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Volume 70

Number 8 - August/2015

CLINICS
Editor
Edmund Chada Baracat

Faculdade de Medicina da Universidade de So Paulo


So Paulo, SP, Brazil

Area Editors
Ana Maria de Ulhoa Escobar
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Jos Luiz Gomes do Amaral


Universidade Federal de So Paulo
So Paulo, SP, Brazil

Paulo Pgo-Fernandes
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Anna Sara Shafferman Levin


Faculdade de Medicina da Universidade de So Paulo
Sao Paulo, SP, Brazil

Ludhmila Abrahao Hajjar


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

Raul Coimbra
University of California, San Diego
La Jolla, CA, USA

Antonio Egidio Nardi


Universidade Federal do Rio de Janeiro
Rio de Janeiro, RJ, Brazil

Luiz Eugenio Garcez-Leme


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

Renato Delascio Lopes


Duke University Medical Center
Durham, NC, USA

Anuar Ibrahim Mitre


Faculdade de Medicina da Universidade de So Paulo
Sao Paulo, SP, Brazil

Luz Fernando Onuchic


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

Ricardo Bassil Lasmar


Universidade Federal Fluminense
Niteri, RJ, Brazil

Berenice Bilharinho Mendonca


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

Lydia Masako Ferreira


Universidade Federal de So Paulo
So Paulo, SP, Brazil

Rosa Maria Rodrigues Pereira


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

Bruno Zilberstein
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Marcos Intaglietta
University of California, San Diego
San Diego, CA, USA

Rossana Francisco
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Carlos Serrano
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Mauricio Etchebehere
Universidade Estadual de Campinas
Campinas, SP, Brazil

Rubens Belfort Jr.


Universidade Federal de So Paulo
So Paulo, SP, Brazil

Carmen Silvia Valente Barbas


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

Michele Correale
University of Foggia
Foggia, Italy

Ruth Guinsburg
Universidade Federal de So Paulo
So Paulo, SP, Brazil

Claudia Regina Furquim de Andrade


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

Naomi Kondo Nakagawa


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

Emilia Inoue Sato


Universidade Federal de So Paulo
So Paulo, SP, Brazil

Nelson Wolosker
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Sandro Esteves
ANDROFERT - Andrology & Human
Reproduction Clinic
Campinas, SP, Brazil

Fulvio Alexandre Scorza


Universidade Federal de So Paulo
So Paulo, SP, Brazil

Newton Kara-Junior
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Sergio Paulo Bydlowski


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

Geraldo Busatto
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Olavo Pires de Camargo


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

Simone Appenzeller
Universidade Estadual de Campinas
Campinas, SP, Brazil

Heitor Franco de Andrade Jr.


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

Oswaldo Keith Okamoto


Universidade de So Paulo
So Paulo, SP, Brazil

Suely Kazue Nagahashi Marie


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

Jesus Paula Carvalho


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

Patricia Rieken Macedo Rocco


Universidade Federal do Rio de Janeiro
Rio de Janeiro, RJ, Brazil

Valeria Aoki
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Joaquim Prado Moraes-Filho


Faculdade de Medicina da Universidade de So Paulo
Scio Paulo, SP, Brazil

Paulo Hoff
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Ruy Jorge Cruz Junior


University of Pittsburgh
Pittsburgh, PA, USA

Editorial Board
Abhijit Chandra
King Georges Medical College
Lucknow, India

Arnaldo Valdir Zumiotti


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

Ernest Eugene Moore


University of Colorado Denver
Denver, CO, USA

Adamastor Humberto Pereira


Universidade Federal do Rio Grande
do Sul
Porto Alegre, RS, Brazil

Artur Brum-Fernandes
Universit de Sherbrooke
Qubec, Canad

Euclides Ayres Castilho


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

Adauto Castelo
Universidade Federal de So Paulo
So Paulo, SP, Brazil

Carmita Helena Najjar Abdo


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

Ademar Lopes
Fundao Antnio Prudente, Hospital
do Cncer
So Paulo, SP, Brazil

Cesar Gomes Victora


Faculdade de Medicina da Universidade
Federal de Pelotas
Pelotas, RS, Brasil

Alberto Azoubel Antunes


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

Daniel Romero Muoz


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

Alexandre Roberto Precioso


Faculdade de Medicina da Universidade
de So Paulo
So Paulo, SP, Brazil
Andrea Schmitt
University of Goettingen
Goettingen, Germany

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

Ivan Cecconello
Faculdade de Medicina da Universidade
de So Paulo
So Paulo, SP, Brazil
Ke-Seng Zhao
Southern Medical University
Guangzhou, China

