and lung. Gastrointestinal peristalsis during radiation is now recognized as a major cause of both geographic miss and colonic overdosage in human prostate cancer patients undergoing radiation therapy. Through the use of fiducial markers or infrared sensors, there are now techniques that can actually adjust the radiation field to match patient motion. While organ

motion was once difficult to study, 4-dimensional CT can provide extensive information about the dynamic nature of tumors and internal organs (Webb, 2006). Linear accelerators are now available with integrated imaging devices to provide target identification, real time monitoring for motion and delivery modification, verification, dose reconstruction, and adaptive therapy. Imaging devices that allow for respiratory gating, breathing control, and adapt therapy to account for motion will allow further sparing of normal tissue and finer tumor targeting (Giraud et al, 2006). These techniques will become extremely important for techniques such as IMRT with extremely conformal dose distributions and gradients at the boundary of target volumes and organs at risk (Keall et al, 2006). Refining these techniques will likely allow for dose escalations resulting in higher rates of local tumor control and survival.

IX. Treatment planning A. Hand planning When treating an area surrounding a scar from an incompletely excised soft tissue sarcoma or mast cell tumor for example, a hand calculated plan is often sufficient and saves cost to the owner. This is done by placing an appropriate margin around the scar and then calculating an appropriate dose to the desired depth, taking into account the skin and exit doses. This becomes rapidly impractical if multiple beams, complex blocking or beam 172

Cancer Therapy Vol 6, page 173 modifying devices are used. The dose delivered also may not match the dose calculated across the radiation field, particularly if the patient’s body contour in the radiation field is not flat.

X. 3-D conformal radiation therapy and IMRT 3-D conformal radiation therapy is similar to conventional 3-D planning except that multiple beam angles and conformal blocks are used to shape the dose closely to the target volume and simultaneously allow sparing of normal tissues. These beam angles and blocks are selected using reconstructed imaging data and treatment planning software capable of performing thousands of calculations in a short amount of time. IMRT is a technique that takes advantage of exact patient positioning, multileaf collimators (MLC) and treatment planning software to shape and modify the intensity of the beam so that the delivered dose conforms to the tumor (Purdy, 2007). There are two fundamental differences between IMRT and 3-D conformal radiation therapy. The first fundamental difference is the use of the computer driven small mobile tungsten leaves of the MLC to various positions around the patient. The “step and shoot” technique of IMRT involves moving the leaves to multiple fixed positions around the patient. What is more common and useful is dynamic treatment in which the leaves of the MLC move while the linear accelerator beam is on, modulating the fluence of the beam and effectively treating thousands of “beamlets” per field. The calculation algorithms for dynamic IMRT are so complex that they cannot be planned by conventional methods (Bortfeld, 2006). The second fundamental difference between IMRT and conformal radiotherapy is the use of “inverse planning.” Recall that in conventional and conformal treatment planning, the radiation oncologist or dosimetrist creates the beam orientation, weight, shape, field size and modifiers. The treatment planning system then calculates the resulting dose. The parameters can then be adjusted by modifying beam weighting, adding or removing beams,

B. Computer planning With the advent of computer planning, radiation plans with multiple beams can be easily made using images from a CT or MRI. Most treatment planning systems use CT scans for treatment planning since it provides density information for dose calculation and because it provides better geometric accuracy than MRI. However many of these programs allow fusing an MRI to a CT scan, taking advantage of the superior soft tissue detail seen with MRI. This is particularly useful when planning brain tumors, which are much better visualized on MRI than CT. For most other tumor types usually a CT alone is sufficient. For treatment planning, the patient is scanned in the position that they will be treated in using any patient positioning devices that will be used. This allows for the most accurate dose calculation and delivery. The CT is then imported into the treatment planning software program. The area to be irradiated is then identified and marked and the CTV is drawn. The margin for the CTV is based on the tumor’s type, location, and biological behavior. The PTV is then drawn around the CTV. The radiation oncologist then adds beams onto the tumor and calculates the dose. 3-D treatment planning systems let you visualize dose on a 3-D image and also allow creation of dose volume histograms, which are graphs that show what volume of tissue is receiving what dose of radiation (Figure 6). This is useful in ensuring that the tumor is receiving the desired dose and that normal structures (such as lung, spinal cord or eyes) are getting the minimal possible dose. Individual plans can then be compared and the best one chosen.

Figure 6. (a) A plan from a dog with a pituitary tumor and (b) the dose volume histogram from the same plan showing dose to the CTV (purple), PTV (red) and to the brain (blue) and eyes (green).


