CLINICS October 2017 by Clinics

Volume 72 Number 10 - October/2017

CLINICS Editor Edmund Chada Baracat

Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Anna Sara Shafferman Levin Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Antonio Egidio Nardi Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Berenice Bilharinho Mendonca Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Bruno Zilberstein Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Carlos Serrano Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Claudio Roberto Cernea Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Emilia Inoue Sato Universidade Federal de São Paulo São Paulo, SP, Brazil Flair José Carrilho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Fulvio Alexandre Scorza Universidade Federal de São Paulo São Paulo, SP, Brazil Geraldo Busatto Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Heloisa de Andrade Carvalho Hospital das Clı´nicas da Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Jesus Paula Carvalho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil José Guilherme Cecatti Universidade Estadual de Campinas Campinas, SP, Brazil José Maria Soares Júnior Hospital das Clı´nicas da Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Luiz Eugenio Garcez-Leme Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Luíz Fernando Onuchic Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de São Paulo São Paulo, SP, Brazil Marcos Intaglietta University of California, San Diego San Diego, CA, USA Maria José Carvalho Carmona Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Mauricio Etchebehere Universidade Estadual de Campinas Campinas, SP, Brazil Michele Correale University of Foggia Foggia, Italy Naomi Kondo Nakagawa Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Olavo Pires de Camargo Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Paulo Hoff Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Paulo Pêgo-Fernandes Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Duke University Medical Center Durham, NC, USA Ricardo Bassil Lasmar Universidade Federal Fluminense Niterói, RJ, Brazil Ricardo Nitrini Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Rosa Maria Rodrigues Pereira Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Rossana Francisco Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de São Paulo São Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de São Paulo São Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sandro Esteves ANDROFERT - Andrology & Human Reproduction Clinic Campinas, SP, Brazil Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Sigmar de Mello Rode Universidade Estadual Paulista Júlio de Mesquita Filho São José dos Campos, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Valeria Aoki Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Editorial Board Abhijit Chandra King George’s Medical College Lucknow, India Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil Adauto Castelo Universidade Federal de São Paulo São Paulo, SP, Brazil Ademar Lopes Fundação Antônio Prudente, Hospital do Câncer São Paulo, SP, Brazil Alberto Azoubel Antunes Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Alexandre Roberto Precioso Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Andrea Schmitt University of Goettingen Goettingen, Germany

Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Artur Brum-Fernandes Université de Sherbrooke Québec, Canadá Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil Daniel Romero Muñoz Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Eduardo Ferreira Borba Neto Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany

Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Ernest Eugene Moore University of Colorado Denver Denver, CO, USA Euclides Ayres Castilho Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Fábio Biscegli Jatene Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Francisco Laurindo Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil

Irshad Chaudry University of Alabama Birmingham, AL, USA Ivan Cecconello Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Ke-Seng Zhao Southern Medical University Guangzhou, China Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK Marcelo Zugaib Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Marco Martins Amatuzzi Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Mauro Perretti William Harvey Research Institute London, UK

Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Michael Gregory Sarr Mayo Clinic Rochester, MN, USA

Pedro Puech-Leão Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA Philip Cohen University of Houston Health Center Houston, Texas, USA Rafael Andrade-Alegre Santo Tomás Hospital Republic of Panamai, Panamá Ricardo Antonio Reﬁnetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Roberto Chiesa San Raffaele Hospital Milan, Italy

Milton de Arruda Martins Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA Naomi Kondo Nakagawa Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Navantino Alves Faculdade de Ciências Médicas de Minas Gerais Belo Horizonte, MG, Brazil

Samir Rasslan Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Valentim Gentil Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Board of Governors Alberto José da Silva Duarte Aluisio Augusto Cotrim Segurado Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonça Carlos Alberto Buchpiquel Carlos Roberto Ribeiro de Carvalho Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa Daniel Romero Muñoz Edivaldo Massazo Utiyama Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfá Euripedes Constantino Miguel Fábio Biscegli Jatene Flair José Carrilho Geraldo Busatto Gerson Chadi Gilberto Luis Camanho Giovanni Guido Cerri Irene de Lourdes Noronha Irineu Tadeu Velasco

Ivan Cecconello Jorge Elias Kalil José Antonio Franchini Ramires José Antonio Sanches José Eduardo Krieger José Otávio Costa Auler José Ricardo de Carvalho Mesquita Ayres Linamara Rizzo Battistella Luiz Augusto Carneiro D’Albuquerque Luiz Fernando Onuchic Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Miguel Srougi Milton de Arruda Martins Mirian Nacagami Sotto Nelson de Luccia Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hilário Nascimento Saldiva Paulo Manuel Pêgo Fernandes Paulo Marcelo Gehm Hoff Paulo Rossi Menezes

Editorial Director

Kavita Kirankumar Patel-Rolim Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Pedro Puech-Leão Remo Susanna Ricardo Ferreira Bento Ricardo Nitrini Roberto Kalil Roberto Zatz Roger Chammas Rolf Gemperli Rosa Maria Rodrigues Perreira Sandra Joseﬁna Ferraz Ellero Grisi Selma Lancman Tarcísio Eloy Pessoa de Barros Uenis Tannuri Umbertina Conti Reed Valentim Gentil Vanderson Geraldo Rocha Venâncio Avancini Ferreira Alves Vicente Odone Wagner Farid Gattaz Werther Brunow de Carvalho William Carlos Nahas Wilson Jacob

Editorial Assistants

Nair Gomes Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de São Paulo São Paulo, SP, Brazil

Editorial Office: Rua Dr. Ovídio Pires de Campos, 225 - 6˚ Andar CEP 05403-010 São Paulo/SP Tel.: +55-11-2661-6235 Email: clinics@hc.fm.usp.br Website: www.scielo.br/clinics Submission: http://mc04.manuscriptcentral.com/clinics-scielo Indexations: LILACS; MEDLINE; PubMed; PubMed Central; SciELO; Science Citation Index Expanded (ISI Web of Knowledge); Scopus; Ulrich’s Periodical Directory; Qualis/Capes - Classified as an International Circulation Journal in Medicine. Clinics. São Paulo: Scientific Journal of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, 2005Monthly Periodical: January to December ISSN 1807-5932 printed version ISSN 1980-5322 online version Formerly Revista do Hospital das Clínicas da FMUSP, 1946–2004. 1. Medicine-scientific production. 2.Medical Sciences I. Hospital das Clínicas da Faculdade deMedicina da Universidade de São Paulo. CDD 610

PUBLICATION INFORMATION AND EDITORIAL POLICIES CLINICS publishes peer-reviewed articles of interest to clinicians and researchers in the medical sciences. CLINICS is registered with PubMed Central (http://www.ncbi.nlm.nih.gov/pmc/journals/ 795/) and SciELO (http://www.scielo.br/clinics) and complies with the policies of funding agencies which request or require deposition of the published articles that they fund into publicly available databases. CLINICS supports the position of the International Committee of Medical Journal Editors (ICMJE) on trial registration. Visit http://www.icmje.org for further details and http://www.who.int/ictrp/network/en/ for the WHOâ&#x20AC;&#x2122;s list of approved registries. Please make sure to submit your manuscript in the exact format that is described below. Failure to do so will cause the submission to be returned to you during the preliminary examination by the Editorial OfďŹ ce. - Manuscripts involving human subjects or the use of laboratory animals must clearly state adherence to appropriate guidelines and approval of protocols by their institutional review boards. Photographs that may identify patients or other human participants of studies shall be acceptable only when a legally valid consent form is signed by the participating patient, other human participant, or his/her legally constituted representative. - Authors are strongly advised to use abbreviations sparingly whenever possible to avoid jargon and improve readability of the manuscript. All abbreviations must be defined the first time that they are used. Only terms or expressions that are used at least 5 times throughout the text should be abbreviated. Never use abbreviations that spell common English words, such as FUN, PIN, SCORE, and SUN.

clinical research. Original studies must conform to the following format: Title: Up to 250 characters. Running title: Up to 40 characters. Authors' full names and contribution: Authors should

PROFESSIONAL ENGLISH REVISION Please keep in mind that ALL accepted manuscripts must be sent by the authors to the company American Journal Experts (https://www.aje.com) for premium editing prior to publication.

Publication FEES CLINICS uses a business model whereby expenses are partly recovered by charging the authors, or research sponsors, a publication fee for each published article. We have updated our 2016 prices, effective for articles submitted from 05/01/2016 onwards, as follows: original articles, review articles and rapid communications: US$ 1,500.00 (International authors) and R$ 1.500,00 (Brazilian authors). There are no charges for Invited reviews, editorials and letters to the editors. Waiver Policy: Most institutions and funding agencies have set up funding sources to cover Article Publication Costs. Unfortunately CLINICS cannot grant fee waivers due to its 2016 policies.

MANUSCRIPT CATEGORIES ORIGINAL STUDY: Complete original studies should be submitted in this category. Two sections are offered: basic and

have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Such participation must be declared in this section of the manuscript. Any financial or other relationships that may lead to a conflict of interest. Abstract: Abstracts are limited to 250 words and structured into objectives, method, results, and conclusions. Citations or abbreviations (except internationally recognized abbreviations, such as weights, measures, and physical or chemical abbreviations) are not permitted. Authors are strongly encouraged not to display numerical statistical information but to merely state what is significantly different (or not) between the described parameters. Please make sure to always include your Abstract in the main document file. Keywords: For keywords, 3â&#x20AC;&#x201C;6 items from the Medical Subject Headings (MeSh) should be used. Introduction: The introduction should set the purpose of the study, provide a brief summary (not a review) of previous relevant studies, and state the new advances in the current investigation. The introduction should not include data or conclusions from the work being reported. A final sentence summarizing the novel finding to be presented is permissible. Materials and Methods: This section should briefly give clear and sufficient information to permit the study to be repeated by others. Standard techniques only need to be referenced. Previously published methods may be briefly described following the reference. Ethics: When reporting experiments on human subjects, indicate whether the procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, which was revised in 1983. When reporting experiments on animals, indicate whether the institutionâ&#x20AC;&#x2122;s guide, a national research councilâ&#x20AC;&#x2122;s guide, or any national law on the care and use of laboratory animals was followed. Results: The results section should be a concise account of the new information that was discovered, with the least personal judgment. Do not repeat in text all the data in the tables and illustrations but briefly describe what these data comprise. Discussion: The discussion should include the significance of the new information and relevance of the new findings in light of existing knowledge. Only unavoidable citations should be included. Citation to review articles are not encouraged in this section. Acknowledgments: This section should be short, concise, and restricted to acknowledgments that are necessary.

References in text: CLINICS adopts the Vancouver

format. Cite references in the text using Arabic numerals in the order of appearance, within parentheses, (1) after the previous word, with spacing as in this example: â&#x20AC;&#x2DC;â&#x20AC;&#x2DC;Diabetes (2), hypertension (3,4) and alcoholism (5â&#x20AC;&#x201C;9) are complex medical problems (10).â&#x20AC;&#x2122;â&#x20AC;&#x2122; Under exceptional circumstances, authorsâ&#x20AC;&#x2122; names may appear in text: Single author: â&#x20AC;&#x2DC;â&#x20AC;&#x2DC;Einstein (11) proposed a new theory â&#x20AC;Śâ&#x20AC;&#x2122;â&#x20AC;&#x2122;, Two authors: â&#x20AC;&#x2DC;â&#x20AC;&#x2DC;Watson & Crick (12) reported on the structure of â&#x20AC;Śâ&#x20AC;&#x2122;â&#x20AC;&#x2122;, or Three or more authors: â&#x20AC;&#x2DC;â&#x20AC;&#x2DC;Smith et al. (13) described â&#x20AC;Śâ&#x20AC;&#x2122;â&#x20AC;&#x2122; Reference List: Only citations that appear in the text should be referenced. References must be restricted to directly relevant published works, papers, or abstracts. Unpublished papers, unless accepted for publication, should not be cited. Work that is accepted for publication should be referred to as â&#x20AC;&#x2DC;â&#x20AC;&#x2DC;in pressâ&#x20AC;&#x2122;â&#x20AC;&#x2122; and a letter of acceptance of the journal must be provided. Authors are responsible for the accuracy and completeness of their references and for correct text citation. Usually the total number of references should not exceed 35. For up to 6 authors, list all authors. For more than 6 authors, list first 6 authors followed by â&#x20AC;&#x2DC;â&#x20AC;&#x2DC;et al.â&#x20AC;&#x2122;â&#x20AC;&#x2122;. Tables and Figures: Tables: Should be constructed using the table feature in your word processor or using a spreadsheet program such as Excel. The tables should be numbered in order of appearance in the text, using Arabic numerals. Each table should have a title and an explanatory legend, if necessary. All tables must be referenced and succinctly described in the text. Under no circumstances should a table repeat data that are presented in an illustration. Statistical measures of variation (i.e., standard deviation or standard error) should be identified, and decimal places in tabular data should be restricted to those with mathematical and statistical significance. Figures: Photographs, illustrations, charts, drawings, line graphs, etc. are all defined as figures. Number figures consecutively using Arabic numerals in order of appearance. Figure legend(s) should be descriptive and should allow examination of the figure without reference to text. Images must be of professional quality and uploaded as *.tiff files. Generally, figures will be reduced to fit one column of text. The actual magnification of all photomicrographs should be provided, preferably by placing a scale bar on the print. Line graphs and charts should never be sent as *.jpeg illustrations. We recommend preparing line graphs and charts as ExcelH files and copying these files into a Word *.doc sheet. REVIEW ARTICLES: Review articles should cover themes that are relevant to medical practice. Spontaneously submitted reviews are welcome; however, potential authors should bear in mind that they are expected to have expertise in the reviewed ďŹ eld. The sections should be arranged as follows: Title: Up to 250 characters. Running title: Up to 40 characters. Authors' full names and contribution: Authors should

have participated sufficiently in the work to take public

responsibility for appropriate portions of the content. Such participation must be declared in this section of the manuscript. Any financial or other relationships that may lead to a conflict of interest. Abstract, keywords and text should be arranged to cover the subject that is being reviewed. If appropriate, the method of reference collection should be described. The use of headings, subheadings, and paragraph titles is encouraged to improve clarity. Abbreviations, acknowledgments, tables and figures should be formatted as described in the Original Study section. The number of references is at the discretion of the authors. No publication fee discount is allowed for spontaneously submitted review articles that are accepted for publication.

RAPID COMMUNICATIONS: Title: Up to 250 characters. Running title: Up to 40 characters. Authors' full names and contribution: Authors should

LETTERS TO THE EDITOR: Letters to the editor expressing comments or dissenting opinions concerning articles that have been recently published in CLINICS are published at the discretion of the editor. A letter is a single section containing untitled text concerning the article under discussion, followed by references. No publication fee is charged for this class of manuscripts. EDITORIAL: Editorials should cover broad aspects of medical or biological sciences. Such manuscripts are published at the discretion of the editor. No publication fee is charged for this class of manuscripts. COMMENTARY: A commentary is an invited text with respect to an article that is being published by CLINICS. No publication fee is charged for this class of manuscripts.

INVITED REVIEW: These reviews are by invitation only and follow the format proposed for general reviews. No publication fee is charged for this class of manuscripts. SPECIAL ISSUE ARTICLE: Special issue articles are by invitation only and follow a speciﬁc format that is set by the editor in charge of the collection. Currently CLINICS does not accept: case reports, technical notes, translations and validations of questionnaires, and articles referring to ﬁrst demonstration in Brazil.

SUBMISSION A copyright transfer form, signed by all authors, must be uploaded as a complementary ﬁle as soon as the manuscript is submitted.

When the authors are satisfied that the manuscript complies with the journal format, our site should be accessed using the website www.clinics.org.br. The system will guide authors through the manuscript submission process and will prompt authors to input information into specific fields as they are submitting their manuscript. The editorial office and authors will be automatically notified of the submission. Progress of the manuscript through the Editorial Office’s procedures will be available to authors at all times.

PEER REVIEW Manuscripts are reviewed by at least two expert consultants. Accepted manuscripts are edited to comply with the journal’s format, remove redundancies, and improve clarity and understanding without altering meaning. The edited text will be presented to authors for approval.

ISSN-1807-5932

CLINICS CONTENTS Clinics 2017 72(10)588–648

CLINICAL SCIENCE

Next-generation Sequencing-based genomic proﬁling: Fostering innovation in cancer care? Gustavo S. Fernandes, Daniel F. Marques, Daniel M. Girardi, Maria Ignez F. Braghiroli, Renata A. Coudry, Sibele I. Meireles, Artur Katz, Paulo M. Hoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588

Longitudinal study of lung function in pregnant women: Inﬂuence of parity and smoking Luciana Duzolina Manfré Pastro, Miriam Lemos, Frederico Leon Arrabal Fernandes, Silvia Regina Dias Médici Saldiva, Sandra Elisabete Vieira, Beatriz Mangueira Saraiva Romanholo, Paulo Hilário Nascimento Saldiva, Rossana Pulcineli Vieira Francisco . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

Identiﬁcation of the difference in the pathogenesis in heart failure arising from different etiologies using a microarray dataset Guodong Yang, Shuping Chen, Aiqun Ma, Jun Lu, Tingzhong Wang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600

Comparison of short-segment monoaxial and polyaxial pedicle screw ﬁxation combined with intermediate screws in traumatic thoracolumbar fractures: a ﬁnite element study and clinical radiographic review Hongwei Wang, Yiwen Zhao, Zhongjun Mo, Jianda Han, Yu Chen, Hailong Yu, Qi Wang, Jun Liu, Changqing Li, Yue Zhou, Liangbi Xiang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609

Effect of prophylactic non-invasive mechanical ventilation on functional capacity after heart valve replacement: a clinical trial Amaro Afrânio de Araújo-Filho, Manoel Luiz de Cerqueira-Neto, Lucas de Assis Pereira Cacau, Géssica Uruga Oliveira, Telma Cristina Fontes Cerqueira, Valter Joviniano de Santana-Filho . . . . . . . . . . . . . . . . . . 618

BASIC RESEARCH

Local and systemic effects of ﬁbrin and cyanoacrylate adhesives on lung lesions in rabbits Marcus V.H. Carvalho, Evaldo Marchi, Andre J. Fruchi, Bruno V.B. Dias, Clovis L. Pinto, Geovane R. dos Santos, Milena M.P. Acencio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624

ORIGINAL RESEARCH

Impact assessment of an automated drug-dispensing system in a tertiary hospital Débora de-Carvalho, José Luiz Alvim-Borges, Cristiana Maria Toscano . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629

REVIEW

Review of magnetic resonance-guided focused ultrasound in the treatment of uterine ﬁbroids Pedro Felipe Magalhães Peregrino, Marcos de Lorenzo Messina, Ricardo dos Santos Simões, José Maria Soares-Júnior, Edmund Chada Baracat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637

RAPID COMMUNICATION

Plasmid-mediated mcr-1 in carbapenem-susceptible Escherichia coli ST156 causing a blood infection: an unnoticeable spread of colistin resistance in Brazil? Flavia Rossi, Raquel Girardello, Carlos Morais, Ana Paula Cury, Layla Farage Martins, Aline Maria da Silva, Edson Abdala, João Carlos Setubal, Alberto José da Silva Duarte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642

Reduction of venous pressure during the resection of liver metastases compromises enteric blood ﬂow: IGFBP-1 as a novel biomarker of intestinal barrier injury Hermes Vieira Barbeiro, Marcel Autran César Machado, Heraldo Possolo de Souza, Fabiano Pinheiro da Silva, Marcel Cerqueira César Machado. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645

CLINICAL SCIENCE

Next-generation Sequencing-based genomic profiling: Fostering innovation in cancer care? Gustavo S. Fernandes,I,* Daniel F. Marques,I Daniel M. Girardi,II Maria Ignez F. Braghiroli,I,II Renata A. Coudry,I Sibele I. Meireles,I Artur Katz,I Paulo M. Hoff I,II I Centro de Oncologia, Hospital Sirio Libanes, Sao Paulo, SP, BR. II Instituto do Cancer do Estado de Sao Paulo, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVES: With the development of next-generation sequencing (NGS) technologies, DNA sequencing has been increasingly utilized in clinical practice. Our goal was to investigate the impact of genomic evaluation on treatment decisions for heavily pretreated patients with metastatic cancer. METHODS: We analyzed metastatic cancer patients from a single institution whose cancers had progressed after all available standard-of-care therapies and whose tumors underwent next-generation sequencing analysis. We determined the percentage of patients who received any therapy directed by the test, and its efficacy. RESULTS: From July 2013 to December 2015, 185 consecutive patients were tested using a commercially available next-generation sequencing-based test, and 157 patients were eligible. Sixty-six patients (42.0%) were female, and 91 (58.0%) were male. The mean age at diagnosis was 52.2 years, and the mean number of pre-test lines of systemic treatment was 2.7. One hundred and seventy-seven patients (95.6%) had at least one identified gene alteration. Twenty-four patients (15.2%) underwent systemic treatment directed by the test result. Of these, one patient had a complete response, four (16.7%) had partial responses, two (8.3%) had stable disease, and 17 (70.8%) had disease progression as the best result. The median progression-free survival time with matched therapy was 1.6 months, and the median overall survival was 10 months. CONCLUSION: We identified a high prevalence of gene alterations using an next-generation sequencing test. Although some benefit was associated with the matched therapy, most of the patients had disease progression as the best response, indicating the limited biological potential and unclear clinical relevance of this practice. KEYWORDS: Molecular Profiling; Targeted Therapy; Precision; Refractory. Fernandes GS, Marques DF, Girardi DM, Braghiroli MI, Coudry RA, Meireles SI, et al. Next-generation Sequencing-based genomic profiling: Fostering innovation in cancer care? Clinics. 2017;72(10):588-594 Received for publication on March 28, 2017; First review completed on May 23, 2017; Accepted for publication on May 23, 2017 *Corresponding author. E-mail: gustavo.hemato@gmail.com

â&#x20AC;&#x2122; INTRODUCTION

very attractive for patients without any remaining conventional therapy available and for whom comprehensive genomic profiling could identify a potential new, targeted therapeutic approach. Next-generation sequencing (NGS) is one of the most advanced technologies applied to deciphering molecular alterations in tumors and enables scientists to rapidly identify numerous mutations in patient tumors (5-7). This growing knowledge has significantly improved pharmaceutical development over the years, leading to some impressive successes in cancer care (1,2). One of the first studies to demonstrate the potential benefit of matched therapy based on molecular profiling in heavily pretreated patients showed that 98% of the patient tumors had a gene alteration (GA) that could be used as a target. That study reported that 27% of the patients had an increase in progression-free survival (PFS) with treatment based on tumor molecular alterations compared with the PFS obtained with their previous treatment (8). Certain other small, retrospective studies have also provided evidence of a high prevalence of GA in patient tumors and suggested the potential clinical benefit of molecular profiling (9,10); however, the recent prospective and randomized phase II SHIVA trial

The enhancement of molecular biology techniques in the past decades and the subsequent understanding of cell-cycle control mechanisms have helped to define the hallmarks of cancer and initiate the era of targeted therapy (1,2). The development of imatinib for the treatment of patients with chronic myeloid leukemia has led to an impressive improvement in the clinical management of this disease, initiating a race to develop and clinically test small-molecule inhibitors and monoclonal antibodies targeting fundamental effectors involved in cell carcinogenesis (3,4). Personalized medicine involves matching the right drugs to the right patients. The potential benefit of this approach is

Copyright & 2017 CLINICS â&#x20AC;&#x201C; This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. No potential conflict of interest was reported. DOI: 10.6061/clinics/2017(10)01

588

Genomic profiling: Fostering innovation? Fernandes GS et al.

CLINICS 2017;72(10):588-594

assess the prevalence of genomic and targetable alterations and to determine the PFS and OS obtained with the directed therapies. All statistical analyses were performed using Predictive Analytics Software (PASW 18) and R Project for Statistical Computing. Tumor response was evaluated through computed tomography imaging and retrospectively assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (12). The PFS and OS data were summarized using the Kaplan-Meier method and compared using the log-rank test. OS was measured from the first day of treatment with the matched therapy until death or the last follow-up. PFS was measured from the first day of treatment until the date of disease progression, death or the last followup, whichever came first. All the P-values presented were two-sided, and statistical significance was determined when pp0.05. This study was approved by the local institutional review board and was conducted in accordance with state and federal regulations.

suggested that targeted agents matched according to GAs outside of their formal indications did not improve PFS (11). The present retrospective study was conducted to evaluate the role of genomic testing in treatment decisions for patients with heavily pretreated metastatic solid tumors at a single institution.

’ METHODS Patients We evaluated consecutive patients with advanced solid and hematological malignancies, whose tumors were subjected to NGS profiling from July 2013 to December 2015 at all oncology units of Hospital Sírio-Libanês in São Paulo and Brasília. Patients 18 years or older who presented with radiological evidence of metastatic disease and failed to respond or progressed on all recognized standard-of-care therapies were eligible for analysis. Patients with non-metastatic solid tumor(s) who did not receive systemic cancer treatment prior to testing or received targeted therapy based on test results considered standard for their disease were excluded. Additionally, outpatients with a loss to follow-up or lack of data concerning treatment after completing the genomic sequencing via NGS were also excluded. Clinical characteristics, such as age, gender, the location of the primary tumor, performance status based on the Eastern Cooperative Oncology Group (ECOG) score, previous systemic treatment, PFS and overall survival (OS) were obtained from medical records. We also compared patients who received targeted therapy against ERBB2 (HER2) with patients who received targeted therapy for any other alteration with the exception of HER2.

’ RESULTS Patient characteristics From July 2013 to December 2015, 185 patients underwent FM-CGP testing, and 157 patients were eligible for further analysis. Among the 28 patients excluded from the analysis, 19 patients were lost to follow-up or had no data on treatment after the completion of the genomic sequencing test, 6 patients received targeted therapy considered standard for the respective histology, and 3 patients were younger than 18 years old. The baseline characteristics of the 157 patients available for analysis are described in Table 1. The mean age at diagnosis

Analysis of molecular alterations Patients with adequate tumor tissue from archival formalinfixed paraffin-embedded (FFPE) tumor blocks or a minimum of 10 FFPE slides 4 to 5 mm thick were evaluated using commercially targeted NGS assays (FoundationOne and FoundationOne Heme from Foundation Medicine, Massachusetts, USA). This test utilizes the DNA sequencing of 315 cancerrelated genes and 28 genes commonly rearranged in cancer. For hematologic malignancies and sarcomas, the FoundationOne Heme test was performed, with DNA sequencing for 405 genes somatically altered in cancer and 31 genes involved in rearrangements in addition to the RNA sequencing of 265 genes. Genomic alterations simultaneously detected by this assay include base substitutions, short insertions and deletions, focal gene amplifications and homozygous deletions (copy number alterations), and select gene fusions and rearrangements (7).

Table 1 - Characteristics of the population available for analysis (N= 157). Characteristic

Number (%)

Women Men

66 (42.0) 91 (58.0)

ECOG 0 1 2 3 Uninformed Mean Age (years) Mean prior lines of treatment Tumor Origin Lung Colorectal Pancreas Soft tissue Stomach Breast Central nervous system Esophagus Liver Unknown primary site Ovary Adrenal Head and neck Gastric-esophageal junction Uterus Others

Treatment A therapy was considered "matched" if a U.S. Food and Drug Administration (FDA)-approved drug was known to inhibit the functional activity resulting from at least one of the GAs of a patient’s tumor, as indicated in the Foundation Medicine cancer gene panel (FM-CGP) results. The decision whether to use this matched therapy or not was made by the patient’s physician.

Objectives and statistical analyses Our primary objective was to evaluate the response rate of the tested patients who received therapy directed by NGSbased genomic profiling. The secondary objectives were to

589

51 81 16 1 8

(32.5) (51.6) (10.2) (0.6) (5.1)

52.23 years (range 15.3 to 91.25) 2.72 (range 1 to 10) 29 21 17 11 9 9 5 5 5 4 4 3 3 3 3 26

(18.5) (13.4) (10.8) (7.0) (5.7) (5.7) (3.2) (3.2) (3.2) (2.5) (2.5) (1.9) (1.9) (1.9) (1.9) (16.6)

Genomic profiling: Fostering innovation? Fernandes GS et al.

CLINICS 2017;72(10):588-594

Two patients received more than one line of targeted therapy based on the FM-CGP results (one patient received two lines, and the other patient received three lines of different targeted therapies), with the remaining patients receiving only one line of matched therapy (Table 2). Among the patients receiving the 27 different lines of matched therapy, one patient achieved a complete response (CR), and four patients achieved a partial response (PR). Three therapies resulted in stable disease (SD), while the best response to 19 therapies was progression of disease (PD) (Table 2). The overall response rate was 20.8%, and the clinical benefit (CR+PR+SD) was 29.2%. The patient who had a CR was a female with adenocarcinoma of an unknown primary site who had received three previous lines of systemic therapy. The FM-CGP revealed a HER2 mutation, and the patient was treated with trastuzumab combined with cisplatin and gemcitabine. After five months of treatment, CT revealed a complete radiological response. Four months after the end of the trastuzumab-based therapy, the patient experienced a radiological progression, and T-DM1 was initiated, with SD as the best response. Among the patients with PRs, two patients had metastatic non-small-cell lung adenocarcinoma (NSCLA) with wildtype EGFR at diagnosis, which progressed after receiving all available standard therapies. The NGS-based genomic profiling results revealed a mutation in EGFR, and a PR was observed with afatinib+cetuximab in one patient and with afatinib alone in the other patient. The third patient also had metastatic NSCLA without an EGFR mutation or ALK

was 52.2 years (range: 15.3 to 91.2). Sixty-six patients (42.0%) were female, and ninety-one patients (58.0%) were male. Most of the patients had an ECOG performance status of 0 or 1 (84.1%). The mean number of treatment lines was 2.7 (range 1 to 10). The most commonly observed tumor sites were lung (18.5%), colorectal (13.4%) and pancreas (10.8%).

Genomic test results Among the 185 consecutive patients whose tumors were analyzed using the FM-CGP, at least one GA was identified in 177 patients (95.6%). The average number of altered genes per patient was 3.9 (standard deviation 3.28). Among those 185 patients, 128 patients (69.2%) had at least one targetable molecular alteration based on an FDA-approved therapy. Most patients (43.7%) had one, while 25.4% had two or more druggable alterations.

Treatment and response Regarding the 157 patients eligible for treatment analysis, 24 patients (15.2%) received systemic treatment directed by NGS-based genomic profiling testing. Twelve patients (50%) were male, and twelve patients (50%) were female. The mean age was 46.8 years. The majority of the patients had an ECOG performance of 0 or 1 (90.16%). This population was heavily pre-treated. Most patients (62.6%) had received three or more previous lines of treatment. The most common primary sites were lung (12.5%), colorectal (12.5%) and pancreas (12.5%). The most frequently utilized targeted therapies were everolimus (25.9%), trastuzumab (11.1%) and T-DM1 (7.4%).

Table 2 - Patients treated with targeted therapy based on the results of FoundationOne (N=24). First-line Treatment Primary site

Unknown Lung Lung Lung Breast Head and Neck Colorectal Liver Soft tissue Pancreas Pancreas Uterus Ovary Colorectal Colorectal Pancreas Stomach Breast Brain Liver Prostate Leukemia Gastricesophageal junction Pancreas

Second-line Treatment

Third-line Treatment

Treatment

Best Disease Response

Treatment

Best Disease Response

Trastuzumab + Cisplatin + Gemcitabine Cetuximab + Afatinib Afatinib Crizotinib Everolimus + Exemestane Trastuzumab + Docetaxel Cabozantinib + Panitumumab Trastuzumab + Docetaxel Everolimus Everolimus + Mitomycin + Cyclophosphamide Everolimus Everolimus Everolimus T-DM1

TDM-1

Trastuzumab + Pertuzumab + Capecitabine

Treatment

Best Disease Response

Lapatinib + Trastuzumab

PR PR PR PR SD SD PD PD PD PD PD PD PD

Sorafenib Dasatinib Trastuzumab + Carboplatin + Pemetrexed Everolimus Everolimus Pazopanib Everolimus Nilotinib Lapatinib + Paclitaxel

PD PD PD PD PD PD PD PD PD

Palbociclib

Abbreviations: CR, complete response; SD stable disease; PR, partial response; PD, progression of disease.

590

Genomic profiling: Fostering innovation? Fernandes GS et al.

CLINICS 2017;72(10):588-594

therapies. Despite the high prevalence of druggable alterations, only 15% of the patients received a targeted therapy guided by a specific mutation. Although it was not completely clear with all patients, the reasons for not being treated involved patient refusal, treatment cost and no insurance coverage. The outcome analysis revealed a CR in one patient, a PR in four patients and SD in two patients. The survival analysis in these patients was not encouraging with a median PFS of 1.6 months and an OS of 10 months. However, this population was heavily pretreated and had no standard treatment available otherwise. The prevalence of molecular targets and the response rates observed in our study are consistent with those of other nonrandomized trials. A retrospective study of patients with cancer of unknown primary site (CUPS), using the same cancer genome panel as that in our study, found that 96% of the total cases of CUPS harbored at least one alteration, and one patient treated with a matched therapy achieved a CR (10). Similarly, the results of another study indicated that in patients with at least one targetable alteration, a matched therapy compared with treatment without matching was associated with a higher objective response rate (12% vs. 5%; po0.0001) and a significantly longer PFS (median, 3.9 vs. 2.2 months; p=0.001) and survival (median 11.4 vs. 8.6 months; p=0.04) (9). Only a few prospective studies are available in the literature with conflicting results. A prospective trial presented at the 2015 American Society of Clinical Oncology (ASCO) meeting compared the outcomes of patients with advanced malignancies who were on a molecular alteration-matched therapy with those on a non-matched therapy. In total, 95%

translocation at diagnosis, which had progressed despite two different lines of chemotherapy prior to the NGS results. The test results revealed MET/HGF amplification, and this patient experienced a PR with crizotinib. The last patient was a female with metastatic triple-negative breast cancer who had progressed despite eight lines of prior chemotherapy. The NGS test results revealed an NF1 gene mutation, and a PR was observed with everolimus. The median PFS for patients exposed to matched therapy was 1.6 months (range, 0.77-10.3 months), and the median OS was 10 months (range, 0.5-19 months) (Figure 1). We also evaluated the role of targeted therapy directed against HER2 alterations and performed a comparison between these patients and the remaining patients treated with a matched therapy. A total of 6 patients were treated with a matched therapy against HER2 alterations, and 19 patients were treated with therapies directed at other targets. The median PFS was 2 months for patients who underwent anti-HER2 therapy and 1.5 months for patients who underwent other targeted therapies (p=0.332). The OS for patients with anti-HER2 treatment was not reached, and the OS for those treated with non-HER2 targeted therapies was 9 months (p=0.866) (Figure 2).

â&#x20AC;&#x2122; DISCUSSION In this cohort of heavily pretreated patients, we observed a high prevalence of at least one targetable alteration in tumor samples (95.6%) with an average of 3.9 mutated genes per patient. The majority of these molecular alterations (approximately 70%) were targetable with FDA-approved

Figure 1 - A = Progression-free survival for patients with matched therapy based on the results of the FM-CGP. B = Overall survival for patients with matched therapy based on the results of the FM-CGP.

591

Genomic profiling: Fostering innovation? Fernandes GS et al.

CLINICS 2017;72(10):588-594

Figure 2 - A = Progression-free survival for patients with targeted therapy against HER2 versus any other targeted therapy. B = Overall survival for patients with targeted therapy against HER2 versus any other targeted therapy.

data (16,17). These technologies have potential applications for many purposes, such as the identification of multiple GAs that can be targeted in personalized therapy (18). However, despite the latest progress in this technology, our study suggests that only a few patients experienced clinical benefit from targeted therapy based solely on NGS results. Therefore, many points must be considered regarding the clinical use of these data. First, as shown in our study, most tumor samples have more than one targetable GA, and the prioritization of the choices for targeted therapy taking into account the different mechanisms of resistance remains a challenging task (11,19). Second, it is important to emphasize that not all alterations of the genes involved in carcinogenesis will act as ‘‘drivers’’ with the potential to respond to targeted therapy. In addition, many of the rare oncogene variants are of uncertain functional and clinical significance and require further studies (19). The costs associated with NGS and the difficulty in estimating cost effectiveness are also complicating factors. The evolution of genomics knowledge has consistently demonstrated that solid cancers have extensive heterogeneity among individual tumors and among different regions of the same tumor, which could contribute to treatment failure and drug resistance (20-22). Therefore, as Marco Gerlinger has stated, "reconstructing tumor clonal architectures and the identification of common mutations located in the trunk of the phylogenetic tree may contribute to more robust biomarkers and therapeutic approaches" (20). Matched therapy appears to benefit a few patients whose cancers have progressed despite all available standard

of 339 tested patients had at least one GA indicated by NGS. Patients treated with a matched therapy had significantly improved PFS (median, 3.9 vs. 3.3 months; p=0.002) and OS (median, 10.8 vs. 7.5 months; p=0.013) (13). A recent multicenter, open-label, randomized, controlled phase 2 trial of molecularly targeted therapy based on tumor molecular profiling versus treatment based on the physician’s choice in patients with refractory cancer (the SHIVA trial) revealed no difference in PFS between the two arms (median, 2.3 vs. 2.0 months, hazard ratio: 0.88, 95% CI 0.65-1.19, p=0.41). The objective responses also did not differ between the two groups (4.1% vs. 3.4%, p=0.19). In a subgroup analysis of patients with alterations in the RAF/MEK pathway, the HR was 0.58. However, this value was not significant, possibly due to the small number of patients in the subgroup (11). The most interesting result in our series was a patient with an adenocarcinoma of unknown primary site with a HER2 mutation who experienced a CR with trastuzumab-based therapy. Based on this observation, we compared targeted therapies against a HER2 alteration with other therapies. Although clearly exploratory, our data failed to demonstrate a significant difference between targeted therapies directed against HER2 versus those directed at other targets. Comprehensive genomic profiling has the potential to significantly change clinical practice by individualizing patient treatment. For more than two decades, molecular alterations in tumor cells have been guiding anticancer drug development with increasing success (14,15). The recent advent of NGS technologies has revolutionized the field of human genetics, enabling the fast and cost-effective generation of

592

Genomic profiling: Fostering innovation? Fernandes GS et al.