Fbio Biscegli Jatene


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

Laura Cunha Rodrigues


London School of Hygiene and
Tropical Medicine - University
of London London, UK

Francisco Laurindo
Faculdade de Medicina da Universidade
de So Paulo
So Paulo, SP, Brazil

Marcelo Zugaib
Faculdade de Medicina da Universidade
de So Paulo
So Paulo, SP, Brazil

Hiroyuki Hirasawa
Chiba University School of Medicine
Chiba, Japan

Marco Martins Amatuzzi


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

Irismar Reis de Oliveira


Faculdade de Medicina da Universidade
Federal da Bahia
Salvador, BA, Brasil
Irshad Chaudry
University of Alabama
Birmingham, AL, USA

Maria Aparecida Shikanai Yasuda


Faculdade de Medicina da Universidade
de So Paulo So Paulo, SP, Brazil
Mauro Perretti
William Harvey Research Institute
London, UK

Michael Gregory Sarr


Mayo Clinic
Rochester, MN, USA
Milton de Arruda Martins
Faculdade de Medicina da Universidade de So Paulo
Sao 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
Navantino Alves
Faculdade de Cincias Mdicas de Minas Gerais
Belo Horizonte, MG, Brazil
Noedir Antonio Groppo Stolf
Faculdade de Medicina da Universidade de So Paulo
Sao Paulo, SP, Brazil

Pedro Puech-Leo
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Peter Libby
Brigham and Womens Hospital
Boston, Boston, MA, USA
Philip Cohen
University of Houston Health Center
Houston, Texas, USA
Rafael Andrade-Alegre
Santo Toms Hospital
Republic of Panamai, Panam
Ricardo Antonio Renetti
Faculdade de Medicina da Universidade Federal do
Rio de Janeiro Rio de Janeiro, RJ, Brazil
Roberto Chiesa
San Raffaele Hospital
Milan, Italy

Ronald A. Asherson
Netcare Rosebank Hospital
Rosebank, Johannesburg, South ifrica
Samir Rasslan
Faculdade de Medicina da Universidade de
So Paulo So Paulo,
SP, Brazil
Tarcisio Eloy Pessoa de Barros
Faculdade de Medicina da Universidade de
So Paulo
So Paulo, SP, Brazil
Valentim Gentil
Faculdade de Medicina da Universidade de
So Paulo So Paulo,
SP, Brazil
Wagner Farid Gattaz
Faculdade de Medicina da Universidade de
Scio Paulo So Paulo, SP, Brazil

Board of Governors
Alberto Jos da Silva Duarte
Aluisio Augusto Cotrim Segurado
Ana Claudia Latronico Xavier
Berenice Bilharinho de Mendona
Carlos Roberto Ribeiro de Carvalho
Clarice Tanaka
Claudia Regina Furquim de Andrade
Cyro Festa
Dalton de Alencar Fischer Chamone
Daniel Romero Muoz
Edmund Chada Baracat
Eduardo Massad
Eloisa Silva Dutra de Oliveira Bonf
Euripedes Constantino Miguel
Fbio Biscegli Jatene
Flair Jos Carrilho
Gerson Chadi
Gilberto Luis Camanho
Giovanni Guido Cerri
Irene de Lourdes Noronha
Irineu Tadeu Velasco
Ivan Cecconello

Jorge Elias Kalil


Jos Antonio Franchini Ramires
Jos Antonio Sanches
Jos Eduardo Krieger
Jos Otvio Costa Auler
Jos Ricardo de Carvalho Mesquita Ayres
Lenine Garcia Brando
Luiz Augusto Carneiro DAlbuquerque
Luiz Fernando Onuchic
Magda Maria Sales Carneiro-Sampaio
Manoel Jacobsen Teixeira
Marcelo Zugaib
Marcus Castro Ferreira
Maria Aparecida Shikanai Yasuda
Miguel Srougi
Milton de Arruda Martins
Nelson de Luccia
Olavo Pires de Camargo
Paulo Andrade Lotufo
Paulo Hilrio Nascimento Saldiva
Paulo Manuel Pgo Fernandes
Paulo Marcelo Gehm Hoff

Editorial Director

Kavita Kirankumar Patel-Rolim


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

Paulo Rossi Menezes


Pedro Puech-Leo
Remo Susanna
Ricardo Ferreira Bento
Ricardo Nitrini
Roberto Kalil
Roberto Zatz
Roger Chammas
Samir Rasslan
Sandra Josena Ferraz Ellero Grisi
Selma Lancman
Tarcsio Eloy Pessoa de Barros
Uenis Tannuri
Umbertina Conti Reed
Valentim Gentil
Venncio 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 So Paulo
So Paulo, SP, Brazil
Daniela Aquemi Higa
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Ariane Maris Gomes
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil

Editorial Ofce: Rua Dr. Ovdio Pires de Campos, 225 - 6 Andar CEP 05403-010 So 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; Ulrichs Periodical Directory; Qualis/Capes - Classied as an International Circulation Journal in Medicine.
Clinics. So Paulo: Scientic Journal of Hospital das Clnicas da Faculdade de Medicina da Universidade de So Paulo, 2005Monthly Periodical: January to December
ISSN 1807-5932 printed version
ISSN 1980-5322 online version
Formerly Revista do Hospital das Clnicas da FMUSP, 19462004.
1. Medicine-scientic production. 2.Medical Sciences I. Hospital das Clnicas da Faculdade deMedicina da Universidade de So 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 and SciELO and complies with the policies
of funding agencies, such as the Wellcome Trust, the Research
CouncilsUK- (RCUK), the National Institutes of Health (NIH),
and the German Research Foundation (DFG), 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
(http://www.icmje.org/) on trial registration. All trials initiated
after January 1, 2012 must be prospectively registered (before
patient recruitment begins) in a publicly accessible registry. Trials
initiated before January 1, 2012 must be registered before
submission to our journals. See the ICMJE FAQ regarding trial
registration for further details. Visit http://www.who.int/ictrp/
network/list_registers/en/index.html for the WHOs list of
approved registries. CLINICS suggests: http://www.clinicaltrials.
gov as a user friendly site.

Title page:
 Title (up to 250 characters);
 Running title (up to 40 characters, letters and spaces);
 Full address of corresponding author only;
 Authors names (without titles or degrees). 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.
Manuscript:
 Abstract: Abstracts are limited to 250 words and

Publication Fees
CLINICS uses a business model in which expenses are recoveredin
part by charging a publication fee to the authors or research
sponsors for each published article. Our 2015 prices are as
follows: original articles, review articles and rapid communications: US$ 1,500.00 or R$ 3.000,00. Invited reviews, editorials
and letters to the editors: no charge.
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.
Manuscripts should be digitalized using aWord *.doc-compatible
software program and submitted online in English.
Authors are strongly advised to submit the manuscript in its
nal form to a spell check for English (US). Submissions with
excessive spelling or syntax mistakes as well as articles in which
the meaning is not sufciently clear shall be returned to authors
for correction. Authors are also strongly advised to use
abbreviations sparingly whenever possible to avoid jargon
and improve the readability of the manuscript. All abbreviations must be dened the rst time that they are used. Only
terms or expressions that are used at least 5 times through
out the text should be abbreviated. Never use abbreviations
that spell common English words, such as FUN, PIN, SCORE
and SUN.
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 Ofce.




Manuscripts are invited in the following categories:


ORIGINAL STUDY: Complete original studies should be
submitted in this category. Three sections are offered: basic,
clinical, and surgical research. Original studies must conform to
the following format:

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.
Keywords: For keywords, 36 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 institutions guide, a
national research councils guide, orany 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
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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

 Title page: As described in the Original Study section.

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: Diabetes (2), hypertension (3,4) and
alcoholism (59) are complex medical problems (10).
Under exceptional circumstances, authors names may
appear in text: Single author: Einstein (11) proposed a
new theory , Two authors: Watson & Crick (12)
reported on the structure of , or Three or more
authors: Smith et al. (13) described

 Manuscript: Abstract, keywords and text should be

 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 inpress 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 six
authors, list all authors. For more than 6 authors, list first
six authors followed by et al..
 Tables and Figures: The maximum number of tables

and/or figures is six tables and/or figures. Tables:


Should be constructed using the table feature in your
word processor or using a spread sheet program such
as Excel. The tables should be numbered in order of
appearance in the text, using Arabic numerals.
Eachtable 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
data 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 Arabicnumerals in order of
appearance. Figure legend(s) should be descriptive
and should allow examination of the figure without
reference to text. Images must beof professional
quality and uploaded as *.tiff files. Generally, figures
will be reduced to fit one column of text. The actual
magnification of all photo micrographs should be
provided, preferably by placing a scale bar onthe print.
Line graphs and charts should never be sent as *.jpeg
illustrations. We recommend preparing linegraphs 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
eld. The sections should be arranged as follows:

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 page: As described in the Original Study section.
 Manuscript: Rapid communications are limited to 1,500

words, not including the reference list, abstract and


keywords. Authors should format rapid communications based on the subject at hand. 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. For keywords, 3-6 items from
the Medical Subject Headings (MeSh) should be used.
LETTERS TO THE EDITOR: Letters to the editor expressing
comments or dissenting opinions concerning articles that have
been recently published in CLINICS are not submitted to peer
review and 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 not submitted to
peer review and 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 specic format that is set by the
editor in charge of the collection.
Currently CLINICS does not accept: case reports, technical
notes, retrospective studies, translations and validations of
questionnaires, and articles referring to rst demonstration in
Brazil.
Peer Review: Manuscripts are reviewed by at least two expert
consultants. Accepted manuscripts are edited to comply with
the journals format, remove redundancies, and improve clarity
and understanding without altering meaning. The edited text
will be presented to authors for approval.
Submission: A copyright transfer form, signed by all authors,
must be submitted by fax (55-11-2661-7524) or by mail as soon

as the manuscript is submitted. Any nancial or other


relationships that may lead to a conict of interest must be
disclosed in the copyright transfer form. If the editor considers
this conict of interest relevant to the paper, a footnote will be
added to show the equity interest in or afliation with the
identied commercial rm(s). When the authors are satised
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
specic elds as they are submitting their manuscript. The
editorial ofce and authors will be automatically notied of the
submission. Progress of themanuscript through the Editorial
Ofces procedures will be available to authors at all times.

ISSN-1807-5932

CLINICS
CONTENTS
Clinics 2015 70(8):535599

CLINICAL SCIENCES

The negative prognostic impact of bone metastasis with a tumor mass


Birsen Ycel, Mustafa Grol Celasun, Bilge ztoprak, Zekiye Hasbek, Seher Bahar, Turgut Kacan,
Aykut Bahceci, Mehmet Metin Seker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535

Ocular risk management in patients undergoing general anesthesia: an analysis of 39,431 surgeries
Newton Kara-Junior, Rodrigo Franca de Espindola, Joao Valverde Filho, Christiane Pellegrino Rosa,
Andre Ottoboni, Enis Donizete Silva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541

Flow-through anastomosis using a T-shaped vascular pedicle for gracilis functioning free muscle
transplantation in brachial plexus injury
Yi Hou, Jiantao Yang, Yi Yang, Bengang Qin, Guo Fu, Xiangming Li, Liqiang Gu, Xiaolin Liu,
Qingtang Zhu, Jian Qi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544

Treatment with dasatinib or nilotinib in chronic myeloid leukemia patients who failed to respond to two
previously administered tyrosine kinase inhibitors a single center experience
Beatriz Felicio Ribeiro, Eliana C.M. Miranda, Dulcinia Martins de Albuquerque, Mrcia T. Delamain,
Gislaine Oliveira-Duarte, Maria Helena Almeida, Bruna Verglio, Rosana Antunes da Silveira,
Vagner Oliveira-Duarte, Irene Lorand-Metze, Carmino A. De Souza, Katia B.B. Pagnano . . . . . . . . . . . . . . . . . . . . 550

The effect of elemene on lung adenocarcinoma A549 cell radiosensitivity and elucidation of its mechanism
Kun Zou, Caigang Liu, Zhuo Zhang, Lijuan Zou . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556

Non-alcoholic steatohepatitis-related liver cirrhosis is increasing in China: A ten-year retrospective study


Ji Xiong, Jun Wang, Juan Huang, Wenjing Sun, Jun Wang, Dongfeng Chen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563

Glutamine treatment attenuates hyperglycemia-induced mitochondrial stress and apoptosis in


umbilical vein endothelial cells
Sher Zaman Sa, Kalaivani Batumalaie, Marzida Mansor, Karuthan Chinna, Syam Mohan,
Selva Kumar, Hamed Karimian, Rajes Qvist, Muhammad Aqeel Ashraf, Garcie Ong Siok Yan . . . . . . . . . . . . . . . . 569

BASIC RESEARCH

Effect of hypertonic saline treatment on the inammatory response after hydrochloric acid-induced
lung injury in pigs
Carla Augusto Holms, Denise Aya Otsuki, Marcia Kahvegian, Cristina Oliveira Massoco, Denise Tabacchi Fantoni,
Paulo Sampaio Gutierrez, Jose Otavio Costa Auler Junior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577

REVIEW

Operative versus nonoperative treatment for displaced midshaft clavicle fractures: a meta-analysis
based on current evidence
Xin-Hua Wang, Wei-Jun Guo, A-Bing Li, Guang-Jun Cheng, Tao Lei, You-Ming Zhao. . . . . . . . . . . . . . . . . . . . . . . . . 584

The association between the rs11196218A/G polymorphism of the TCF7L2 gene and type 2 diabetes in
the Chinese Han population: a meta-analysis
Enting Ma, Huili Wang, Jing Guo, Ruirui Tian, Li Wei. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593

CLINICAL SCIENCE

The negative prognostic impact of bone metastasis


with a tumor mass
ztoprak,II Zekiye Hasbek,III Seher Bahar,I Turgut Kacan,IV
Birsen Yucel,I,* Mustafa Gurol Celasun,I Bilge O
Aykut Bahceci,IV Mehmet Metin SekerIV
I

Department of Radiation Oncology


Turkey.