Gordon and Kent: Veterinary radiation oncology and adjusting other dose modifiers in a trial and error process. In inverse planning, the process starts by prescribing a dose to the defined target volume and setting constraints for the tolerable doses to normal tissues. The treatment planning system is responsible for optimizing beam weights, field sizes, and beam fluence to meet those constraints (Bortfeld, 2006). When those constraints cannot be met, inverse planning becomes an interactive process whereby the radiation oncologist or dosimetrist must adjust or prioritize the dose constraints until the most ideal treatment plan is created. Because frequently the ratio of the dose delivered to normal tissues compared to the tumor is reduced to a minimum with IMRT, dose escalation to more effective radiation doses can safely be performed with fewer side effects compared with conventional radiotherapy techniques (Smith et al, 2006). There are several limitations to IMRT. First, not all treatment locations can be improved to a significant extent compared to more conventional techniques. It is much more time consuming to verify radiation dose with IMRT than conventional and conformal techniques. The problems of organ motion (see section on image guided radiation therapy) may also interfere with the ability to accurately carry out an IMRT plan (Purdy, 2004). Of unknown clinical importance at this time is the effect of adding more beams to treatment. In addition to daily treatment time increasing, the volume of normal tissue that receives some dose of radiation increases and the total body radiation dose increases as well. Because these techniques are still relatively new, it is not yet known whether or to what extent those increases may lead to increased rates of carcinogenesis or other adverse effects (Hall, 2006).

use these benefits without losing the benefit of the information provided by a CT scan by overlaying the data. Historically, U/S has found limited application for external beam radiation due to a lack of a well-defined method for using a three dimensional coordinate system. As image quality has improved, U/S is now able to verify patient setup and identify organs at risk in a radiation field (Smith et al, 2006). It also has wide applications in humans for image guided placement of seed implants. While radioactive implants may not be a viable option for many veterinary patients, the placement of fiducial markers into internal sites for daily treatment localization can be readily accomplished (Smith et al, 2006). Techniques such as fMRI are not yet widely available in veterinary medicine but can provide information about local oxygen concentrations, oxygen consumption, and perfusion which may give information about areas within a tumor that may be hypoxic or more resistant to radiation. MRSI has the ability to detect molecules that may reflect different disease states to differentiate normal and malignant tissues (Smith et al, 2006). For 50 years, radiation oncologists have tried to improve the homogeneity of dose throughout a tumor. It is well known that tumors are very heterogeneous. As advances in imaging permit detection of these tumor heterogeneities, it may be found that the best treatment is not one of homogeneous dose throughout the tumor but one of planned heterogeneous dosing with higher risk portions of the tumor boosted to a much increased dose. IMRT techniques can accomplish these dose profiles more readily than traditional methods of radiation therapy.

B. Particle radiotherapy

XI. Moving toward the future in veterinary radiotherapy

1. Neutron therapy The main potential advantage for neutron therapy is the radiobiologic advantage they confer. Due to the characteristics of their dose deposition, they cause less sublethal damage to cells that can be repaired. Additionally, the extent of cell killing by neutrons is not cell cycle dependant and is not decreased by the presence of hypoxia. As neutrons are not charged particles they do not cause direct ionization and are also likely to pass through tissue without interacting. One approach is the use of boronated compounds to capture neutrons in the tumor itself. The concept of â&#x20AC;&#x153;boron-neutron capture therapyâ&#x20AC;? is also an area where significant advances may still be made. There are very few facilities capable of treating veterinary patients with a neutron beam although there is a recent report of boron neutron capture therapy for feline nasal squamous cell carcinoma (Trivillin et al, 2007).

A. Advanced imaging The future of radiation therapy is intimately tied to technology driven and image guided techniques inside and outside of the linear accelerator vault. The ability to accurately delineate target volumes based on physiologic imaging tests represents an area where significant advancement can be made. The ability to register and fuse multiple imaging modalities in combination with immobilization devices reduces both the CTV and PTV by reducing geometric uncertainties. These improvements will incorporate computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (U/S), positron emission tomography and PET/CT, single-positron emission computed tomography (SPECT), functional magnetic resonance imaging (fMRI) and magnetic resonance spectroscopic imaging (MRSI). For 30 years, CT has been the most commonly used imaging technique for radiation treatment planning due to its spatial fidelity and the ability to reconstruct images into a three-dimensional model (Smith et al, 2006). Alternative imaging techniques including MRI and PET may provide superior visualization of tumor and normal structures. Fusion of CT and MRI images is common in veterinary medicine in treatment of brain, spinal and nasal tumors to