CLINICS 2017;72(10):588-594

collected data, reviewed the manuscript and approved the final version of the manuscript. Girardi DM collected the data, wrote the first draft of the manuscript, assisted in interpreting the collected data and approved the final version of the manuscript. Braghiroli MI interpreted the collected data, reviewed the manuscript and approved the final version of the manuscript. Coudry RA Interpreted the collected data, reviewed the manuscript and approved the final version of the manuscript. Meireles SI interpreted the collected data, reviewed the manuscript and approved the final version of the manuscript. Katz A reviewed the manuscript and approved the final version of the manuscript. Hoff PM interpreted the collected data, reviewed the manuscript and approved the final version of the manuscript.

therapies, and how to identify these patients remains elusive. Furthermore, as the development of new drugs progresses, new challenges emerge. In the modern era, immunotherapy has increasingly demonstrated activity in many tissues; therefore, we evaluated the mutational load identified by cancer gene panels, such as the FM-CGP, and the clinical benefit of a PD-1 blockade in non-small-cell lung cancer patients. Patients harboring a high mutational load (more than 7 mutations found in the FM-CGP) had a statistically significant benefit from anti-PD-1 treatment compared with the patients with a low mutational load (median PFS of 14.5 vs. 3.4 months, HR: 0.265, p=0.005), suggesting that mutational load could be used as a predictive clinical marker for immunotherapy (23). The present study is limited by its retrospective nature, the small number of patients included, and a possible patient selection bias. However, our study reflects the outcomes of a new approach in oncology practice in a distinct selective population of advanced, heavily treated patients. Our data are similar to those of other retrospective and non-randomized trials that showed some response in certain individuals when targeted therapies were used based on the results of the genomic sequencing of tumor samples; however, the real benefit remains unclear and is probably smaller than anticipated. Future and ongoing trials will add evidence regarding this matter. The large phase II National Cancer Institute - Molecular Analysis for Therapy Choice (NCI-MATCH) trial will screen 5,000 patients for a targeted treatment guided by tumor GAs (24,25). Similarly, the NCI-MPACT (Molecular Profiling-Based Assignment of Cancer Therapy) trial aims to compare the response rate and/or 4-month PFS for treatment with matched therapy guided by molecular aberrations versus treatment with drugs randomly chosen from a complementary set of agents (24). MyPathway is an ongoing phase II trial evaluating four treatment regimens in a population similar to that in our study, i.e., without available standard treatment. The first analysis of this trial was presented at the 2016 Gastrointestinal Cancer Symposium, and 29 out of 129 patients had some response to targeted therapy, with the most promising results observed with anti-HER2 therapies in patients with colorectal, biliary and bladder cancers. The study is designed to accrue up to 500 patients (26). Similarly, the TAPUR (Targeted Agent and Profiling Utilization Registry) study aims to evaluate the role of commercially available anticancer drugs prescribed for the treatment of patients with advanced cancer that has a potentially actionable genomic variant (NCT02693535). In conclusion, the advancement of cancer genome knowledge associated with the availability of agents that target altered genes has driven us to consider unusual therapeutic approaches in patients with an excellent performance status and without any conventional therapy available. In this setting, large basket trials will provide knowledge of targetable mutations in different cancers. Furthermore, hypermutated tumors could also be suitable candidates for immune checkpoint inhibitors, and this possibility should be evaluated in future clinical trials.

’ REFERENCES 1. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med. 2004;10(8):789-99, http://dx.doi.org/10.1038/nm1087. 2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1): 57-70, http://dx.doi.org/10.1016/S0092-8674(00)81683-9. 3. Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2(5):561-6, http://dx.doi.org/10.1038/ nm0596-561. 4. Bartram CR. bcr Rearrangement without juxtaposition of c-abl in chronic myelocytic leukemia. J Exp Med. 1985;162(6):2175-9, http://dx.doi.org/ 10.1084/jem.162.6.2175. 5. Lawrence MS, Stojanov P, Mermel CH, Robinson JT, Garraway LA, Golub TR, et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature. 2014;505(7484):495-501, http://dx.doi.org/10.1038/ nature12912. 6. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2(5):401-4, http://dx.doi.org/10.1158/2159-8290.CD-12-0095. 7. Frampton GM, Fichtenholtz A, Otto GA, Wang K, Downing SR, He J, et al. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol. 2013; 31(11):1023-31, http://dx.doi.org/10.1038/nbt.2696. 8. Von Hoff DD, Stephenson JJ Jr, Rosen P, Loesch DM, Borad MJ, Anthony S, et al. Pilot study using molecular profiling of patients0 tumors to find potential targets and select treatments for their refractory cancers. J Clin Oncol. 2010;28(33):4877-83, http://dx.doi.org/10.1200/JCO.2009.26.5983. 9. Tsimberidou AM, Wen S, Hong DS, Wheler JJ, Falchook GS, Fu S, et al. Personalized medicine for patients with advanced cancer in the phase I program at MD Anderson: validation and landmark analyses. Clin Cancer Res. 2014;20(18):4827-36, http://dx.doi.org/10.1158/1078-0432.CCR14-0603. 10. Ross JS, Wang K, Gay L, Otto GA, White E, Iwanik K, et al. Comprehensive Genomic Profiling of Carcinoma of Unknown Primary Site: New Routes to Targeted Therapies. JAMA Oncol. 2015;1(1):40-9, http://dx.doi. org/10.1001/jamaoncol.2014.216. 11. Le Tourneau C, Delord JP, Goncalves A, Gavoille C, Dubot C, Isambert N, et al. Molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer (SHIVA): a multicentre, open-label, proof-of-concept, randomised, controlled phase 2 trial. Lancet Oncol. 2015;16(13):1324-34, http://dx.doi.org/10.1016/S1470-2045(15) 00188-6. 12. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228-47, http://dx.doi. org/10.1016/j.ejca.2008.10.026. 13. Wheler JJ, Yelensky R, Stephen B, Hong DS, Zinner R, Subbiah V, et al. Prospective study comparing outcomes in patients with advanced malignancies on molecular alteration-matched versus non-matched therapy. J Clin Oncol. 2015;33:S11019, http://dx.doi.org/10.1200/jco.2015.33. 15_suppl.11019. 14. Friedman AA, Letai A, Fisher DE, Flaherty KT. Precision medicine for cancer with next-generation functional diagnostics. Nat Rev Cancer. 2015;15(12):747-56, http://dx.doi.org/10.1038/nrc4015. 15. Hartwell LH, Szankasi P, Roberts CJ, Murray AW, Friend SH. Integrating genetic approaches into the discovery of anticancer drugs. Science. 1997;278(5340):1064-8, http://dx.doi.org/10.1126/science.278.5340.1064. 16. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005;437(7057):376-80. 17. Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science. 2005;309(5741):1728-32, http://dx.doi.org/10.1126/science. 1117389. 18. Xuan J, Yu Y, Qing T, Guo L, Shi L. Next-generation sequencing in the clinic: promises and challenges. Cancer Lett. 2013;340(2):284-95, http://dx.doi.org/10.1016/j.canlet.2012.11.025.

’ AUTHOR CONTRIBUTIONS Fernandes GS conceived the research, interpreted the collected data, reviewed the manuscript and approved the ﬁnal version of the manuscript. Marques DF wrote the ﬁrst draft of the manuscript, interpreted the

593

Genomic profiling: Fostering innovation? Fernandes GS et al.

CLINICS 2017;72(10):588-594

23. Campesato LF, Barroso-Sousa R, Jimenez L, Correa BR, Sabbaga J, Hoff PM, et al. Comprehensive cancer-gene panels can be used to estimate mutational load and predict clinical benefit to PD-1 blockade in clinical practice. Oncotarget. 2015;6(33):34221-7, http://dx.doi.org/10.18632/ oncotarget.5950. 24. Redig AJ, Janne PA. Basket trials and the evolution of clinical trial design in an era of genomic medicine. J Clin Oncol. 2015;33(9):975-7, http://dx. doi.org/10.1200/JCO.2014.59.8433. 25. Mullard A. NCI-MATCH trial pushes cancer umbrella trial paradigm. Nat Rev Drug Discov. 2015;14(8):513-5, http://dx.doi.org/10.1038/nrd4694. 26. Burris HA, Hurwitz H, Perez EA, Spigel DR, Swanton C, Hainsworth JD, et al. MyPathway: An open-label phase IIa study of trastuzumab/pertuzumab, erlotinib, vemurafenib, and vismodegib in patients who have advanced solid tumors with mutations or gene expression abnormalities targeted by these agents. J Clin Oncol 33. 2015, http://dx.doi.org/ 10.1200/jco.2015.33.15_suppl.tps11111.

19. Dienstmann R, Jang IS, Bot B, Friend S, Guinney J. Database of genomic biomarkers for cancer drugs and clinical targetability in solid tumors. Cancer Discov. 2015;5(2):118-23, http://dx.doi.org/10.1158/2159-8290. CD-14-1118. 20. Gerlinger M, Rowan AJ, Horswell S, Math M, Larkin J, Endesfelder D, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366(10):883-92, http://dx. doi.org/10.1056/NEJMoa1113205. 21. Fojo T, Mailankody S, Lo A. Unintended consequences of expensive cancer therapeutics-the pursuit of marginal indications and a me-too mentality that stifles innovation and creativity: the John Conley Lecture. JAMA Otolaryngol Head Neck Surg. 2014;140(12):1225-36, http://dx.doi. org/10.1001/jamaoto.2014.1570. 22. McGranahan N, Swanton C. Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell. 2015;27(1):15-26, http://dx.doi.org/10.1016/j.ccell.2014.12.001.

594

CLINICAL SCIENCE

Longitudinal study of lung function in pregnant women: Influence of parity and smoking Luciana Duzolina Manfre´ Pastro,I,* Miriam Lemos,II Frederico Leon Arrabal Fernandes,III Silvia Regina Dias Me´dici Saldiva,IV Sandra Elisabete Vieira,V Beatriz Mangueira Saraiva Romanholo,VI,VII Paulo Hila´rio Nascimento Saldiva,II Rossana Pulcineli Vieira FranciscoI I Departamento de Ginecologia e Obstetricia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Departamento de Patologia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. III Laboratorio de Funcao Pulmonar, Disciplina de Pulmonologia, Instituto do Coracao (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. IV Instituto de Saude, Secretaria de Estado da Saude, Sao Paulo, SP, BR. V Departamento de Pediatria, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. VI Departamento de Medicina (LIM 20), Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. VII Universidade Cidade de Sao Paulo (UNICID), Sao Paulo, SP, BR.

OBJECTIVES: To evaluate pulmonary function in the first and third trimesters of pregnancy and analyze the influence of parity and smoking on spirometry parameters. METHODS: This longitudinal prospective study included a cohort of 120 pregnant women. The inclusion criteria were as follows: singleton pregnancy, gestational age less than 13.86 weeks, and no preexisting maternal diseases. The exclusion criteria were as follows: change of address, abortion, and inadequate spirometry testing. ClinicalTrials.gov: NCT02807038. RESULTS: A decrease in values of forced vital capacity and forced expiratory volume were noted in the first second from the first to third trimester. In the first and third trimesters, multiparous women demonstrated lower absolute forced vital capacity and forced expiratory volume values in the first second compared with nulliparous women (po0.0001 and p=0.001, respectively). Multiparous women demonstrated reduced forced expiratory flow in 25% to 75% of the maneuver compared with nulliparous women in the first (p=0.005) and third (p=0.031) trimesters. The absolute values of forced expiratory flow in 25% to 75%, forced expiratory volume in the first second and predicted peak expiratory flow values in the third trimester were higher in smokers compared with nonsmokers (p=0.042, p=0.039, p=0.024, and p=0.021, respectively). CONCLUSION: There was a significant reduction in forced vital capacity and forced expiratory volume values in the first second during pregnancy. Parity and smoking significantly influence spirometric variables. KEYWORDS: Spirometry; Pregnancy; Pulmonary Function Test; Parity; Smoking. Pastro LD, Lemos M, Fernandes FL, Saldiva SR, Vieira SE, Romanholo BM, et al. Longitudinal study of lung function in pregnant women: Influence of parity and smoking. Clinics. 2017;72(10):595-599 Received for publication on February 17, 2017; First review completed on April 11, 2017; Accepted for publication on May 10, 2017 *Corresponding author. E-mail: lucianapastro@usp.br

’ INTRODUCTION

vital capacity (FVC), forced expiratory volume in the first second (FEV1), and the FEV1/FVC ratio with respect to whether they remain the same, increase, or decrease slightly (1,3,4). The predictors for decreasing lung function during pregnancy remain unknown. Multiparity and smoking status may be associated with decreased lung function.

Pregnancy is a period during which the female body undergoes functional and anatomical alterations, including changes in lung function. These findings can be assessed by spirometry, a simple, inexpensive, and effective method (1,2). There is some controversy regarding the results of spirometry in pregnancy, particularly in terms of tobacco use and parity. Several studies measured lung function during pregnancy and after delivery. Mixed results have been reported for forced

Objectives This study aimed to use spirometry to evaluate changes in pulmonary function in pregnant women between the first (T1) and third trimester (T3) of pregnancy and to evaluate the influence of smoking and parity during this period.

Copyright & 2017 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited.

’ METHODS This longitudinal prospective study was conducted from August 2011 to May 2013 at the University of São Paulo Medical School (FMUSP), Brazil. This study was approved

No potential conflict of interest was reported. DOI: 10.6061/clinics/2017(10)02

595

Study of lung function in pregnant women Pastro LDM et al.

CLINICS 2017;72(10):595-599

Table 2 presents the spirometric values obtained in T1 and T3. The absolute and relative values of FVC and absolute and % predicted values of FEV1 were significantly reduced in T3 compared with T1. In terms of parity, as shown in Table 3, 54 patients (45%) were nulliparous, and the other 66 patients (55%) were multiparous. In T1, the multiparous women (0.816±0.059) had smaller FEV1/FVC ratios compared with the nulliparous women (0.863±0.049) (po0.0001) and smaller forced expiratory flow in 25% to 75% (FEF25-75%), at p=0.005 (multiparous women (3.207±0.911) and nulliparous women (3.657±0.797)). In T3, the multiparous women exhibited reductions in the same parameters compared with the nulliparous women: FEV1/FVC (multiparous women (30.827±0.047) and nulliparous women (0.856±0.047)) and FEF25-75% (multiparous women (3.253±0.788) and nulliparous women (3.558± 0.727)) (p=0.00.1 and p=0.031, respectively). Maternal age differed among nulliparous (23.00±5.57) and multiparous (29.17±5.96) women at pp0.001. To eliminate the confounding effect of age on absolute spirometry results, patients were categorized according to age: less or equal to 25 and more than 25 years age (Table 4). Regarding smoking, as shown in Table 5, 17 of the 120 (14.17%) women smoked one to ten cigarettes a day throughout their pregnancies. Some parameters were significantly higher in nonsmokers compared with smokers. Smokers had lower absolute values of FVC in T1 (3.48± 0.37) when compared to nonsmokers (3.70±0.47), p=0.042 lower absolute values of FEV1 in T3 (2.78±0.29) when compared to nonsmokers (2.99±0.39), p=0.039 lower absolute values of FEF25-75% in T3 (3.00±0.69) when compared to nonsmokers (3.46±0.76), p=0.024 and lower values of predicted peak expiratory flow (PEF) in T3 (smokers (71.71±11.39) and nonsmokers (80.36±12.53), p=0.021.

by the Ethics Committee for Research Project Analysis at the Hospital of the Faculty of Medicine under number 193/11, and written informed consent was obtained from the subjects. ClinicalTrials.gov: NCT02807038.

Study population We included 220 pregnant women who were consecutively recruited from the three primary care units located in the west of the city of São Paulo. The women were part of a larger cohort of 400 patients who were recruited to study the effects of environmental air pollution on pregnancy. The following inclusion criteria were applied: receipt of prior informed consent, singleton pregnancy, gestational age of less than 13.86 weeks at the first evaluation, and absence of preexisting maternal diseases. The exclusion criteria included withdrawal from the project, abortion, and inadequate spirometry testing performed at T1 and T3.

Study design After the confirmation of pregnancy, community health workers made the first contact with the pregnant women. During this first meeting, the research project was explained to the pregnant women, who were invited to participate in the study and provide informed consent. If the women agreed to participate, they received a card with the first assessment date and information about spirometry. The measurement techniques and spirometry parameters were selected in accordance with the Guidelines for Pulmonary Function Test of the Brazilian Thoracic Association (5). All spirometric values were adjusted according to sex, age, height, and weight at the time of the exam to estimate the predicted values (6). At the first assessment, the patients were asked about previous pregnancies and tobacco use during pregnancy according to the questionnaire in the guideline cited above.

’ DISCUSSION

Statistical analysis

In this study, it was possible to evaluate the spirometry results of 120 pregnant women during the first and third trimesters of pregnancy. In addition, the results were compared according to parity and smoking. Spirometry is the most widely used pulmonary function test, although it is estimated that approximately 15% of spirometry tests are inadequate (7,8). Because we required two complete examinations (i.e., in T1 and T3), some patients were excluded (22%). However, in one study of the long-term effect of air pollution on respiratory health in adult Swiss woman (9), a 28% loss to follow-up (related to inadequate spirometry tests) was reported, which was greater than the loss during follow-up in the current study. All spirometric values in the pregnant subjects were within normal ranges. The FVC and FEV1 values decreased significantly in the third trimester. Redivo (10) described no changes in pulmonary function in pregnancy (10); however, Grindheim et al. (7) demonstrated differences, particularly in FVC and PEF (7). The results of recent studies agree with our findings (1,11). These reduced values may be due to decreased negative intrapleural pressure due to an upward tilt of the diaphragm caused by the enlarging uterus. Another explanation is the reduction in the alveolar partial pressure of carbon gas, which is caused by hyperventilation during pregnancy (12). Our results also revealed differences in the spirometric values between smokers and nonsmokers and nulliparous

For the characterization and summary of the study variables, the mean and standard deviation and median, minimum and maximum values were used for quantitative variables, and absolute and relative frequency were used for qualitative variables. A paired t-test was used for comparison of spirometric variables in the first and third trimesters. The t-test was applied to compare the groups of nulliparous and multiparous women in relation to spirometric variables in each trimesters, and the Mann-Whitney-test was used for comparing smokers and nonsmokers.

’ RESULTS The initial sample included 220 patients. In total, 11.4% of the patients were excluded due to abortion, 12.7% were excluded due to loss to follow-up, and 22% were excluded due to unsatisfactory spirometric test results. The final sample consisted of 120 pregnant women with 240 valid spirometric exams. The maternal characteristics are presented in Table 1. We analyzed the mean BMI in the first and third trimesters in the two groups, with no significant difference found (first trimester: nulliparous=25.65±5.02; multiparous=26.75±5.81 p=0.273); (third trimester: nulliparous=28.70±4.73, multiparous= 29.55±5.84 p=0.390). There also was no significant difference in the percentage of obese patients in the two groups studied (nulliparous=11/54 (20.4%), multiparous=13/66 (19.7%) p=0.927).

596

Study of lung function in pregnant women Pastro LDM et al.

CLINICS 2017;72(10):595-599

Table 1 - Maternal characteristics. Mean±SD Age (years) Height (meters) Weight (kg)

T1= T3 = T1 = T3 =

Gestational age (weeks)

26.39±6.54 1.60±0.06 68.55±13.86 75.97±12.86 10.60±1.80 31.70±0.80

Ethnicity

White Brown Black

38 (31.17%) 66 (55.00%) 16 (13.33%)

Marital status

Single Married Cohabitating Separated

39 44 35 2

(32.50%) (36.67%) (29.17%) (1.67%)

Education level

Incomplete primary education Complete primary education Incomplete secondary education High school Incomplete higher education University graduates

30 9 21 47 4 9

(25.00%) (7.5%) (17.50%) (39.17%) (3.33%) (7.50%)

Occupation

Housewife Housemaid General Services Assistant Hairdresser/Manicurist Unemployed Other

35 9 7 5 8 56

(29.2%) (7.5%) (5.8%) (4.2%) (6.7%) (46.6%)

Monthly income (Minimum wage (MW): $ 34,700)

Less than half MW Half to equal to one MW Between one and three times MW Between three and five times MW Could not inform Would not inform

1 13 68 15 6 17

(0.80%) (10.80%) (56.70%) (12.50%) (5.00%) (14.20%)

Exclusions

Abortion Discontinued participation Change of address Inappropriate spirometry test

25 18 10 48

(11.14%) (8.20%) (4.50%) (21.80%)

Table 2 - Comparison of spirometric variables between the first and third trimesters of pregnancy. Variable

T1 Mean±SD

T3 Mean±SD

Sig ( p)

Absolute values

FVC FEV1 FEV1/FVC FEF25-75% PEF

3.670±0.467 3.070±0.397 0.837±0.059 3.409±0.887 6.179±1.113

3.532±0.459 2.964±0.387 0.840±0.049 3.390±0.773 6.042±0.964

o0.001 o0.001 0.486 0.667 0.044

% predicted values

FVC FEV1 FEV1/FVC FEF25-75% PEF

101.31±10.352 99.43±10.880 97.02±6.563 96.00±24.961 80.63±14.918

97.20±10.694 95.90±11.040 97.53±6.075 95.80±23.624 79.23±12.800

o0.001 o0.001 0.212 0.881 0.142

paired t-test.

that for this analysis, there was a significant decrease in the number of patients in each group, which can directly influence the calculation of the p value. It can be observed that the average of these spirometric variables for different age groups remained similar to the general analysis. The % predicted value of FEV1/FVC and FEF25-75%., which took into account individual characteristics (sex, age, and body mass index), was not significantly different between the two groups. Grindheim et al. (7) found similar results in

and multiparous women. Compared with the nulliparous women, the multiparous pregnant women had significantly reduced absolute values of FEV1/FVC and FEF25-75%. This reduction was maintained among the younger multiparous women (p25 years age) when compared to the younger nulliparous women, suggesting that the decrease found is due to parity. In relation to multiparous women who are more than 25 years old, only the FEV1/FVC ratio in T1 maintained a significant effect. It is important to consider

597

Study of lung function in pregnant women Pastro LDM et al.

CLINICS 2017;72(10):595-599

Table 3 - Comparison of spirometric variables in the first and third trimesters between nulliparous and multiparous women. Variable

Absolute

Nulliparous (n=54)

FVC FEV1 FEV1/FVC FEF25-75% PEF

% predicted values

FVC FEV1 FEV1/FVC FEF25-75% PEF

Mean±SD

1 3 1 3 1 3 1 3 1 3

3.645±0.537 3.531 ±0.509 3.142±0.419 3.015 ±0.407 0.863±0.049 0.856 ±0.047 3.657±0.797 3.558 ±0.727 6.346±1.176 6.137 ±1.032

1 3 1 3 1 3 1 3 1 3

101.48±11.881 98.00 ±12.349 101.57±11.661 97.43 ±11.721 98.22±5.784 97.52 ±6.433 99.46±22.094 96.59 ±21.913 83.22±14.392 80.61 ±11.794

3.67 3.57 3.18 3.01 0.87 0.85 3.71 3.59 6.17 6.30 101.00 97 102.00 97.00 99.00 99.00 98.00 98.50 82.00 81.00

(2.65 (2.67 (2.29 (2.24 (0.75 (0.75 (2.10 (1.96 (3.87 (3.77

Sig ( p)

Multiparous (n=66) Mean±SD

Median (Min - Max) -

Median (Min - Max)

3.688±0.400 3.533 ±0.416 3.010±0.317 2.923 ±0.367 0.816±0.059 0.827 ±0.047 3.207±0.911 3.253 ±0.788 6.043±1.049 5.965 ±0.905

4.89) 5.00) 4.07) 4.00) 0.96) 0.98) 5.29) 5.33) 9.40) 8.84)

(79 - 126.00) (74.00 - 128.00) (80.00 - 130.00) (75.00 - 125.00) (84.00 - 108.00) (83.00 - 107.00) (51.00 - 161.00) (46.00 - 151.00) (50.00 - 114.00) (51.00 - 108.00)

3.70 3.49 2.97 2.91 0.82 0.84 3.24 3.26 6.07 6.12

101.17±9.002 96.55 ±9.169 97.68±9.945 94.65 ±10.374 96.03±7.027 97.55 ±5.816 93.17±26.918 95.15 ±25.085 78.90±15.014 78.21 ±13.263

101.50 98.00 98.50 95.00 96.00 98.50 90.00 94.00 80.00 79.00

(2.89 (2.72 (2.15 (2.12 (0.70 (0.72 (1.63 (1.54 (3.78 (3.72

(83.00 (75.00 (78.00 (73.00 (83.00 (85.00 (53.00 (50.00 (49.00 (48.00

4.70) 4.89) 4.12) 4.05) 0.97) 0.96) 5.79) 4.84) 9.76) 9.06) -

123.00) 121.00) 121.00) 122.00) 116.00) 114.00) 173.00) 150.00) 127.00) 117.00)

0.646 0.979 0.071 0.196 o0.0001 0.001 0.005 0.031 0.140 0.330 0.873 0.474 0.051 0.172 0.069 0.981 0.170 0.741 0.152 0.352

Table 4 - Comparison of spirometric variables in the first and third trimesters between nulliparous and multiparous women at p 25 and 4 25 years old. Variable

Absolute

FVC FEV1 FEV1/FVC FEF25-75% PEF

% predicted values

FVC FEV01 FEV1/FVC FEF25-75% PEF

Women p25 years

sig ( p)

Women 425 years Nulliparous (n=14) Mean±SD

sig ( p)

Nulliparous (n=40) Mean±SD

Multiparous (n=20) Mean±SD

Multiparous (n=46) Mean±SD

1 3 1 3 1 3 1 3 1 3

3.626±0.532 3.515±0.493 3.143±0.419 3.016±0.411 0.870±0.053 0.860±0.050 3.721±0.825 3.564±0.755 6.292±0.987 6.121±1.088

3.670±0.315 3.516±0.318 3.038±0.265 2.911±0.269 0.829±0.054 0.830±0.045 3.230±0.673 3.220±0.650 6.169±1.125 5.762±0.911

0.691 0.993 0.310 0.302 0.007 0.026 0.025 0.088 0.666 0.210

3.712±0.569 3.579±0.573 3.141±0.438 3.014±0.412 0.847±0.032 0.845±0.036 3.474±0.707 3.542±0.667 6.502±1.639 6.185±0.892

3.696±0.436 3.542±0.456 2.999±0.412 2.928±0.409 0.812±0.062 0.826±0.049 3.197±1.004 3.268±0.849 5.989±1.023 6.052±0.899

0.910 0.801 0.269 0.499 0.043 0.185 0.342 0.273 0.284 0.630

1 3 1 3 1 3 1 3 1 3

101.250±10.970 97.450±11.031 101.050±10.651 96.625±10.860 97.375±6.270 96.475±6.752 96.950±22.368 92.450±20.551 81.929±11.857 80.111±13.195

99.800±7.031 94.450±7.577 96.050±7.715 91.300±7.349 94.350±5.613 95.300±5.516 83.150±18.082 82.750±17.235 75.867±14.202 71.063±11.710

0.538 0.223 0.065 0.053 0.074 0.504 0.020 0.075 0.144 0.029

101.143±14.623 99.571±15.907 103.071±14.715 99.571±14.095 100.643±3.153 100.500±11.202 106.643±20.353 108.429±22.065 86.000±19.022 81.571±8.847

101.761±9.746 97.457±9.715 98.391±10.771 96.109±11.202 96.761±7.499 98.522±5.726 97.522±29.061 100.543±26.181 79.911±15.294 80.756±12.958

0.928 0.644 0.198 0.325 0.007 0.239 0.279 0.312 0.273 0.827

relation to smoking (active and passive smokers) and investigated repercussions on spirometric parameters. A significant association was found between smoking and spirometric parameters (ANOVA), which was lower in smokers: FVC (p=0.001), FEV1 (p=0.0001), FEV1/FVC (p=0.004), PEF (p=0.0001), and FEF25-75% (p=0.0001). Prasad et al. (14) reported a study involving over 100 female smokers and 100 female non-smokers in the age group of 30-40 years in which three lung function tests, FEVI, FVC and PEFR, were employed, and it was observed that all parameters of the three lung function tests were significantly reduced among the smokers compared to the non-smokers.

their study, which analyzed a cohort of 87 healthy pregnant women. The authors concluded that the predicted FVC values in multiparous pregnant women decreased by 4.4% compared with those in nulliparous women (p=0.039). No statistically significant differences were observed in the absolute and predicted PEF values. Although obesity was related to changes in spirometry and also to multiparity, there was no difference between the groups in relation to this maternal characteristic. The absolute values of FVC, FEF25-75%, and FEV1 were significantly decreased in smokers compared with nonsmokers. Vergara et al. (13) studied lung function in 136 girls (45.5%) in

598

Study of lung function in pregnant women Pastro LDM et al.

CLINICS 2017;72(10):595-599

Table 5 - Comparison of spirometric variables in the first and third trimesters between pregnant smokers and nonsmokers. Variable

Absolute

Nonsmokers (n=103)

FVC FEV1 FEV1/FVC FEF25-75% PEF

% predicted values

FVC FEV1 FEV1/FVC FEF25-75% PEF

Mean±SD

1 3 1 3 1 3 1 3 1 3

3.70±0.47 3.56±0.47 3.10±0.40 2.99±0.39 0.84±0.06 0.84±0.04 3.44±0.90 3.46±0.76 6.25±1.14 6.11±0.96

1 3 1 3 1 3 1 3 1 3

101.95±10.67 97.78±11.00 100.07±11.11 96.70±11.10 97.02±6.62 97.79±6.09 96.86±25.15 97.43±23.54 81.63±15.02 80.36±12.53

3.71 3.53 3.11 2.95 0.84 0.84 3.43 3.42 6.22 6.20

(2.71 (2.67 (2.15 (2.12 (0.70 (0.73 (1.63 (1.55 (3.78 (3.72

Sig ( p)

Smokers (n=17)

Median (Min - Max) -

4.89) 5.00) 4.12) 4.05) 0.97) 0.98) 5.59) 5.33) 9.76) 9.06)

102 (79 - 126) 98 (78 - 128) 100 (78 - 130) 96 (73 - 125) 97 (83 - 116) 99 (83 - 114) 94 (51 - 173) 98 (50 - 151) 82 (49 - 127) 80 (48 - 117)

Mean±SD 3.48±0.37 3.36±0.29 2.91±0.32 2.78±0.29 0.84±0.05 0.83±0.05 3.22±0.75 3.00±0.69 5.73±0.81 5.65±0.85 97.41±7.15 93.71±7.92 95.59±7.63 91.06±9.53 97.00±6.39 96.00±5.88 90.76±23.79 85.94±22.30 84.57±12.47 71.71±11.39

Median (Min - Max) 3.45 3.32 2.96 2.84 0.84 0.83 3.26 3.07 5.92 5.58 96 94 99 95 99 97 93 93 78 70

(2.65 (2.92 (2.29 (2.12 (0.71 (0.72 (1.63 (1.54 (4.02 (3.89

(87 (78 (80 (75 (85 (85 (53 (46 (50 (51

110) 111) 108) 106) 105) 103) 156) 135) 97) 88)

4.39) 3.94) 3.38) 4.05) 0.96) 0.95) 4.21) 4.09) 7.24) 6.83)

0.042 0.074 0.115 0.039 0.842 0.154 0.447 0.024 0.055 0.078 0.064 0.131 0.144 0.081 0.734 0.252 0.416 0.076 0.092 0.021

Mann-Whitney U-test.

preparation of the project and the ﬁnal data analysis. Francisco RP assisted in the preparation of the project, manuscript writing and ﬁnal data analysis.

One limitation is the fact that smoking research was performed only through a structured questionnaire with the possibility of one response (yes or no). Currently, the use of monoximetry is suggested to more accurately discriminate smokers who, for any reason, will declare themselves to be non-smokers. Nonetheless, we must point out that the great majority of studies in the literature that use spirometry for lung function evaluation do not advocate the performance of monoximetry to exclude smoking. In this study, we concluded that the absolute values of FVC, FEF25-75%, and FEV1 were significantly decreased in smokers compared with nonsmokers, with significant reductions in absolute and in % predicted FVC and FEV1 values during pregnancy (T1 to T3). Parity and smoking significantly influenced spirometric parameters, and these are important factors that must be considered when analyzing spirometry results during pregnancy. FVC and FEV1 values decreased significantly in the third trimester. The reductions in these parameters may be explained by the upward tilt of the diaphragm caused by the enlarging uterus, causing a decreased negative intrapleural pressure, and because of a reduction in the alveolar partial pressure of carbon gas, which is caused by hyperventilation during pregnancy. Decreased lung function at both the beginning and end of the gestational period is more evident in multiparous women and those who smoke.

’ REFERENCES 1. Gupta L, Dixit R. A linear study of pulmonary function tests in normal pregnant and non-pregnant women. J Indian Med Assoc. 2013;111(10): 666-9. 2. Tan EK, Tan EL. Alterations in physiology and anatomy during pregnancy. Best Pract Res Clin Obstet Gynaecol. 2013;27(6):791-802, http://dx. doi.org/10.1016/j.bpobgyn.2013.08.001. 3. Hegewald MJ, Crapo RO. Respiratory physiology in pregnancy. Clin Chest Med. 2011;32(1):1-13, http://dx.doi.org/10.1016/j.ccm.2010.11.001. 4. Kolarzyk E, Szot WM, Lyszczarz J. Lung function and breathing regulation parameters during pregnancy. Arch Gynecol Obstet. 2005;272(1):53-8, http://dx.doi.org/10.1007/s00404-004-0691-1. 5. Pereira C. Diretrizes para o teste de função pulmonar da Sociedade Brasileira de pneumologia e tisiologia–Espirometria. J Pneumol. 2002;1-82. 6. American Thoracic Society. Standardization of Spirometry, 1994 Update. Am J Respir Crit Care Med. 1995;152(3):1107-36, http://dx.doi.org/ 10.1164/ajrccm.152.3.7663792. 7. Grindheim G, Toska K, Estensen ME, Rosseland LA. Changes in pulmonary function during pregnancy: a longitudinal cohort study. BJOG. 2012;119(1):94-101, http://dx.doi.org/10.1111/j.1471-0528.2011.03158.x. 8. Mbatchou Ngahane BH, Afane Ze E, Chebu C, Mapoure NY, Temfack E, Nganda M, et al. Effects of cooking fuel smoke on respiratory symptoms and lung function in semi-rural women in Cameroon. Int J Occup Environ Health. 2015;21(1):61-5, http://dx.doi.org/10.1179/2049396714Y.00000 00090. 9. Schikowski T, Schaffner E, Meier F, Phuleria HC, Vierkötter A, Schindler C, et al. Improved air quality and attenuated lung function decline: modification by obesity in the SAPALDIA cohort. Environ Health Perspect. 2013;121(9):1034-9, http://dx.doi.org/10.1289/ehp.1206145. 10. Redivo MB. Avaliação da função pulmonar em gestantes no período gestacional entre 28 a 36 semanas. Universidade do Sul de Santa Catarina. 2007. 11. Neeraj, Sodhi C, Pramod J, Singh J, Kaur V. Effect of advanced uncomplicated pregnancy on pulmonary function parameters of North Indian subjects. Indian J Physiol Pharmacol. 2010;54(1):69-72. 12. Ogbodo S, Nwagha U, Okaka A, Okeke A, Chukwurah F, Ezeonu P. Low levels of some nutritional parameters of pregnant women in a rural community of South East Nigeria: implications for the attainment of the millennium developmental goal. Ann Med Health Sci Res. 2012;2(1): 49-55, http://dx.doi.org/10.4103/2141-9248.96939 13. Suárez López de Vergara RG, Fernández C, Oliva Hernández C, Doménech Martínez E, Dorta Delgado JM, Dorta Suárez M. Lung function and exposure to tobacco smoke among adolescents. An Pediatr. 2007;67(6): 559-66, http://dx.doi.org/10.1016/S1695-4033(07)70804-4 14. Prasad BK, Sahay AP, Singh AK. Smoking women and their lung function tests. Kathmandu Univ Med J (KUMJ). 2004;2(2):142-4.

’ ACKNOWLEDGMENTS Financial support: FAPESP, process number 2009/17315-9 and CNPq, process number 140260/2011-0.

’ AUTHOR CONTRIBUTIONS Pastro LD performed spirometry and data analyses and was responsible for the ﬁnal preparation of the manuscript. Lemos M assisted in the design of the study and spirometry. Fernandes FL provided training regarding spirometry and analyzed spirometric tests. Saldiva SR assisted in the preparation of the project and the recruitment of pregnant women. Vieira SE assisted in the preparation of the project and the recruitment of pregnant women. Romanholo BM performed spirometry. Saldiva PH assisted in the

599

CLINICAL SCIENCE

Identification of the difference in the pathogenesis in heart failure arising from different etiologies using a microarray dataset Guodong Yang,I Shuping Chen,I Aiqun Ma,I,II,III,* Jun Lu,IV Tingzhong WangI,II,III,* I Department of Cardiovascular Medicine, First Affiliated Hospital of Xi’an Jiaotong University, China. II Key Laboratory of Molecular Cardiology, Shaanxi Province, China. III Key Laboratory of Environment and Genes Related to Diseases (Xi’an Jiaotong University), Ministry of Education, China. IV Clinical Research Center, First Affiliated Hospital of Xi’an Jiaotong University, China.