II

Department of Radiology

III

Department of Nuclear Medicine

IV

Department of Medical Oncology, Sivas,

OBJECTIVE: Typically, bone metastasis causes osteolytic and osteoblastic lesions resulting from the interactions
of tumor cells with osteoclasts and osteoblasts. In addition to these interactions, tumor tissues may grow inside
bones and cause mass lesions. In the present study, we aimed to demonstrate the negative impact of a tumor
mass in a large cohort of patients with bone metastatic cancer.
METHODS: Data from 335 patients with bone metastases were retrospectively reviewed. For the analysis, all
patients were divided into three subgroups with respect to the type of bone metastasis: osteolytic, osteoblastic,
or mixed. The patients were subsequently categorized as having bone metastasis with or without a tumor mass,
and statistically significant differences in median survival and 2-year overall survival were observed between
these patients (the median survival and 2-year overall survival were respectively 3 months and 16% in patients
with a tumor mass and 11 months and 26% in patients without a tumor mass; po0.001).
RESULTS: According to multivariate analysis, the presence of bone metastasis with a tumor mass was found to
be an independent prognostic factor (p=0.011, hazard ratio: 1.62, 95% confidence interval: 1.111.76). Bone
metastasis with a tumor mass was more strongly associated with osteolytic lesions, other primary diseases
(except for primary breast and prostate cancers), and spinal cord compression.
CONCLUSION: Bone metastasis with a tumor mass is a strong and independent negative prognostic factor for
survival in cancer patients.
KEYWORDS: Bone metastasis; Bone metastasis with a tumor mass; Prognostic factor; Survival.
ztoprak B, Hasbek Z, Bahar S, Kacan T, et al. The negative prognostic impact of bone metastasis with a tumor mass.
Yucel B, Celasun MG, O
Clinics. 2015;70(8):535-540
Received for publication on February 6, 2015; First review completed on March 23, 2015; Accepted for publication on March 31, 2015
E-mail: yucelbirsen@yahoo.com
*Corresponding author

INTRODUCTION

bone lesions have been described (2,3). The first is an osteolytic


lesion that progresses with bone resorption as a result of
osteoclast activation; the second is an osteoblastic lesion that
triggers bone formation and osteoblastic cell activation. These
two types of lesions may be present concomitantly in certain
patients (mixed type) following stimulation of the two different
types of bone cells. Alternatively, the tumor itself may grow
inside the bone tissue and destroy the bone directly (4). These
mass lesions may cause an increase in complications (e.g., spinal
cord compression, pathologic fracture) due to metastasis-related
bone destruction and suggest the presence of a significant tumor
burden. Examples of computerized tomography images of
osteolytic lesions, osteoblastic lesions, and bone metastasis with
a tumor mass are shown in Figure 1.
Although the duration of survival varies according to the
primary tumor, bone metastases are usually incurable (5).
General treatment procedures for patients with bone
metastasis include bisphosphonate administration, chemotherapy, and palliative radiation therapy. However,
responses to these treatment modalities are relatively poor,
and the patients quality of life is generally impaired.

Bone metastasis is the most frequent complication of cancer,


occurring in up to 70% of patients with breast or prostate cancer
and in approximately 1530% of patients with carcinoma of the
lung, colon, stomach, bladder, rectum, thyroid, or kidney (1).
Although the exact incidence of bone metastasis remains
unknown, this type of metastasis is an attractive area of study
given its high prevalence in cancer patients.
Bone metastases develop as a result of interactions between
tumor cells and bone cells. Cancer cells can induce various
metastatic bone lesions through different mechanisms that
depend on the primary disease, and two types of metastatic

Copyright & 2015 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.
No potential conflict of interest was reported.
DOI: 10.6061/clinics/2015(08)01

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Figure 1 - Types of bone metastasis (white arrows). A) Osteolytic metastasis. B) Osteoblastic metastasis. C) Bone metastasis with a tumor
mass.

recategorized into two groups: bone metastasis with or


without a tumor mass.
Pain intensity was evaluated using visual analog scales in
139 (41%) of the cases (9). Patients were routinely asked to
rate their pain intensity by placing a mark on a 10-mm visual
analog scale at the start of radiotherapy and at 1 month after
radiotherapy. This scaling system was used to evaluate the
intensity of pain only in the radiotherapy-affected region.
The response to radiotherapy was determined by calculating
the difference between the pain intensity on the visual analog
scale before and 1 month after the initiation of radiotherapy.
Statistical Package for Social Sciences (SPSS) for Windows
14.0 (SPSS, Inc., Chicago, IL, USA) was used for the statistical
analysis. For descriptive statistics, the mean, standard
deviation, frequency, and median were used. Categorical
data were compared statistically using the chi-square test or
Fishers exact test. Survival rates were calculated according
to the Kaplan-Meier method. A multivariate analysis (Cox
regression analysis) was used to evaluate independent risk
factors affecting survival. P-values p0.05 were accepted as
statistically significant.