2. Proton therapy The main potential advantage for protons is that as a proton passes through tissue, the dose deposited increases slowly with depth until it reaches a sharp peak at its maximum depth of penetration, known as the Bragg peak. No dose is delivered beyond this peak. The depth of the peak can be adjusted by varying the energy of the beam or through the use of compensating material and specialized filters. This property makes it possible to create a dose 174

Cancer Therapy Vol 6, page 175 profile that is precisely confined to a tumor volume with extremely sharp dose fall off deep to the tumor in normal tissue. There are only scattered reports of proton therapy in veterinary medicine, including the treatment of brain tumors. The technique and dose distributions have also been evaluated for canine nasal tumors and may have some dosimetric advantages to photons (Kaser-Hotz et al, 2002; Bley et al, 2005).

tumors (Lester et al, 2001; Farese et al, 2004). Many other potential applications exist and there are anecdotal reports of effective SRS treatments for urinary bladder and urethral transitional cell carcinoma, pituitary dependant Cushingâ&#x20AC;&#x2122;s disease, and spinal tumors.

XII. Radiation training and expertise As radiation therapy becomes more complex and technology driven, the role of the medical physicist and board certified radiation oncologist has become far more important. Because the specialty of veterinary radiation oncology is still relatively new and small, there are many facilities that rely on radiologists, medical oncologists, general practitioners, and human radiotherapists to perform radiation. Implementation of more complex techniques with appropriate care requires more advanced training and better knowledge of radiation equipment, radiation safety, radiation physics, and radiobiology. The role of the medical physicist includes calibration of the machine, collection of dosing data, assuring radiation safety and assisting in treatment planning and assuring that dose delivered is the dose planned. The law only requires yearly testing of the equipment in California for veterinary facilities. In human facilities the requirements are much more strict, with a physicist on-site or on call at all times. As veterinary facilities begin to implement treatments that have similar complexity, regular quality assurance evaluations by a medical physicist are needed to prevent serious errors. Similarly, as target margins become smaller with the goals of decreasing toxicity and dose escalation, the risks associated with subtle errors in calculation and treatment become much greater. Having an on-site radiation oncologist to evaluate patients and treatment sites to develop an integrative plan for imaging and therapy is important. Even more critically, the radiation oncologist is responsible for verifying accurate positioning and port films before a treatment is administered. Because the consequences of even small errors are markedly increased with finely conformal therapy, the importance of such verification is greatly amplified.

C. Tomotherapy Tomotherapy represents a specific type of advanced IMRT with IGRT. It is a helical machine employing a linear accelerator mounted into a CT style gantry that rotates as a fan beam modulated by an MLC and rotating around the patient as the couch moves the patient into the gantry. Tomotherapy represents an integrated system for treatment planning, patient positioning, and treatment delivery utilizing an inverse planning system. Using this type of image registration for patient positioning provides more detail for not only the 3 translational degrees of freedom but also the 3 rotational degrees of freedom (pitch, roll, and yaw) (Forrest et al, 2004; Hong et al, 2007). The tomotherapy unit is also capable of verifying treatment delivery using a detector to compute the energy fluence delivered with any error outside of the specified tolerance range triggering treatment shutdown. This information can also be used for dose reconstruction to compare the planned dose delivery to the actual delivery for quality assurance and adaptive radiation therapy (Lawrence and Forrest, 2007; Tome et al, 2007).

D. Stereotactic radiosurgery SRS was originally designed by a neurosurgeon with the intent to treat functional disorders of the brain. It is now recognized as a treatment with many indications including tumors, vascular lesions, and pain syndromes, including but not exclusive to the brain and spine (Smith et al, 2006). There are three basic techniques currently available for stereotactic radiosurgery. The underlying principle is the use of many finely collimated beams focused on a specific (and usually small) target to deliver a very high dose to the small target with much lower doses to all surrounding tissue. Initially, all stereotactic radiosurgical treatments were performed with a single or very few fractions. Fractionated SRS is now preferred for many conditions (Smith et al, 2006). Linear accelerator based SRS uses stereotactic cones to modify and collimate the beam of a standard linear accelerator. The gantry rotates similar to 3-D conformal therapy or IMRT to multiple positions. Gamma knife based radiosurgery utilizes up to 201 small Cobalt-60 sources in a heavily shielded apparatus. The third technique is robotically controlled radiosurgery (Cyberknife) which employs a small linear accelerator mounted on a robotic arm to deliver multiple beams. Currently, the few veterinary facilities performing SRS are using primarily linear accelerator based treatments although there is currently one veterinary facility with a Cyberknife. Published veterinary applications for SRS include an alternative to surgery for appendicular osteosarcoma and treatment of intracranial

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