OBJECTIVES: Clinically, patients with chronic heart failure arising from different etiologies receive the same treatment. However, the prognoses of these patients differ. The purpose of this study was to elucidate whether the pathogenesis of heart failure arising from different etiologies differs. METHODS: Heart failure-related dataset GSE1145 was obtained from the Gene Expression Omnibus database. Differentially expressed genes were identified using R. A protein-protein interaction network of the differentially expressed genes was constructed using Search Tool for the Retrieval of Interacting Genes. The modules in each network were analyzed by Molecular Complex Detection of Cytoscape. The Database for Annotation, Visualization and Integrated Discovery was used to obtain the functions of the modules. RESULTS: Samples contained in GSE1145 were myocardial tissues from patients with dilated cardiomyopathy, familial cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, and post-partum cardiomyopathy. The differentially expressed genes, modules, and functions of the modules associated with different etiologies varied. Abnormal formation of extracellular matrix was overlapping among five etiologies. The change in cytoskeleton organization was specifically detected in dilated cardiomyopathy. The activation of the Wnt receptor signaling pathway was limited to hypertrophic cardiomyopathy. The change in nucleosome and chromatin assembly was associated with only familial cardiomyopathy. Germ cell migration and disrupted cellular calcium ion homeostasis were solely detected in ischemic cardiomyopathy. The change in the metabolic process of glucose and triglyceride was detected in only post-partum cardiomyopathy. CONCLUSION: These results indicate that the pathogenesis of heart failure arising from different etiologies varies, which may provide molecular evidence supporting etiology-based treatment for heart failure patients. KEYWORDS: Heart Failure; Different Etiologies; Microarray; Expression Profile; Pathogenesis; Data Mining. Yang G, Chen S, Ma A, Lu J, Wang T. Identification of the difference in the pathogenesis in heart failure arising from different etiologies using a microarray dataset. Clinics. 2017;72(10):600-608 Received for publication on February 17, 2017; First review completed on May 24, 2017; Accepted for publication on July 19, 2017 *Corresponding authors. E-mails: maaiqun@medmail.com.cn / tingzhong.wang@mail.xjtu.edu.cn

’ INTRODUCTION

failure is a very serious global public health problem in the 21st century, and there is an urgent need to improve the outcome of such patients (6,7). Currently, patients with chronic heart failure arising from different etiologies are given the same treatment in clinical practice, and the differences in etiology are ignored (6,7). Although the clinical presentation of heart failure has a similar appearance among such cases, the prognoses are different, suggesting that the pathogeneses of heart failure arising from different etiologies are different. This implies that the etiologies should be taken into consideration when deciding on treatment options, and etiology-based treatments should be given to patients with heart failure. The purpose of this study was thus to elucidate whether the pathogeneses of various types of heart failure arising from different etiologies is different. The pathogenesis of diseases is usually not determined by a single gene but by the interactions among multiple genes forming a pathogenetic network, which is characterized by determining changes in the

Heart failure is the end stage of various heart diseases, including dilated cardiomyopathy, hypertension, hypertrophic cardiomyopathy, and myocardial infarction. Patients with heart failure suffer from a high mortality and a poor prognosis. Worldwide, the mortality of patients within 5 years of chronic heart failure is greater than 50% (1,2). Recently, the incidence of heart failure has been increasing due to improvement in the treatment of underlying diseases, changes of life style, and the aging of the population (3-5). Thus, heart

600

The different etiologies of heart failure Yang G et al.

CLINICS 2017;72(10):600-608

Analysis of the modules in the PPI network

gene expression profile (8-11). In a pathogenetic network, some gene products have similar or the same functions. They are, thus, located in the same functional unit of the network, called a module, and work together to carry out their biological functions (12). A microarray globally detects the expression profile of the genome and is helpful for uncovering the pathogenesis of diseases. In this study, microarray data from the myocardial tissues of patients with heart failure arising from different etiologies were thus compared with those from normal controls to identify the differentially expressed genes (DEGs). Then, we constructed a protein-protein interaction (PPI) network of the DEGs and analyzed the modules of each PPI network. Finally, we explored the functions of these modules.

Modules are functional units of a network. Genes with a similar or the same function make up a certain module. Here, Cytoscape was used to visualize the PPI network and the molecular complex detection (MCODE), a plugin of Cytoscape, was used to identify the modules in each network. The parameters of MCODE were as follows: the degrees of each node in a module were no less than 2, and subgraphs of each node were greater than 2 (12).

Enrichment analysis of the function of modules The Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/) was applied to perform the functional enrichment analysis for the modules associated with the different etiologies (19). Po0.05 was set as the cut-off.

â&#x20AC;&#x2122; MATERIALS AND METHODS Microarray data

â&#x20AC;&#x2122; RESULTS

GSE1145 was downloaded from the Gene Expression Omnibus (GEO) database. The samples contained in GSE1145 were myocardial tissues from patients with heart failure arising from five different etiologies, including 12 samples arising from dilated cardiomyopathy (DCM), 5 from familial cardiomyopathy (FCM), 5 from hypertrophic cardiomyopathy (HCM), 20 from ischemic cardiomyopathy (ISCM), and 4 from post-partum cardiomyopathy (PPCM), as well as 11 normal controls. The myocardial samples of heart failure were collected from patients undergoing cardiac transplantation whose failure arose from the different etiologies mentioned above. The normal control myocardial samples were collected from normal organ donors whose hearts could not be used for transplants. The platform used was GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array, which contains a total of 54,675 probes. These probes correspond to 20,283 genes. When multiple probes corresponded to one gene, the variance of the expression levels of the probes was averaged as the expression value of the gene.

Data preprocessing and screening of the DEGs After normalization, the median gene expression value in each box was approximately at the same level, which indicated an excellent degree of normalization (Figure 1). Compared with the normal controls, the numbers of DEGs in DCM, HCM, FCM, ISCM, and PPCM were different, namely, 331 (320 upregulated, 11 downregulated), 298 (198 upregulated, 100 downregulated), 685 (667 upregulated, 18 downregulated), 747 (731 upregulated, 16 downregulated), and 343 (306 upregulated, 37 downregulated), respectively (Table 1). The numbers of genes overlapping among the different etiologies are shown in Figure 2.

PPI network construction Based on the DEGs screened previously, a PPI network of each etiology was constructed. The number of pairs of genes included in the PPI networks of DCM, HCM, FCM, ISCM, and PPCM were 128, 153, 507, 719, and 160, respectively. Figure 3 shows the PPI network visualized by Cytoscape.

Data preprocessing

Analysis of the modules in the PPI network

The raw data from the original CEL files were preprocessed and transformed into expression values by Affy, an R package. Then, the expression values were normalized using the robust multiarray averaging (RMA) algorithm, and the missing data were filled in using the k-Nearest Neighbor (KNN) algorithm (13,14). Box plots were drawn before and after the normalization to estimate the degree of normalization (15,16).

The number of modules in the PPI networks of DCM, HCM, FCM, ISCM, and PPCM was also different, namely, 7, 7, 9, 12, and 6, respectively (Figure 4). We analyzed the overlapping and unique module-related genes of the different etiologies and found that FMOD, LUM, OMD, and OGN were overlapping genes among the five etiologies, and they formed a module with overlap for all of the five etiologies. The numbers of unique module-related DEGs of DCM, HCM, FCM, ISCM, and PPCM were 8, 13, 29, 41, and 11, respectively, which was almost half of the total modulerelated DEGs (Table 2).

Analysis of DEGs Limma, a package of R, was applied to identify the DEGs between each etiology and the normal controls (10). The statistical method that we used was the empirical Bayes method, which was implemented in limma (17). The threshold for the DEGs was set as an adjusted p-value o0.05, and the fold change values X2 (16).

Functional annotation of the modules The functions of the modules in each PPI network were annotated using DAVID (Table 3). We found that the functions of the modules in each etiology were not exactly the same. The modules not only shared similarities but also exhibited differences. The modules related to immune/ inflammatory response and the formation of the extracellular matrix were overlapping in the five etiologies. The genes in the modules related to immune response and inflammatory response were not equal among the different etiologies. However, FMOD, LUM, OMD, and OGN, which participate

Construction of the PPI network Search Tool for the Retrieval of Interacting Genes (STRING, http://string-db.org/), a database containing both direct (physical) and indirect (functional) associations of protein interactions, was used to predict the interactions between the identified DEGs and to construct the proteinprotein interaction (PPI) network (18).

601

The different etiologies of heart failure Yang G et al.

CLINICS 2017;72(10):600-608

Figure 1 - Box plots for the expression value data before and after normalization. The horizontal axis indicates the different samples, and the vertical axis represents the expression values of the genes. The black line in each box is the median of the expression value. Before normalization, the median gene expression value in each box was not at the same level. After normalization, the median of the expression value was almost on the same line, suggesting an excellent performance of the normalization.

Table 1 - The number of DEGs in the different etiologies. Etiologies DCM HCM FCM ISCM PPCM

DEGs

Upregulated

Downregulated

331 298 685 747 343

320 198 667 731 306

11 100 18 16 37

PPCM, with upregulated G6PC, GPAM, and PCK1. The genes in the modules related to the functions mentioned above are shown in Table 3.

â&#x20AC;&#x2122; DISCUSSION The immune response and inflammatory response play important roles in heart failure, leading to the development of this disease. Circulating inflammatory cytokines are elevated in heart failure and are used as predictors of clinical outcome (20-22). In our research, the immune/inflammatory response participated in heart failure arising from all five etiologies. Although the immune/inflammatory response was associated with heart failure induced by all five etiologies, the genes related to the immune/inflammatory response in the different etiologies were not exactly the same, which further indicates the differences in the pathogenesis in heart failure arising from these different etiologies. In addition to the immune/inflammatory response, our data showed that the formation of extracellular matrix overlapped among the types of heart failure arising from the five etiologies, and the genes related to this were the same. Cardiac remodeling is a key feature of heart failure, characterized by reduced myocytes and increased extracellular matrix, which finally

in the formation of the extracellular matrix, overlapped among the five etiologies. The modules related to cytoskeleton organization were detected specifically in heart failure arising from DCM, with increased KIF18A and TUBE1. The modules related to the Wnt receptor signaling pathway were limited to heart failure arising from HCM, with increased WIF1 and FRZB. The modules related to nucleosome and chromatin assembly were associated only with heart failure arising from FCM, with increased HIST1H2BN, CENPA, HIST1H1A and HIST1H2AK. The modules related to germ cell migration and disrupted cellular calcium ion homeostasis were solely detected in heart failure arising from ISCM, with increased CXCR4, CCL5, and CXCL12. The modules related to the metabolic process of glucose and triglycerides were detected only in heart failure arising from

602

The different etiologies of heart failure Yang G et al.

CLINICS 2017;72(10):600-608

Figure 2 - Overlapping DEGs among the groups of different etiologies. The Venn graph displays the number of differentially expressed genes overlapping among the different etiologies. The differentially expressed genes (DEGs) shown in the figure differ significantly in their expression value with an adjusted p-value o0.05 and a fold change 42. DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; FCM, familial cardiomyopathy; ISCM, ischemic cardiomyopathy; PPCM, post-partum cardiomyopathy.

are accompanied by cytoskeletal changes. Such changes are not only the cause but also the consequence of reduced systolic function in patients with heart failure. The cytoskeleton forms a complex network that extends through the cytoplasm and connects the nucleus, the plasma membrane, and even the extracellular matrix. The cytoskeleton participates in the dilatation and contraction of the heart (32,33). DCM is a primary etiology of heart failure with cardiac dilatation and decreased cardiac function (34). We found that the changes in cytoskeleton organization were detected specifically in heart failure arising from DCM. KIF18A belongs to the kinesin superfamily of microtubule-associated molecular motors and regulates microtubule dynamics (35). TUBE1 is a member of the tubulin superfamily and plays a central role in the organization of microtubules (36). These results indicate that the cytoskeleton may play an important role in the pathogenesis of heart failure resulting from DCM. We should, thus, focus on the changes in the cytoskeleton in patients with heart failure arising from DCM. Administering related treatment to these patients may reverse cytoskeletal abnormalities and KIF18A and TUBE1 may be potential therapeutic targets. Wnt signaling is involved in various biological processes. Previous studies have shown that such signaling is reactivated under pathological conditions but mostly remains silent in a normal state. Increasing evidence suggests that Wnt signaling participates in the progression of heart failure and is related to adverse cardiac remodeling (37,38). HCM is a primary myocardial disease that commonly causes thickening of the myocardium (39). In our study, the activation of the Wnt receptor signaling pathway was limited to heart failure arising from HCM. The protein encoded by WIF1

result in cardiac fibrosis (23,24). The FMOD gene is a member of the family of small interstitial proteoglycans. The encoded protein may participate in the assembly of extracellular matrix due to interaction with type I and type II collagen fibrils (25). LUM, OMD, and OGN all belong to the family of small leucine-rich proteoglycans. LUM may regulate collagen fibril organization in the murine heart by coordinating multiple factors of collagen assembly, and OMD may reduce the diameter and change the shape of collagen fibrils by directly interacting with collagen (26,27). The upregulation of OGN may protect against cardiac fibrosis by inhibiting the proliferation and migration of cardiac fibroblasts (28). According to our results, we should take the immune/inflammatory response and cardiac fibrosis into consideration, and administering related treatments for patients with heart failure may be useful. FMOD, LUM, OMD, and OGN may be potential therapeutic targets. The activation of the neurohormonal and sympathetic systems has been demonstrated in heart failure, and blocking these pathways using angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and b-adrenergic blockers is useful to reduce the progression of heart failure and improve clinical outcomes (29-31). However, no evidence of abnormal neurohormonal and sympathetic systems was observed in our data. Considering that the samples used for GSE1145 were cardiac tissue rather than a single type of cardiac cell, we hypothesized that changes in the neurohormonal and sympathetic systems could occur in a certain cell type and that the expression level of related genes may be diluted when detected in cardiac tissue that is a mixture of multiple cardiac cell types. About half of patients with heart failure present an enlarged heart and reduced cardiac pump function, which

603

The different etiologies of heart failure Yang G et al.

CLINICS 2017;72(10):600-608

Figure 3 - The PPI network of each etiology. The node represents the differentially expressed genes (DEGs), and the edge represents the interaction relationship among the products of the DEGs. The modules of each network are colored yellow. DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; FCM, familial cardiomyopathy; ISCM, ischemic cardiomyopathy; PPCM, postpartum cardiomyopathy.

replication-dependent histone, which belongs to the histone H2B family. They are both linked to histone H1 and participate in the compaction of chromatin into higher order structures in transcriptional regulation (44,45). CENPA encodes a centromere protein that contains a histone H3-related histone fold domain. The protein encoded by CENPA is proposed to be a component of a modified nucleosome in which it replaces 1 or both copies of the conventional histone H3 (46). HIST1H1A encodes a replication-dependent histone that is a member of the histone H1 family, which interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures (47). The results suggest that epigenetic regulation is important for heart failure arising from FCM. Epigenetic changes in FCM, such as increased HIST1H2BN, CENPA, HIST1H1A and HIST1H2AK, may thus be useful for distinguishing FCM from HCM and DCM and may also be a useful therapeutic target for patients with heart failure arising from FCM. ISCM is caused by myocardial infarction, which eventually develops into heart failure. It is characterized by a loss of cardiomyocytes and the disruption of cellular calcium ion homeostasis in the infarcted region, which leads to ventricular reconstruction and cardiac dysfunction. Stem cell-based

functions to inhibit Wnt signaling and may impair the function and structure of the heart (40). FRZB is a type of secreted Wnt antagonist that may inhibit fibrosis in vitro (41). This result indicates that Wnt signaling may be extremely important for the pathogenesis of heart failure resulting from HCM. Wnt signaling-related treatment may have potential benefits to patients with heart failure arising from HCM, and WIF1 and FRZB may be potential therapeutic targets. Epigenetic regulation plays an important role in various pathological and physiological conditions. The main mechanisms of epigenetic regulation include DNA methylation and histone modifications, which influence gene expression by affecting the assembly of the nucleosome and chromatin. Studies have shown that epigenetic regulatory mechanisms participate in heart failure and modulate the expression of multiple genes that are essential for the development of heart failure (42). FCM is a genetic disorder that is difficult to recognize until advanced phenotypic manifestations occur. The late phenotypes of FCM, such as an enlarged atrium, are subtle in comparison with those of HCM and DCM (43). Our data showed that changes in nucleosome and chromatin assembly were associated only with heart failure arising from FCM. HIST1H2BN and HIST1H2AK encode a

604

The different etiologies of heart failure Yang G et al.

CLINICS 2017;72(10):600-608

Figure 4 - Modules in the PPI network of the five different etiologies. The node represents the module-related differentially expressed genes (DEGs), and the edge represents the interaction relationship among the products of the DEGs. DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; FCM, familial cardiomyopathy; ISCM, ischemic cardiomyopathy; PPCM, post-partum cardiomyopathy.

Table 2 - Overlapping and unique DEGs in the modules of the different etiologies. Etiologies

Total

Overlapping HCM (27) DCM (8) FCM (55)

4 13 8 29

ISCM (67)

PPCM (24)

Genes FMOD LUM OMD OGN WIF1 MSTN PRELP HLA-DPB1 MYBPC1 MYOC HTR2B THBS2 FRZB HLA-DQA1 PTGFR THBS4 COL10A1 TNNI1 TUBE1 KCNV1 TAS2R5 KCNB1 SSTR5 KCNS1 KIF18A HIST1H2AK POLK CLIC2 ABCG2 CX3CR1 CD74 HIST1H2BN HLA-DRA ZEB1 HLA-DMA MSH3 GABRR3 CCL18 ALDH1A1 GPR18 RBM43 SNAI2 IL10 CCL2 HIST1H1A PROM1 PIK3CG MSH2 EPCAM SCARF1 GNG11 GP5 GABRG3 KITLG SPARC FOS RGS2 RPL26 CXCR4 TTC8 HTR2A VCAM1 EIF1AY CD69 FAM134B HMGN3 NDC80 IFITM1 GADD45G FST RPS4Y1 HMGN1 PLEC HMGN2 BBS10 ARL14 SPC25 XAF1 GADD45A C11orf70 RAP1B RAB33B PRKX KIF20A GHR LIFR SESN1 RGS1 BET1 NSA2 IFNK CCL4 CCDC111 IFI27 BBS5 MMRN1 IGF1 CLU PROS1 COL21A1 DKK2 KRT6A PCK1 REM2 GPAM G6PC

The numbers in parentheses indicate the total number of module-related DEGs.

thus, may enhance stem cell migration (55). CXCL12 stimulates the release of intracellular calcium in a dose-dependent manner. CXCL12-stimulated epithelial cell migration can be abrogated by intracellular calcium chelation (56). Based on our study, a disrupted cellular calcium ion homeostasis was solely detected in ISCM. These results indicate that the regulation of disrupted cellular calcium ion homeostasis may have potential advantages for treating heart failure arising from ISCM, and CXCR4, CCL5, and CXCL12 may be potential therapeutic targets. However, CXCR4 and CXCL12 are also involved in germ cell migration, and further studies are required to clarify the potential efficacy of the CXCR4 and CXCL12 in clinical settings. Heart failure is accompanied by energy metabolic remodeling, which in turn exacerbates heart failure. Free fatty acids (FFAs) are the main substrate for the normal heart to produce ATP. Approximately 60%-90% of ATP is derived from the aerobic oxidation of FFAs. Upon the development

treatments in heart failure have been tested in many trials, and the results are promising (48). Our data showed that germ cell migration was solely detected in heart failure arising from ISCM, suggesting that a stem cell-based treatment may be especially beneficial for ISCM-induced heart failure. CXCR4 encodes a CXC chemokine receptor, which has 7 transmembrane regions and is located on the cell surface. CXCR4 can induce stem cell migration through the FAK/ PI3K/Akt and GSK3b/b-catenin pathways (49). The protein encoded by CXCL12 functions as the ligand for the G-protein coupled receptor and can bind to CXCR4 and regulate the migration of stem cells (50-52). Calcium ion homeostasis is broken in ISCM. Previous studies have indicated that Ca2+ signaling is related to the regulation of cardiac remodeling and turnover (53). CCL5 is one of several chemokine genes clustered on the q-arm of chromosome 17 and can activate calcium signals through a multistep cascade (54). CXCR4 protein expression is influenced by extracellular calcium and,

605

The different etiologies of heart failure Yang G et al.

CLINICS 2017;72(10):600-608

Table 3 - The annotated functions of the modules in the PPI network of each etiology. Etiology

Description

p-value

Gene

immune response inflammatory response cytoskeleton-dependent intracellular transport muscle contraction muscle organ development cytoskeleton organization cell cycle process potassium ion transport extracellular matrix

0.0099 5.75E-4 0.0077 0.0225 0.0310 0.0010 0.0017 1.39E-4 1.95E-5

RSAD2, IFI44L S100A9k, SERPINA3k, LYZ MYL1, MYH6k MYL1, MYH6k MYL1, MYH6k CENPA, KIF18A, TUBE1 CENPA, KIF18A, TUBE1 KCNS1, KCNB1, KCNV1 FMOD, LUM, OMD, OGN

muscle organ development muscle contraction cytoskeleton-dependent intracellular transport Wnt receptor signaling pathway immune response extracellular matrix

7.19E-4 3.78E-4 0.0115 0.0098 0.0026 1.95E-5

MYL1, MSTN, MYH6k MYBPC1k, MYL1, MYH6k MYL1, MYH6k WIF1, FRZB HLA-DRB1, HLA-DPB1, HLA-DQA1 FMOD, LUM, OMD, OGN

immune response nucleosome assembly chromatin assembly positive regulation of protein amino acid phosphorylation inflammatory response negative regulation of programmed cell death JAK-STAT cascade anion transport extracellular matrix

0.0258 2.31E-7 2.57E-7 0.0196 7.03E-4 9.40E-4 0.0228 0.0210 1.95E-5

CCL21, P2RY14, CCL5, CXCL12, HLA-DRB1, HLA-DMA, CD74, HLA-DRA HIST1H2BN, CENPA, HIST1H1A, HIST1H2AK HIST1H2BN, CENPA, HIST1H1A, HIST1H2AK BMP4, KITLG CCL2, IL10, CCL18, F2R PIK3CG, CCL2, IL10, F2R CCL2, F2R GABRG3, CLIC2 FMOD, LUM, OMD, OGN

immune response cellular calcium ion homeostasis germ cell migration patterning of blood vessels inflammatory response ncRNA processing regulation of cell proliferation microtubule cytoskeleton organization cellular response to stress regulation of cell cycle extracellular matrix

1.68E-5 0.0048 0.0071 0.0124 0.0146 0.0409 0.0027 0.0216 0.0017 0.0483 1.95E-5

RGS1, CCL21, CXCR4, P2RY14, CCL5, CXCL12 CXCR4, CCL5, CXCL12 CXCR4, CXCL12 CXCR4, CXCL12 CCL21, CXCR4, CCL5/FOS, CCL4, F2R RPL26, NSA2 VCAM1, BMP4, SPARC, F2R, HTR2A SPC25, NDC80 GADD45G, SESN1, GADD45A GADD45G, GADD45A FMOD, LUM, OMD, OGN

response to wounding blood coagulation anti-apoptosis inflammatory response triglyceride metabolic process glucose metabolic process extracellular matrix

5.98E-5 0.0225 0.0450 5.75E-4 9.87E-6 0.0225 1.95E-5

CLU, IGF1, MMRN1, PROS1 MMRN1, PROS1 CLU, IGF1 S100A9k, SERPINA3k, LYZ G6PC, GPAM, PCK1 G6PC, PCK1 FMOD, LUM, OMD, OGN

DCM

HCM

FCM

ISCM

PPCM

The downregulated genes are marked ‘‘k’’, and the other genes are upregulated.

glycerol-3-phosphate acyltransferase and is located on the outer mitochondrial membrane. GAPM is required to catalyze de novo synthesized fatty acids into triacylglycerol and, thus, to divert them away from oxidation (62). PCK1 is a key player in the initial step of gluconeogenesis and can decrease circulating free fatty acids (63). Energy metabolic remodeling related molecules, such as G6PC, GPAM, and PCK1, may emerge as potential therapeutic targets for these patients. The major strength of this study was that based on the microarray dataset, we showed different pathogeneses of heart failure arising from different etiologies and found the similarities and differences in the DEGs among the different types of heart failure. However, because there are only 4 or 5 samples for FCM, HCM and PPCM, our results of these three groups may have been due to chance and must be confirmed by increasing the sample size.

of heart failure, the substrate utilization changes. The aerobic oxidation of FFAs diminishes, and the uptake of glucose increases. The metabolic pattern also changes from the aerobic oxidation of FFAs to anaerobic glycolysis (57-59). PPCM is a secondary myocardial disease in women with left ventricular failure and occasionally right ventricular failure. It occurs more frequently during the last month of pregnancy or within the first 6 months after delivery (60). Our study found that changes in the metabolic process of glucose and triglycerides were only detected in PPCM-induced heart failure, indicating that energy metabolic remodeling may be more important in PPCM-induced heart failure. G6PC is a multisubunit integral membrane protein that is composed of a catalytic subunit and transporters for glucose. The protein encoded by G6PC is a key enzyme in glucose homeostasis and catalyzes the hydrolysis of D-glucose 6-phosphate to D-glucose and orthophosphate (61). GPAM is an isoform of

606

The different etiologies of heart failure Yang G et al.

CLINICS 2017;72(10):600-608

Our results indicate that the pathogenesis of heart failure arising from different etiologies not only shares similarities but also exhibits differences, which may provide molecular evidence supporting the concept that etiology-based treatment is required for patients with heart failure.

18. Szklarczyk D, Franceschini A, Kuhn M, Simonovic M, Roth A, Minguez P, et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 2011; 39(Database issue):D561-8, http://dx.doi.org/10.1093/nar/gkq973. 19. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44-57, http://dx.doi.org/10.1038/nprot.2008.211. 20. Edelmann F, Holzendorf V, Wachter R, Nolte K, Schmidt AG, KraigherKrainer E, et al. Galectin-3 in patients with heart failure with preserved ejection fraction: results from the Aldo-DHF trial. Eur J Heart Fail. 2015; 17(2):214-23, http://dx.doi.org/10.1002/ejhf.203. 21. Torre-Amione G, Kapadia S, Lee J, Durand JB, Bies RD, Young JB, et al. Tumor necrosis factor-alpha and tumor necrosis factor receptors in the failing human heart. Circulation. 1996;93(4):704-11, http://dx.doi.org/ 10.1161/01.CIR.93.4.704. 22. Vasan RS, Sullivan LM, Roubenoff R, Dinarello CA, Harris T, Benjamin EJ, et al. Inflammatory markers and risk of heart failure in elderly subjects without prior myocardial infarction: the Framingham Heart Study. Circulation. 2003;107(11):1486-91, http://dx.doi.org/10.1161/01.CIR. 0000057810.48709.F6. 23. Beltrami CA, Finato N, Rocco M, Feruglio GA, Puricelli C, Cigola E, et al. The cellular basis of dilated cardiomyopathy in humans. J Mol Cell Cardiol. 1995;27(1):291-305, http://dx.doi.org/10.1016/S0022-2828(08)80028-4. 24. Unverferth DV, Baker PB, Swift SE, Chaffee R, Fetters JK, Uretsky BF, et al. Extent of myocardial fibrosis and cellular hypertrophy in dilated cardiomyopathy. Am J Cardiol. 1986;57(10):816-20, http://dx.doi.org/10.1016/ 0002-9149(86)90620-X. 25. Mormone E, Lu Y, Ge X, Fiel MI, Nieto N. Fibromodulin, an oxidative stress-sensitive proteoglycan, regulates the fibrogenic response to liver injury in mice. Gastroenterology. 2012;142(3):612-21.e5, http://dx.doi. org/10.1053/j.gastro.2011.11.029. 26. Dupuis LE, Berger MG, Feldman S, Doucette L, Fowlkes V, Chakravarti S, et al. Lumican deficiency results in cardiomyocyte hypertrophy with altered collagen assembly. J Mol Cell Cardiol. 2015;84:70-80, http://dx. doi.org/10.1016/j.yjmcc.2015.04.007. 27. Tashima T, Nagatoishi S, Sagara H, Ohnuma S, Tsumoto K. Osteomodulin regulates diameter and alters shape of collagen fibrils. Biochem Biophys Res Commun. 2015;463(3):292-6, http://dx.doi.org/10.1016/j.bbrc.2015. 05.053. 28. Zuo C. Os 36-06 Osteoglycin Inhibits Hypertensive Cardiac Fibrosis Though Suppression of Egfr Signaling. J Hypertens. 2016;34:e403, http:// dx.doi.org/10.1097/01.hjh.0000501035.16494.b8. 29. Granger CB, McMurray JJ, Yusuf S, Held P, Michelson EL, Olofsson B, et al. Effects of candesartan in patients with chronic heart failure and reduced left-ventricular systolic function intolerant to angiotensinconverting-enzyme inhibitors: the CHARM-Alternative trial. Lancet. 2003; 362(9386):772-6, http://dx.doi.org/10.1016/S0140-6736(03)14284-5. 30. Kober L, Torp-Pedersen C, Carlsen JE, Bagger H, Eliasen P, Lyngborg K, et al. A clinical trial of the angiotensin-converting-enzyme inhibitor trandolapril in patients with left ventricular dysfunction after myocardial infarction. Trandolapril Cardiac Evaluation (TRACE) Study Group. N Engl J Med. 1995;333(25):1670-6, http://dx.doi.org/10.1056/NEJM 199512213332503. 31. Lichstein E, Hager WD, Gregory JJ, Fleiss JL, Rolnitzky LM, Bigger JT Jr. Relation between beta-adrenergic blocker use, various correlates of left ventricular function and the chance of developing congestive heart failure. The Multicenter Diltiazem Post-Infarction Research Group. J Am Coll Cardiol. 1990;16(6):1327-32, http://dx.doi.org/10.1016/0735-1097(90)90372-V. 32. Huxley AF. Muscle structure and theories of contraction. Prog Biophys Biophys Chem. 1957;7:255-318. 33. McKillop DF, Geeves MA. Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. Biophys J. 1993;65(2):693-701, http://dx.doi.org/10.1016/S0006-3495(93) 81110-X. 34. Japp AG, Gulati A, Cook SA, Cowie MR, Prasad SK. The Diagnosis and Evaluation of Dilated Cardiomyopathy. J Am Coll Cardiol. 2016; 67(25):2996-3010, http://dx.doi.org/10.1016/j.jacc.2016.03.590. 35. Kevenaar JT, Bianchi S, van Spronsen M, Olieric N, Lipka J, Frias CP, et al. Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity. Curr Biol. 2016;26(7): 849-61, http://dx.doi.org/10.1016/j.cub.2016.01.048. 36. Chang P, Giddings TH Jr, Winey M, Stearns T. Epsilon-tubulin is required for centriole duplication and microtubule organization. Nat Cell Biol. 2003;5(1):71-6, http://dx.doi.org/10.1038/ncb900. 37. Kamimura D, Uchino K, Ishigami T, Hall ME, Umemura S. Activation of Peroxisome Proliferator-activated Receptor gamma Prevents Development of Heart Failure With Preserved Ejection Fraction; Inhibition of Wntbeta-catenin Signaling as a Possible Mechanism. J Cardiovasc Pharmacol. 2016;68(2):155-61, http://dx.doi.org/10.1097/FJC.0000000000000397. 38. Okada K, Naito AT, Higo T, Nakagawa A, Shibamoto M, Sakai T, et al. Wnt/beta-Catenin Signaling Contributes to Skeletal Myopathy in Heart Failure via Direct Interaction With Forkhead Box O. Circ Heart Fail. 2015; 8(4):799-808.

’ ACKNOWLEDGMENTS This work was supported by grants from the Science and Technology Program for Public Wellbeing of China [2012GS610101].

’ AUTHOR CONTRIBUTIONS Ma A, Yang G and Wang T participated in the design of the study. Yang G and Lu J carried out the study. Yang G and Chen S analyzed the data. Yang G and Wang T drafted the manuscript.

’ REFERENCES 1. Cowie MR, Wood DA, Coats AJ, Thompson SG, Suresh V, Poole-Wilson PA, et al. Survival of patients with a new diagnosis of heart failure: a population based study. Heart. 2000;83(5):505-10, http://dx.doi.org/ 10.1136/heart.83.5.505. 2. Mosterd A, Cost B, Hoes AW, de Bruijne MC, Deckers JW, Hofman A, et al. The prognosis of heart failure in the general population: The Rotterdam Study. Eur Heart J. 2001;22(15):1318-27, http://dx.doi.org/ 10.1053/euhj.2000.2533. 3. Braunwald E. The war against heart failure: the Lancet lecture. Lancet. 2015;385(9970):812-24, http://dx.doi.org/10.1016/S0140-6736(14)61889-4. 4. Kenchaiah S, Evans JC, Levy D, Wilson PW, Benjamin EJ, Larson MG, et al. Obesity and the risk of heart failure. N Engl J Med. 2002;347(5): 305-13, http://dx.doi.org/10.1056/NEJMoa020245. 5. Rodgers A, Ezzati M, Vander Hoorn S, Lopez AD, Lin RB, Murray CJ, et al. Distribution of major health risks: findings from the Global Burden of Disease study. PLoS Med. 2004;1(1):e27, http://dx.doi.org/10.1371/ journal.pmed.0010027. 6. Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE Jr, Colvin MM, et al. 2017 ACC/AHA/HFSA Focused Update of the 2013 ACCF/AHA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Failure Society of America. J Am Coll Cardiol. 2017;70(6):776-803, http://dx.doi.org/10.1016/j.jacc.2017.04.025. 7. Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE Jr, Drazner MH, et al. 2013 ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2013; 62(16):e147-239, http://dx.doi.org/10.1016/j.jacc.2013.05.019. 8. Ayers D, Day PJ. Systems Medicine: The Application of Systems Biology Approaches for Modern Medical Research and Drug Development. Mol Biol Int. 2015;2015:698169, http://dx.doi.org/10.1155/2015/698169. 9. Bjornson E, Boren J, Mardinoglu A. Personalized Cardiovascular Disease Prediction and Treatment-A Review of Existing Strategies and Novel Systems Medicine Tools. Front Physiol. 2016;7:2, http://dx.doi.org/ 10.3389/fphys.2016.00002. 10. Louridas GE, Lourida KG. Systems biology and biomechanical model of heart failure. Curr Cardiol Rev. 2012;8(3):220-30, http://dx.doi.org/ 10.2174/157340312803217238. 11. Van Laere S, Dirix L, Vermeulen P. Molecular profiles to biology and pathways: a systems biology approach. Chin J Cancer. 2016;35(1):53, http://dx.doi.org/10.1186/s40880-016-0112-4. 12. Min F, Gao F, Liu Z. Screening and further analyzing differentially expressed genes in acute idiopathic pulmonary fibrosis with DNA microarray. Eur Rev Med Pharmacol Sci. 2013;17(20):2784-90. 13. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31(4):e15, http://dx.doi.org/10.1093/nar/gng015. 14. Wang JM, Wu JT, Sun DK, Zhang P, Wang L. Pathway crosstalk analysis based on protein-protein network analysis in prostate cancer. Eur Rev Med Pharmacol Sci. 2012;16(9):1235-42. 15. Gao S, Ou J, Xiao K. Using R and Bioconductor in Bioinformatics (in Chinese). Tianjin: Tianjin Science and Technology Translation Publishing Co. 2014. 16. Vargas JE, Porto BN, Puga R, Stein RT, Pitrez PM. Identifying a biomarker network for corticosteroid resistance in asthma from bronchoalveolar lavage samples. Mol Biol Rep. 2016;43(7):697-710, http://dx.doi.org/ 10.1007/s11033-016-4007-x. 17. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004;3:Article3, http://dx.doi.org/10.2202/1544-6115.1027.

607

The different etiologies of heart failure Yang G et al.

CLINICS 2017;72(10):600-608

51. Belmadani A, Jung H, Ren D, Miller RJ. The chemokine SDF-1/CXCL12 regulates the migration of melanocyte progenitors in mouse hair follicles. Differentiation. 2009;77(4):395-411, http://dx.doi.org/10.1016/j.diff.2008. 10.015. 52. Fukuda S, Broxmeyer HE, Pelus LM. Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1alpha (CXCL12)/CXCR4 axis. Blood. 2005;105(8):3117-26, http://dx.doi.org/ 10.1182/blood-2004-04-1440. 53. Sandmann S, Claas R, Cleutjens JP, Daemen MJ, Unger T. Calcium channel blockade limits cardiac remodeling and improves cardiac function in myocardial infarction-induced heart failure in rats. J Cardiovasc Pharmacol. 2001;37(1):64-77, http://dx.doi.org/10.1097/00005344-200101000-00008. 54. Shideman CR, Hu S, Peterson PK, Thayer SA. CCL5 evokes calcium signals in microglia through a kinase-, phosphoinositide-, and nucleotidedependent mechanism. J Neurosci Res. 2006;83(8):1471-84, http://dx.doi. org/10.1002/jnr.20839. 55. Wu Q, Shao H, Darwin ED, Li J, Li J, Yang B, et al. Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy. J Cell Mol Med. 2009;13(9B):3764-73, http://dx. doi.org/10.1111/j.1582-4934.2009.00691.x. 56. Agle KA, Vongsa RA, Dwinell MB. Calcium mobilization triggered by the chemokine CXCL12 regulates migration in wounded intestinal epithelial monolayers. J Biol Chem. 2010;285(21):16066-75, http://dx.doi.org/10.1074/ jbc.M109.061416. 57. Degens H, de Brouwer KF, Gilde AJ, Lindhout M, Willemsen PH, Janssen BJ, et al. Cardiac fatty acid metabolism is preserved in the compensated hypertrophic rat heart. Basic Res Cardiol. 2006;101(1):17-26, http://dx. doi.org/10.1007/s00395-005-0549-0. 58. Nascimben L, Ingwall JS, Lorell BH, Pinz I, Schultz V, Tornheim K, et al. Mechanisms for increased glycolysis in the hypertrophied rat heart. Hypertension. 2004;44(5):662-7, http://dx.doi.org/10.1161/01.HYP.0000144292.69599.0c. 59. Oâ&#x20AC;&#x2122;Donnell JM, Fields AD, Sorokina N, Lewandowski ED. The absence of endogenous lipid oxidation in early stage heart failure exposes limits in lipid storage and turnover. J Mol Cell Cardiol. 2008;44(2):315-22, http:// dx.doi.org/10.1016/j.yjmcc.2007.11.006. 60. Nguyen HD, McKeown B. Levosimendan for post-partum cardiomyopathy. Crit Care Resusc. 2005;7(2):107-10. 61. Abbadi S, Rodarte JJ, Abutaleb A, Lavell E, Smith CL, Ruff W, et al. Glucose-6-phosphatase is a key metabolic regulator of glioblastoma invasion. Mol Cancer Res. 2014;12(11):1547-59, http://dx.doi.org/10.1158/ 1541-7786.MCR-14-0106-T. 62. Wendel AA, Cooper DE, Ilkayeva OR, Muoio DM, Coleman RA. Glycerol3-phosphate acyltransferase (GPAT)-1, but not GPAT4, incorporates newly synthesized fatty acids into triacylglycerol and diminishes fatty acid oxidation. J Biol Chem. 2013;288(38):27299-306, http://dx.doi.org/10.1074/jbc. M113.485219. 63. Franckhauser S, Munoz S, Pujol A, Casellas A, Riu E, Otaegui P, et al. Increased fatty acid re-esterification by PEPCK overexpression in adipose tissue leads to obesity without insulin resistance. Diabetes. 2002;51(3): 624-30, http://dx.doi.org/10.2337/diabetes.51.3.624.