Prognosis may vary among patients depending on factors


such as the primary disease type, age, the patients
performance status, the metastatic interval, and the number
of metastatic sites (6,7). Nevertheless, these factors are not
particularly helpful with respect to decision making in
routine clinical practice. Moreover, data on both the
prognostic impact of the mechanism type on bone metastasis
and the additional role of tumor masses in these patients are
lacking.
Therefore, we designed a retrospective analysis to evaluate
the impact of bone metastasis-related tumor mass on patient
survival. We also evaluated differences in the response to
radiation therapy, in complications, and in the pain response
in our cohort according to the type of metastasis.

MATERIALS AND METHODS


This study was conducted at the Department of Radiation
Oncology at Cumhuriyet University Hospital in Sivas,
Turkey, in accordance with the principles of the Declaration
of Helsinki. A total of 335 cancer patients with bone
metastasis who were admitted to the department between
2007 and 2013 were evaluated retrospectively.
All patients were treated with palliative radiotherapy and
bisphosphonate. During the treatment period, all patients
were examined by a radiation oncologist immediately before
and 1 month after radiotherapy. The physical examination
findings as well as body weight; Eastern Cooperative
Oncology Group (ECOG) performance scores; and histopathological, radiological, and laboratory data (alkaline
phosphatase [ALP] and calcium levels) were recorded. The
patients survival data were obtained from hospital records,
and patients lost to follow-up were contacted to obtain
information about their condition. Survival was defined as
the time between the date of the first detection of bone
metastasis and the date of last contact or death.
The cancer type was classified based on the primary site:
head and neck, lung, breast, prostate, gastrointestinal
system, genitourinary system, or other. Prior to palliative
radiotherapy, each patients performance status was scored
according to the ECOG scoring system (8). Weight loss was
defined as loss of 410% of body weight in 1 month.
Bone metastasis was revealed by computerized tomography or magnetic resonance imaging and was confirmed by
bone scintigraphy and positron emission tomography.
All patients were divided into three subgroups with
respect to the type of bone metastasis: osteolytic, osteoblastic, or mixed type. All patients were subsequently

RESULTS
The study group comprised 234 (70%) men and 101 (30%)
women. The median age at the time of cancer diagnosis was
59 years (range, 2182 years). The primary disease distribution was as follows: lung cancer in 107 (32%) patients, breast
cancer in 64 (19%), prostate cancer in 62 (19%), gastrointestinal system tumors in 40 (12%), genitourinary system
tumors in 20 (6%), head and neck tumors in 11 (3%), and
tumors in other organs in 31 (9%).
Osteolytic bone metastasis was observed in 99 (30%)
patients, whereas 155 (46%) had osteoblastic bone metastasis,
and 71 (21%) had mixed-type bone metastasis. Ten (3%)
patients had bone metastasis and only a tumor mass,
without any other lesions; these 10 patients were excluded
when categorizing the patients with respect to the type of
bone lesion (i.e., osteolytic, osteoblastic, or mixed). Bone
metastasis with a tumor mass was present in 73 (22%) cases.
Eleven (3%) patients had a single bone metastatic lesion, and
324 (97%) had two or more lesions. The 11 patients with
single bone lesions had no metastases in other organs. The
locations and frequencies of bone metastases were as follows:
vertebral column metastasis in 283 (84%) patients, pelvic
bone metastasis in 246 (73%), long bone metastasis in 189
(56%), costal metastasis in 189 (56%), and skull metastasis in
63 (19%).

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Bone metastasis with a tumor mass was observed more


frequently in patients with osteolytic lesions than in those
with other bone lesions. Spinal cord compression was
observed more frequently in cases of bone metastasis with
a tumor mass compared to cases without a tumor mass;
when occurring in the latter, the compression was mostly
due to compression fracture, as observed for osteolytic
metastases, or to new bone formation, as observed in
osteoblastic lesions. However, serum ALP levels were higher
in patients without tumor masses. In addition, bone
metastases with tumor masses were observed less frequently
in patients with primary breast or prostate cancer compared
with patients with other primary diseases, such as lung or
gastrointestinal system tumors. With respect to pathologic
fractures, pain severity, and responses to radiotherapy, no
differences were observed between cases of bone metastases
with tumor masses and cases of other bone metastases
(Table 1).
The median survival duration was 10 months (range, 1
147 months), and the 1- and 2-year survival rates were 46%
and 24%, respectively. The median survival duration was 3
months and the 1- and 2-year survival rates were 28% and
16%, respectively, among patients who had bone metastases
with tumor masses and 11 months and 50% and 26%,
respectively, in patients who had bone metastasis without
tumor masses. The survival curves of the patients with or
without a tumor mass are shown in Figure 2. Univariate

Spinal cord compression was observed in 20 patients, or


7% of all patients with vertebral column metastases (N: 283),
whereas 49 (15%) patients had pathologic fractures, 26 (8%)
had neurological deficits, and 16 (5%) had hypercalcemia.
Surgical interventions were performed for pathologic fractures in 19 (39%) patients with pathologic fractures (N: 49).
The types of bone metastasis with respect to primary
disease were as follows. Among patients with lung cancer, 42
(39%) had osteolytic lesions, 44 (41%) had osteoblastic
lesions, 19 (18%) had mixed lesions, and 2 (2%) had bone
metastases with only tumor masses. For patients with breast
cancer, 22 (34%), 19 (30%), and 22 (34%) had osteolytic,
osteoblastic, and mixed lesions, respectively; 1 (2%) had a
bone metastasis with only a tumor mass. Osteolytic,
osteoblastic, and mixed lesions developed in 2 (3%), 53
(86%), and 7 (11%), respectively, patients with prostate
cancer. Regarding patients with gastrointestinal system
tumors, 9 (22%), 18 (45%), and 11 (28%) had osteolytic,
osteoblastic, and mixed lesions, respectively, and 2 (5%)
showed bone metastases with only tumor masses. Among
patients with genitourinary system tumors, 8 (40%), 5 (25%),
and 5 (25%) had osteolytic, osteoblastic, and mixed lesions,
respectively, with 2 (10%) exhibiting bone metastases with
only tumor masses. The incidence of osteolytic, osteoblastic,
and mixed lesions was 2 (18%), 5 (46%), and 2 (18%),
respectively, for the patients with head and neck tumors; 2
(18%) had bone metastases with only tumor masses.

Table 1 - Comparison of features associated with bone metastases with or without tumor masses.

Type of bone metastasis


Osteolytic
Osteoblastic
Mixed
Bone metastasis with only a tumor mass
Primary disease
Lung
Breast
Prostate
Gastrointestinal system
Genitourinary system
Head and neck
Other
Serum ALP1 level
p129 U/L
4129 U/L
Serum calcium level
p10.6 mg/dL
410.6 mg/dL
Spinal cord compression
No
Yes
Pathologic fracture
No
Yes
Surgery
No
Yes
Severity of pain
Mild
Moderate
Severe
Response to radiotherapy
No
Yes

Bone metastasis without a tumor mass


(N: 262, 78%)

Bone metastasis with a tumor mass


(N: 73, 22%)

p-value

60 (61)
151 (97)
51 (72)
-

39 (39)
4 (3)
20 (28)
10 (100)

o0.001

(75)
(89)
(94)
(68)
(65)
(64)
(65)

27 (25)
7 (11)
4 (6)
13 (32)
7 (35)
4 (36)
11 (35)

o0.001

137 (75)
119 (84)

47 (25)
23 (16)

0.028

246 (79)
10 (63)

64 (21)
6 (37)

0.103

219 (83)
6 (30)

44 (17)
14 (70)

o0.001

228 (80)
34 (69)

58 (20)
15 (31)

0.079

249 (79)
13 (68)

67 (21)
6 (32)

0.213

12 (86)
25 (66)
57 (65)

2 (14)
13 (34)
30 (35)

0.312

23 (64)
71 (69)

13 (36)
32 (31)

0.306

80
57
58
27
13
7
20

Abbreviation: 1ALP, alkaline phosphatase

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CLINICS 2015;70(8):535-540

Survival Functions

Tumor mass

100

No
Ye
Yes
NoNo-censored
Yes-censore
Yes-censored

Cum survival

80

60

40

20

0
0

20

40

60

80

100

120

140

Months

Figure 2 - Survival curves of patients with or without a tumor mass.