39. American College of Cardiology Foundation/American Heart Association Task Force on Practice; American Association for Thoracic Surgery; American Society of Echocardiography; American Society of Nuclear Cardiology; Heart Failure Society of America, Heart Rhythm Society, et al. 2011 ACCF/AHA guideline for the diagnosis and treatment of hypertrophic cardiomyopathy: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Thorac Cardiovasc Surg. 2011;142(6):e153-203, http://dx.doi.org/ 10.1016/j.jtcvs.2011.10.020. 40. Lu D, Dong W, Zhang X, Quan X, Bao D, Lu Y, et al. WIF1 causes dysfunction of heart in transgenic mice. Transgenic Res. 2013;22(6):1179-89, http://dx.doi.org/10.1007/s11248-013-9738-z. 41. De Langhe E, Aznar-Lopez C, De Vooght V, Vanoirbeek JA, Luyten FP, Lories RJ. Secreted frizzled related proteins inhibit fibrosis in vitro but appear redundant in vivo. Fibrogenesis Tissue Repair. 2014;7:14, http:// dx.doi.org/10.1186/1755-1536-7-14. 42. Movassagh M, Choy MK, Knowles DA, Cordeddu L, Haider S, Down T, et al. Distinct epigenomic features in end-stage failing human hearts. Circulation. 2011;124(22):2411-22, http://dx.doi.org/10.1161/CIRCULA TIONAHA.111.040071. 43. Judge DP, Johnson NM. Genetic evaluation of familial cardiomyopathy. J Cardiovasc Transl Res. 2008;1(2):144-54, http://dx.doi.org/10.1007/ s12265-008-9025-1. 44. Liao YP, Chen LY, Huang RL, Su PH, Chan MW, Chang CC, et al. Hypomethylation signature of tumor-initiating cells predicts poor prognosis of ovarian cancer patients. Hum Mol Genet. 2014;23(7):1894-906, http://dx.doi.org/10.1093/hmg/ddt583. 45. Srivastava A, Ritesh KC, Tsan YC, Liao R, Su F, Cao X, et al. De novo dominant ASXL3 mutations alter H2A deubiquitination and transcription in Bainbridge-Ropers syndrome. Hum Mol Genet. 2016;25(3):597-608, http://dx.doi.org/10.1093/hmg/ddv499. 46. Roulland Y, Ouararhni K, Naidenov M, Ramos L, Shuaib M, Syed SH, et al. The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity. Mol Cell. 2016;63(4):674-85, http://dx.doi.org/10.1016/j.molcel. 2016.06.023. 47. Hutchinson JB, Cheema MS, Wang J, Missiaen K, Finn R, Gonzalez Romero R, et al. Interaction of chromatin with a histone H1 containing swapped N- and C-terminal domains. Biosci Rep. 2015;35(3). pii: e00209, http://dx.doi.org/10.1042/BSR20150087. 48. Nguyen PK, Rhee JW, Wu JC. Adult Stem Cell Therapy and Heart Failure, 2000 to 2016: A Systematic Review. JAMA Cardiol. 2016;1(7):831-41, http://dx.doi.org/10.1001/jamacardio.2016.2225. 49. Li M, Sun X, Ma L, Jin L, Zhang W, Xiao M, et al. SDF-1/CXCR4 axis induces human dental pulp stem cell migration through FAK/PI3K/Akt and GSK3beta/beta-catenin pathways. Sci Rep. 2017;7:40161, http://dx. doi.org/10.1038/srep40161. 50. Golan K, Kollet O, Lapidot T. Dynamic Cross Talk between S1P and CXCL12 Regulates Hematopoietic Stem Cells Migration, Development and Bone Remodeling. Pharmaceuticals (Basel). 2013;6(9):1145-69, http:// dx.doi.org/10.3390/ph6091145

608

CLINICAL SCIENCE

Comparison of short-segment monoaxial and polyaxial pedicle screw fixation combined with intermediate screws in traumatic thoracolumbar fractures: a finite element study and clinical radiographic review Hongwei Wang,I,II,*,# Yiwen Zhao,II,# Zhongjun Mo,III Jianda Han,II Yu Chen,I Hailong Yu,I Qi Wang,I Jun Liu,I Changqing Li,IV Yue Zhou,II Liangbi XiangI I Department of Orthopedics, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang, Liaoning, 110016, China. II State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Science, Shenyang, Liaoning, 110016, China. III National Research Center for Rehabilitation Aids, Beijing, 100176, China. IV Department of Orthopedics, Xinqiao Hospital, the Third Military Medical University, Chongqing, 400037, China.

OBJECTIVES: No studies have compared monoaxial and polyaxial pedicle screws with regard to the von Mises stress of the instrumentation, intradiscal pressures of the adjacent segment and adjacent segment degeneration. METHODS: Short-segment monoaxial/polyaxial pedicle screw fixation techniques were compared using finite element methods, and the redistributed T11-L1 segment range of motion, largest maximal von Mises stress of the instrumentation, and intradiscal pressures of the adjacent segment under displacement loading were evaluated. Radiographic results of 230 patients with traumatic thoracolumbar fractures treated with these fixations were reviewed, and the sagittal Cobb’s angle, vertebral body angle, anterior vertebral body height of the fractured vertebrae and adjacent segment degeneration were calculated and evaluated. RESULTS: The largest maximal values of the von Mises stress were 376.8 MPa for the pedicle screws in the shortsegment monoaxial pedicle screw fixation model and 439.9 MPa for the rods in the intermediate monoaxial pedicle screw fixation model. The maximal intradiscal pressures of the upper adjacent segments were all greater than those of the lower adjacent segments. The maximal intradiscal pressures of the monoaxial pedicle screw fixation model were larger than those in the corresponding segments of the normal model. The radiographic results at the final follow-up evaluation showed that the mean loss of correction of the sagittal Cobb’s angle, vertebral body angle and anterior vertebral body height were smallest in the intermediate monoaxial pedicle screw fixation group. Adjacent segment degeneration was less likely to be observed in the intermediate polyaxial pedicle screw fixation group but more likely to be observed in the intermediate monoaxial pedicle screw fixation group. CONCLUSION: Smaller von Mises stress in the pedicle screws and lower intradiscal pressure in the adjacent segment were observed in the polyaxial screw model than in the monoaxial pedicle screw fixation spine models. Fracture-level fixation could significantly correct kyphosis and reduce correction loss, and adjacent segment degeneration was less likely to be observed in the intermediate polyaxial pedicle screw fixation group. KEYWORDS: Biomechanics; Finite Element Analysis; Spine Fracture; Monoaxial Pedicle Screw; Polyaxial Pedicle Screw. Wang H, Zhao Y, Mo Z, Han J, Chen Y, Yu H, et al. Comparison of short-segment monoaxial and polyaxial pedicle screw fixation combined with intermediate screws in traumatic thoracolumbar fractures: a finite element study and clinical radiographic review. Clinics. 2017;72(10):609-617 Received for publication on February 20, 2017; First review completed on April 11, 2017; Accepted for publication on July 21, 2017 *Corresponding author. E-mail: cplawhw@163.com #

These authors contributed equally to this work.

’ INTRODUCTION

unstable spine, mainly due to trauma, and approximately 10-20% of such injuries are burst fractures (1,2). Despite the advantages of a short-segment spinal instrumentation approach, such as improved correction of the spinal deformity, this procedure has also been associated with instrumentation failure, such as screw loosening, screw breakage and correction loss in some cases (3-7). Reinforcement of fixation at the fracture level can help to improve kyphosis correction and biomechanical stability (8-14). As a result, improved design and implantation techniques of pedicle screws have reduced the rate of pedicle screw and rod breakage and facilitated efficient application of the connecting rod without undue

The posterior transpedicular pedicle screw fixation technique has been widely adopted for the management of an

609

Monoaxial and polyaxial pedicle screw fixation Wang H et al.

CLINICS 2017;72(10):609-617

the calculation model presented in this paper is rational (14), but the pattern of mechanical load presented here is different from the previous study. The redistributed ROM of the T11L1 segment, the largest maximal von Mises stress (LMS) of the instrumentation, and the IDPs of the adjacent segment under displacement loading were evaluated.

stress on the construct (15-19). Compared to a monoaxial screw design, the compression and bending strength at the polyaxial head is reduced due to its specific structural design (17,18), but no studies have compared monoaxial and polyaxial pedicle screws with regard to the range of motion (ROM), von Mises stress (VMS) of the internal fixation devices, intradiscal pressures (IDPs) of the adjacent segment, or adjacent segment degeneration (ASD) using radiographic reviews. In the current study, the biomechanical characteristics of fixation techniques (SM4+2/SP4+2: short-segment monoaxial/polyaxial pedicle screw fixation; IM6+2/IP6+2: intermediate monoaxial/polyaxial pedicle screw fixation) were compared, using finite element methods, with regard to the redistributed ROM, the VMS of internal fixation devices, and the IDPs of the adjacent segment under displacement loading. Two hundred thirty patients presenting with traumatic thoracolumbar fractures and treated with these types of fixation techniques were reviewed, and ASD was evaluated.

Radiographic Review Two hundred thirty patients presenting with a single traumatic thoracolumbar fracture with a fractured vertebral body between T11 and L3 underwent surgery in our hospitals using posterior short segment fixation techniques. The inclusion criteria consisted of a traumatic T11-L3 fracture, a type A fracture according to the AO classification (26), the absence of neurologic deficits, age of 18 to 50 years, a time interval between trauma and surgery of less than 15 days, and a follow-up period longer than 12 months. The exclusion criteria were a pathologic or osteoporotic fracture, lumbar degenerative diseases, or a history of thoracic or lumbar surgery. The SP4+2, IP6+2, SM4+2 and IM6+2 groups included 27, 50, 53 and 100 patients, respectively (Table 1). The sagittal Cobb’s angle (SCA), vertebral body angle (VBA) and anterior vertebral body height (AVBH) of the fractured vertebrae were calculated as described previously (19,27,28). The correction loss refers to the loss of the SCA, VBA and AVBH during the follow-up period compared to the immediate postoperative results. ASD was defined, according to a previous study (28), as one or more of the following conditions: 1) a preoperative disc height with more than 20% correction loss, 2) retrolisthesis or anterolisthesis of more than 3 mm, or 3) an osteophyte greater than 3 mm. The ethics committee of Xinqiao Hospital approved the procedures (registry number: 20100030), and written informed consent was obtained from patients.

’ MATERIALS AND METHODS Biomechanical Study A finite element model including seven vertebrae and six discs between T9 and L3 of the spine was reconstructed and analyzed using finite element analysis software (14,20-22). The fixation models are described as SM4+2, IM6+2, SP4+2 and IP6+2. In the current study, SM4+2/SP4+2 is defined as short-segment monoaxial/polyaxial pedicle screw fixation, and IM6+2/IP6+2 is defined as intermediate monoaxial/polyaxial pedicle screw fixation. Surface-tosurface contact was defined between articulation facets. The diameter and length of the screws were 6 mm and 45 mm, respectively. The pedicle screws in the current study included monoaxial and polyaxial pedicle screws. The constraint was defined between polyaxial pedicle screw heads and shafts. However, a load limitation was defined. Surface-tosurface contact was defined between polyaxial pedicle screw heads and shafts. The screw tilt (the maximal deviation of the long axis of the screw away from perpendicular to the longitudinal rod) was 25o, and the static torque was 8 Nm, which indicated that the polyaxial pedicle screw heads will move relative to the shafts when the torque between the heads and shafts reaches 8 Nm. In our previous study (14), which used only monoaxial pedicle screws, we only applied a pure moment of 10 Nm combined with a precompressive load of 150 N to the fixation models (23-25) before measuring the ROM of the T11-L1 segment. In the current study, we measured the ROM of the intact spine model of T9-L3 under flexion, extension, left/right lateral bending and left/right axial rotation and then applied ROM displacement loading to the four fixation models. Our previous study showed that

Statistical Analysis We used SPSS 15.0 software (SPSS Inc., Illinois, USA) to perform all statistical analyses, and a value of po0.05 was considered significant (two-tailed). Independent samples t-tests and one-way ANOVA were used to compare group means. The chi-square test was conducted to assess frequency data when appropriate.

’ RESULTS Biomechanical Study The SM4+2, IM6+2 and IP6+2 fixation models presented a decreased ROM compared to the intact normal spine model in all states of motion, and these results are illustrated in Figure 1. The redistributed ROM was smallest in the IM6+2 fixation model for flexion, extension and axial rotation, and the redistributed ROM was largest in the SP4+2 fixation

Table 1 - Preoperative demographic and clinical data [Mean±SD]. Variable Patients (n) Mean age (years) Sex ratio (M/F) AO fracture classification (A1/A2/A3) Time interval from injury to operation (days) Mean ISS Follow-up (months)

SM4+2

SP4+2

IM6+2

IP6+2

53 37.4±9.3 36/17 33/9/11 6.5±2.9 13.7±7.1 22.4±8.1

27 36.0±10.3 19/8 15/5/7 6.7±2.9 12.7±6.5 21.3±10.5

100 38.7±8.3 65/35 55/20/25 6.9±3.1 13.8±7.1 22.4±8.0

50 37.5±9.5 36/14 27/9/14 6.6±3.0 13.6±7.2 24.9±5.9

SM4+2/SP4+2: short-segment monoaxial/polyaxial pedicle screw fixation; IM6+2/IP6+2: intermediate monoaxial/polyaxial pedicle screw fixation; ISS: injury severity score.

610

Monoaxial and polyaxial pedicle screw fixation Wang H et al.

CLINICS 2017;72(10):609-617

Figure 1 - Range of motion (ROM). SM4+2/SP4+2: short-segment monoaxial/polyaxial pedicle screw fixation; IM6+2/IP6+2: intermediate monoaxial/polyaxial pedicle screw fixation.

presented with pedicle screws breakage, and one patient (1/27, 3.7%) presented with screw loosening. No patients in the IP6+2 group presented with pedicle screw breakage, rod breakage, or screw loosening. Illustrative cases with pedicle screw or rod breakage are presented in Figure 5. Two patients (1/53, 1.9%) in the SM4+2 group, two patients (3/100, 3.0%) in the IM6+2 group, and no patients in the SP4+2 and IP6+2 groups presented with ASD.

model, especially for flexion, for which the ROM was larger than in the intact spine model. The LMS of the pedicle screws was observed in flexion and axial rotation in the monoaxial pedicle screw fixation models (MPSF) and in flexion and extension in the polyaxial pedicle screw fixation models (PPSF). The upper pedicle screw presented the LMS in the SM4+2, SP4+2 and IP6+ 2 models, and the lower pedicle screw presented the LMS in the IM6+2 model (Figure 2). The LMS values of the pedicle screws were 376.8 MPa, 332.8 MPa, 163.8 MPa and 145.0 MPa for the SM4+2, IM6+2, SP4+2, and IP6+ 2 models, respectively. The LMS of the rods was observed during flexion in the MPSF models and during lateral bending and rotation in the PPSF models (Figure 3). The LMS values of the rods were 409.9 MPa, 439.9 MPa, 143.1 MPa and 340.3 MPa for the SM4+2, IM6+2, SP4+2, and IP6+ 2 models, respectively. The results of stress distribution within the instruments demonstrated that the largest stress occurred at the pedicle screw root during flexion. Maximal IDPs of the adjacent segment were observed during lateral bending. The maximal IDPs of the upper adjacent segments were all larger than those of the lower adjacent segments. In the MPSF models, the maximal IDPs were larger than those in the corresponding segment of the normal model. In the PPSF models, some IDPs of the adjacent segments were less than those of the normal model (Figure 4).

â&#x20AC;&#x2122; DISCUSSION Reinforcement with fixation at the fracture level can help to improve kyphosis correction and biomechanical stability (8-14). However, no studies have compared monoaxial and polyaxial pedicle screws with regard to the VMS of the instrumentation and the IDP of the adjacent segment. Our previous study suggested that the intermediate screw fixation technique can significantly increase the stability of the spine in both the MPSF and PPSF groups. However, the MPSF group exhibited more stability in flexion and extension than the PPSF group (13). Because we previously could not measure the VMS of the instrumentation or the IDP of the adjacent segment (13), we performed a finite analysis study to determine the biomechanical comparisons between monoaxial or polyaxial pedicle screws in thoracolumbar fractures. Fixation models, including the SM4+2, IM6+2 and IP6+2 models, exhibited a lower ROM than the intact normal spine model. The ROM was smallest in the IM6+2 model. In contrast, the ROM was largest in the SP4+2 model, especially during flexion, during which the ROM was larger than that in the intact spine model. The biomechanical results were consistent with our radiological results. The postoperative SCA, VBA and AVBH were significantly improved compared to the preoperative values in all four groups. The final follow-up evaluations showed that the mean loss of correction of the SCA, VBA and AVBH was smallest in the IM6+2 group and largest in the SP4+2 group. This phenomenon can be explained because polyaxial pedicle screws heads are vulnerable to fatigue failure; the region between the screw head and

Radiographic Review The postoperative SCA, VBA and AVBH were significantly improved compared to the preoperative values in all four groups. The postoperative SCA in the SP4+2 group and the AVBH in the IM6+2 group were significantly larger than the preoperative values. The final follow-up evaluation showed that the mean loss of correction of the SCA, VBA and AVBH were smallest in the IM6+2 group (Table 2). In the SM4+2 group, screw loosening occurred in four patients (4/53, 7.5%). In the IM6+2 group, breakage of pedicle screws (4 patients) or rods (1 patient) was observed in five patients (5/100, 5.0%). In the SP4+2 group, two patients (2/27, 7.4%)

611

Monoaxial and polyaxial pedicle screw fixation Wang H et al.

CLINICS 2017;72(10):609-617

Figure 2 - von Mises stress of the pedicle screws. A. Upper pedicle screws. B. Lower pedicle screws. C. Ratio of von Mises stress of the upper to the lower pedicle screws. SM4+2/SP4+2: short-segment monoaxial/polyaxial pedicle screw fixation; IM6+2/IP6+2: intermediate monoaxial/polyaxial pedicle screw fixation.

612

Monoaxial and polyaxial pedicle screw fixation Wang H et al.

CLINICS 2017;72(10):609-617

Figure 3 - von Mises stress of the rods. SM4+2/SP4+2: short-segment monoaxial/polyaxial pedicle screw fixation; IM6+2/IP6+2: intermediate monoaxial/polyaxial pedicle screw fixation.

(fracture stress) were 550 MPa, 869 MPa, 924 MPa, respectively. In the current study, the LMS values were 376.8 MPa in all pedicle screws and 439.9 MPa in all rods. We suggest that this phenomenon may be related the patients all being manual workers who often bend over, and the lordosis angles of the rods were so large that the stress of the pedicle screws increased. The LMS value of the rods was largest in the IM6+2 group. In the SP6+2 group, no patients presented with pedicle screw breakage, rod breakage, or screw loosening. By comparison, additional intermediate polyaxial pedicle screws may result in a stiffer construct than the normal spine model and less VMS of the pedicle screws than in the MPSF models. The LMS of the pedicle screws was observed in flexion and axial rotation in the MPSF models and in flexion and extension in the PPSF models; the LMS of the rods was observed in flexion in the MPSF models but in lateral bending and rotation in the PPSF models. Therefore, we suggested that patients should be prohibited from bending over, rotating abruptly or bearing heavy weight on their back during the early postoperative stage. In the current study, the maximal IDPs of the adjacent segment were observed during lateral bending. The maximal IDPs of the upper adjacent segments were all larger than those of the lower adjacent segments in the MPSF models. These results were consistent with those of previous studies (35-39). Rahm et al. (35) noted that upper ASD can develop more easily than lower ASD after lumbar fusion with instrumentation. Chen et al. (37) observed that the upper disc adjacent to the interbody fusion presented with a larger stress than the lower disc. In the MPSF models, the maximal IDPs were larger than in the corresponding segments of the normal model. These results were consistent with previous studies that noted that fusion accelerates ASD compared to the natural history (40-42), although Radcliff KE (43) reported that spinal operations are associated with ASD. However, whether such operations accelerate the natural history of ASD remains unknown. In the current PPSF model, some IDPs of the adjacent segments were lower than those of the normal model. Additional intermediate polyaxial pedicle screws may result in lower IDPs of adjacent segments than in

shaft were found to fail first in many biomechanical studies (29-31). The failure may then result in loss of correction of the SCA, VBA and height of the vertebral bodies. The LMS of the upper pedicle screw was larger than that of the lower pedicle screw in the SM4+2, SP4+2 and IP6+ 2 models. The LMS values of the instrumentation in the MPSF group were larger than those in the PPSF models. These results may suggest that the PPSF technique can decrease the VMS of the instrumentation. Upon suspecting that a pedicle screw is broken, the upper pedicle screw must be the focus of attention for patients subjected to SM4+2, SP4+2 and IP6+2 fixation techniques, and the lower pedicle screw is the focus for the IM6+2 fixation technique. Clinically, 12 patients presented with fixation failure, including instrument breakage and screw loosening. Six patients presented with pedicle screw breakage, all of which occurred at the root of the pedicle screw because the results of stress distribution within the instruments demonstrated that the largest stress occurred at the pedicle screw root. In the SP4+2 group, pedicle screw breakage was observed in 2 patients (one upper pedicle screw, one lower pedicle screw), and screw loosening occurred in 1 patient (upper pedicle screw). In the SM 4+2 group, screw loosening occurred in 4 patients (three upper pedicle screws, one lower pedicle screw). We suggest that the phenomenon may be related to the fact that the upper pedicle screw presented the LMS in the SP4+2 and SM4+2 groups, and the redistributed ROM was largest in these two fixation models. The rate of screw loosening in the SM 4+2 group (7.5%) was significantly higher than in the SP 4+2 group (3.7%). It is difficult to precisely align the side opening of two monoaxial pedicle screw heads for insertion of the rod, and small discrepancies in the fixation of monoaxial pedicle screws may increase the rotational forces and intervertebral translational forces with negative effects at the screw-to-vertebralbody interface, which may lead to screw cut-out or loosening (29,32,33). In the IM6+2 group, pedicle screw breakage (two upper pedicle screws, two lower pedicle screw) and rod (1 patient) was observed in 5 patients. According to a previous study (34), the fatigue stress, yield stress, and ultimate stress

613

Monoaxial and polyaxial pedicle screw fixation Wang H et al.

CLINICS 2017;72(10):609-617

Figure 4 - Intradiscal pressure of the adjacent segment. A. Maximal intradiscal pressure of the adjacent segment. B. Ratio of the IDP of the fixation model to that of the normal model. SM4+2/SP4+2: short-segment monoaxial/polyaxial pedicle screw fixation; IM6+2/ IP6+2: intermediate monoaxial/polyaxial pedicle screw fixation; U: upper adjacent segment; L: lower adjacent segment; IDP: intradiscal pressure.

including different patient conditions, muscle forces, ribs, and the length and diameter of pedicle screws, for a more clinically feasible conclusion because these factors can influence finite element analysis results. Fourth, the stress of the screw-to-vertebral-body interface was not analyzed in the current study; we will conduct further research specifically considering spinal models with different bone quality in the near future.

MPSF models. Our radiological results showed that ASD was less likely to be observed in the IP6+2 group but more likely to be observed in the IM6+2 group. This study has many limitations. First, the number of patients included in the study was small, and this was a retrospective study. Second, a selection bias may exist because this study included patients referred to our teaching hospitals. Third, it is necessary to discuss several factors,

614

Monoaxial and polyaxial pedicle screw fixation Wang H et al.

CLINICS 2017;72(10):609-617

Table 2 - Radiological assessment of the deformity correction [Mean ± SD]. Variable Patients (n) SCA (o) Preoperative Postoperative Correction loss VBA (o) Preoperative Postoperative Correction loss AVBH (%) Preoperative Postoperative Correction loss

SM4+2

SP4+2

IM6+2

IP6+2

100

11.3±6.6 1.8±7.1 3.6±2.8

12.1±8.2 6.1±6.4 3.8±1.8

12.7±9.0 1.5±5.1 1.2±1.1

11.7±6.7 2.3±4.4 1.5±1.8

16.2±5.7 7.0±5.2 1.4±2.2

15.0±5.2 7.5±4.1 1.5±1.9

16.1±5.7 7.0±4.6 0.7±1.0

15.3±5.5 6.8±3.7 0.8±1.2

65.5±12.5 89.1±11.2 1.8±4.4

70.3±8.8 87.4±9.3 3.0±2.3

66.7±15.5 96.0±9.7 1.5±1.2

68.8±14.0 89.0±10.0 2.3±1.6

SM4+2/SP4+2: short-segment monoaxial/polyaxial pedicle screw fixation; IM6+2/IP6+2: intermediate monoaxial/polyaxial pedicle screw fixation; SCA: sagittal Cobb’s angle; VBA: vertebral body angle; AVBH: anterior vertebral body height.

Figure 5 - Illustrative cases presented with pedicle screw and rod breakage. A. Pedicle screw breakage occurred 14 months after surgery in a 14-year-old girl with an L1 fracture. B. Pedicle screw breakage occurred 10 months after surgery in a 47-year-old man with an L2 fracture. C. Pedicle screw breakage occurred 18 months after surgery in a 39-year-old woman with a T12 fracture. D. Pedicle screw breakage occurred 8 months after surgery in a 35-year-old man with an L2 fracture. E. Pedicle screw breakage occurred 12 months after surgery in a 45-year-old man with a T12 fracture. F. Rod breakage occurred 24 months after surgery in a 27-year-old man with an L3 fracture.

segment than MPSF models. Fracture-level fixation could significantly correct kyphosis and reduce correction loss, and ASD was less likely to occur in the IP6+2 group. Our results provided a theoretical basis for the treatment of spinal fractures using minimally invasive intermediate polyaxial pedicle screw fixation.

The use of additional intermediate monoaxial pedicle screws may result in a stiffer construct and reduced VMS of the pedicle screws than in SPSF models. In comparison, use of additional intermediate polyaxial pedicle screws may result in a stiffer construct than the normal spine model and lower VMS of the pedicle screws and IDPs of the adjacent

615

Monoaxial and polyaxial pedicle screw fixation Wang H et al.

CLINICS 2017;72(10):609-617

’ ACKNOWLEDGMENTS

15. McKinley TO, McLain RF, Yerby SA, Sharkey NA, Sarigul-Klijn N, Smith TS. Characteristics of pedicle screw loading. Effect of surgical technique on intravertebral and intrapedicular bending moments. Spine. 1999;24(1): 18-24, http://dx.doi.org/10.1097/00007632-199901010-00005. 16. Fogel GR, Reitman CA, Liu W, Esses SI. Physical characteristics of polyaxial-headed pedicle screws and biomechanical comparison of load with their failure. Spine. 2003;28(5):470-3. 17. Stanford RE, Loefler AH, Stanford PM, Walsh WR. Multiaxial pedicle screw designs: static and dynamic mechanical testing. Spine. 2004;29(4): 367-75, http://dx.doi.org/10.1097/01.BRS.0000092369.50397.85. 18. Shepard MF, Davies MR, Abayan A, Kabo JM, Wang JC. Effects of polyaxial pedicle screws on lumbar construct rigidity. J Spinal Disord Tech. 2002;15(3):233-6, http://dx.doi.org/10.1097/00024720-20020600000012. 19. Wang H, Zhou Y, Li C, Liu J, Xiang L. Comparison of open versus percutaneous pedicle screw fixation using the sextant system in the treatment of traumatic thoracolumbar fractures. Clin Spine Surg. 2017;30(3):E239E246, http://dx.doi.org/10.1097/BSD.0000000000000135. 20. Akamaru T, Kawahara N, Sakamoto J, Yoshida A, Murakami H, Hato T, et al. The transmission of stress to grafted bone inside a titanium mesh cage used in anterior column reconstruction after total spondylectomy: a finite-element analysis. 2005;30(24):2783-7. 21. Kim Y, Kim TW. Finite element analysis of the effects of pedicle screw fixation nut loosening on lumbar interbody fusion based on the elastoplateau plasticity of bone characteristics. Spine. 2010;35(6):599-606, http://dx.doi.org/10.1097/BRS.0b013e3181b6258a. 22. Kim HJ, Chun HJ, Moon SH, Kang KT, Kim HS, Park JO, et al. Analysis of biomechanical changes after removal of instrumentation in lumbar arthrodesis by finite element analysis. Med Biol Eng Comput. 2010;48(7): 703-9, http://dx.doi.org/10.1007/s11517-010-0621-2. 23. Denozière G, Ku DN. Biomechanical comparison between fusion of two vertebrae and implantation of an artificial intervertebral disc. J Biomech. 2006;39(4):766-75, http://dx.doi.org/10.1016/j.jbiomech.2004.07.039. 24. Yamamoto I, Panjabi MM, Crisco T, Oxland T. Three-dimensional movements of the whole lumbar spine and lumbosacral joint. Spine. 1989; 14(11):1256-60, http://dx.doi.org/10.1097/00007632-198911000-00020. 25. Chen SH, Lin SC, Tsai WC, Wang CW, Chao SH. Biomechanical comparison of unilateral and bilateral pedicle screws fixation for transforaminal lumbar interbody fusion after decompressive surgery -- a finite element analysis. BMC Musculoskelet Disord. 2012;13:72, http://dx.doi. org/10.1186/1471-2474-13-72. 26. Magerl F, Aebi M, Gertzbein SD, Harms J, Nazarian S. A comprehensive classification of thoracic and lumbar injuries. Eur Spine J. 1994;3(4):184-201, http://dx.doi.org/10.1007/BF02221591. 27. Mumford J, Weinstein JN, Spratt KF, Goel VK. Thoracolumbar burst fractures. The clinical efficacy and outcome of nonoperative management. 1993;18(8):955-70. 28. Tsuji T, Watanabe K, Hosogane N, Fujita N, Ishii K, Chiba K, et al. Risk factors of radiological adjacent disc degeneration with lumbar interbody fusion for degenerative spondylolisthesis. J Orthop Sci. 2016;21(2):133-7, http://dx.doi.org/10.1016/j.jos.2015.12.007. 29. Kubosch D, Kubosch EJ, Gueorguiev B, Zderic I, Windolf M, Izadpanah K, et al. Biomechanical investigation of a minimally invasive posterior spine stabilization system in comparison to the Universal Spinal System (USS). BMC Musculoskelet Disord. 2016;17:134, http://dx.doi.org/10.1186/ s12891-016-0983-1. 30. Fogel GR, Reitman CA, Liu W, Esses SI. Physical characteristics of polyaxial-headed pedicle screws and biomechanical comparison of load with their failure. Spine. 2003;28(5):470-3. 31. Stanford RE, Loefler AH, Stanford PM, Walsh WR. Multiaxial pedicle screw designs: static and dynamic mechanical testing. Spine. 2004;29(4): 367-75, http://dx.doi.org/10.1097/01.BRS.0000092369.50397.85. 32. Wan S, Lei W, Wu Z, Liu D, Gao M, Fu S. Biomechanical and histological evaluation of an expandable pedicle screw in osteoporotic spine in sheep. Eur Spine J. 2010;19(12):2122-9, http://dx.doi.org/10.1007/s00586-0101489-4. 33. Wang X, Aubin CE, Larson AN, Labelle H, Parent S. Biomechanical analysis of pedicle screw density in spinal instrumentation for scoliosis treatment: first results. Stud Health Technol Inform. 2012;176:303-6. 34. Brown SA. Synthetic biomaterials for spinal application. In: Kurtz SM, Edidin AA (eds) Spine technology handbook., Burlington. 2006:11-33. 35. Rahm MD, Hall BB. Adjacent-segment degeneration after lumbar fusion with instrumentation: a retrospective study. J Spinal Disord. 1996;9(5): 392-400, http://dx.doi.org/10.1097/00002517-199610000-00005. 36. Pellisé F, Hernández A, Vidal X, Minguell J, Martínez C, Villanueva C. Radiologic assessment of all unfused lumbar segments 7.5 years after instrumented posterior spinal fusion. Spine. 2007;32(5):574-9, http://dx. doi.org/10.1097/01.brs.0000256875.17765.e6. 37. Chen CS, Cheng CK, Liu CL, Lo WH. Stress analysis of the disc adjacent to interbody fusion in lumbar spine. Med Eng Phys. 2001;23(7):483-91, http://dx.doi.org/10.1016/S1350-4533(01)00076-5. 38. Nakashima H, Kawakami N, Tsuji T, Ohara T, Suzuki Y, Saito T, et al. Adjacent Segment Disease After Posterior Lumbar Interbody Fusion:

This work was supported by the Foundation of the State Key Laboratory of Robotics (2017-O01), the Foundation of the State Key Laboratory of Materials Processing and Die & Mold Technology (P2018-011), the Open Project Program of the State Key Laboratory of CAD & CG (A1718), the Liaoning Province Doctor Startup Fund (201601389) and the Research Foundation for Clinical Diagnosis and Treatment of Anemia for Young Physicians (LIM-SS-16001).

’ AUTHOR CONTRIBUTIONS Wang H, Zhao Y and Mo Z conceived of the study, collected the data, participated in the data analysis, drafted the manuscript and performed the statistical analysis. Han J conceived the study and participated in its design, coordination and drafting. Chen Y, Yu H, Wang Q, Liu J and Li C participated in the data analysis and interpretation. Zhou Y and Xiang L participated in the review, coordination and drafting of the manuscript and contributed to the analysis via constructive discussions.

’ REFERENCES 1. Verlaan JJ, Diekerhof CH, Buskens E, van der Tweel I, Verbout AJ, Dhert WJ, et al. Surgical treatment of traumatic fractures of the thoracic and lumbar spine: a systematic review of the literature on techniques, complications, and outcome. Spine. 2004;29(7):803-14, http://dx.doi.org/ 10.1097/01.BRS.0000116990.31984.A9. 2. Wang H, Zhang Y, Xiang Q, Wang X, Li C, Xiong H, et al. Epidemiology of traumatic spinal fractures: experience from medical university-affiliated hospitals in Chongqing, China, 2001-2010. J Neurosurg Spine. 2012;17(5): 459-68, http://dx.doi.org/10.3171/2012.8.SPINE111003. 3. Alanay A, Acaroglu E, Yazici M, Oznur A, Surat A. Short-segment pedicle instrumentation of thoracolumbar burst fracture: does transpedicular intracorporeal grafting prevent early failure? Spine. 2001;26(2):213-7, http://dx.doi.org/10.1097/00007632-200101150-00017. 4. Kramer DL, Rodgers WB, Mansfield FL. Transpedicular instrumentation and short-segment fusion of thoracolumbar fractures: a prospective study using single instrumentation system. J Orthop Trauma. 1995;9(6):499-506, http://dx.doi.org/10.1097/00005131-199509060-00007. 5. McCormack T, Karaikovic E, Gaines RW. The load sharing classification of spine fractures. Spine. 1994;19(15):1741-4, http://dx.doi.org/10.1097/ 00007632-199408000-00014. 6. McLain RF, Sparling E, Benson DR. Early failure of short-segment pedicle instrumentation for thoracolumbar fractures. A preliminary report. J Bone Joint Surg Am. 1993;75(2):162-7, http://dx.doi.org/10.2106/00004623199302000-00002. 7. Shen WJ, Liu TJ, Shen YS. Nonoperative treatment versus posterior fixation for thoracolumbar junction burst fractures without neurologic deficit. Spine. 2001;26(9):1038-45. 8. Mahar A, Kim C, Wedemeyer M, Mitsunaga L, Odell T, Johnson B, et al. Short-segment fixation of lumbar burst fractures using pedicle fixation at the level of the fracture. Spine. 2007;32(14):1503-7, http://dx.doi.org/ 10.1097/BRS.0b013e318067dd24. 9. Anekstein Y, Brosh T, Mirovsky Y. Intermediate screws in short segment pedicular fixation for thoracic and lumbar fractures: a biomechanical study. J Spinal Disord Tech. 2007;20(1):72-7, http://dx.doi.org/10.1097/ 01.bsd.0000211240.98963.f6. 10. Guven O, Kocaoglu B, Bezer M, Aydin N, Nalbantoglu U. The use of screw at the fracture level in the treatment of thoracolumbar burst fractures. J Spinal Disord Tech. 2009;22(6):417-21, http://dx.doi.org/10.1097/ BSD.0b013e3181870385. 11. Baaj AA, Reyes PM, Yaqoobi AS, Uribe JS, Vale FL, Theodore N, et al. Biomechanical advantage of the index-level pedicle screw in unstable thoracolumbar junction fractures. J Neurosurg Spine. 2011;14(2):192-7, http://dx.doi.org/10.3171/2010.10.SPINE10222. 12. Tian JW, Wang L, Xia T, Liu CY, Zhao QH, Dong SH. Posterior shortsegmental fixation combined with intermediate screws vs conventional intersegmental fixation for monosegmental thoracolumbar fractures. Orthopedics. 2011;34(8):e389-96, http://dx.doi.org/10.3928/0147744720110627-08. 13. Wang H, Li C, Liu T, Zhao WD, Zhou Y. Biomechanical efficacy of monoaxial or polyaxial pedicle screw and additional screw insertion at the level of fracture, in lumbar burst fracture: An experimental study. Indian J Orthop. 2012;46(4):395-401, http://dx.doi.org/10.4103/0019-5413. 98835. 14. Li C, Zhou Y, Wang H, Liu J, Xiang L. Treatment of unstable thoracolumbar fractures through short segment pedicle screw fixation techniques using pedicle fixation at the level of the fracture: a finite element analysis. PLoS One. 2014;9(6):e99156, http://dx.doi.org/10.1371/journal. pone.0099156.

616

Monoaxial and polyaxial pedicle screw fixation Wang H et al.

CLINICS 2017;72(10):609-617

with increased adjacent segment disc degeneration but without influence on clinical outcome: results of a combined follow-up from 4 randomized controlled trials. Spine. 2014;39(17):1373-83, http://dx.doi.org/10.1097/ BRS.0000000000000437. 42. Schulte TL, Leistra F, Bullmann V, Osada N, Vieth V, Marquardt B, et al. Disc height reduction in adjacent segments and clinical outcome 10 years after lumbar 360 degrees fusion. Eur Spine J. 2007;16(12):2152-8, http:// dx.doi.org/10.1007/s00586-007-0515-7. 43. Radcliff KE, Kepler CK, Jakoi A, Sidhu GS, Rihn J, Vaccaro AR, et al. Adjacent segment disease in the lumbar spine following different treatment interventions. Spine J. 2013;13(10):1339-49, http://dx.doi.org/10.1016/ j.spinee.2013.03.020.

Based on Cases With a Minimum of 10 Years of Follow-up. Spine. 2015; 40(14):E831-41, http://dx.doi.org/10.1097/BRS.0000000000000917. 39. Pellisé F, Hernández A, Vidal X, Minguell J, Martínez C, Villanueva C. Radiologic assessment of all unfused lumbar segments 7.5 years after instrumented posterior spinal fusion. Spine. 2007;32(5):574-9, http://dx. doi.org/10.1097/01.brs.0000256875.17765.e6. 40. Ekman P, Möller H, Shalabi A, Yu YX, Hedlund R. A prospective randomised study on the long-term effect of lumbar fusion on adjacent disc degeneration. Eur Spine J. 2009;18(8):1175-86, http://dx.doi.org/10.1007/ s00586-009-0947-3. 41. Mannion AF, Leivseth G, Brox JI, Fritzell P, Hägg O, Fairbank JC. ISSLS Prize winner: Long-term follow-up suggests spinal fusion is associated

617

CLINICAL SCIENCE

Effect of prophylactic non-invasive mechanical ventilation on functional capacity after heart valve replacement: a clinical trial Amaro Afraˆnio de Arau´jo-Filho,I,II,* Manoel Luiz de Cerqueira-Neto,III,IV Lucas de Assis Pereira Cacau,II Ge´ssica Uruga Oliveira,III Telma Cristina Fontes Cerqueira,IV Valter Joviniano de Santana-FilhoIII I Nucleo de Pos Graduacao em Ciencias da Saude, Universidade Federal de Sergipe, Aracaju, SE, BR. II Departamento de Fisioterapia, Universidade Tiradentes UNIT, Aracaju, SE, BR. III Departamento de Fisioterapia, Universidade Federal de Sergipe - UFS, Aracaju, SE, BR. IV Departamento de Fisioterapia, Universidade Federal de Sergipe - UFS, Lagarto, SE, BR.

OBJECTIVE: During cardiac surgery, several factors contribute to the development of postoperative pulmonary complications. Non-invasive ventilation is a promising therapeutic tool for improving the functionality of this type of patient. The aim of this study is to evaluate the functional capacity and length of stay of patients in a nosocomial intensive care unit who underwent prophylactic non-invasive ventilation after heart valve replacement. METHOD: The study was a controlled clinical trial, comprising 50 individuals of both sexes who were allocated by randomization into two groups with 25 patients in each group: the control group and experimental group. After surgery, the patients were transferred to the intensive care unit and then participated in standard physical therapy, which was provided to the experimental group after 3 applications of non-invasive ventilation within the first 26 hours after extubation. For non-invasive ventilation, the positive pressure was 10 cm H2O, with a duration of 1 hour. The evaluation was performed on the 7th postoperative day/discharge and included a 6-minute walk test. The intensive care unit and hospitalization times were monitored in both groups. Brazilian Registry of Clinical Trials (REBeC): RBR number 8bxdd3. RESULTS: Analysis of the 6-minute walk test showed that the control group walked an average distance of 264.34±76 meters and the experimental group walked an average distance of 334.07±71 meters (p=0.002). The intensive care unit and hospitalization times did not differ between the groups. CONCLUSION: Non-invasive ventilation as a therapeutic resource was effective toward improving functionality; however, non-invasive ventilation did not influence the intensive care unit or hospitalization times of the studied cardiac patients. KEYWORDS: Thoracic Surgery; Continuous Positive Airway Pressure; Walk Test. de Arau´jo-Filho AA, de Cerqueira-Neto ML, Cacau LA, Oliveira GU, Cerqueira TC, de Santana-Filho VJ. Effect of prophylactic non-invasive mechanical ventilation on functional capacity after heart valve replacement: a clinical trial. Clinics. 2017;72(10):618-623 Received for publication on April 4, 2017; First review completed on May 7, 2017; Accepted for publication on August 2, 2017 *Corresponding author. E-mail: amarofisio@yahoo.com.br

’ INTRODUCTION

capacity, and contribute to the appearance of atelectasis and to changes in the ventilation/perfusion ratio, which reduces the cardiorespiratory capacity and promotes physical inactivity, loss of muscle strength and loss of physical conditioning (4,5). Thus, cardiac rehabilitation plays an important role in the safe and effective treatment of these patients, reduces mortality, hospitalizations and hospital costs and improves symptoms and quality of life. A cardiac rehabilitation program should focus on improving the physiological state, functional ability and psychology of the patient and be based on a multidisciplinary approach (3-6). As an important component of this multidisciplinary approach, physical therapy is used to both prevent and treat complications by employing techniques and therapeutic procedures to restore a functional respiratory pattern and the physical independence of the patient, either in the hospital bed or on an ambulatory basis (4,7,8). Among the therapeutic resources used, non-invasive ventilation (NIV) has shown

Cardiac surgeries can cause a series of clinical and functional complications that are linked to specific factors, such as general anesthesia, median sternotomy, cardiopulmonary bypass and thoracic manipulation. Postoperative pulmonary complications are the most common complication type and, in turn, contribute directly to increased morbidity and mortality and longer hospital stays (1-3). These dysfunctions alter breathing patterns, which reduces the lung volume and

618

Non-invasive ventilation after heart valve replacement de Arau´jo-Filho et al.

CLINICS 2017;72(10):618-623

Brazilian Unified Health System (Sistema Único de Saúde – SUS) as a referral for the care of cardiovascular surgery in the state of Sergipe and in the cities of the states of Bahia and Alagoas.

satisfactory results. NIV has the main objective of providing ventilatory assistance to improve oxygenation and/or decrease CO2 retention, which reduces breathing efforts, anaerobic metabolism and the dyspnea index, avoiding endotracheal intubation and increasing residual volume and alveolar recruitment, thus preventing the presence of atelectasis and maintaining adequate gas exchange (4,9-12). NIV can increase physical performance in such patients by increasing oxygenation in the microcirculation of peripheral muscles, which reflects a better contractile performance of the heart muscle and subsequently provides better perfusion of peripheral tissues, including muscles; thus, exercise tolerance is increased (9,13-17). Given the aforementioned data describing the use of NIV during the cardiac postoperative period, information regarding the mechanisms of action of NIV in the functional capacity of these patients is lacking, and whether there are advantages to NIV use as a preventive therapy during the postoperative period is unknown. Thus, this study aimed to evaluate the functional capacity and length of stay of patients in the nosocomial intensive care unit (ICU) and the lengths of hospital stays of patients after heart valve replacement who had undergone three prophylactic one-hour applications of NIV in CPAP mode with a pressure level of 10 cm H2O. We hypothesized that this therapeutic resource would improve the functional performance of these patients.

Study Design The study was a randomized clinical trial. A quantitative approach was used to assess the use of non-invasive mechanical ventilation as a prophylactic therapeutic resource for the functional capacity of patients in a cardiac postoperative period. The sample consisted of adult patients of both sexes who were randomized into two groups (an experimental group and a control group, with 25 patients in each group) through an electronic system (http://randomizer.org/form.htm) as the patients underwent hospital admission to undergo heart valve replacement surgery. To guarantee concealment of the allocation list, the randomization was performed by an independent researcher (Figure 1).

Blinding Considering the type of our intervention, it was not possible to blind the patients and the investigators that performed the prophylactic CPAP. However, the investigators that assessed the outcomes were blinded.

Outcomes

’ MATERIALS AND METHODS

The primary outcome of this study was the maximum walked distance in the six-minute walking test (6MWT), and the secondary outcome was the length of the hospital stay. The walking test was assessed at hospital discharge. The length of stay was assessed at intensive care discharge and at hospital discharge.

Research Setting The study was conducted at the Cardiovascular ICU and Cardiology Office of Charitable Foundation of Surgery Hospital (Fundac¸ão de Beneficência Hospital de Cirurgia – FBHC) located in Aracaju-SE, which participates in the

Figure 1 - Algorithm for patient distribution (CONSORT). Source: research data.

619

Non-invasive ventilation after heart valve replacement de Arau´jo-Filho et al.

CLINICS 2017;72(10):618-623

Population

and adjusted with a PEEP of 10 cm of H2O (9,18). In addition to the NIV application, patients in the experimental group underwent the physical therapy protocol, which was performed by the hospital’s physiotherapy team based on the guidelines of the Brazilian Society of Cardiology, throughout their hospitalization. The control group did not undergo NIV therapy; these patients participated in respiratory and motor therapy only.

This study was carried out in a reference hospital for cardiac surgery of the Brazilian Public Health System (Sistema Único de Saúde - SUS, Fundac¸ão de Beneficência Hospital de Cirurgia - FBHC, Aracaju, Sergipe). The eligibility criteria were as follows: patients were admitted to the hospital for heart valve replacement surgery; comprised both genders; and were between 20 and 70 years old. The exclusion criteria were as follows: patients with motor disabilities that prevented walking and patients with contraindications to CPAP, such as a decreased level of consciousness, an ineffective cough, an airway obstruction, abdominal distention, vomiting, upper gastrointestinal bleeding, acute coronary syndrome, complex arrhythmias, facial trauma, esophageal surgery and an undrained barotrauma. Sources of lost tracking (discontinuity) included the following: early discharge from the hospital; need for reintubation; need for invasive mechanical ventilation more than 24 hours after surgery; non-invasive need for ventilation therapeutically for reversal of respiratory failure; surgical complications or reintervention; hemodynamic instability; death; and removal of free and informed consent. At any time in the study, patients from the control group who showed a need for invasive or non-invasive ventilatory support for dyspnea (i.e., increased respiratory rate 425 bpm, paradoxical pattern, use of accessory muscles and decrease in peripheral saturation oxygen) were removed from the study. Based on the eligibility criteria, 104 patients were included in this study, and 30 patients were removed after the exclusion criteria analysis. A total of 74 patients were randomized; 36 were allocated to the experimental group, and 38 were allocated to the control group. Of these patients, 11 from the experimental group and 13 from the control group were affected by the research discontinuation criteria; 50 patients remained for the final analysis of the present study and were distributed as follows: 25 in the experimental group, and 25 in the control group (Figure 1).

Instrument The 6MWT. The 6MWT is a simple test that is easy to perform, safe, inexpensive and consists of a patient walking 30 meters for 6 minutes in a corridor. To assess his or her tolerance to physical exertion, the test was performed with a brisk walk, but no running, and the patient was verbally encouraged during each minute of the test to complete the maximum distance. The Borg Scale was used before and after the 6 minutes to verify the patient’s subjective fatigue (19). To prepare for the test, checks were performed at zero, 3 and 6 minutes. At zero minutes, vital signs such as blood pressure (BP), heart rate (HR), respiratory rate (RR) and oxygen saturation (SpO2) were measured. At the third minute, only SpO2 and HR were measured because they needed to be checked while walking, without affecting the patient’s performance. At no time did the evaluator interrupt the patient, which was possible with use of a pulse oximeter. At the sixth minute, all variables were verified (BP, RR, HR and SpO2), and the patient’s fatigue level was evaluated immediately after termination (20,21).

Ethical Aspects Patients only participated in the study after providing either signed or fingerprint-marked informed consent directly or by proxy through a guardian (family and/or companion). The informed consent form consisted of a declaration informing the subject of the research, objectives, evaluation methods and procedures, risks and benefits. Each patient received an explanation regarding how the research could contribute positively to his or her recovery process and to clarify possible doubts. The patients were informed that they could withdraw from research participation whenever they wanted. The patients were also warned that, at the time of the NIV application, respiratory distress and fatigue may occur. The study was approved by the Ethics Committee on Human Research of Tiradentes University in accordance with the Declaration of Helsinki under Protocol number 021211R. The study was published in the Brazilian Registry of Clinical Trials (REBeC) with the RBR number 8bxdd3.

Sampling Technique Patients were randomized into two groups (the experimental group and the control group) using a randomization process produced by an electronic system: http://randomizer.org/form.htm. At the time of hospitalization, each patient underwent a preoperative assessment and an assessment protocol that consisted of patient identification, vital signs, clinical diagnosis, surgery type, medical history, medications and the preoperative ejection fraction (EF). Postoperative revaluation was performed on the 7th postoperative day (POD) or at discharge. The 6MWT was performed, and the operative times, extracorporeal circulation (ECC), ICU and hospitalization times, as well as the postoperative EFs of both groups, were assessed. After surgery, the patients were transferred to the ICU, where they were subjected to the interruption process of mechanical ventilation. The patients in the experimental group received three applications of NIV within the first 26 hours after extubation, and the first application was performed within the first 2 hours after the removal of the tube, maintaining an interval of 12 hours between the other applications. For therapy, a positive pressure device, ResMed S7 Lightweight in Continuous Positive pressure mode (CPAP), and an oronasal interface were used for one hour

Statistical Analysis Data were categorized in Microsoft Excel 2007s spreadsheet files, and for the statistical analysis, BioEstat 5.0 was used. The normality condition was assessed using the ShapiroWilk test. Student’s t-test was used to compare the parametric data, and the Mann-Whitney test was used for nonparametric samples. For comparisons of categorical variables, such as sex, a chi-squared test was used, and p values o0.05 were considered statistically significant. The study by Oliveira et al. (3) was used as the basis for the calculation of sample size. The distance walked in the

620

Non-invasive ventilation after heart valve replacement de Arau´jo-Filho et al.

CLINICS 2017;72(10):618-623

6MWT was considered the main parameter. Assuming that the effect size=50 meters, variability=50.66 meters, significance level=5%, and power test=95%, a minimum sample size of 19 patients for each group was determined.

’ RESULTS Table 1 shows the data for the characterization of the sample relating to the experimental group and control group, considering age, gender, pre-surgical EF of the left ventricle (LVEF) and ECC time. The results demonstrated that there was no statistically significant difference between the groups, even with the homogeneity of the patients included in the study. Table 1 shows the length of stay in the ICU and total hospital stay times of the experimental and control groups. Regarding the ICU times, the length of stay for the experimental group length was 3 days (2, 3), and the length of stay for the control group was 3 days (2, 3) (p=0.41). In relation to hospitalization times, the length of stay for the experimental group was 7 days (7, 9), and the length of stay for the control group was 8 days (6, 5, 8) (p=0.55). These data showed that there was no statistically significant difference between the groups. Figure 2 shows the 6MWT results. The experimental group (334.07±71 meters) covered a significantly greater distance than the control group (264.34±76 meters).

’ DISCUSSION In this study, we evaluated the functional capacities, lengths of stay in the ICU and lengths of stay in the hospital of the patients after heart valve replacement and three applications of prophylactic NIV within the first 26 hours after extubation. The patients in the experimental group walked 334.07 meters in the 6MWT, whereas the patients from the control group walked 264.34 meters, which represented a significant difference. As a secondary outcome, no difference was detected between the groups for length of stay in the hospital or in the ICU. These findings demonstrate that the use of CPAP in the immediate postoperative period in patients subjected to heart valve replacement improves functional activity, as evidenced by a longer distance walked in the 6MWT at hospital discharge. NIV has been extensively used in early-stage acute respiratory failure (ARF), and the success of treatment has been associated with the ARF type and severity, underlying disease, application time, treatment site, and the experience of the

Figure 2 - Comparison of the distance covered IN the 6MWT by the experimental group (EG) and control group (CG). A t-test was performed for the independent samples (*po0.05).

team (14,15,18,22-25). However, reports describing the prophylactic use of NIV are lacking. The study by Zarbock et al. (10) demonstrated that administration of NIV for an extended period of time after cardiac surgery improved arterial oxygenation, reduced the incidence of pulmonary complications, including pneumonia and the need for reintubation, and reduced the rate of readmission to the ICU, which may explain the superior performance in the walking test of the patients who underwent NIV in our study. Using a similar methodological design and population, Mazullo Filho et al. (26) carried out a randomized, controlled study with 32 patients during the immediate postoperative period after cardiac surgery. The experimental group used NIV for 2 hours after extubation. The authors found an average reduction in HR and a lower cardiac output, thus reducing the need for energy expenditure and respiratory effort, compared to the control group, which did not use NIV. With lower energy expenditure and lower RR than patients without NIV, the cardiopathy patient with NIV can exhibit better cardiopulmonary fitness and perform his or her activities in a more functional way, which is the primary outcome of our research. Functional capacity has been evaluated in research studies of patients with heart failure. The 6MWT is a useful tool for this purpose because the test is simple and cost-effective. The 6MWT can be used to reproduce daily activities, evaluate exercise tolerance, assess the degree of functional limitations, and enable prognostic stratification (27,28). Thus, the 6MWT was chosen as an evaluation tool in this study. Additionally, the 6MWT is sensitive for the detection of better exercise

Table 1 - Study participant characteristics. Control

42.44 ± 11

46.08 ± 14.23

0.31

16 (64.0) 9 (36.0) 3 [2, 3] 7 [7, 9] 56.84 ± 11.0 92.6 ± 41.9

11 (44.0) 14 (56.0) 3 [2, 3] 8 [6, 5, 8] 57.36 ± 12.1 81.65 ± 26.8

0.42 0.41 0.49 0.12 0.87 0.51

NIV Age Gender Male n (%) Female n (%) ICU* time (days) Hospitalization time (days) Preoperative LVEF** (%) ECC*** time (min.)

*ICU: Intensive care unit; **LVEF: left ventricular ejection fraction; ***ECC: extracorporeal circulation. Held Test t for parametric variables (age and LVEF) and Mann-Whitney test for nonparametric ones (ICU time, hospitalization time and ECC time) and Chi-squared test for the categorical variable (gender) (*po0.05).

621

Non-invasive ventilation after heart valve replacement de Arau´jo-Filho et al.

CLINICS 2017;72(10):618-623

When evaluating the secondary outcomes of our study, such as length of stay in the ICU and the total hospital stay of patients during the postoperative period after heart valve replacement surgery, no significant differences in these times were evident between the groups. Trevisan et al. (35), in a randomized clinical trial, and Landoni et al. (9) and Cabrini et al. (24), in systematic reviews, showed that the use of NIV when applied post-extubation does not interfere with the length of stay in the ICU or in the hospital. In our study, we found a slight, but not significant, reduction in the number of days. These data may be explained by the profiles of the studied patients, who had low EFs, hypoactivity, and important muscle strength deficits that were linked to heart failure, which are factors that the therapeutic tool cannot fully revert. This study was limited by the absence of a preoperative evaluation of the distance covered in a 6MWT because the patients were in a serious health state, debilitated and with preoperative restrictions. Another limitation was that the average time for application of CPAP was 60 minutes. This timeframe suggested the use of BPAP to promote greater ventilation comfort and thus apply NIV for a longer time and perhaps under higher pressure. Another limitation was the absence of variables to evaluate lung function so that we could correlate the improvement in functionality with the likely improvement in lung volume and capacity. Thus, we suggest further research to answer these questions and develop new therapeutic approaches in cardiac rehabilitation. As a prophylactic therapeutic approach, NIV has been confirmed to be effective in improving the functional capacity of patients during the postoperative period of heart valve replacement surgery, as measured by the distance covered in a 6MWT. However, NIV as a therapeutic resource did not affect the length of stay in the ICU or the length of the total hospital stay of the studied cardiac patients.

tolerance in patients who are subjected to CPAP during the postoperative stage of heart valve replacement. A meta-analysis by Bundchen et al. (16) revealed only three studies that used NIV after heart failure surgery. The primary outcome was the distance traveled during the 6MWT, and all of the studies showed positive results. Wittmer et al. (29) compared a program of breathing exercises associated with 30 minutes of CPAP (8 cm H2O) versus only breathing exercises for a period of 14 days. Chermont (30) compared 30 minutes of CPAP (4-6 cm H2O) versus placebo before the 6MWT, and Lima et al. (31) showed that the use of CPAP (10 cm H2O) for 30 minutes before the 6MWT increased the distance traveled compared to that of the control group without the use of CPAP. With the same primary outcome, our study used a different methodological design, comprising three one-hour CPAP applications (10 cm H2O) in the patient who was still hospitalized in the ICU, and obtained positive results, evidenced by an increase in the distance walked during the 6MWT carried out at discharge. This reinforces the idea of making CPAP a standard procedure in the immediate postoperative period of cardiac surgery. Functional capacity is limited in patients with cardiac disease, not only due to cardiac factors but also due to the interaction between the cardiopulmonary and musculoskeletal systems. Abnormal ventilatory responses are common in these patients, and combined with peripheral muscle dysfunction, these responses result in greater difficulty in performing the activities of daily life, especially in the cardiac postoperative period, which is the focus of our research. NIV can reduce respiratory effort and increase physical performance in such patients by increasing oxygenation in the microcirculation of peripheral muscles that can lead to increased intrathoracic pressure, reducing the transmural pressure of the left ventricle and consequently the pre- and post-load pressure. This process reflects better contractile performance of the heart muscle, providing better perfusion of peripheral tissues, including muscles, and thus causes an increase in exercise tolerance (9,14-17). This process has been demonstrated as our primary outcome when patients who received prophylactic CPAP walked longer distances than patients who did not receive prophylactic CPAP. According to Morales-Blanhir et al. (32), through a systematic review of the 6MWT, the average distance traveled by healthy people is 550 meters. Oliveira et al. (3) affirms that patients who perform the 6MWT during the cardiac postoperative stage have an average distance of 375 meters. A literature review by Zielinska et al. (33) shows that the 6MWT for cardiac patients is a reliable measure and a predictor of mortality, demonstrating that patients who travel a distance of fewer than 350 meters have a higher mortality rate. In a retrospective study using data from 330 surgical cardiac patients, with 97 of them undergoing heart valve replacement, La Rovere et al. (34) analyzed pre- and postsurgery 6MWT results and obtained values of 256 meters and 381 meters before and after heart valve replacement surgery, respectively. Our research showed that patients in the NIV group traveled 334.04 meters, while patients in the control group reached 264.34 meters. Our results are within the reference values of the literature; however, these values are very different from the values for healthy individuals, demonstrating the great functional loss of cardiac patients, highlighting the need to maintain cardiac rehabilitation (phase two) of these patients after hospital discharge.

’ ACKNOWLEDGMENTS We thank all patients who participated in this study.

’ AUTHOR CONTRIBUTIONS De Araújo-Filho AA conceived, designed, and executed the study and prepared the manuscript. De Cerqueira-Neto ML, Cacau LA, Cerqueira TC assisted in the design and critical review of the study. De Santana-Filho VJ supervised the study, conceived, designed and critically reviewed the manuscript. Oliveira GU participated in the design, analysis and interpretation of the data collected.

’ REFERENCES 1. Chiumello D, Chevallard G, Gregoretti C. Non-invasive ventilation in postoperative patients: a systematic review. Intensive Care Med. 2011; 37(6):918-29, http://dx.doi.org/10.1007/s00134-011-2210-8. 2. Ferreira LL, Souza NM, Vitor AL, Bernardo AF, Valenti VE, Vanderlei LC. Ventilação mecânica não-invasiva no pós-operatório de cirurgia cardíaca: atualização da literatura. Rev Bras Cir Cardiovasc. 2012;27(3):446-52, http://dx.doi.org/10.5935/1678-9741.20120074. 3. Oliveira EK, Silva VZM, Turquetto ALR. Relação do teste de caminhada pós-operatório e função pulmonar com o tempo de internação da cirurgia cardíaca. Rev Bras Cir Cardiovasc. 2009;24(4):478-84, http://dx.doi.org/ 10.1590/S0102-76382009000500008. 4. Cavenaghi S, Ferreira LL, Marino LH, Lamari NM. Fisioterapia respiratória no pré e pós-operatório de cirurgia de revascularização do miocárdio. Rev Bras Cir Cardiovasc. 2011;26(3):455-61, http://dx.doi.org/ 10.5935/1678-9741.20110022. 5. Herdy AH, López-Jimenez F, Terzic CP, Milani M, Stein R, Carvalho T, et al. Diretriz Sul-Americana de Prevenção e Reabilitação Cardiovascular. Arq Bras Cardiol. 2014;103(2Supl.1):1-31, http://dx.doi.org/10.5935/abc. 2014S003.

622

Non-invasive ventilation after heart valve replacement de Arau´jo-Filho et al.

CLINICS 2017;72(10):618-623

21. Oliveira GU, Oliveira Carvalho V, de Assis Cacau LP, de Araújo Filho AA, de Cerqueira Neto ML, da Silva WM Jr, et al. Determinants of distance walked during the six-minute walk test in patients undergoing cardiac surgery at hospital discharge. J Cardiothorac Surg. 2014;9:95, http://dx.doi.org/10.1186/1749-8090-9-95. 22. Lord S, Rochester L. Walking in the real world: concepts related to functional gait. New Zealand Journal of Physiotherapy. 2007;35(3):126-30. 23. Nava S, Navalesi P, Carlucci A. Non-invasive ventilation. Minerva Anestesiol. 2009;75(1-2):31-6. 24. Cabrini L, Plumari VP, Nobile L, Olper L, Pasin L, Bocchino S, et al. Noninvasive ventilation in cardiac surgery: a concise review. Heart Lung Vessel. 2013;5(3):137-41. 25. Preisig A, Lagni VB, Almeida VL, Vieira FN, Lucio EA, Santos LJ, et al. Ventilac¸ão não Invasiva após Cirurgia Cardiovascular: um Ensaio Clínico Randomizado. Rev Bras Cardiol. 2014;27(1):539-48. 26. Mazullo Filho JB, Bonfim VJ, Aquim EE. Ventilac¸ão mecânica não invasiva no pós-operatório imediato de cirurgia cardíaca. Rev Bras Ter Intensiva. 2010;22(4):363-8, http://dx.doi.org/10.1590/S0103-507X2010000400009. 27. Rubim VS, Drumond Neto C, Romeo JL, Montera MW. Valor prognóstico do teste de caminhada de seis minutos na insuficiência cardíaca. Arq Bras Cardiol. 2006;86(2):120-5, http://dx.doi.org/10.1590/S0066-782X200 6000200007. 28. Guimarães GV, Carvalho VO. Bocchi E A Reproducibility of the selfcontrolled six-minute walking test in heart failure patients. Clinics. 2008;63(2):201-6, http://dx.doi.org/10.1590/S1807-59322008000200008. 29. Wittmer VL, Simoes GM, Sogame LC, Vasquez EC. Effects of continuous positive airway pressure on pulmonary function and exercise tolerance in patients with congestive heart failure. Chest. 2006;130(1):157-63, http:// dx.doi.org/10.1378/chest.130.1.157. 30. Chermont S, Quintão MM, Mesquita ET, Rocha NN, Nóbrega AC. Noninvasive ventilation with continuous positive airway pressure acutely improves 6-minute walk distance in chronic heart failure. J Cardiopulm Rehabil Prev. 2009;29(1):44-8, http://dx.doi.org/10.1097/HCR.0b013e318 1927858. 31. Lima Eda S, Cruz CG, Santos FC, Gomes-Neto M, Bittencourt HS, Reis FJ, et al. Suporte ventilatório na capacidade funcional de pacientes com insuficiência cardíaca: estudo piloto. Arq Bras Cardiol. 2011;96(3):227-32, http://dx.doi.org/10.1590/S0066-782X2011005000002. 32. Morales-Blanhir JE, Palafox Vidal CD, Rosas Romero Mde J, García Castro MM, Londoño Villegas A, Zamboni M. Six-minute walk test: a valuable tool for assessing pulmonary impairment. J Bras Pneumol. 2011;37(1): 110-7, http://dx.doi.org/10.1590/S1806-37132011000100016. 33. Zielinska D, Bellwon J, Rynkiewicz A, Elkady MA. Prognostic value of the six-minute walk test in heart failure patients undergoing cardiac surgery: a literature review. Rehabil Res Pract. 2013;2013:965494, http://dx.doi. org/10.1155/2013/965494. 34. La Rovere MT, Pinna GD, Maestri R, Olmetti F, Paganini V, Ricardi G, et al. The 6-minute walking test and all-cause mortality in patients undergoing a post-cardiac surgery rehabilitation program. Eur J Prev Cardiol. 2015;22(1):20-6, http://dx.doi.org/10.1177/2047487313502405. 35. Trevisan CE, Vieira SR; Research Group in Mechanical Ventilation Weaning. Noninvasive mechanical ventilation may be useful in treating patients who fail weaning from invasive mechanical ventilation: a randomized clinical trial. Crit Care. 2008;12(2):R51, http://dx.doi.org/ 10.1186/cc6870

6. Morais DB, Lopes AC, Sá VM, Junior WM, Neto ML. Avaliação do Desempenho Funcional em Pacientes Submetidos à Cirurgia Cardíaca. Rev Bras Cardiol. 2010;23(5):263-9. 7. Arcêncio L, Souza MD, Bortolin BS, Fernandes AC, Rodrigues AJ, Evora PR. Cuidados pré e pós-operatórios em cirurgia cardiotorácica: uma abordagem fisioterapêutica. Rev Bras Cir Cardiovasc. 2008;23(3):400-10, http://dx.doi.org/10.1590/S0102-76382008000300019. 8. Renault JA, Costa-Val R, Rossetti MB. Respiratory physiotherapy in the pulmonary dysfunction after cardiac surgery. Rev Bras Cir Cardiovasc. 2008;23(4):562-9, http://dx.doi.org/10.1590/S0102-76382008000400018. 9. Landoni G, Zangrillo A, Cabrini L. Noninvasive ventilation after cardiac and thoracic surgery in adult patients: a review. J Cardiothorac Vasc Anesth. 2012;26(5):917-22, http://dx.doi.org/10.1053/j.jvca.2011.06.003. 10. Rahal L, Garrido AG, Cruz Jr RJ. Ventilação Não Invasiva: quando utilizar? Rev Assoc Med Bras. 2005;51(5):241-55, http://dx.doi.org/10.1590/ S0104-42302005000500007. 11. Zarbock A, Mueller E, Netzer S, Gabriel A, Feindt P, Kindgen-Milles D. Prophylactic nasal continuous positive airway pressure following cardiac surgery protects from postoperative pulmonary complications: a prospective, randomized, controlled trial in 500 patients. Chest. 2009;135(5): 1252-9, http://dx.doi.org/10.1378/chest.08-1602. 12. Lopes CR, Brandão CM, Nozawa E, Auler JO Jr. Benefits of non-invasive ventilation after extubation in the postoperative period of heart surgery. Rev Bras Cir Cardiovasc. 2008;23(3):344-50, http://dx.doi.org/10.1590/ S0102-76382008000300010. 13. Reis HV, Borghi-Silva A, Catai AM, Reis MS. Impact of CPAP on physical exercise tolerance and sympathetic-vagal balance in patients with chronic heart failure. Braz J Phys Ther. 2014;18(3):218-27, http://dx.doi.org/ 10.1590/bjpt-rbf.2014.0037. 14. Cabrini L, Nobile L, Plumari VP, Landoni G, Borghi G, Mucchetti M, et al. Intraoperative prophylactic and therapeutic non-invasive ventilation: a systematic review. Br J Anaesth. 2014;112(4):638-47, http://dx.doi.org/ 10.1093/bja/aet465. 15. Esquinas AM, Jover JL, Úbeda A, Belda FJ. Non-invasive mechanical ventilation in postoperative patients. A clinical review. Rev Esp Anestesiol Reanim. 2015;62(9);512-22. 16. Bündchen DC, Gonzáles AI, Noronha MD, Brüggemann AK, Sties SW, Carvalho TD. Noninvasive ventilation and exercise tolerance in heart failure: A systematic review and meta-analysis. Braz J Phys Ther. 2014; 18(5):385-94, http://dx.doi.org/10.1590/bjpt-rbf.2014.0039. 17. Franco AM, Torres FC, Simon IS, Morales D, Rodrigues AJ. Avaliação da ventilação não-invasiva com dois níveis de pressão positiva nas vias aéreas após cirurgia cardíaca. Rev Bras Cir Cardiovasc. 2011;26(4):582-90, http://dx.doi.org/10.5935/1678-9741.20110048. 18. Cabrini L, Zangrillo A, Landoni G. Preventive and therapeutic noninvasive ventilation in cardiovascular surgery. Curr Opin Anesthesiol. 2015; 28(1):67–72, http://dx.doi.org/10.1097/ACO.0000000000000148. 19. ATS Committee on Proficiency Standards for Clinical Pulmonary Function Laboratories. ATS statement: guidelines for the six minute walk test. Am J Respir Crit Care Med. 2002;166(1):111-7, http://dx.doi.org/10.1164/ ajrccm.166.1.at1102. 20. Fumagalli E, Ribeiro MA, Ferreira MS, Santos CI. Utilização do teste de caminhada de 6 minutos no manejo da hipertensão pulmonar. Arq Bras Cardiol. 2010;95(1):e10-3, http://dx.doi.org/10.1590/S0066-782X2010001 100024.

623

BASIC RESEARCH

Local and systemic effects of fibrin and cyanoacrylate adhesives on lung lesions in rabbits Marcus V.H. Carvalho,I,* Evaldo Marchi,I,III Andre J. Fruchi,I Bruno V.B. Dias,I Clovis L. Pinto,II Geovane R. dos Santos,II Milena M.P. AcencioIII I Departamento de Cirurgia Toracica, Faculdade de Medicina de Jundiai, Jundiai, SP, BR. II Departamento de Patologia, Faculdade de Medicina de Jundiai, Jundiai, SP, BR. III Laboratorio de Pleura, Divisao Pulmonar, Instituto do Coracao (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVES: Tissue adhesives can be used to prevent pulmonary air leaks, which frequently occur after lung interventions. The objective of this study is to evaluate local and systemic effects of fibrin and cyanoacrylate tissue adhesives on lung lesions in rabbits. METHODS: Eighteen rabbits were submitted to videothoracoscopy + lung incision alone (control) or videothoracoscopy + lung incision + local application of fibrin or cyanoacrylate adhesive. Blood samples were collected and assessed for leukocyte, neutrophil and lymphocyte counts and interleukin-8 levels preoperatively and at 48 hours and 28 days post-operatively. After 28 days, the animals were euthanized for gross examination of the lung surface, and lung fragments were excised for histopathological analysis. RESULTS: Fibrin and cyanoacrylate produced similar adhesion scores of the lung to the parietal pleura. Microscopic analysis revealed uniform low-cellular tissue infiltration in the fibrin group and an intense tissue reaction characterized by dense inflammatory infiltration of granulocytes, giant cells and necrosis in the cyanoacrylate group. No changes were detected in the leukocyte, neutrophil or lymphocyte count at any timepoint, while the interleukin-8 levels were increased in the fibrin and cyanoacrylate groups after 48 hours compared with the pre-operative control levels (po0.01). CONCLUSION: Both adhesive agents promoted normal tissue healing, with a more pronounced local inflammatory reaction observed for cyanoacrylate. Among the serum markers of inflammation, only the interleukin-8 levels changed post-operatively, increasing after 48 hours and decreasing after 28 days to levels similar to those of the control group in both the fibrin and cyanoacrylate groups. KEYWORDS: Thoracic Surgery; Cyanoacrylate; Fibrin; Tissue Adhesives; Systemic Effects. Carvalho MV, Marchi E, Fruchi AJ, Dias BV, Pinto CL, dos Santos Gr, Acencio MM. Local and systemic effects of fibrin and cyanoacrylate adhesives on lung lesions in rabbits. Clinics. 2017;72(10):624-628 Received for publication on May 31, 2017; First review completed on July 7, 2017; Accepted for publication on July 18, 2017 *Corresponding author. E-mail: marcus.carvalho@sbccv.org.br

’ INTRODUCTION

it is considered highly reactive to tissues and does not degrade over time (7). To our knowledge, no literature addresses the systemic effects of tissue adhesives applied to lung lesions. This study aims to evaluate the local and systemic effects of the application of FB and CA tissue adhesives in an experimental model using videothoracoscopy (VATS) in rabbits with lung lesions.

Prolonged air leak (PAL) after lung resections is a frequent and challenging complication, increasing postoperative morbidity and the duration of hospital stays and resulting in high costs to the health system (1,2). When suturing and stapling fail to prevent air leaks, topical sealants may be considered (3-6). Fibrin (FB) sealants have been used for more than a decade in lung resections, but their effectiveness is debated (3,4). In contrast, cyanoacrylate (CA) has been demonstrated to have a strong capacity for bonding tissues together (5,6); however,

’ MATERIALS AND METHODS This study was performed in accordance with human and animal care statements and Brazilian National Research Council guidelines for animal care, and it was approved by the Ethical Committee for Animal Research of Jundiai Medical School. Eighteen adult male New Zealand rabbits weighing 2.5 to 3.0 kg were randomized into three groups of six rabbits each. Each rabbit was anesthetized with 35 mg/kg of ketamine hydrochloride (Cristalia, Itapira, SP, Brazil) and 5 mg/kg of Xilazine (Bayer, Sao Paulo, SP, Brazil) that was infused through a catheter placed into the auricular vein.

624

Local and systemic effects of tissue adhesives Carvalho MVH et al.

CLINICS 2017;72(10):624-628

method, the thin fibers (immature collagen, typed III) stain green, while the thick fibers (mature collagen, type I) stain orange-red. The same pathologist analyzed the samples in a blinded fashion.

The animals were then positioned in the left lateral decubitus position, and their right chest areas were shaved and cleansed with iodopovidone (Rioquimica, ́ S.J. Rio Preto, SP, Brazil). A mask adapted to each rabbits muzzle provided continuous oxygen during the entire experiment. Each rabbit underwent a videothoracoscopy procedure involving a lateral 2 cm skin incision in the right thorax, followed by muscle division and the intercostal insertion of a 5 mm trocar (Thoracoport, Medline, IL, USA) for accessing the right pleural cavity. Another 2 cm medial incision was made to introduce the second port for the lung incision procedure. The treatment groups were as follows: a control group (VATS + lung incision without treatment), an FB group (VATS + lung incision + FB adhesive application - Evicels; Omrix Biopharmaceuticals, Ethicon, Israel) and a CA group (VATS + lung incision + CA adhesive application Omnexs; Omrix Biopharmaceuticals, Ethicon, Israel). All lung incisions were 1 cm in length and performed in the middle lobe. The amount of sealant applied to each lung incision was 1 mL. Before the trocars were withdrawn, the residual air in the pleural space was completely aspirated, and the skin was sutured with 4-0 monofilament nylon. During the experiment, the animals were carefully monitored using pulse oximetry and electrocardiography. The same surgeon and surgical team performed all surgical interventions.

’ RESULTS All procedures were well tolerated, and all animals were stable with respect to hemodynamics and oxygen saturation during the interventions. There were no deaths at any point of the study in the groups, including the control group, which received a lung lesion without any treatment.

Macroscopic Findings No difference in the medium scores of pleural adhesions was observed in the control, FB or CA group. However, the CA group produced slightly more pleural adhesions than did the control group (p=0.051). The macroscopic examination at 28 days post-operatively revealed that no residual FB adhesive remained on the lung surface and that the operated area was whitish, with no texture modification. In contrast, at 28 days postoperatively in the CA group, a hard consistency of the lung parenchyma was observed, and residual adhesive was present over an area of approximately 0.5 cm around the application site (Figure 1).

Euthanasia and Macroscopic Analysis

Histological Analysis

On the 28th postoperative day, the animals were anesthetized and euthanized with a lethal dose of pentobarbital. Each thorax was removed en bloc, and 60 mL of 10% formalin was infused through the trachea to maintain lung expansion. The thorax was maintained in the formalin solution for 48 hours and subsequently opened by a longitudinal sternal incision to access both pleural cavities for macroscopic examination. Adhesions between the visceral and parietal pleura were scored (8) based on a 1-4 grade scale, as follows: 1 - no adhesions; 2 - fewer than three localized adhesions; 3 - more than three localized adhesions, and 4 - complete obliteration of the pleural cavity.

For the histological analysis, the FB group presented with locally rich neo-angiogenesis and a mix of inflammatory infiltration and edema, similar to that observed in the normal phases of scar formation (Figure 1a). In contrast, in the CA group, a dense lymph-mononuclear inflammatory infiltration with lymphoid aggregates was observed, permeated by cavitation areas. Covering the cavitation areas, a histiocytic ‘in palisade’ infiltration was observed, as were some multinucleated giant cells with cytoplasmic vacuolization and residues of phagocytosed CA adhesive (Figure 1b and 2a). The interior of the cavities presented with an amorphous content and necrosis. In addition, the perilesional stroma showed edema with minimum deposition of collagen fibers (Figure 2b).

Leukocyte, Neutrophil and Cytokine Measurements Blood samples were collected preoperatively and 48 hours and 28 days after surgery to evaluate the systemic effects of the interventions. Total blood leukocytes and neutrophils were measured by an automated hematological analyzer that included a specific module for rabbit blood analysis (ADVIA, Bayer, Germany). Blood samples were centrifuged at 1,000 rpm for 10 minutes at 4o C. The supernatant was removed and stored at -80o C for later quantification of the cytokine interleukin-8 (IL-8) (Pharmingen, San Diego, CA); quantification was performed by ELISA using a 450 nm Power Wave filter (Bio-Tek, Winooski, VT, USA). The minimum detection level for IL-8 was 15.6 pg/mL.

Systemic Effects There was no significant variation in the total leukocyte, neutrophil or lymphocyte count in the pre-operative phases or 48 hours or 28 days post-operatively in any group (Table 1). Serum IL-8 levels were significantly elevated after 48 hours in both the FB and CA groups compared with those in the control group (po0.01). However, at 28 days postoperatively, the IL-8 levels in both groups had decreased to levels comparable to those of the control group.

’ DISCUSSION

Histopathological Analysis

The incidence of intra-operative air leak may be as high as 60 to 70% in patients after lung resection (9-11), and it can be prolonged in approximately 8 to 15% of patients (12). The incidence of PAL depends on several factors, such as the presence of damaged or fragile lung parenchyma (13), the use of steroids (12-14), whether upper lobectomy is performed (15), and the presence of pleural adhesions (16). Thus, PAL remains a frequent and challenging complication.

For microscopic analysis, lung tissue from the operated area was sectioned, fixed in 10% buffered formalin and embedded in paraffin. The 3-mm-thick sections were processed and stained with hematoxylin-eosin (HE) for microscopic analysis. To identify collagen fibers in the visceral pleural samples, the slides were stained with 2% Sirius red solution dissolved in aqueous saturated picric acid. By this

625

Local and systemic effects of tissue adhesives Carvalho MVH et al.

CLINICS 2017;72(10):624-628

Figure 1 - Photomicroscopy image of rabbit lung parenchyma stained by HE (X100 magnification). The image in a (FB group) shows intense neoangiogenesis and inflammatory cell infiltration, which are findings compatible with normal wound healing. The image in b (CA group) shows a palisade of histiocytic cells and lymphocytes (white arrow) and a central area of necrosis (black arrow).

Figure 2 - Photomicroscopy image of rabbit lung parenchyma from the CA group. The image in a shows a multinucleated giant cell (arrow) with cytoplasmic vacuoles containing CA residues with HE staining (X400 magnification) (A). The image in b shows an absence of collagen deposits and accentuated stromal edema with picrosirius red staining (X100 magnification).

Table 1 - Values (median and quartiles [25-75%]) of total leukocyte, neutrophil, and lymphocyte counts (cells/mm3) and IL-8 levels (pg/mL) at 48 hours and 28 days in the control, fibrin and cyanoacrylate groups. Control Leukocytes Neutrophils Lymphocytes IL-8

48 28 48 28 48 28 48 28

h d h d h d h d

8,800 8,100 4,168 3,413 3,868 4,087 38.6 69.1

(7,300 – 10,500) (6,100 – 9,400) (1,339 – 6,997) (2,040 – 4,050) (3,504 – 4,800) (3,888-4,753) (38.5-49.6) (32.3-72.9)

Fibrin 9,100 7,400 3,491 3,300 4,940 3,784 75.3 78.4

(7,800 – 9,500) (7,000 – 8,800) (2,380 – 4,602) (2,170-4,361) (3,666 – 5,096) (3,774-4,183) (67.3-86.0) (49.8-80.3)

Cyanoacrylate 6,800 6,850 3,664 3,146 5.126 3,070 72.2 67.6

(6,300 – 8,200) (6,700-7,100) (1,599 – 4,775) (1,800 – 3,645) (4.284 – 6.120) (3,932-4,761) (64.5-83.5) (28.3-79.1)

p value 0.138 0.214 0.745 0.846 0.085 0.450 o0.010 0.297

There was a significant difference in the IL-8 level between the control and treatment groups at 48 hours but not at 28 days (po0.010). No difference was found in the leukocyte, neutrophil or lymphocyte count.

also raises concerns, specifically regarding biocompatibility and local toxicity (20). The FB tissue adhesive consists of a combination of human plasma fibrinogen, thrombin and an anti-fibrinolytic product. In the presence of thrombin, fibrinogen is converted into a fibrin clot. FB glue has been the most widely used tissue adhesive in thoracic surgery over the past two decades. However, interest in the product has decreased

To help prevent postoperative lung air leakage, tissue sealants/adhesives can be used. FB is the most common adhesive agent used in surgical practice. As a biological substance, FB allows adequate tissue healing, but concerns about its use have been raised, including its unclear efficiency (17,18) and the possibility of allergic reactions to its proteins (19). In contrast, CA is a synthetic product with known efficiency, although its use

626

Local and systemic effects of tissue adhesives Carvalho MVH et al.

CLINICS 2017;72(10):624-628

present study did not find an increase in blood leukocyte, neutrophil or lymphocyte count following application. Montanaro L et al. (34) found no significant variation in the numbers of total or differential leukocyte counts when CA glue was used. In our study, although the blood cellular profile did not vary among groups, the serum levels of IL-8 increased 48 hours after the application of FB or CA in lung lesions. These results show that the local inflammatory reaction may produce systemic repercussions when more sensitive markers are used than when cellular counts alone are used. The IL-8 levels increased in a similar manner for the two adhesive agents, and the levels of this cytokine decreased at day 28 to levels comparable to those of the control group. In conclusion, either FB or CA applied to the lung lesions of rabbits promoted effective local healing. CA promoted a pronounced local inflammatory response with cell infiltration and the appearance of foreign body giant cells, and FB promoted normal tissue healing. The only systemic effect observed for both agents was an elevation of serum IL-8 levels at 48 hours; these levels had decreased at 28 days after the procedure. These findings may be relevant to surgeons who need to use adhesives as adjuvants to seal complex injuries. In addition, the current study may decrease the concern regarding the potential undesirable adverse effects of CA that have been reported by previous studies.

since randomized trials conducted by Fleisher (17) and Wong (21). In these trials, the use of FB in comparison with the control revealed no differences in outcome, number of postoperative air leaks, chest drain duration or length of hospital stay. In a recent systematic review addressing the application of surgical sealants, primarily FB adhesives, after pulmonary resection, the authors concluded that there are decreases in postoperative air leaks and time to chest drain removal and no reduction in the length of postoperative hospital stay (22). One of the most important benefits of fibrin sealants is their low inflammation and rapid degradation compared with those of other sealants (23). Although some studies have shown that FB sealants allow a superior aerostasis in patients undergoing pulmonary resection (24), other studies have reported a lower resistance bonding property of FB sealants compared with that of CA glue (25). CA adhesive has shown effectiveness in preventing air leaks, but its use is associated with intense local reactions and no signs of degradation over time (26,27). The current study found an intense local reaction to CA applied to lung lesions in rabbits; however, no systemic repercussion, as measured by the response of leukocytes and neutrophils, was observed. Nevertheless, both FB and CA produced an acute elevation of serum IL-8 levels 48 hours after application. To our knowledge, no previous studies have addressed the systemic effects of CA and FB sealants/adhesives applied to lung lesions. Concerning the local reactions to adhesives, the findings of the present study are similar to those of previous reports (23,28). The reactions mainly include a pronounced local inflammatory response to CA, with the infiltration of granulocytes, lymphocytes and foreign body giant cells, and minor signs of degradation over time. However, when FB sealant was used, no residual adhesive was found and microscopy analysis revealed adequate tissue healing. CA derivatives have low viscosity, dry rapidly, gain adhesion rapidly and, most importantly, have strong adhesive capability. Among several available commercial formulations, Omnexs represents a long-chain derivative of octylcyanoacrylate that polymerizes slowly and gradually releases heat to the injured tissue. In theory, newer formulations of CA are less toxic; however, they are not completely free of the adverse inflammatory properties of previous products. The findings of our study show that CA shows persistent adherence to lung tissue, and after drying, it polymerizes and becomes a very stable and inert compound. A rodent model of CA injection showed that this polymer persisted inside the abdominal cavity up to 12 months after local application (27). Although CA does not appear to degrade over time, local toxicity does not appear to be a concern. In a study using electron microscopy, CA showed no inhibition of tissue regeneration and no histotoxicity (29). Another study with nanoparticles composed of CA revealed that the substance was stable and showed no toxicity (30). In addition, amnion cell cultures from fetal membranes showed no toxicity after exposure to CA (31). In another in vitro study using mouse fibroblasts, it was found that when a large number of cells was used, cell proliferation overcame the cytotoxic effect of CA (32). Another study with cardiomyoblasts using an extract dilution assessment showed that CA had no toxic effect on the cells (33). Regarding the possible systemic inflammatory reaction related to the application of FB or CA to lung lesions, the

’ ACKNOWLEDGMENTS We thank Maria L. de Oliveira, Technician, Surgical Laboratory, Medical College of Jundiai, for helping with the surgical procedures and for handling animal care. Financial support was provided by Foundation to Support Research from the State of Sao Paulo, Grant 12/21192-2.

’ AUTHOR CONTRIBUTIONS Carvalho MV and Marchi E conceived and designed the study, they were responsible for the animal procedures, the acquisition and analysis of the data, and the drafting and revision of the manuscript. Fruchi AJ collaborated in all phases of the study. Dias BV, Pinto CL, dos Santos GR and Acencio MM analyzed and interpreted the samples and helped in writing the manuscript.

’ REFERENCES 1. Singhal S, Ferraris VA, Bridges CR, Clough ER, Mitchell JD, Fernando HC, et al. Management of alveolar air leaks after pulmonary resection. Ann Thorac Surg. 2010;89(4):1327-35, http://dx.doi.org/10.1016/j.athoracsur. 2009.09.020. 2. Varela G, Jimenez MF, Novoa N, Aranda JL. Estimating hospital costs attributable to prolonged air leak in pulmonary lobectomy. Eur J Cardiothorac Surg. 2005; 27(2):329-33, http://dx.doi.org/10.1016/j.ejcts.2004. 11.005. 3. Tambiah J, Rawlins R, Robb D, Treasure T. Can tissue adhesives and glues significantly reduce the incidence and length of postoperative air leaks in patients having lung resections? Interact Cardiovasc Thorac Surg. 2007;6(4):529-33, http://dx.doi.org/10.1510/icvts.2007.153080. 4. Pedersen TB, Honge JL, Pilegaard HK, Hasenkan JM. Comparative study of lung sealants in a porcine ex vivo model. Ann Thorac Surg. 2012;94(1): 234-40, http://dx.doi.org/10.1016/j.athoracsur.2012.03.050. 5. Kosar A, Kapicibaci HO, Alpay AL, Misirlioglu AK, Sonmez H, Iskender I, et al. The experimental use of N-butyl cyanoacrylate tissue adhesive in pulmonary wedge resections. Heart Lung Circ. 2012;21(11):711-4, http://dx.doi.org/10.1016/j.hlc.2012.06.020. 6. Cagirici U, Cetin Y, Cakan A, Samancilar O, Veral A, Askar FZ. Experimental use of N-butyl cyanoacrylate tissue adhesive on lung parenchyma after pulmonary resection. Thorac Cardiovasc Surg. 2007;55(3):180-1, http://dx.doi.org/10.1055/s-2006-924579. 7. Januchowski R, Ferguson WJ. The clinical use of tissue adhesives: a review of the literature. Ostheopathic Family Physician. 2014;6(2):25-9.

627

Local and systemic effects of tissue adhesives Carvalho MVH et al.

CLINICS 2017;72(10):624-628

23. Petter-Puchner AH, Simunek M, Redl H, Puchner KU, Van Griensven M. A comparison of a cyanoacrylate [corrected] glue (Glubran) vs. fibrin sealant (Tisseel) in experimental models of partial pulmonary resection and lung incision [corrected] in rabbits. J Invest Surg. 2010;23(1):40-7, http://dx.doi.org/10.3109/08941930903469383. 24. Belboul A, Dernevik L, Aljassim O, Skrbic B, Radberg G, Roberts D. The effect of autologous fibrin sealant (Vivostat) on morbidity after pulmonary lobectomy: a prospective randomised, blinded study. Eur J Cardiothorac Surg. 2004;26(6):1187-91, http://dx.doi.org/10.1016/j.ejcts.2004. 08.009. 25. Kull S, Martinelli I, Briganti E, Losi P, Spiller D, Tonlorenzi S, et al. Glubran 2 surgical glue: in vitro evaluation of adhesive and mechanical properties. J Surg Res. 2009;157(1):e15-21, http://dx.doi.org/10.1016/ j.jss.2009.01.034. 26. Miyano G, Yamataka A, Kato Y, Tei E, Lane GJ, Kobayashi H, et al. Laparoscopic injection of dermabond tissue adhesive for the repair of inguinal hernia: short- and long-term follow-up. J Pediatr Surg. 2004; 39(12):1867-70, http://dx.doi.org/10.1016/j.jpedsurg.2004.08.018. 27. Carr JA. The intracorporeal use of 2-octyl cyanoacrylate resin to control air leaks after lung resection. J Cardiothorac Surg. 2011;39(4):579-83, http://dx.doi.org/10.1016/j.ejcts.2010.07.036. 28. Kaplan M, Bozkurt S, Kut MS, Kullu S, Demirtas MM. Histopathological effects of ethyl 2-cyanoacrylate tissue adhesive following surgical application: an experimental study. Eur J Cardiothorac Surg. 2004;25(2):167-72, http://dx.doi.org/10.1016/j.ejcts.2003.11.016. 29. Nitsch A, Pabyk A, Honig JF, Verheggen R, Merten HA. Cellular, histomorphologic, and clinical characteristics of a new octyl-2-cyanoacrylate skin adhesive. Aesthetic Plast Surg. 2005;29(1):53-8, http://dx.doi.org/ 10.1007/s00266-004-0096-3. 30. Huang CY, Lee YD. Core-shell type of nanoparticles composed of poly[(nbutyl cyanoacrylate)-co-(2-octyl cyanoacrylate)] copolymers for drug delivery application: synthesis, characterization and in vitro degradation. Int J Pharm. 2006;325(1-2):132-9, http://dx.doi.org/10.1016/j.ijpharm. 2006.06.008. 31. Bilic G, Brubaker C, Messersmith PB, Mallik AS, Quinn TM, Haller C, et al. Injectable candidate sealants for fetal membrane repair: bonding and toxicity in vitro. Am J Obstet Gynecol. 2010;202(1):85.e1-9, http://dx.doi. org/10.1016/j.ajog.2009.07.051. 32. Kaplan M, Baysal K. In vitro toxicity test of ethyl 2-cyanaocrylate, a tissue adhesive used in cardiovascular surgery, by fibroblast cell culture method. Heart Surg Forum. 2005;8(3):E169-72, http://dx.doi.org/10.1532/HSF98. 20041126. 33. Abtahi S, Behravan J, Ehtesham Gharaee M, Aghasizadeh Sharbaf M, Vafaeey Z, Afsharnezhad S. Evaluation of the cytotoxic effect of tissue glue (octyl 2-cyanaoacrylate) on H9C2 cardiomyoblast cells using extract dilution assay. Razavi Int J Med 2015;3(4):e22411, http://dx.doi.org/ 10.17795/rijm22411. 34. Montanaro L, Arciola CR, Cenni E, Ciapetti G, Savioli F, Filippini F, et al. Cytotoxicity, blood compatibility and antimicrobial activity of two cyanoacrylate glues for surgical use. Biomaterials. 2001;22(1):59-66, http://dx.doi.org/10.1016/S0142-9612(00)00163-0.

8. Marchi E, Vargas FS, Acencio MM, Antonangelo L, Teixeira LR, Genofre EH, et al. Talc and silver nitrate induce systemic inflammatory effects during the acute phase of experimental pleurodesis in rabbits. Chest. 2004;125(6):2268-77, http://dx.doi.org/10.1378/chest.125.6.2268. 9. Wain JC, Kaiser LR, Johnstone DW, Yang SC, Wright CD, Friedberg JS, et al. Trial of a novel synthetic sealant in preventing air leaks after lung resection. Ann Thorac Surg. 2001;71(5):1623-8, http://dx.doi.org/ 10.1016/S0003-4975(01)02537-1. 10. Alphonso N, Tan C, Utley M, Cameron R, Dussek J, Lan-Lazdunski L, et al. A prospective randomized controlled trial of suction versus nonsuction to the under-water seal drains following lung resection. Eur J Cardiothorac Surg. 2005;27(3):391-4, http://dx.doi.org/10.1016/j.ejcts. 2004.12.004. 11. Mueller MR, Marzluf BA. The anticipation and management of air leaks and residual spaces post lung resection. J Thorac Dis. 2014;6(3):271-84, http://dx.doi.org/10.3978/j.issn.2072-1439.2013.11.29. 12. Cerfolio RJ, Bass CS, Pask AH, Katholi CR. Predictors and treatment of persistent air leaks. Ann Thorac Surg. 2002;73(6):1727-30, http://dx.doi. org/10.1016/S0003-4975(02)03531-2. 13. Linden PA, Bueno R, Colson YL, Jaklitsch MT, Lukanich J, Mentzer S, et al. Lung resection in patients with preoperative FEV1 o 35% predicted. Chest. 2005;127(6):1984-90, http://dx.doi.org/10.1378/chest.127.6.1984. 14. Cerfolio RJ. Chest tube management after pulmonary resection. Chest Surg Clin N Am. 2002;12(3):507-27, http://dx.doi.org/10.1016/S10523359(02)00015-7. 15. Abolhoda A, Liu D, Brooks A, Burt M. Prolonged air leak following radical upper lobectomy: an analysis of incidence and possible risk factors. Chest. 1998;113(6):1507-10, http://dx.doi.org/10.1378/chest.113. 6.1507. 16. Brunelli A, Monteverde M, Borri A, Salati M, Marasco RD, Fianchini A. Predictors of prolonged air leak after pulmonary lobectomy. Ann Thorac Surg. 2004;77(4):1205-10, http://dx.doi.org/10.1016/j.athoracsur.2003.10.082. 17. Fleisher AG, Evans KG, Nelems B, Finley RJ. Effect of routine fibrin glue use on the duration of air leaks after lobectomy. Ann Thorac Surg. 1990; 49(1):133-4, http://dx.doi.org/10.1016/0003-4975(90)90371-C. 18. Mandel SP, Gibran NS. Fibrin sealants: surgical hemostat, sealant and adhesive. Expert Opin Biol Ther. 2014;14(6):821-30, http://dx.doi.org/ 10.1517/14712598.2014.897323. 19. Spotnitz WD. Fibrin Sealant: The Only Approved Hemostat, Sealant, and Adhesiveâ&#x20AC;&#x201C;a Laboratory and Clinical Perspective. ISRN Surg. 2014; 2014:203943, http://dx.doi.org/10.1155/2014/203943. 20. Mehdizadeh M, Yang J. Design strategies and applications of tissue bioadhesives. 2013;13(3):271-88, http://dx.doi.org/10.1002/mabi.2012 00332. 21. Wong K, Goldstraw P. Effect of fibrins glue in the reduction of postthoracotomy alveolar air leak. Ann Thorac Surg. 1997;64(4):979-81, http://dx.doi.org/10.1016/S0003-4975(97)00820-5. 22. Belda-Sanchis J, Serra-Mitjans M, Iglesias Sentis M, Rami R. Surgical sealant for preventing air leaks after pulmonary resections in patients with lung cancer. Cochrane Database Syst Rev. 2010;(1):CD003051, http://dx.doi.org/10.1016/j.jtcvs.2010.09.019.

628

ORIGINAL RESEARCH

Impact assessment of an automated drug-dispensing system in a tertiary hospital De´bora de-Carvalho,I Jose´ Luiz Alvim-Borges,II,* Cristiana Maria ToscanoIII I Farmacia, Hospital Sirio-Libanes (HSL), Sao Paulo, SP, BR. II Instituto de Ensino e Pesquisa, Hospital Sirio-Libanes (HSL), Sao Paulo, SP, BR. III Departamento de Saude Coletiva, Instituto de Patologia Tropical e Saude Publica, Universidade Federal de Goias, Goiania, GO, BR.

OBJECTIVE: To evaluate the costs and patient safety of a pilot implementation of an automated dispensing cabinet in a critical care unit of a private tertiary hospital in Sa˜o Paulo/Brazil. METHODS: This study considered pre- (January-August 2013) and post- (October 2013-October 2014) intervention periods. We considered the time and cost of personnel, number of adverse events, audit adjustments to patient bills, and urgent requests and returns of medications to the central pharmacy. Costs were evaluated based on a 5-year analytical horizon and are reported in Brazilian Reals (R$) and US dollars (USD). RESULTS: The observed decrease in the mean number of events reported with regard to the automated drugdispensing system between pre- and post-implementation periods was not significant. Importantly, the numbers are small, which limits the power of the mean comparative analysis between the two periods. A reduction in work time was observed among the nurses and administrative assistants, whereas pharmacist assistants showed an increased work load that resulted in an overall 6.5 hours of work saved/day and a reduction of R$ 33,598 (USD 14,444) during the first year. The initial investment (R$ 206,065; USD 88,592) would have been paid off in 5 years considering only personnel savings. Other findings included significant reductions of audit adjustments to patient hospital bills and urgent requests and returns of medications to the central pharmacy. CONCLUSIONS: Evidence of the positive impact of this technology on personnel time and costs and on other outcomes of interest is important for decision making by health managers. KEYWORDS: Pharmacy; Cost and Cost Analysis; Automated Drug-Dispensing Systems; Impact. De-Carvalho D, Alvim-Borges JL, Toscano CM. Impact assessment of an automated drug-dispensing system in a tertiary hospital. Clinics. 2017; 72(10):629-636 Received for publication on February 8, 2017; First review completed on April 25, 2017; Accepted for publication on May 30, 2017 *Corresponding author. E-mail: jose.borges@hsl.org.br

’ INTRODUCTION

country estimates that ADEs entail a total annual cost of h1.058 billion (3). A prospective observational study found that 61% (170/ 277) of serious medical errors – which cause or have the potential to cause damage or injury – are drug prescription or administration errors (4). Another prospective observational study of intensive care reported one error, resulting in a potential or actual ADE, for every 5 prescription items administered (5). The occurrence of drug prescription errors is 7%, affecting 2% of patients/day and 50% of hospital admissions (6). Conversely, a systematic review indicated that approximately 10% of errors involving drugs corresponded to administration errors (7). In Brazil, a prospective cohort study, which was conducted in a tertiary teaching hospital with blinded nursing staff, found that 21% (238/1,119) of the doses of drugs prescribed and dispensed by the pharmacy were not administered (8). The application of information technology to healthcare has increased the safety of hospital prescription and administration procedures. Computerized physician order-entry systems with a drug menu standardized by the hospital institution, a clinical decision support system, and electronic

Healthcare-related errors are common (1). In the United States between 2000 and 2002, 37 million admissions occurred in the Medicare system, and 1.14 million patient safetyrelated incidents were reported. In an extrapolation to all admissions in American hospitals during this period, 575,000 preventable deaths would result from patient safety-related incidents (2). A German study addressing three public hospitals, totaling 1,208 beds and 49,462 patients showed that the mean cost of patients (n 1,891) who suffered an adverse drug event (ADE) was h5,113-h10,059. The mean hospital stay of patients with an ADE was 2.9 days longer than that of patients without an ADE. An extrapolation for the entire

629

Impact assessment of automated drug in hospital de-Carvalho D et al.

CLINICS 2017;72(10):629-636

Table 1 - Characterization of the Central Pharmacy of Hospital Sı´rio-Libaneˆs regarding the products administered and dispensed as well as the number of staff. 2013. Sa˜o Paulo, Brazil.

medication administration record are key tools for patient drug safety (9,10). Automated dispensing devices (ADDs) are increasingly present in healthcare organizations. The transition of the pharmaceutical profession to direct patient care, changes in healthcare systems and cost-reduction pressures have promoted the use of ADDs. The American Society of HealthSystem Pharmacists (ASHP) approves the use of automated drug distribution because the system frees pharmacists from labor-intensive distribution functions. Those pharmacists then begin to share responsibility for drug inventory with nurses, which improves the accuracy and timeliness of drug availability and enhances patient care (11). A recent systematic review showed that ADDs decrease drug storage errors and increase resource management efficiency. The nursing staff spent significantly less time managing controlled drugs. The results related to other drugs are inconclusive. Similarly, there was no clear evidence that these devices reduced drug errors resulting in patient harm, increased nursing or pharmacy staff time spent on patient care or reduced hospital costs (12). Although the literature indicates a high usage rate of this technology in tertiary hospitals within developed countries, studies specifically addressing ADDs in Brazilian hospitals remain scarce. This study was conducted to assess the economic and patient-safety impact of the implementation of the Pyxiss automated materials and drug-dispensing system in an inpatient unit for critically and semi-critically ill patients in a tertiary hospital.

Characteristic Products administered Products dispensed Professionals Pharmaceutical manager Pharmacy coordinators Materials supervisors Pharmacists Pharmacy technicians Pharmacy assistants Messengers Assistants and administrative assistants Trainees Young apprentices Total

Monthly mean (number) 1,994,973 270,421 Number of professionals 1 4 5 35 11 102 12 38 4 1 213

Intervention The Pyxiss ADD system was pilot-deployed at an HSL unit in September 2013. The unit in which the Pyxiss was implemented consisted of eleven cardiology beds, including seven semi-critical beds and four intensive care beds. The demographic characterizations of the unit in the periods before (January-August 2013) and after (October 2013-October 2014) the introduction of the ADD system were similar. Drug dispensing via the HSL pharmacy was performed using a hybrid model. Regularly prescribed drugs were dispensed by the central pharmacy. Drugs and disposable materials used during the first hours of patient admission as well as those that are used because of changes in drug prescriptions were dispensed from the peripheral stock located within each care unit. Each care unit also had a cart stocked with drugs and materials for emergency care. The patient profile and medical specialties of the care unit determined the composition of the drugs and disposable materials in this peripheral stock. The quantity of each product was based on the historical quantitative consumption provided by the hospital information system. The products were barcoded to ensure the traceability, stock write-off, and invoicing of the drugs and materials included in the peripheral stocks. The peripheral stock structure changed significantly with the implementation of the ADD system. Previously, products were placed in conventional cabinets in the drug room in the care unit, and only controlled drugs were stored in lockable drawers (13). The other products were stored in various places, according to the convenience of the team managing the stock. Resupply was performed once per day and was automatically generated by the hospital information system. Electronic prescription was not integrated with this stock, which together with the possibility of introducing or removing products from cabinets without a mandatory barcode reading, led to the need for many inventory adjustments and increased potential error. Administrative processes (invoicing, stock write-off for resupply and control, and key procedures in drug use safety) depended on the adherence of the nursing staff (who were directed to access the hospital information system using a password) to select the patient and perform the barcode reading of each product.

’ METHODS Study site, period and design

The study was conducted at ‘‘Hospital Sírio-Libanês’’ (HSL), which is a private, tertiary hospital that has 368 beds, including 44 intensive care beds; averages 18,840 admissions per year; and is located in São Paulo, Brazil. The hospital pharmacy department consists of a central pharmacy, 23 storage units, and 5 satellite pharmacies located in the following specific hospital units: the oncology unit, the emergency room, the surgery unit, the intensive care unit, and the diagnostic and imaging center. The hospital adopted an individual medication order-distribution system. The oncology department and the main intensive care unit used the unit-dose system. The Pyxiss ADD system was gradually introduced at HSL units in São Paulo from 2013 to 2015, primarily to promote drug-use safety by integrating drug prescription with dispensing. This new technology was also expected to reduce the time spent by the nursing and administrative staff on managing the inventory of the drug room and to improve urgent care performance. Improvements in the quality of patient billing (through the adequate invoicing of drugs and disposable medical supplies in the unit) and stock accuracy to reduce inventory adjustments were also expected. The effect of introducing the ADD system for the storage and control of the peripheral stock located in an 11-bed unit housing semi-critically and critically ill patients was assessed. Clinical and cost outcomes were assessed with cost outcomes considering a 5-year analytical horizon starting in 2013. The characterization of the central pharmacy regarding the products administered, the products dispensed, and the number of professionals in 2013 is outlined in Table 1.

630

Impact assessment of automated drug in hospital de-Carvalho D et al.

CLINICS 2017;72(10):629-636

ADD system. This assessment was performed by a pharmacist who was an HSL pharmacy coordinator. The working time was then estimated as hours per day spent on each activity. The cost per hour of work for each professional category was estimated from the gross monthly salary (including the benefits paid by the hospital), considering a weekly load of 40 hours. In addition, 30% pay for vacation and a 1 month salary bonus at the end of every working year (i.e., a 13th paycheck) were used to estimate annual salary because these benefits are paid by the hospital per Brazilian legislation. The working time per day was multiplied by the number of days in a year to estimate the annual economic impact. The total annual time (in hours) was multiplied by the salary per hour of work resulting in the annual costs for personnel by professional category. Personnel costs during the period before the implementation of the ADD system were then compared with personnel costs during the period after the implementation of the ADD system. The periods pre- and post-introduction of the automated drug-dispensing system were considered January to August 2013 and October 2013 to October 2014, respectively, for all outcomes. Categorical data are described and expressed as absolute (n) and relative (%) frequencies. Continuous variables are described as the mean and standard deviation. Unpaired Student’s t-test was used to compare the means of continuous variables with normal distributions. Pearson’s chisquared test was used to compare ratios. The significance level was set at 0.05 for all tests. The data were tabulated and analyzed using Microsoft Excel 2010 and SPSS for Windows, version 22. Direct medical costs, including the investment costs for implementing the Pyxiss ADD system and human resource costs, were considered in the cost analysis. All costs were expressed as Brazilian Reals (R$) and converted to US Dollars (USD) considering the official exchange rate in December 2013 (1 BRL=0.43 USD). To estimate the annual cost of human resources for future years (2014-2017), the estimated annual savings resulting from averted costs for human resource salaries for 2013 were then adjusted for inflation considering the Brazilian annual national consumer price index (15) according to the salary-adjustment procedures adopted by the hospital. The following annual cumulative national consumer price indices were used: 5.56% for 2013, 6.23% for 2014, 11.28% for 2015, and 6.58% for 2016. The rate of medical equipment depreciation was set at 10% per year, assuming a useful life of 10 years, according to current Brazilian law (16). The study was reviewed by the Institutional Review Board, which waived the need for approval.

After introducing the ADD system, the peripheral stock began to be placed inside the device within the care unit, resulting in a centralized, organized, and closed stock. Resupply, which was automatically generated by the hospital information system, began to be performed twice daily and was monitored by the team of pharmacy assistants to ensure organization according to the record of each product in the dispensing system. Electronic prescriptions were integrated with the dispensing system with biometric control of stock access for all users and privileges according to professional category. From that time forward, the nursing team had access to only the drugs prescribed for each patient. Emergency drugs, such as those not reviewed by the pharmacist and not part of the patient’s profile, were removed only by a nurse who was previously authorized to execute an override order. Then, the patient and the drugs were selected based on time, and each compartment containing the drug or material opened only for the practitioner to remove the products from the dispensing system after all of these steps were completed. After closing each compartment, the system invoiced the patient and adjusted the inventory. No change was introduced regarding the control of product expiration dates, which was performed monthly by the pharmacy team.

Data analysis The following outcomes were considered in the analysis: number of ADEs, audit adjustments to invoicing of materials and drugs in patient bills, performance in meeting urgent requests, need for central pharmacy services and product returns from the unit to the central pharmacy. According to the World Health Organization, an ADE is a patient injury resulting from a medication, either because of a pharmacological reaction to a normal dose or because of a preventable adverse reaction to a drug resulting from an error (14). Data concerning ADEs were collected from the HSL information system, which stored the details of each event notification, including the date, place, type of occurrence, drug involved, phase of the process, classification, and degree and type of resulting harm. Events that occurred in the unit during the study period were analyzed. Events that occurred during the drug prescription and transcription phases were disregarded. The events were categorized by phase of the process (dispensing or administration) and harm classification. Harm to patients was classified in accordance with the World Health Organization International Classification for Patient Safety. Mild harm was characterized as an event that results in mild symptoms, loss of function, and minimal or intermediate damage, albeit shortlived, without need for major intervention or requiring minimal intervention (observation, investigation, review, or minimal treatment). Moderate harm was characterized as an event resulting in symptoms that required intervention (additional surgical procedure or therapeutic treatment), prolonged hospital stay, permanent long-term damage or loss of function. Severe harm was defined as a symptomatic event requiring life-saving intervention or a major surgical/ medical intervention, shortened life expectancy, or major permanent or long-term harm or loss of function. The time spent by the healthcare professionals involved in the drug and material management process of the care unit was assessed directly by measuring the work time of each activity and healthcare professional involved. This measurement was performed before and after implementing the

’ RESULTS Number of events During the study period, a total of 37 ADEs were reported in the unit. Most (n=31, 83.8%) adverse events occurred during the drug-dispensing phase, whereas the other events (n=6, 16.2%) occurred during the drug administration phase. Although a decrease in the mean number of events reported was observed between the ADD system pre- (2.25±2.19 events/month) and post-implementation (1.46±1.39 events/ month) periods, this difference was not significant (p=0.32). The same trend was also observed when assessing the events that occurred during drug dispensing (1.88 versus 1.23, p=0.34) and

631

Impact assessment of automated drug in hospital de-Carvalho D et al.

CLINICS 2017;72(10):629-636

administration (0.38 versus 0.23, p=0.65) separately. It is worth noting that the numbers are very small, thus limiting the power of the comparative analysis of means between the two periods.

Healthcare professional time The implementation of the ADD system resulted in a change in the distribution of the time and type of healthcare professionals involved in drug management activities (Table 4). The time spent on activities performed by nurses and administrative assistants decreased, whereas the time spent on activities performed by pharmacy assistants increased, resulting in a total reduction of 6.5 work hours per day (Table 4). Nursing time was devoted primarily to drug inventory activities, including counting controlled drugs and performing stock write-offs, which significantly decreased after the implementation of the ADD system. Administrative assistant time, which also decreased, was primarily devoted to counting drugs and materials and resupplying items (Figure 1). Stock inventory and item resupply activities, which were performed by the stock room supervisor and administrative assistants, respectively, began to be performed by another professional, i.e., the pharmacy assistant.

Audit adjustments to hospital bills The number of audit adjustments to the hospital bill of each hospitalized patient was analyzed regarding the inclusion or exclusion of materials and drugs administered. We observed a significant decrease in adjustments to drug inclusions and exclusions and material inclusions. A decrease in material exclusions also occurred, although this change was not significant (Table 2).

Product requests and returns to the central pharmacy A significant decrease (by 71%) in urgent requests was observed after implementing the ADD system when assessing the number of requests and the need for central pharmacy services during both periods (Table 3). Similarly, the number of products returned to the central pharmacy decreased significantly (by 30%) during the ADD system post-implementation period. The observed 15% decrease in routine requests was not significant.

Costs The total cost of the Pyxiss ADD system included the cost of the device (R$ 198,065.88; USD 85,153) and costs associated with cabinet-making and remodeling services (R$ 8,000.00; USD 3,439.40). Information technology (IT) costs

Table 2 - Audit adjustments to material and medication invoicing in the Advanced Heart Failure Unit of Hospital Sı´rio-Libaneˆs before and after implementing the automated medication dispensing system. 2013-2014. Sa˜o Paulo, Brazil. Type of product

Drugs Materials

Procedure requested

Conventional dispensing period

Automated dispensing period

by the audit

Mean

Standard deviation

Mean

Standard deviation

Inclusion (mean/month) Exclusion (mean/month) Inclusion (mean/month) Exclusion (mean/month)

1,215.75 1,635.13 1,061.25 3,008.75

815.49 451.19 668.20 1,425.01

555.08 1,036.92 461.54 2,255.15

210.49 297.21 151.72 786.20

Variation (%)

p-value

-54.30 -36.60 -56.50 -25.10

0.01 o0.01 o0.01 0.13

Table 3 - Urgent and routine requests as well as pharmaceutical product returns to the central pharmacy at Hospital Sı´rio-Libaneˆs before and after implementing the automated medication dispensing system. 2013-2014. Sa˜o Paulo, Brazil. Conventional dispensing period

Total requests/month Routine Urgent Total returns/month

Automated dispensing period

Mean; SD (R$)

Mean; SD (USD)

Mean; SD (R$)

Mean; SD (USD)

1,510 (332.60) 1,519 (220.90) 869 (90.50)

649.18 (142.99) 279.1 (94.97) 373.60 (38.91)

1,279 (385.10) 431 (88.90) 603 (136.30)

549.87 (165.53) 185.30 (38.22) 259.11 (58.60)

% Reduction

p-value

-15.3 -71.6 -30.60

0.17 o0.001 o0.001

SD = standard deviation.

Table 4 - Activity and time associated with healthcare professional involvement in drug and material management in the Advanced Assistance Unit of the Hospital Sı´rio-Libaneˆs before and after implementing the automated medication dispensing system, according to the type of professional. 2013-2014. Sa˜o Paulo, Brazil. Type of healthcare professional and activities

Nurse Daily inventory count – controlled substances Adjustments/ corrections of the inventory counts Material supervisor Stock inventory Administrative assistant Daily inventory count – drugs Daily inventory count – materials Item resupply Pharmacy assistant Item resupply Stock inventory

Conventional dispensing period

Automated dispensing period

Hours/day

3.75 2.25 1.5 0.07 0.07 5 2 2 1 0 0 0

0 0 0 0 0 2 0 0 2 4 1 3

-3.75 -2.25 -1.5 -0.07 -0.07 -3 -2 -2 1 4 1 3

-100 -100 -100 -100 -100 -60 -100 -100 100 New New New

632

Variation

Impact assessment of automated drug in hospital de-Carvalho D et al.

CLINICS 2017;72(10):629-636

Figure 1 - Drug dispensing before and after implementing the automated dispensing system. Hospital Sı´rio-Libaneˆs, Sa˜o Paulo. Brazil. 2013-2014.

assistant (R$ 1,598; USD 687), materials supervisor (R$ 3,526; USD 1,516), and pharmacy assistant (R$ 1,710; USD 735). Thus, the reduction in personnel costs totaled R$ 33,598 (USD 14,444) per year during the first year after introducing the ADD system (Table 5). For the 5-year period between 2013 and 2017, considering the salary adjustment based on the Broad National Consumer Price Index between 2013 and 2016 and assuming a

were disregarded because the server was included in the cost of the device and the user interface software between the central pharmacy computer system and the ADD system was provided by the IT service of the HSL. The 2013 average mid-career salary, considering the gross monthly salary for a 40-hour weekly workload, was used in the analysis of healthcare personnel costs as follows: fulltime nurse (R$ 5,869; USD 2,523), administrative/hospitality

633

USD

-15,730.67 -176.41 -3,426.49 4,888.87 -14,444.72

-36,589.55 -410.34 -7,970.03 11,371.50 -33,598.41 0.00 0.00 2,284.33 4,888.87 7,173.19

Cost/year (USD) Cost/year (R$)

0.00 0.00 5,313.35 11,371.50 16,684.85 0.00 0.00 6.35 13.58 19.93

Cost/day (USD) Cost/day (R$)

0.00 0.00 14.76 31.59 46.35 15.730.67 176.41 5,710.82 0.00 21,617.91

Cost/year (USD)

This investigation is the first study to assess the impact and costs resulting from implementing automated dispensing systems in Latin America. Implementing this technology resulted in reduced human resource costs, reduced audit adjustments regarding drug inclusions and exclusions and materials inclusions, and a lower number of urgent requests and product returns to the central pharmacy. Our results corroborate evidence from developed countries. Overall human resource time and costs decreased because of a reduction in nursing time and activity assignment, whereas the work time of pharmacy assistants increased. A study assessing the implementation of the Pyxiss system in the United States in 1995 showed a significant reduction in personnel time related to the use of the ADD system that resulted in a savings of approximately USD 1 million during a 5-year period for the institution (17). Similar findings were observed in Spain, where the authors reported significant increases in clinical activities by pharmacists despite not having identified significant reductions in medicationrelated activities (18). This result might be related to the type of work attributed to pharmacists and nurses; however, it also reinforces the finding that the ADD system provides pharmacists with more time for other types of clinical work. Other studies have assessed the economic effect of ADDs using various methods (19-24). Although those authors showed a reduction in human resource personnel time and costs, in the United States, Klein et al. (24) concluded that this reduction was insufficient because the costs of the bulk drugs purchased to supply the local ADD system were higher than those for the same drugs in conventional packaging. However, the drugs in Brazil are included in the ADD system in unit-dose packaging, which is the same as in manual systems. Financial impact studies of ADDs in Spain (21-23) have indicated a reduction in workload and personnel costs. Poveda et al. (23) conducted a cost-benefit analysis for the implementation of 11 ADDs in clinical and surgical intensive and emergency care units of a university hospital center consisting of two hospital units in Albacete, Spain. These authors found a positive cost-benefit ratio (1.95) that favored the implemented technology. Another publication from the same study that considered a longer follow-up time reported an estimated h32,390 in annual personnel savings, which resulted in an even higher cost-benefit ratio (2.19); in that case, the initial investment was paid off in an estimated 44 months (21). This result is similar to the findings of our study, which estimated that the initial investment would be paid off in 5 years or 60 months, considering the reduction in personnel costs alone. Although the aforementioned studies consistently indicated the positive economic impact of implementing ADD systems, a systematic review conducted by the Canadian Agency for Drugs and Technologies in Health (CADTH) in 2009 to assess the impact of new technologies for drug dispensing and administration regarding their effectiveness,

36,589.55 410.34 13,283.38 0.00 50,283.26

Cost/year (R$)

’ DISCUSSION

43.70 0.49 15.86 0.00 60.05

Cost/day (USD)

constant inflation rate of 10% starting in 2016, the reduction in personnel costs alone totaled R$ 35,480 (USD 15,254) in 2014, R$ 37,690 (USD 16,204) in 2015, R$ 41,942 (USD 18,032) in 2016, and R$ 44,702 (USD 19,218) in 2017. Thus, the initial investment would be paid off in 5 years, considering only personnel savings.

101.64 1.14 36.90 0.00 139.68

Cost/day (R$)

CLINICS 2017;72(10):629-636

Nurse Materials supervisor Administrative assistant Pharmacy assistant TOTAL

Variation/ year Automated dispensing period Conventional dispensing period Type of healthcare professional

Table 5 - Human resource costs of the Advanced Assistance Unit of the Hospital Sı´rio-Libaneˆs before and after implementing the automated medication dispensing system, according to the type of healthcare professional. 2013-2014. Sa˜o Paulo, Brazil.

Impact assessment of automated drug in hospital de-Carvalho D et al.

634

Impact assessment of automated drug in hospital de-Carvalho D et al.

CLINICS 2017;72(10):629-636

automated Pyxiss drug-dispensing system in another 16 HSL units starting in 2013. Previous studies have indicated the widespread use of ADD systems in several countries. In the United States, a nationwide survey conducted by the American Society of Health-Systems Pharmacists in 2011 found that 89% of the 1,401 hospitals assessed used ADD systems (29). A Spanish study from 2013 on the adoption of safe drug practices included 36 hospitals and showed that 60.9% of the hospitals used ADD systems coupled with electronic prescription systems (30). In Brazil, reports indicate that this technology has been gradually incorporated into hospital departments. However, little evidence on this subject is available in the literature. Although this study was performed in a tertiary hospital located in São Paulo (and therefore precludes direct extrapolations of the results to other hospitals, particularly Brazilian private hospitals), we believe that our results are relevant, especially in the Brazilian context, considering the lack of studies on the subject. The results from this study contribute to the body of available evidence showing the positive effect of ADD systems in reducing healthcare personnel time and costs and other outcomes of interest related to healthcare services. Studies assessing the cost and effect of implementing technologies related to pharmaceutical care in hospital departments remain scarce in Brazil and should be encouraged, considering the importance of their results to the decisionmaking process. This analysis, in the context of a Brazilian tertiary private hospital, is useful and may help Brazilian healthcare decision-makers and managers regarding the use of this technology.

cost-effectiveness and budget impact indicated a lack of robust and high-quality evidence related to ADD systems (25,26). Since that time, other studies have been performed using the before-and-after method and were therefore subject to various biases. A study assessing the economic effect of implementing an ADD system in a 12-bed resuscitation/ intensive care unit in a French university hospital using the cash flow method showed reductions in personnel time costs and drug-storage costs. This cost reduction resulted in an estimated 5-year savings of h71,586 (19,20). A more complete and recent analysis of the financial effect of ADD use in a surgical intensive care unit of a French university hospital also showed a decrease in drug-related nursing time and an increase in time dedicated by pharmacy technicians to the local stock. This study also considered the decrease in the stock of expired drugs and their respective costs. The annual cost of the drug stock decreased by h44,298, and the cost of expired drugs decreased by h14,772. Using the cash flow method, the study showed that the overall cash flow was h148,229, and the current net value of the project 5 years after the initial investment was positive by h510,404. The authors concluded that the implementation of ADD systems results in a high return on the initial investment (19). The impact of the ADD system on the number and value of drugs in stock or on expired drugs could not be assessed in our study. It is worth noting that most institutions use the model of fully meeting drug needs using the stock of the ADD system. In the ‘‘hybrid’’ model implemented at the HSL described above, the peripheral stocks available in the care units had minimal product types and enough quantity to minimize financial losses and care failures. The implementation of the ADD enabled the unit to increase the number and availability of certain types of products, thereby increasing process agility. Packaging, dispensing, and barcoding prescribed items using an ADD system for prescription items can decrease the occurrence of ADEs (10). Although a decrease in the number of ADEs was observed in this study, this decrease was nonsignificant. Considering that the number of events that occurred during the study period was small, this finding might result from the low power of the study, which failed to show an effect on ADEs. However, recently published evidence indicates controversy regarding the effectiveness of ADD systems to reduce ADEs related to drug administration. Only one of 3 systematic reviews published recently showed that ADD systems effectively reduce the number of ADEs, albeit modestly (RR=0.72, therefore, 28% effectiveness) (27). The other studies reported no evidence of decreased ADEs from using this technology. However, all reviews emphasized the small number of studies and the high risk of bias in the studies reviewed (28). Thus, similar to most studies on this subject, our study has several limitations that should be reported. First, the study was performed retrospectively and using secondary data available in the hospital. Thus, several outcomes of interest, including stock control and expired drugs, could not be assessed, as mentioned previously. Furthermore, costs related to the necessary planning, training and information system adaptations in the stage of preparation for implementing the automated drug-dispensing system were not included in the cost analysis. Despite these limitations, the preliminary results from this study support the decision to gradually implement the

’ AUTHOR CONTRIBUTIONS de-Carvalho D and Alvim-Borges JL conceived and designed the study. Toscano CM designed the study methods as well as analyzed and interpreted the data. All authors were responsible for writing the manuscript and have approved the ﬁnal version.

’ REFERENCES 1. Makary MA, Daniel M. Medical error-the third leading cause of death in the US. BMJ. 2016;353:i2139. 2. The Healthcare Quality Experts. HealthGrades Quality Study Patient Safety in American Hospitals July 2004. 22p. [cited 2016 May]. Available from: http://www.providersedge.com/ehdocs/ehr_articles/Patient_Safety_ in_American_Hospitals-2004.pdf 3. Rottenkolber D, Hasford J, Stausberg J. Costs of adverse drug events in German hospitals--a microcosting study. Value Health. 2012;15(6):868-75, http://dx.doi.org/10.1016/j.jval.2012.05.007. 4. Rothschild JM, Landrigan CP, Cronin JW, Kaushal R, Lockley SW, Burdick E, et al. The Critical Care Safety Study: The incidence and nature of adverse events and serious medical errors in intensive care. Crit Care Med. 2005; 33(8):1694-700, http://dx.doi.org/10.1097/01.CCM.0000171609.91035.BD. 5. Kopp BJ, Erstad BL, Allen ME, Theodorou AA, Priestley G. Medication errors and adverse drug events in an intensive care unit: direct observation approach for detection. Crit Care Med. 2006;34(2):415-25, http:// dx.doi.org/10.1097/01.CCM.0000198106.54306.D7. 6. Lewis PJ, Dornan T, Taylor D, Tully MP, Wass V, Ashcroft DM. Prevalence, incidence and nature of prescribing errors in hospital inpatients: a systematic review. Drug Saf. 2009;32(5):379-89, http://dx.doi.org/10.2165/ 00002018-200932050-00002. 7. Berdot S, Gillaizeau F, Caruba T, Prognon P, Durieux P, Sabatier B. Drug administration errors in hospital inpatients: a systematic review. PLoS One. 2013;8(6):e68856, http://dx.doi.org/10.1371/journal.pone.0068856. 8. Leite B, Mistro S, Carvalho C, Mehta SR, Badaro R. Cohort study for evaluation of dose omission without justification in a teaching general hospital in Bahia, Brazil. Int J Qual Health Care. 2016;28(3):288-93, http:// dx.doi.org/10.1093/intqhc/mzw016.

635

Impact assessment of automated drug in hospital de-Carvalho D et al.

CLINICS 2017;72(10):629-636

21. Poveda Andrés JL, Hernández Sansalvador M, Díez Martínez AM, García Gómez C. Análisis coste-beneficio del proceso de semiautomatizacion en la pre-paracion de dosis unitarias por el Servicio de Farmacia. Farm Hosp. 2004;28(2):76-83. 22. Alvarez Rubio L, Martín Conde JA, Alberdi Léniz A, Plasencia García I, Cáceres González F, Martín Martín A. Evaluación de un sistema automatico de dispensacion en el Servicio de Urgencias de un hospital de tercer nivel. Farm Hosp. 2003;27(2):72-7. 23. Poveda Andrés JL, García Gómez C, Hernández Sansalvador M, Valladolid Walsh A. Analisis coste-beneficio de la implantacion de los sistemas automati-cos de dispensación de medicamentos en las Unidades de Criticos y Urgenci-as. Farm Hosp. 2003;27(1):4-11. 24. Klein EG, Santora JA, Pascale PM, Kitrenos JG. Medication cart-filling time, accuracy, and cost with an automated dispensing system. Am J Hosp Pharm. 1994;51(9):1193-6. 25. Canadian Agency for Drugs and Technologies in Health (CADTH). Technologies to reduce errors in dispensing and administration of medication in hospitals: clinical and economic analyses. CADTH Technol Overv. 2010;1(3):e0116. 26. Perras C, Jacobs P, Boucher M, Murphy G, Hope J, Lefebvre P, et al. Technologies to reduce errors in dispensing and administration of medication in hospitals: clinical and economic analyses. Ottawa: Canadian Agency for Drugs and Technologies in Health; 2009. [Technology report; no.121]. 27. Keers RN, Williams SD, Cooke J, Walsh T, Ashcroft DM. Impact of interventions designed to reduce medication administration errors in hospitals: a systematic review. Drug Saf. 2014;37(5):317-32, http://dx.doi. org/10.1007/s40264-014-0152-0. 28. Berdot S, Roudot M, Schramm C, Katsahian S, Durieux P, Sabatier B. Interventions to reduce nurses’ medication administration errors in inpatient settings: A systematic review and meta-analysis. Int J Nurs Stud. 2016;53:342-50, http://dx.doi.org/10.1016/j.ijnurstu.2015.08.012. 29. Pedersen CA, Schneider PJ, Scheckelhoff DJ. ASHP national survey of pharmacy practice in hospital settings: dispensing and administration2011. Am J Health Syst Pharm. 2012;69(9):768-85, http://dx.doi.org/10.2146/ ajhp110735. 30. Otero López MJ, Bermejo Vicedo T, Moreno Gómez AM, Aparicio Fernández MA, Palomo Cobos L; Grupo de Trabajo TECNO de la SEFH. Análisis de la implantación de prácticas seguras en los sistemas automatizados de dispensa-ción de medicamentos. Farm Hosp. 2013;37(6): 469-81, http://dx.doi.org/10.7399/FH.2013.37.6.1053.

9. Seidling HM, Bates DW. Evaluating the impact of health IT on medication safety. Stud Health Technol Inform. 2016;222:195-205, http://dx.doi.org/ 10.3233/978-1-61499-635-4-248. 10. Agrawal A. Medication errors: prevention using information technology systems. Br J Clin Pharmacol. 2009;67(6):681-6, http://dx.doi.org/10.1111/ j.1365-2125.2009.03427.x. 11. ASHP Guidelines on the safe use of automated dispensing devices. Am J Health Syst Pharm. 2010;67(6):483-90, http://dx.doi.org/10.2146/sp100004. 12. Tsao NW, Lo C, Babich M, Shah K, Bansback NJ. Decentralized automated dispensing devices: systematic review of clinical and economic impacts in hospitals. Can J Hosp Pharm. 2014;67(2):138-48. 13. Brasil. Ministério da Saúde. Agência Nacional de Vigilância Sanitária. Portaria n.o 344, de 12 de maio de 1998. Aprova o Regulamento Técnico sobre substâncias e medicamentos sujeitos a controle especial. Brasília, DF; 1998. [cited 2016 May]. Available from: http://www.anvisa.gov.br/ hotsite/talidomida/legis/Portaria_344_98.pdf 14. Conceptual Framework for the International Classification for Patient Safety Version 1.1. Final Technical Report January 2009 Available from: http://www.who.int/patientsafety/taxonomy/icps_full_report.pdf 15. Brasil. Instituto Brasileiro de Geografia e Estatística (IBGE). Índice Nacional de Preços ao Consumidor (INPC). Série Histórica. [cited 2017 Apr 20]. Available from: http://www.ibge.gov.br/home/estatistica/indicadores/ precos/inpc_ipca/defaultseriesHist.shtm 16. Brasil. Ministério da Fazenda. Sistema Normas Gestão da Informação. Instrução Normativa SRF No 162, de 31 de dezembro de 1998. [cited 2016 Sep]. Available from: http://normas.receita.fazenda.gov.br/sijut2consulta/ link.action?visao=anotado&idAto=15004 17. Schwarz HO, Brodowy BA. Implementation and evaluation of an automated dispensing system. Am J Health Syst Pharm. 1995;52(8):823-8. 18. Guerrero RM, Nickman NA, Jorgenson JA. Work activities before and after implementation of an automated dispensing system. Am J Health Syst Pharm. 1996;53(5):548-54. 19. Chapuis C, Bedouch P, Detavernier M, Durand M, Francony G, Lavagne P, et al. Automated drug dispensing systems in the intensive care unit: a financial analysis. Crit Care. 2015;19:318, http://dx.doi.org/10.1186/ s13054-015-1041-3. 20. Kheniene F, Bedouch P, Durand M, Marie F, Brudieu E, Tourlonnias MM, et al. Impact economique de la mise en place d’un automate de distribution des medicaments dans un service de reanimation. Ann Fr Anesth Reanim. 2008;27(3):208-15, http://dx.doi.org/10.1016/j.annfar.2007.11.026.

636

REVIEW

Review of magnetic resonance-guided focused ultrasound in the treatment of uterine fibroids Pedro Felipe Magalha˜es Peregrino, Marcos de Lorenzo Messina, Ricardo dos Santos Simo˜es, Jose´ Maria Soares-Ju´nior,* Edmund Chada Baracat Disciplina de Ginecologia, Departamento de Obstetricia e Ginecologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

Uterine leiomyoma is the most frequently occurring solid pelvic tumor in women during the reproductive period. Magnetic resonance-guided high-intensity focused ultrasound is a promising technique for decreasing menorrhagia and dysmenorrhea in symptomatic women. The aim of this study is to review the role of Magnetic resonance-guided high-intensity focused ultrasound in the treatment of uterine fibroids in symptomatic patients. We performed a review of the MEDLINE and Cochrane databases up to April 2016. The analysis and data collection were performed using the following keywords: Leiomyoma, High-Intensity Focused Ultrasound Ablation, Ultrasonography, Magnetic Resonance Imaging, Menorrhagia. Two reviewers independently performed a quality assessment; when there was a disagreement, a third reviewer was consulted. Nineteen studies of Magnetic resonance-guided high-intensity focused ultrasound-treated fibroid patients were selected. The data indicated that tumor size was reduced and that symptoms were improved after treatment. There were few adverse effects, and they were not severe. Some studies have reported that in some cases, additional sessions of Magnetic resonance-guided high-intensity focused ultrasound or other interventions, such as myomectomy, uterine artery embolization or even hysterectomy, were necessary. This review suggests that Magnetic resonance-guided high-intensity focused ultrasound is a safe and effective technique. However, additional evidence from future studies will be required before the technique can be recommended as an alternative treatment for fibroids. KEYWORDS: Leiomyoma; High-Intensity Focused Ultrasound Ablation; Ultrasonography; Magnetic Resonance Imaging; Menorrhagia. Peregrino PF, Messina ML, Simo˜es RS, Soares-Ju´nior JM, Baracat EC. Review of magnetic resonance-guided focused ultrasound in the treatment of uterine fibroids. Clinics. 2017;72(10):637-641 Received for publication on December 15, 2016; First review completed on February 16, 2017; Accepted for publication on April 4, 2017 *Corresponding author. E-mail: jsoares415@hotmail.com

’ INTRODUCTION

and urinary habits of the patient, in addition to causing discomfort (3). The classic treatments for fibroids include excision of the tumors and hysterectomy. However, such techniques may be very costly due to the length of surgery, increased bleeding risk, longer hospitalization, and possible postoperative complications. Therefore, researchers are developing new techniques such as magnetic resonance-guided high-intensity focused ultrasound (MRgFUS) (4-6). MRgFUS is a minimally invasive therapy capable of producing necrosis by thermal coagulation of myometrial nodules at a precise location in the uterus (4). It is primarily used to decrease the volume of fibroids and to reduce complaints about symptoms. However, in many centers, MRgFUS is currently neither advocated nor established as an alternative therapy. Initial studies comprising various cohort sizes reported significant symptom improvement in response to MRgFUS. Even with partial results, MRgFUS has been shown to be an efficient and minimally invasive method for treating uterine fibroids (4-6). Most of the studies in which MRgFUS has been used are case studies. Therefore, it is necessary to perform a systematic review of the studies to better assess MRgFUS as a

A uterine fibroid is a benign neoplasia originating from smooth muscle tissue. Uterine fibroids are classified as subserosal, intramural, and submucosal based on their location within the uterus. These different types of uterine fibroids may have diverse manifestations. The main symptoms are intense or prolonged menstrual bleeding (menorrhagia), dysmenorrhea, dyspareunia, adjacent organ compression (bladder and bowel), urinary incontinence, increased abdominal volume, infertility, and repeated miscarriages (1-3). Menorrhagia may cause anemia, which together with dysmenorrhea and dyspareunia, has a negative effect on the quality of life of these women. The increase in abdominal volume and the compression of adjacent organs may affect the intestinal

637

Focused ultrasound and uterine fibroids Peregrino PFM et al.

CLINICS 2017;72(10):637-641

Most studies used assessment questionnaires that have been validated in the literature. These assessments indicated that MRgFUS might improve the patients’ symptoms and quality of life (7-14,17-19,23-24). Some articles used only the symptom severity score (SSS), which is part of the Uterine Fibroid Symptom and Health-Related Quality of Life Questionnaire (UFS-QOL) to assess symptom improvement in patients. In the UFS-QOL, the SSS mainly analyzes symptoms such as menorrhagia and bulk-related symptoms. Three studies did not use validated questionnaires to evaluate quality of life or symptom improvement (14,20,25) or did not administer any specific questionnaires to evaluate patients’ symptoms and quality of life. These studies were included only in the analysis of the fibroid volume reduction. The decrease in the SSS ranged from 32% to 74%. Four studies used the complete UFS-QOL and reported increases in quality of life ranging from 20% to 47%. The only clinical trial included in this review analyzed 109 women with fibroids and showed a significant reduction of severe symptoms (51%) at 12 months of follow-up, which was statistically significant (po0.001). No severe complications were observed in the studies included in the analysis. The main complaints after MRgFUS were abdominal pain and lumbar pain, which improved with a mild analgesic. Two studies reported abdominal pain due to the accumulation of contrast material in the muscular and subcutaneous layers of the abdominal wall in 9% and 11% of the patients, respectively (7,13). In some cases, the necrotic material was expelled via the vagina after treatment over the subsequent menstrual cycles (11). The most severe complications occurred in a patient who developed deep vein thrombosis and required longer hospitalization (14) and in 2 patients who had endometritis with subsequent hysterectomy (15). In some studies, new interventions were required because the patients’ symptoms persisted or worsened (8,12,15,17,21). For these reasons, Mindjuk et al. (8) reported a 12.7% rate of additional treatments such as hysterectomy, myomectomy, or embolization of uterine blood vessels. In the Funaki et al. (17) study, 1 patient underwent a hysterectomy, 5 patients underwent a myomectomy, and 5 others underwent a repeat MRgFUS after the primary treatment.

therapeutic modality for uterine fibroids in symptomatic women during the reproductive period.

’ METHODS A review was conducted using articles in English, Spanish and Portuguese without publication year restriction up to April 2016 and retrieved from the MEDLINE and Cochrane databases. The following inclusion criteria were used: a) studies including patients of reproductive age with uterine fibroids; b) studies including patients with menorrhagic cycles, dysmenorrhea, and an increase in abdominal volume; c) the use of MRgFUS for the treatment of uterine fibroids. The exclusion criteria were as follows: a) studies of animal models; b) studies that included asymptomatic women; c) narrative reviews; d) case reports; e) cost assessments of therapeutic methods; f) comparisons of different techniques of the MRgFUS method; g) evaluation or study of the MRgFUS technique; h) studies with different protocols for the use of MRgFUS; i) studies that used a non-validated questionnaire. Articles were retrieved using the search strategies described in Box 1 and were grouped and structured according to the PICO strategy (the initials for ‘‘Patient’’, ‘‘Intervention,’’ ‘‘Control’’ and ‘‘Outcome’’). The search strategy is described in Figure 1. The references cited in the articles retrieved from the database were also evaluated. Systematic reviews and metaanalyses were consulted, but only the original articles were used. Two reviewers independently assessed the quality of the studies; when there was a disagreement, a third reviewer was consulted. The JADAD scale was used for the critical analysis of Randomized clinical trial.

’ RESULTS During the investigated period, a total of 207 articles were found in MEDLINE and 8 articles were found the in the Cochrane database (Figure 1). After the titles and abstracts were read and the eligibility criteria were applied, 188 articles were excluded (incomplete reviews, case reports, studies of therapy costs, studies in animal models, and studies of the comparisons of the different techniques of the MRgFUS method or studies with different protocols for the use of MRgFUS). The remaining 19 papers were included in this review (7-25). The effects of MRgFUS on fibroid volume and on the patients’ symptoms and quality of life are summarized in Tables 1 and 2. In the 19 selected studies, patient age ranged from 32 to 50 years, and follow-up lasted from 3 to 36 months. This review comprised case studies and one clinical trial (24). Fibroid volume decreased in all the included studies; however, there was a discrepancy in the percentage reduction after MRgFUS. The decrease in fibroid volume ranged from 9,3% to 90% (11,19), as shown in Table 1.

’ DISCUSSION The American College of Obstetricians and Gynecologists (ACOG) recommends that hysterectomies be included as an option for the treatment of benign conditions such as leiomyoma (26). Otherwise, myomectomy is the treatment of choice (1-3). However, recovery time, prolonged hospitalization, high rates of complications that may occur intraoperatively, and possible hysterectomies can be avoided. In this systematic review, MRgFUS was evaluated and found to be effective, promising, and safe for the treatment of benign uterine tumors. Hence, minimally invasive techniques such

Box 1 - Databases and search strategies.

638

Focused ultrasound and uterine fibroids Peregrino PFM et al.

CLINICS 2017;72(10):637-641

Figure 1 - Flowchart: Selection of studies included in the review.

Table 1 - Studies assessing the decrease in fibroid volume and the necrosis volume immediately after treatment in symptomatic women. Author and Year of Publication Park et al., 2014 Kim et al., 2012 Ikink et al., 2013 LeBlang et al., 2010 Morita et al., 2008 Rabinovic et al., 2007 Hindley et al., 2004 Lenard et al., 2008 Dobrotwir et al., 2012 Wang et al., 2012* Funaki et al., 2009 Kim et al., 2011

Patient Follow-up Time

Number of Patients

Age, Mean and Standard Deviation

Mean Reduction in Fibroid Volume

NPV Mean (%)

3 months 3 months 6 months 6 months 6 months 6 months 6 months 12 months 12 months 24 months 24 months 36 months

79 27 46 80 48 35 109 66 74 78 91 40

43.6±4.4 44.5±3.8 45.3±4.1 44±3.2 42.6±5.8 46.4±4.7 44.8±4.9 45.4±4.4 42±7.0 38.2±6.4 40.4±4.6 46±4.5

23.1% 64.2% 29% 31% 33% 15% 13.5% 9.3% 38% 90.1% 39.5% 32%

62.7±25.5 64.2±19.9 40±22 55±25 60±18 31±23 25±6.0 16.3±13.3 67±25 80±12 67±25 32.1±6.2

NPV: nonperfused volume. * Study of women with submucosal fibroids.

639

Focused ultrasound and uterine fibroids Peregrino PFM et al.

CLINICS 2017;72(10):637-641

Table 2 - Studies assessing the improvement of symptoms and the quality of life of symptomatic women. Author and Year of Publication

Patient Follow-up Time

Number of Patients

Mean Age and Standard Deviation

Mean Reduction in SSS

Increase in the Overall UFS-QOL Score

Symptom Improvement

NPV Mean (%)

Park et al., 2014 Park et al., 2012** Kim et al., 2012 Harding et al., 2008 Ikink et al., 2013 Mikami et al., 2008 Hindley et al., 2004 Lenard et al., 2008 Dobrotwir et al., 2012 Gorny et al., 2011 Stewart et al., 2006* Funaki et al., 2009 Wang et al., 2012 Stewart et al., 2007

3 months 3 months 3 months 6 months 6 months 6 months 6 months 12 months 12 months 12 months 12 months 24 months 24 months 24 months

79 9 27 102 46 48 109 66 74 130 109 91 78 359

43.6±4.4 39.8±6.2 44.5±3.8 45±4.8 45.3±4.1 45±5.2 44.8±4.9 45.4±4.4 42±7.0 45.6±5.5 44.8±4.9 40.4±4.6 38.2±6.4 45.4±5.0

35.6% 55.5% 35.8 44.8% 31.8% 38.8% 51% 51% 65.5% 35.6%

33.6% 19.5% 31.25% -

SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM

Mindjuk et al., 2014 Kim et al., 2011

24 months 36 months

252 40

42.1±6.9 45.98±4.52

70% 73.7%

47.4%

SIM SIM

62.7±25.5 66.9±10.6 64.2±19.9 40±22 47±13 25±6.0 16.3±13.3 67±25 45.4±22.5 67±25 80±12 19.9±17.2/ 21.9±18.7 88.7±14.4 32.1±6.2

SSS: Symptom Severity Score. * Clinical Trial. ** Study of women with pedunculated subserosal fibroids.

intense symptoms (15,21,25). A low SSS (a component of the UFS-QOL) indicates an improvement in symptoms, whereas an overall low UFS-QOL score indicates an enhancement of the patient’s quality of life. The articles in this review showed that the use of MRgFUS favors improvements in severe symptoms, as well as in the overall quality of life, despite the wide range of the follow-up time and the age of the study patients. The SSS questionnaire primarily evaluates symptoms such as menorrhagia; therefore, its scores are more meaningful in studies of patients with intramural or submucosal fibroids. Taking this into consideration, Park et al. (10) studied 9 women with subserosal fibroids whose main complaints were mass effect and increased abdominal volume. They concluded that there was a significant reduction in volume and in mass effect (89%) but only a slight improvement in the SSS. A better quality of life and improvements in bleeding and dysmenorrhea are also related to a larger NPV (11-13,19,23). Stewart et al. (23), whose study included more patients than any of the other studies in this review, analyzed 359 patients from all the clinical trials of MRgFUS in the treatment of fibroids and found that symptom improvement was significant. Furthermore, after a 24-month follow-up, they concluded that the SSS improvement was, on average, 6 points larger in the group of patients with the greater NPV. The relationship between NPV and symptom improvement may also be explained by the association of an MRI T2 hyposignal and a larger NPV, as found in some of the previously mentioned studies. In all the studies included in the present analysis, the rates of adverse effects and complications following MRgFUS were low. The most common symptoms were abdominal pain, lumbar pain, first-degree burns, and light vaginal bleeding. The abdominal pain and lumbar pain were relieved with a common, mild analgesic. There was also pain caused by the accumulation of contrast material in the muscle and subcutaneous layers of the abdominal wall (7,13). However, neither hospitalization nor intervention were subsequently required to treat such complications. In general, tumors are

as MRgFUS may be an option, but further studies with a longer follow-up are necessary. Since the introduction of MRgFUS and its approval by the FDA as a therapeutic modality (2004), several studies have been conducted and have reported symptom improvement in leiomyoma patients (6), as discussed in this review. The articles included in this study were mostly case series because it is difficult to conduct double-blind controlled studies with MRgFUS. This fact limits the design and performance of these studies. Additionally, the technique used with MRgFUS is very different from minimally invasive techniques such as embolization of uterine arteries (27). Patient follow-up after MRgFUS lasted at least 3 months (7,10,13) and at most 36 months (14). Regardless of the variability in the length of follow-up, the studies under investigation reported reduced fibroid volume and a reduction in the patients’ symptoms. To assess the volume reduction, the nonperfused volume (NPV) after MRgFUS was considered. Only 9 of the 19 studies included in this review had an NPV over 50%. Although higher NPVs are associated with greater efficacy, symptom improvement was similar among the studies. It is possible that unevaluated uterine factors occurred after MRgFUS. Potential endometrial alterations may have a considerable impact on the uterus, primarily on a woman’s reproductive future. The studies included in this review did not evaluate this potential impact, i.e., they did not attempt to determine the feasibility of using the MRgFUS method in women with a desire to have children. The differences in the previously mentioned results may be explained by the variety of fibroid types and the variation in signal intensity in the MRI T2 images soon after treatment (20,24). Tumors generating an MRI T2 hyposignal tend to exhibit higher necrosis rates and, consequently, larger reductions in fibroid volume. The reason for this is still unknown, but it seems to depend on both tumor volume and factors such as fibroid vascularization, intramural components, lesion density, and tumor-specific hormone expression (23). Quality of life and obvious symptoms were assessed using the UFS-QOL. One potential limitation of the present study is that three of the included articles did not address variables of

640

Focused ultrasound and uterine fibroids Peregrino PFM et al.

CLINICS 2017;72(10):637-641

highly vascularized and may undergo a parturition process after necrosis. In a few cases, the necrotic material was expelled via the vagina after treatment and disappeared after 4 menstrual cycles (11). The most severe complications were a case of deep vein thrombosis in 1 patient, which entailed a longer hospital stay (15), and 2 cases of endometritis requiring subsequent hysterectomy (16). Cases requiring intervention immediately after MRgFUS were rare. Hysterectomy, myomectomy, or uterine artery embolization was required for patients whose condition worsened or whose dysmenorrhea or hypermenorrhea relapsed. To date, there are no randomized clinical trials that are suitable for inclusion in a meta-analysis, which would provide a more robust analysis. Our review suggests that MRgFUS is an effective and promising therapeutic technique for decreasing myoma volume and patients’ symptoms. However, further controlled and randomized studies will be required before MRgFUS can be recommended as an alternative fibroid treatment (7-25).

10.

11.

12. 13.

14.

15.

’ AUTHOR CONTRIBUTIONS

16.

Peregrino PF designed the study and was responsible for the analysis of data and manuscript writing. Messina ML designed the study, wrote and revised the manuscript. Simões RS designed the study and was responsible for the analysis of data and manuscript writing. Soares-Júnior JM designed the study and was responsible for the analysis of data and manuscript writing. Baracat EC designed the study and wrote and revised the manuscript.

17.

18.

’ REFERENCES

19.

1. Vollenhoven BJ, Lawrence AS, Healy DL. Uterine fibroids: a clinical review. Br J Obstet Gynaecol. 1990;97(4):285-98, http://dx.doi.org/10.1111/ j.1471-0528.1990.tb01804.x. 2. Borsari R, Bozzini N, Junqueira CR, Soares JM Jr, Hilário SG, Baracat EC. Genic expression of the uterine leiomyoma in reproductive-aged women after treatment with goserelin. Fertil Steril. 2010;94(3):1072-7, http://dx.doi.org/10.1016/j.fertnstert.2009.03.112. 3. Bittencourt CA, Dos Santos Simões R, Bernardo WM, Fuchs LF, Soares Júnior JM, Pastore AR, et al. Accuracy of saline contrast sonohysterography in detection of endometrial polyps and submucosal leiomyomas in women of reproductive age with abnormal uterine bleeding: systematic review and meta-analysis. Ultrasound Obstet Gynecol. 2017;50(1):32-9, http://dx.doi.org/10.1002/uog.17352. 4. Cline HE, Hynynen K, Watkins RD, Adams WJ, Schenck JF, Ettinger RH, et al. Focused US system for MR imaging-guided tumor ablation. Radiology. 1995;194(3):731-7, http://dx.doi.org/10.1148/radiology.194.3. 7862971. 5. Fennessy FM, Tempany CM, McDannold NJ, So MJ, Hesley G, Gostout B, et al. Uterine leiomyomas: MR imaging-guided focused ultrasound surgery—results of different treatment protocols. Radiology. 2007;243(3): 885-93, http://dx.doi.org/10.1148/radiol.2433060267. 6. Hesley GK, Felmlee JP, Gebhart JB, Dunagan KT, Gorny KR, Kesler JB, et al. Noninvasive treatment of uterine fibroids: early Mayo Clinic experience with magnetic resonance imaging-guided focused ultrasound. Mayo Clin Proc. 2006;81(7):936-42, http://dx.doi.org/10.4065/81.7.936. 7. Park MJ, Kim YS, Rhim H, Lim HK. Safety and therapeutic efficacy of complete or near-complete ablation of symptomatic uterine fibroid tumors by MR imaging-guided high-intensity focused US therapy. J Vasc Interv Radiol. 2014;25(2):231-9, http://dx.doi.org/10.1016/j.jvir.2013. 11.011. 8. Mindjuk I, Trumm CG, Herzog P, Stahl R, Matzko M. MRI predictors of clinical success in MR-guided focused ultrasound (MRgFUS) treatments of uterine fibroids: results from a single centre. Eur Radiol. 2015;25(5): 1317-28, http://dx.doi.org/10.1007/s00330-014-3538-6. 9. Ikink ME, Voogt MJ, Verkooijen HM, Lohle PN, Schweitzer KJ, Franx A, et al. Mid-term clinical efficacy of a volumetric magnetic resonance-guided

20.

21.

22.

23.

24.

25.

26. 27.

641

high-intensity focused ultrasound technique for treatment of symptomatic uterine fibroids. Eur Radiol. 2013;23(11):3054-61, http://dx.doi.org/10.1007/ s00330-013-2915-x. Park H, Yoon SW, Kim KA, Jung Kim D, Jung SG. Magnetic resonance imaging-guided focused ultrasound treatment of pedunculated subserosal uterine fibroids: a preliminary report. J Vasc Interv Radiol. 2012; 23(12):1589-93, http://dx.doi.org/10.1016/j.jvir.2012.08.018. Wang W, Wang Y, Wang T, Wang J, Wang L, Tang J. Safety and efficacy of US-guided high-intensity focused ultrasound for treatment of submucosal fibroids. Eur Radiol. 2012;22(11):2553-8, http://dx.doi.org/10.1007/s00330012-2517-z. Dobrotwir A, Pun E. Clinical 24 month experience of the first MRgFUS unit for treatment of uterine fibroids in Australia. J Med Imaging Radiat Oncol. 2012;56(4):409-16, http://dx.doi.org/10.1111/j.1754-9485.2012.02376.x. Kim YS, Kim JH, Rhim H, Lim HK, Keserci B, Bae DS, et al. Volumetric MR-guided high-intensity focused ultrasound ablation with a one-layer strategy to treat large uterine fibroids: initial clinical outcomes. Radiology. 2012;263(2):600-9, http://dx.doi.org/10.1148/radiol.12111707. Kim HS, Baik JH, Pham LD, Jacobs MA. MR-guided high-intensity focused ultrasound treatment for symptomatic uterine leiomyomata: long-term outcomes. Acad Radiol. 2011;18(8):970-6, http://dx.doi.org/ 10.1016/j.acra.2011.03.008. Gorny KR, Woodrum DA, Brown DL, Henrichsen TL, Weaver AL, Amrami KK, Hangiandreou NJ, et al. Magnetic resonance-guided focused ultrasound of uterine leiomyomas: review of a 12-month outcome of 130 clinical patients. J Vasc Interv Radiol. 2011;22(6):857-64, http://dx.doi. org/10.1016/j.jvir.2011.01.458. LeBlang SD, Hoctor K, Steinberg FL. Leiomyoma shrinkage after MRI-guided focused ultrasound treatment: report of 80 patients. AJR Am J Roentgenol. 2010;194(1):274-80, http://dx.doi.org/10.2214/AJR.09.2842. Funaki K, Fukunishi H, Sawada K. Clinical outcomes of magnetic resonance-guided focused ultrasound surgery for uterine myomas: 24-month follow-up. Ultrasound Obstet Gynecol. 2009;34(5):584-9, http://dx.doi. org/10.1002/uog.7455. Harding G, Coyne KS, Thompson CL, Spies JB. The responsiveness of the uterine fibroid symptom and health-related quality of life questionnaire (UFS-QOL). Health Qual Life Outcomes. 2008;6:99, http://dx.doi.org/ 10.1186/1477-7525-6-99. Lénárd ZM, McDannold NJ, Fennessy FM, Stewart EA, Jolesz FA, Hynynen K, et al. Uterine leiomyomas: MR imaging-guided focused ultrasound surgery--imaging predictors of success. Radiology. 2008;249(1): 187-94, http://dx.doi.org/10.1148/radiol.2491071600. Morita Y, Ito N, Hikida H, Takeuchi S, Nakamura K, Ohashi H. Non-invasive magnetic resonance imaging-guided focused ultrasound treatment for uterine fibroids - early experience. Eur J Obstet Gynecol Reprod Biol. 2008;139(2):199-203, http://dx.doi.org/10.1016/j.ejogrb. 2007.10.018. Mikami K, Murakami T, Okada A, Osuga K, Tomoda K, Nakamura H. Magnetic resonance imaging-guided focused ultrasound ablation of uterine fibroids: early clinical experience. Radiat Med. 2008;26(4):198-205, http://dx.doi.org/10.1007/s11604-007-0215-6. Rabinovici J, Inbar Y, Revel A, Zalel Y, Gomori JM, Itzchak Y, et al. Clinical improvement and shrinkage of uterine fibroids after thermal ablation by magnetic resonance-guided focused ultrasound surgery. Ultrasound Obstet Gynecol. 2007;30(5):771-7, http://dx.doi.org/10.1002/uog.4099. Stewart EA, Gostout B, Rabinovici J, Kim HS, Regan L, Tempany CM. Sustained relief of leiomyoma symptoms by using focused ultrasound surgery. Obstet Gynecol. 2007;110(2 Pt 1):279-87, http://dx.doi.org/ 10.1097/01.AOG.0000275283.39475.f6. Stewart EA, Rabinovici J, Tempany CM, Inbar Y, Regan L, Gostout B, et al. Clinical outcomes of focused ultrasound surgery for the treatment of uterine fibroids. Fertil Steril. 2006;85(1):22-9, http://dx.doi.org/10.1016/ j.fertnstert.2005.04.072. Hindley J, Gedroyc WM, Regan L, Stewart E, Tempany C, Hynyen K, et al. MRI guidance of focused ultrasound therapy of uterine fibroids: early results. AJR Am J Roentgenol. 2004;183(6):1713-9, http://dx.doi. org/10.2214/ajr.183.6.01831713. Broder MS, Kanouse DE, Mittman BS, Bernstein SJ. The appropriateness of recommendations for hysterectomy. Obstet Gynecol. 2000;95(2): 199-205, http://dx.doi.org/10.1016/S0029-7844(99)00519-0. Froeling V, Meckelburg K, Schreiter NF, Scheurig-Muenkler C, Kamp J, Maurer MH, et al. Outcome of uterine artery embolization versus MRguided high-intensity focused ultrasound treatment for uterine fibroids: long-term results. Eur J Radiol. 2013;82(12):2265-9, http://dx.doi.org/ 10.1016/j.ejrad.2013.08.045.

RAPID COMMUNICATION

Plasmid-mediated mcr-1 in carbapenem-susceptible Escherichia coli ST156 causing a blood infection: an unnoticeable spread of colistin resistance in Brazil? Flavia Rossi,I,* Raquel Girardello,I Carlos Morais,II,III Ana Paula Cury,I Layla Farage Martins,II Aline Maria da Silva,II Edson Abdala,IV Joa˜o Carlos Setubal,II,III Alberto Jose´ da Silva DuarteI I

Divisao Laboratorio Central – LIM03, Setor de Microbiologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sao Paulo, Sao Paulo, SP, BR. III Nucleo de Apoio a Pesquisa em Biologia Computacional e Genomica (NUBIC), Universidade de Sao Paulo, Sao Paulo, SP, BR. IV Instituto do Cancer do Estado de Sao Paulo, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVE: We describe an IncX4 pHC891/16mcr plasmid carrying mcr-1 in a colistin-resistant and carbapenemsusceptible E. coli isolate (HC891/16), ST156, which caused a blood infection in a Brazilian patient with gallbladder adenocarcinoma. METHODS: Strain HC891/16 was subjected to whole genome sequencing using the MiSeq Platform (Illumina, Inc., USA). Assembly was performed using Mira and ABACAS. RESULTS: The isolates showed resistance only to ciprofloxacin, ampicillin and cefoxitin, and whole-genome sequencing revealed the presence of aac(6’)Ib-cr and blaTEM1. CONCLUSION: Our findings warn of the possible silent dissemination of colistin resistance by carbapenemsusceptible mcr-1 producers, as colistin susceptibility is commonly tested only among carbapenem-resistant isolates. KEYWORDS: Colistin Resistance; Polymyxin Resistance; Escherichia Coli; Carbapenem-Susceptible; ST156. Rossi F, Girardello R, Morais C, Cury AP, Martins LF, Silva AM, et al. Plasmid-mediated mcr-1 in carbapenem-susceptible Escherichia coli ST156 causing a blood infection: an unnoticeable spread of colistin resistance in Brazil? Clinics. 2017;72(10):642-644 Received for publication on March 7, 2017; First review completed on May 23, 2017; Accepted for publication on May 30, 2017 *Corresponding author. E-mail: flaviarossi61@gmail.com

carbapenem-susceptible E. coli isolate that caused a blood infection in a cancer patient from Brazil. ECF, a 75-year-old female with a gallbladder adenocarcinoma diagnosed in January 2016, was admitted to Hospital das Clínicas, São Paulo for investigation, and a hepatic drain was placed. Bile cultures were positive for Enterococcus faecalis susceptible to all drugs. One month later, she developed fever and presented to the emergency room (ER). Blood cultures were positive for E. coli phenotypically classified as an ESbL-producer that was susceptible to carbapenem and aminoglycosides, resistant to ciprofloxacin (MIC44 mg/L) and with a colistin MICp0.5 mg/L. She received meropenem with good clinical evolution. One month later, she began ambulatory chemotherapy (cisplatin and gemcitabine). On April 11, 2016, she returned to the ER with a high fever, leukocytosis, and elevated CRP, and her bloodcultures were positive for E. coli with a different phenotypic profile. It was susceptible to amikacin (MIC, p2 mg/L), gentamicin (MIC, 2 mg/mL), cefuroxime (MIC, 4 mg/L), ceftazidime (MIC, p1 mg/L), ceftriaxone (MIC, p1 mg/L), cefepime (MIC, p1 mg/L), ertapenem (MIC, p0.5 mg/L), imipenem (MIC, p0.25 mg/L), meropenem (MIC, p0.25 mg/L), piperacillin/tazobactam (MIC, p4 mg/L) and tigecycline (MIC, p0.5 mg/L); intermediate to cefoxitin (MIC, 16 mg/L);

Liu et al. (1) reported the emergence of an mcr-1 plasmidial resistance mechanism to colistin in E. coli from China, which was followed by many reports worldwide (2-7). In Brazil, Fernandes et al. (8) reported an IncX4 plasmid carrying mcr-1 in E. coli isolated from a soft tissue infection in a human. These authors also described evidence that E. coli harboring mcr-1 has been occurring in food-producing animals in Brazil since at least 2012 (9). However, to date, there is no consensus in the scientific community regarding the clinical importance of this new colistin resistance mechanism, as it confers a low level of colistin resistance compared with chromosomal mechanisms and appears to have been present for a long time (10). Epidemiological data can be used to elucidate the impact of this plasmidial mechanism for the dissemination of colistin resistance. In this study, we describe a plasmid-mediated mcr-1 in a colistin-resistant and

642

mcr-1 producer-Escherichia coli in Brazil Rossi F et al.

CLINICS 2017;72(10):642-644

and resistant to ampicillin (MIC, 432 mg/L) and ciprofloxacin (MIC, 44 mg/L). The colistin MIC was 4 mg/L. This strain was positive for mcr-1 by PCR using specific primers as previously described (1) and was denominated HC891/16. The patient received antibiotics and support medication and was discharged with no infection. The identification and antimicrobial susceptibility tests of the isolates were performed by Vitek-MS and Vitek-2 (bioMeriéux, France), respectively, and colistin susceptibility was confirmed by CLSI broth microdilution (11). Strain HC891/16 was subjected to whole genome sequencing using the MiSeq Platform (Illumina, Inc., USA). The sequencing library was prepared from 40 ng of HC891/16 strain total DNA using the Illumina Nextera DNA library preparation kit (Illumina, Inc., USA). After quantification of the resulting library using the KAPA Library Quantification Kit, a sequencing run was performed in the Illumina MiSeq to generate 250 bp pairedend reads. Assembly was performed using Mira (12) and ABACAS (13), resulting in 71 contigs encompassing the main chromosome (5,053,377 bp) and at least one plasmid (33,227 bp). Genome sequences were deposited in GenBank under accession number MRDN01000000. The presence of resistance genes was determined with ResFinder (14). The strain HC891/16 belongs to sequence type (ST) 156. E. coli ST156 harboring the mcr-1 gene was previously detected in one duck sample, from China, showing a carbapenemresistant profile (15). The mcr-1 gene is in pHC891/16mcr, a 33.2-kb plasmid, which is classified as incompatibility group IncX4. This plasmid shows 100% nucleotide identity to pICBEC7Pmcr (CP017246.1), and it is found in animal samples from Latin America. Furthermore, 99% nucleotide identity was observed when this plasmid was compared with the following previously described conjugative plasmids harboring the mcr-1 gene: pOW3E1 (KX129783.1), detected in environmental and animal samples; pMCR1.2IT (KX236309.1), harboring a mcr-1.2 variant of K. pneumoniae from a human infection in Italy; pmcr1_IncX4 (KU761327.1), found in clinical isolates from China; pMCR1-NJ-IncX4 (KX447768.1), found in a carbapenem-resistant E. coli isolate harboring mcr-1 and blaNDM-5, causing a complicated urinary tract infection in the USA (16-19). The IncX4 plasmid appears to be globally disseminated among environmental, food and human mcr-1 producer strains, including KPC-2 and a mcr-1 producer-K. pneumoniae strain that have been recently reported in Brazil (20). In addition to mcr-1, we also detected the presence of genes aac(6')Ib-cr and blaTEM1 in the HC891/16 strain. In a previous study from our institution, carbapenem susceptibility was observed in 21.5% of all colistin-resistant Gram-negative isolates examined in the last five years (21). It is important to highlight that the carbapenem-susceptible and mcr-1-positive phenotype may easily go unnoticed because many clinical laboratories in Brazil only test or release colistin susceptibility to clinicians when the isolates are carbapenem-resistant. Olaitan et al. (22) state that clinicians and microbiologists should be vigilant for the possible existence of colistin-resistant bacteria not only in patients undergoing colistin therapy but also in patients who are not receiving the drug, as resistant bacteria could later be selected if colistin is used. In Brazil, colistin has been used as a first-line drug in many critical care units due to high rates of carbapenem-resistance among the OXA23-producer Acinetobacter baumannii and KPC- producer K. pneumoniae (23,24).

The dissemination of colistin resistance is a serious problem for infection control, as this antimicrobial is one of the last therapeutic options for infections caused by multidrugresistant Gram-negative isolates. At this time, attention should be paid to this carbapenem-susceptible-mcr-1 phenotype, as these isolates may be silently contributing to the spread of colistin resistance.

’ ACKNOWLEDGMENTS We would like to thank the Microbiology Laboratory group of HC-FMUSP for its signiﬁcant contributions to the clinical routine.

’ AUTHOR CONTRIBUTIONS Rossi F and Girardello R were responsible for the data analysis and preparation of the manuscript. Morais C, Martins LF, Silva AM, Setubal JC were responsible for the bioinformatics analysis. Cury AP was responsible for the data analysis. Abdala E was responsible for the description of clinical case. Duarte AJ was responsible for the preparation of the manuscript.

’ REFERENCES 1. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16(2):161-8, http://dx.doi.org/10.1016/S14733099(15)00424-7. 2. Hasman H, Hammerum AM, Hansen F, Hendriksen RS, Olesen B, Agersø Y, et al. Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015. Euro Surveill. 2015;20(49), http://dx.doi. org/10.2807/1560-7917.ES.2015.20.49.30085. 3. Du H, Chen L, Tang YW, Kreiswirth BN. Emergence of the mcr-1 colistin resistance gene in carbapenem-resistant Enterobacteriaceae. Lancet Infect Dis. 2016;16(3):287-8, http://dx.doi.org/10.1016/S1473-3099(16)00056-6. 4. Falgenhauer L, Waezsada SE, Yao Y, Imirzalioglu C, Käsbohrer A, Roesler U, et al. Colistin resistance gene mcr-1 in extended-spectrum b-lactamaseproducing and carbapenemase-producing Gram-negative bacteria in Germany. Lancet Infect Dis. 2016;16(3):282-3, http://dx.doi.org/10.1016/ S1473-3099(16)00009-8. 5. Olaitan AO, Chabou S, Okdah L, Morand S, Rolain JM. Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis. 2016;16(2):147, http://dx.doi.org/10.1016/S1473-3099(15)00540-X. 6. Stoesser N, Mathers AJ, Moore CE, Day NP, Crook DW. Colistin resistance gene mcr-1 and pHNSHP45 plasmid in human isolates of Escherichia coli and Klebsiella pneumoniae. Lancet Infect Dis. 2016;16(3):285-6, http://dx. doi.org/10.1016/S1473-3099(16)00010-4. 7. Rapoport M, Faccone D, Pasteran F, Ceriana P, Albornoz E, Petroni A, et al. First Description of mcr-1-Mediated Colistin Resistance in Human Infections Caused by Escherichia coli in Latin America. Antimicrob Agents Chemother. 2016;60(7):4412-3, http://dx.doi.org/10.1128/AAC.00573-16. 8. Fernandes MR, McCulloch JA, Vianello MA, Moura Q, Pérez-Chaparro PJ, Esposito F, et al. First Report of the Globally Disseminated IncX4 Plasmid Carrying the mcr-1 Gene in a Colistin-Resistant Escherichia coli Sequence Type 101 Isolate from a Human Infection in Brazil. Antimicrob Agents Chemother. 2016;60(10):6415-7, http://dx.doi.org/10.1128/AAC. 01325-16. 9. Fernandes MR, Moura Q, Sartori L, Silva KC, Cunha MP, Esposito F, et al. Silent dissemination of colistin-resistant Escherichia coli in South America could contribute to the global spread of the mcr-1 gene. Euro Surveill. 2016;21(17), http://dx.doi.org/10.2807/1560-7917.ES.2016.21.17.30214. 10. Skov RL, Monnet DL. Plasmid-mediated colistin resistance (mcr-1 gene): three months later, the story unfolds. Euro Surveill. 2016;21(9):30155, http://dx.doi.org/10.2807/1560-7917.ES.2016.21.9.30155. 11. CLSI. Clinical Laboratory Standard Institute Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard— Document M07-A9. Wayne, Pa. 2012, http://dx. doi.org/10.1016/j.jpha.2015.11.005. 12. Chevreux B, Wetter T, Suhai S. Genome Sequence Assembly Using Trace Signals and Additional Sequence Information. Computer Science and Biology: Proceedings of the German Conference on Bioinformatics (GCB). 1999;99:45-56. 13. Assefa S, Keane TM, Otto TD, Newbold C, Berriman M. ABACAS: algorithm-based automatic contiguation of assembled sequences. Bioinformatics. 2009;25(15):1968-9, http://dx.doi.org/10.1093/bioinforma tics/btp347.

643

mcr-1 producer-Escherichia coli in Brazil Rossi F et al.

CLINICS 2017;72(10):642-644

14. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67(11):2640-4, http://dx.doi.org/10.1093/ jac/dks261. 15. Yang RS, Feng Y, Lv XY, Duan JH, Chen J, Fang LX, et al. Emergence of NDM-5- and MCR-1-Producing Escherichia coli Clones ST648 and ST156 from a Single Muscovy Duck (Cairinamoschata). Antimicrob Agents Chemother. 2016;60(11):6899-6902, http://dx.doi.org/10.1128/ AAC.01365-16. 16. Zurfluh K, Klumpp J, Nüesch-Inderbinen M, Stephan R. Full-Length Nucleotide Sequences of mcr-1-Harboring Plasmids Isolated from Extended-Spectrum-b-Lactamase-Producing Escherichia coli Isolates of Different Origins. Antimicrob Agents Chemother. 2016;60(9):5589-91, http://dx.doi.org/10.1128/AAC.00935-16. 17. Di Pilato V, Arena F, Tascini C, Cannatelli A, Henrici De Angelis L, Fortunato S, et al. mcr-1.2, a New mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella pneumoniae Strain of Sequence Type 512. Antimicrob Agents Chemother. 2016;60(9):5612-5, http://dx.doi.org/10.1128/AAC.01075-16. 18. Li A, Yang Y, Miao M, Chavda KD, Mediavilla JR, Xie X, et al. Complete Sequences of mcr-1-Harboring plasmids from Extended-Spectrum-bLactamase- and Carbapenemase-Producing Enterobacteriaceae. Antimicrob Agents Chemother. 2016;60(7):4351-4, http://dx.doi.org/10.1128/ AAC.00550-16. 19. Mediavilla JR, Patrawalla A, Chen L, Chavda KD, Mathema B, Vinnard C, et al. Colistin- and Carbapenem-Resistant Escherichia coli Harboring mcr-1 and blaNDM-5, Causing a Complicated Urinary Tract Infection in a

20.

21.

22.

23.

24.

644

Patient from the United States. MBio. 2016;7(4).pii:e01191-16, http://dx. doi.org/10.1128/mBio.01191-16. Aires CAM, da Conceição-Neto OC, Tavares E Oliveira TR, Dias CF, Montezzi LF, Picão RC, et al. Emergence of the Plasmid-Mediated mcr-1 Gene in Clinical KPC-2-Producing Klebsiella pneumoniae Sequence Type 392 in Brazil. Antimicrob Agents Chemother. 2017;61(7).pii:e00317-17, http://dx.doi.org/10.1128/AAC.00317-17. Rossi F, Girardello R, Cury AP, Di Gioia TS, Almeida JN Jr, Duarte AJ. Emergence of colistin resistance in the largest university hospital complex of São Paulo, Brazil, over five years. Braz J Infect Dis. 2017;21(1):98-101, http://dx.doi.org/10.1016/j.bjid.2016.09.011. Olaitan AO, Morand S, Rolain JM. Emergence of colistin-resistant bacteria in humans without colistin usage: a new worry and cause for vigilance. Int J Antimicrob Agents. 2016;47(1):1-3, http://dx.doi.org/10.1016/ j.ijantimicag.2015.11.009. Agência Nacional de Vigilância Sanitária (ANVISA). Boletim Informativo: Segurança e qualidade em serviço de saúde no 12: Relatório da resistência microbiana em infecções primárias de corrente sanguínea confirmadas laboratorialmente relacionadas ao uso de cateter venoso central em unidades de terapia intensiva (2014). Acessado em 05 de agosto 2016. Disponível em: http://www20.anvisa.gov.br/segurancadopaciente/index. php/publicacoes/item/12 Vasconcelos AT, Barth AL, Zavascki AP, Gales AC, Levin AS, Lucarevschi BR, et al. The changing epidemiology of Acinetobacter spp. producing OXA carbapenemases causing bloodstream infections in Brazil: a BrasNet report. Diagn Microbiol Infect Dis. 2015;83(4):382-5, http://dx.doi.org/ 10.1016/j.diagmicrobio.2015.08.006.

RAPID COMMUNICATION

Reduction of venous pressure during the resection of liver metastases compromises enteric blood flow: IGFBP-1 as a novel biomarker of intestinal barrier injury Hermes Vieira Barbeiro,I Marcel Autran Ce´sar Machado,II Heraldo Possolo de Souza,I Fabiano Pinheiro da Silva,I Marcel Cerqueira Ce´sar MachadoI,II,* I Departamento de Emergencias Clinicas, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Hospital Sirio Libanes, Sao Paulo, SP, BR.

OBJECTIVES: Disruption of the intestinal barrier and bacterial translocation commonly occur when intestinal blood flow is compromised. The aim of this study was to determine whether liver resection induces intestinal damage. METHODS: We investigated intestinal fatty-acid binding protein and insulin-like growth factor binding protein levels in the plasma of patients who underwent liver resection. RESULTS: We show that liver resection is associated with significant intestinal barrier injury, even if the Pringle maneuver is not performed. CONCLUSION: We propose the use of insulin-like growth factor binding protein-1 as a novel biomarker of intestinal damage in such situations. KEYWORDS: Liver; Inflammation; Intestinal Barrier; Bacterial Translocation; IGFBP-1. Barbeiro HV, Machado MA, de Souza HP, Pinheiro da Silva F, Machado MC. Reduction of venous pressure during the resection of liver metastases compromises enteric blood flow: IGFBP-1 as a novel biomarker of intestinal barrier injury. Clinics. 2017;72(10):645-648 Received for publication on April 3, 2017; First review completed on May 25, 2017; Accepted for publication on July 12, 2017 *Corresponding author. E-mail: mccm37@uol.com.br

’ INTRODUCTION

whether lowering of the central venous pressure is sufficient to promote acute intestinal barrier dysfunction in patients subjected to liver resection without the Pringle maneuver.

Several reports have revealed that in several clinical situations, such as burns (1,2), hemorrhagic shock (3,4), acute pancreatitis (5,6), aortic dissection and aortic surgery (7), sepsis (8) and after a Pringle maneuver during liver resection surgery (9), the intestinal barrier is disrupted when intestinal blood flow is compromised. Because blood transfusion secondary to intraoperative blood loss is associated with early- and long-term complications in patients subjected to liver resections, several strategies, such as total (10) or intermittent (9) Pringle maneuvers and the reduction of central venous pressure, have been used to minimize blood loss. During the Pringle maneuver, however, reduction of intestinal perfusion results in intestinal barrier dysfunction and endotoxemia (9). Similarly, we hypothesized that reduction of central venous pressure during liver resection followed by reduction of arterial pressure would also reduce intestinal blood flow and damage the intestinal barrier. The aim of this study was to investigate

’ PATIENTS AND METHODS Eighteen patients who underwent liver surgery at Hospital Sirio Libanes in São Paulo (Brazil) were eligible for inclusion in this study. All patients had colon cancer. Surgeries were performed to resect one or multiple liver metastases. Only three patients, who required the Pringle maneuver, were excluded. Perioperative profile of the study population is shown in Table 1. Informal consent was obtained from every patient. The protocols employed followed the Hospital Sirio Libanes guidelines, were approved by their ethics committee (protocol number 2013-54) and were performed in accordance with the principles of the National Council of Animal Experiment Control (Concea). Central venous pressure was maintained at a low level (below 10 mmHg) during liver resection to reduce bleeding and to facilitate liver tissue manipulation. Seventeen patients underwent major liver resection (43 segments), and one patient underwent a minor resection (segmentectomy). During the procedure, catheters were inserted to monitor the central venous and radial arterial pressures. Parenchymal resection was performed using bipolar cautery, sutures and vascular staples. Blood samples were obtained from the

645

IGFBP-1 and intestinal injury Barbeiro HV et al.

CLINICS 2017;72(10):645-648

venous pressure. However, we hypothesized that this strategy would reduce the arterial blood pressure, which might also compromise the intestinal blood flow. As previously demonstrated, reduction of intestinal blood flow is followed by intestinal barrier dysfunction and bacterial translocation (11), and several reports have shown that IFABP is an accurate biomarker of intestinal damage (8,12). In the present study, we observed a significant increase in IFABP blood levels, at 8h after hepatic resection, with a reduction of the central venous pressure during liver transection even without the Pringle maneuver (Figure 1A), and a similar trend was observed for the IL-6 plasma levels. These results indicate that liver resection induces a significant local and systemic inflammatory response, even affecting organs located at sites distant from the surgical procedure, such as the intestines. IGFBPs are important physiological regulators of the interaction of insulin-like growth factors (IGFs) with their receptors within the gastrointestinal tract and liver (13,14), and thus, we decided to investigate them as novel potential biomarkers of acute intestinal dysfunction. The seven cloned mammalian IGFBPs are implicated in cell proliferation and survival as well as in several other cellular responses. IGFBPs are secreted by human intestinal epithelial cells and may leak into the systemic circulation in situations involving intestinal damage. In the present study, we observed a significant increase in IGFBP-1 blood levels after liver resection (Figure 2A). However, other IGFBPs presented a postoperative blood level decrease after hepatic resection (Figure 2). In a previous study, intestinal epithelial cell damage and endotoxemia were demonstrated to occur after the Pringle maneuver during liver resection (9), but patients submitted to liver resection without the Pringle maneuver did not present intestinal damage. Those patients, however, were more hemodynamically stable and did not require the Pringle maneuver. In our study, all patients were operated on without the Pringle maneuver, independent of their hemodynamic conditions. In our patients, central venous pressure was maintained below 10 cm H2O by pharmacological manipulation, and intestinal barrier dysfunction was detected. Elevation of IGFBP-1 has also been observed during critical illnesses (15), most likely due to systemic inflammation. However, in our study, the IGFBP-1 levels had returned to normal (data not shown) at 24h after liver resection. Elevations of IGFBP-1 after burn injury (16) may also (at least in part) be related to acute intestinal dysfunction (17).

arterial line at the start of the procedure and 8h later. Blood was collected in EDTA-containing vacuum tubes, kept on ice, and centrifuged at 4oC; the plasma was stored at -70oC. Intestinal fatty acid binding protein (IFABP), a specific marker of intestinal barrier dysfunction (8), was measured by an enzyme-linked immunosorbent assay (ELISA kit, MyBIOSource, USA), according to instructions provided in the manual. Insulin-like growth factor binding protein (IGFBP) plasma measurements were performed using Milliplex technology (Merck, Genese Diagnostics, Darmstadt, Germany). Plasma IL-6 levels were determined by an enzyme-linked immunosorbent assay (R&D Biosystems, USA).

Statistical analysis

Data are expressed as the means±SD. The results were compared using Student’s t-test. A p value of o0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 5.0 for Windows.

’ RESULTS AND DISCUSSION We found a significant increase in the plasma levels of IFABP (Figure 1A), IL-6 (Figure 1B) and IGFBP-1 (Figure 2A) at 8h after the start of the surgeries. In contrast, the IGFBP-3, IGFBP-4, IGFBP-6 and IGFBP-7 plasma levels were decreased after the surgical procedure (Figure 2 C, D, F and G). Previous studies have shown that, in patients undergoing liver resection, total or intermittent Pringle maneuvers are associated with an increase in IFABP plasma levels due to the loss of enterocyte membrane integrity. In addition to the Pringle maneuver, alternative strategies have been used during hepatic resection to minimize intraoperative blood loss, such as the pharmacological reduction of central Table 1 - Perioperative profile of the study population (means). Standard deviations are listed in parenthesis. Age Gender male/female Blood loss (mL) Number of resected segments Postoperative aspartate aminotransferase IU/L Postoperative alanine aminotransferase IU/L Postoperative gamma-glutamyl transpeptidase IU/L Postoperative total bilirubin mg/dL Postoperative creatinine mg/dL

60.6 (29-77) 7/9 515 (100-1500) 3 (1-4) 503 (76-1306) 366 (59-697) 144 (28-405) 1.97 (0.22-4.93) 1.51 (0.65-3.30)

Figure 1 - Intestinal fatty-acid binding protein (IFABP, Figure 1A) and IL-6 (Figure 1B) plasma levels at the beginning of liver resection (in white) and 8 h later (in black).

646

IGFBP-1 and intestinal injury Barbeiro HV et al.

CLINICS 2017;72(10):645-648

Figure 2 - Insulin-like growth factors binding proteins (IGFBP) 1 to 7 plasma levels (Figures A to G) at the start of liver resections (in white) and 8 hours later (in black).

In conclusion, the present study demonstrated that, even without the Pringle maneuver, intestinal barrier dysfunction most likely occurs secondary to the reduction of intestinal blood flow induced by pharmacological manipulation of the central venous and arterial pressure. In the modern era of liver surgery, the Pringle maneuver has been restricted to situations in which major bleeding occurs. Future investigations are necessary to establish whether intestinal barrier dysfunction plays a role in the etiology of infectious or noninfectious perioperative complications. Intestinal barrier dysfunction may be an important concern in patients with small liver remnants. In fact, endotoxin translocation, secondary to intestinal barrier dysfunction, may induce liver damage (18) and further jeopardize liver function. An alternative approach would be to perform a selective intrahepatic Glissonian approach (19) and proceed to liver resection without a central venous pressure reduction, thereby maintaining the intestinal perfusion and epithelial cell integrity. However, this approach still needs to be further investigated. A previous study in patients with biliary malignancy demonstrated that bacterial translocation can predict the occurrence of postoperative infectious complications after liver resection (20). Thus, we propose that novel techniques to perform liver resections without the Pringle maneuver and without reduction of the central venous pressure should be explored because both of the latter procedures compromise the intestinal integrity. IFAPB is a reliable marker of intestinal injury even before such injury can be detected by histological analysis. The gut is the main producer of IGFBP-1, although other organs can produce it in smaller amounts. We suggest that IGFBP-1 is a novel marker of intestinal damage, but further studies are necessary to confirm this finding.

2. Costantini TW, Loomis WH, Putnam JG, Drusinsky D, Deree J, Choi S, et al. Burn-induced gut barrier injury is attenuated by phosphodiesterase inhibition: effects on tight junction structural proteins. Shock. 2009;31(4): 416-22, http://dx.doi.org/10.1097/SHK.0b013e3181863080. 3. Rhodes RS, Depalma RG, Robinson AV. Intestinal barrier function in hemorrhagic shock. J Surg Res. 1973;14(4):305-12, http://dx.doi.org/10.1016/ 0022-4804(73)90032-2. 4. Deitch EA, Bridges W, Berg R, Specian RD, Granger DN. Hemorrhagic shock-induced bacterial translocation: the role of neutrophils and hydroxyl radicals. J Trauma. 1990;30(8):942-51; discussion 951-2. 5. Barbeiro DF, Koike MK, Coelho AM, da Silva FP, Machado MC. Intestinal barrier dysfunction and increased COX-2 gene expression in the gut of elderly rats with acute pancreatitis. Pancreatology. 2016;16(1):52-6, http://dx.doi.org/10.1016/j.pan.2015.10.012. 6. Pan L, Wang X, Li W, Li N, Li J. The intestinal fatty acid binding protein diagnosing gut dysfunction in acute pancreatitis: a pilot study. Pancreas. 2010;39(5):633-8, http://dx.doi.org/10.1097/MPA.0b013e3181c79654. 7. Gu J, Hu J, Qian H, Shi Y, Zhang E, Guo Y, et al. Intestinal Barrier Dysfunction: A Novel Therapeutic Target for Inflammatory Response in Acute Stanford Type A Aortic Dissection. J Cardiovasc Pharmacol Ther. 2016;21(1):64-9, http://dx.doi.org/10.1177/1074248415581176. 8. Machado MC, Barbeiro HV, Pinheiro da Silva F, de Souza HP. Circulating fatty acid binding protein as a marker of intestinal failure in septic patients. Crit Care. 2012;16(6):455, http://dx.doi.org/10.1186/cc11653. 9. Dello SA, Reisinger KW, van Dam RM, Bemelmans MH, van Kuppevelt TH, van den Broek MA, et al. Total intermittent Pringle maneuver during liver resection can induce intestinal epithelial cell damage and endotoxemia. PloS One. 2012;7(1):e30539, http://dx.doi.org/10.1371/journal. pone.0030539. 10. Pringle JH V. Notes on the Arrest of Hepatic Hemorrhage Due to Trauma. Ann Surg. 1908;48(4):541-9, http://dx.doi.org/10.1097/00000658-19081000000005. 11. Holland J, Carey M, Hughes N, Sweeney K, Byrne PJ, Healy M, et al. Intraoperative splanchnic hypoperfusion, increased intestinal permeability, down-regulation of monocyte class II major histocompatibility complex expression, exaggerated acute phase response, and sepsis. Am J Surg. 2005;190(3):393-400, http://dx.doi.org/10.1016/j.amjsurg.2005. 03.038. 12. Derikx JP, van Waardenburg DA, Thuijls G, Willigers HM, Koenraads M, van Bijnen AA, et al. New Insight in Loss of Gut Barrier during Major Non-Abdominal Surgery. PloS One. 2008;3(12):e3954, http://dx.doi.org/ 10.1371/journal.pone.0003954. 13. Kuemmerle JF. Insulin-like growth factors in the gastrointestinal tract and liver. Endocrinol Metab Clin North Am. 2012;41(2):409-23, http://dx.doi. org/10.1016/j.ecl.2012.04.018. 14. Bach LA. Insulin-Like Growth Factor Binding Proteins–an Update. Pediatr Endocrinol Rev. 2015;13(2):521-30. 15. Baxter RC. Changes in the IGF-IGFBP axis in critical illness. Best Pract Res Clin Endocrinol Metab. 2001;15(4):421-34, http://dx.doi.org/10.1053/ beem.2001.0161. 16. Mendoza AE, Maile LA, Cairns BA, Maile R. Burn injury induces high levels of phosphorylated insulin-like growth factor binding protein-1. Int J Burns Trauma. 2013;3(4):180-9. 17. Costantini TW, Peterson CY, Kroll L, Loomis WH, Putnam JG, Wolf P, et al. Burns, inflammation, and intestinal injury: protective effects of an

’ AUTHOR CONTRIBUTIONS Barbeiro HV performed the experiments. The remaining authors designed the project and wrote the manuscript.

’ REFERENCES 1. Deitch EA. Intestinal permeability is increased in burn patients shortly after injury. Surgery. 1990;107(4):411-6, http://dx.doi.org/10.1002/bjs.1800770541.

647

IGFBP-1 and intestinal injury Barbeiro HV et al.

CLINICS 2017;72(10):645-648

study over 7 years. Surgery. 2016;160(3):643-51, http://dx.doi.org/10.1016/ j.surg.2016.01.017. 20. Mizuno T, Yokoyama Y, Nishio H, Ebata T, Sugawara G, Asahara T, et al. Intraoperative bacterial translocation detected by bacterium-specific ribosomal rna-targeted reverse-transcriptase polymerase chain reaction for the mesenteric lymph node strongly predicts postoperative infectious complications after major hepatectomy for biliary malignancies. Ann Surg. 2010;252(6):1013-9, http://dx.doi.org/10.1097/SLA.0b013e3181f3f355.

anti-inflammatory resuscitation strategy. J Trauma. 2009;67(6):1162-8, http://dx.doi.org/10.1097/TA.0b013e3181ba3577. 18. Henao-Mejia J, Elinav E, Thaiss CA, Licona-Limon P, Flavell RA. Role of the intestinal microbiome in liver disease. J Autoimmun. 2013;46:66-73, http://dx.doi.org/10.1016/j.jaut.2013.07.001. 19. Machado MA, Surjan RC, Basseres T, Schadde E, Costa FP, Makdissi FF. The laparoscopic Glissonian approach is safe and efficient when compared with standard laparoscopic liver resection: Results of an observational

648

No Rebouças, pequenos eventos

tornam-se grandes.

Seja pequeno, médio ou grande, o Rebouças tem o espaço ideal para todo o tipo de evento. A melhor logística e infraestrutura de apoio maximizam os recursos operacionais e potencializam os encontros científicos, promocionais, institucionais ou culturais. O Centro de Convenções Rebouças é isso: um dos mais bem planejados e tradicionais espaços receptivos do País, dimensionado para sediar congressos, convenções, exposições, seminários, simpósios, cursos, lançamento de produtos, entre outros, bem no coração da cidade de São Paulo.

Atualmente possui 8 ambientes climatizados e modernizados que atendem até 1.200 participantes, além de uma equipe preparada e empenhada em acompanhar cerca de 280 eventos por ano. EM BREVE, estará ampliando suas instalações, que permitirá o dobro da capacidade de público.

Av. Rebouças, 600 - 05402-000 - São Paulo - Brasil - Tel.: 55 11 3898-7850 / Fax: 55 11 3898-7878 - reboucas@hcnet.usp.br