increase the pain intensity or affect the response to


radiotherapy.
Certain researchers have studied prognostic factors in
patients with bone metastases. In a study of 350 patients with
skeletal metastases, Katagiri et al. (6) reported that the
patients performance status, the primary lesion site, the
presence of multiple skeletal metastases, the presence of
visceral or cerebral metastases, and a history of previous
chemotherapy were important prognostic factors. Van der
Linder et al. (7) reported a median survival time of 7 months
for 342 patients with vertebral metastases, and Karnofsky
stated that the performance score, the primary tumor type,
and absence of visceral metastasis were significant predictors
of survival. In the present study, female gender, the presence
of osteoblastic and/or mixed lesions, and primary breast or
prostate cancer were considered to be good prognostic
predictors. In contrast, the presence of bone metastasis with a
tumor mass as well as male gender, weight loss, primary
lung cancer, the presence of osteolytic lesions, and elevated
ALP and calcium levels were found to be poor prognostic
predictors. Poor performance in a single-variable analysis, a
disease-free interval of o2 years, the presence of extraosseous metastasis, and multiple bone lesions were also poor
prognostic factors.
Circulating metastatic cells in blood become entrapped by
the bone marrow spongiosum. Cancerous bone undergoes
secondary lytic or blastic changes (10), and the type of bone
metastasis is determined by these changes. In the literature,
osteolytic lesions have been reported to be more frequent in
breast cancer cases, whereas osteoblastic lesions are observed
in cases of prostate cancer. In the present study, osteoblastic
lesions (46%) were more frequently observed in the overall
patient population; similar to the findings of other studies,
osteolytic lesions were more frequent in patients with breast
cancer, with osteoblastic lesions being more common in
patients with prostate cancer. In terms of the conventional
classification of bone metastases, the presence of a tumor

analyses showed that the survival duration after metastasis


was affected by the presence of bone metastasis with a tumor
mass as well as by gender, weight loss, performance status,
serum ALP and calcium levels, primary disease, bone
metastasis type, number of bone lesions, the presence of
extraosseous metastasis, and the disease-free interval. The
prognostic factors that affected survival time after the
development of bone metastasis are shown in Table 2.
Multivariate analyses revealed that the presence of bone
metastasis with a tumor mass as well as gender, weight loss,
primary disease, type of bone metastasis, and serum ALP
and calcium levels were independent prognostic factors that
affected survival. The independent prognostic factors that
affected the duration of survival after the development of
bone metastasis are shown in Table 3.

DISCUSSION
The prevalence of bone metastasis is higher in advancedstage cancers. Patients diagnosed with bone metastasis
usually have incurable disease, though the survival duration
does vary based on the primary disease. Accordingly, it is
very important to determine prognostic factors once a
diagnosis of bone metastasis has been made. The present
study investigated the prognostic and clinical importance of
bone metastasis with a tumor mass and found that this
feature was an apparently strong negative prognostic factor
for survival. The higher incidence of these metastases in
association with osteolytic lesions might have contributed to
this result, as the presence of osteolytic lesions was found to
be a poor prognostic factor in a multivariate analysis. In
addition, growth of the tumor itself inside the bone might
indicate a larger tumor burden, which might also contribute
to a shorter survival duration. Given the soft tissue
component of bone metastasis with a tumor mass, spinal
cord compression was observed more frequently in these
patients; nonetheless, the presence of these lesions did not

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CLINICS 2015;70(8):535-540

Table 2 - Prognostic factors affecting patient survival after the development of bone metastasis, as determined by univariate survival
analysis.

Bone metastasis with tumor mass


No
Yes
Gender
Male
Female
Weight loss
No
Yes
ECOG PS1
ECOG0-1
ECOG2 and higher
Serum ALP2 level
p129 U/L
4129 U/L
Serum calcium level
p10.6 mg/dL
410.6 mg/dL
Primary disease
Lung
Breast
Prostate
Gastrointestinal system
Genitourinary system
Head and neck
Type of bone metastasis
Osteolytic
Osteoblastic
Mixed
Number of bone lesions
1 lesion
X2 lesions
Extraosseous metastasis
No
Yes
Disease-free interval
o24 months
X24 months

No. of patients

1-year survival (%)

2-year survival (%)

Median survival (months)

p-value

262
73

50
28

26
16

11
3

*o0.001

234
101

39
61

17
42

8
17

o0.001

248
87

53
24

27
12

12
5

o0.001

168
167

55
36

30
17

13
7

o0.001

184
142

50
39

29
16

12
9

0.004

310
16

46
-

24
-

10
3

0.027

107
64
62
40
20
11

27
72
69
24
20
9

10
47
31
6
10
-

5
18
15
5
5
3

o0.001

99
155
71

29
53
49

14
26
26

4
12
12

0.004

11
324

68
44

68
22

32
10

0.040

176
159

51
40

27
18

12
8

0.032

259
76

41
61

20
35

9
18

0.026

Abbreviations: 1ECOG PS, Eastern Cooperative Oncology Group performance status; 2ALP, alkaline phosphatase

The survival duration in patients with bone metastases


varied quite significantly depending on the primary disease,
and it is reported that the duration is generally longer for
patients with breast or prostate cancer than for those with
other types of cancer (1,7,6,11). Ahn et al. (12) reported a
median survival time of 55.2 months among 110 breast
cancer patients with only bone metastases. In contrast,
survival durations as short as 57 months were reported
among patients with lung cancer and bone metastases
(11,13,14). In our study, the longest survival durations were
observed in patients with breast cancer, followed by those
with prostate cancer (median survival durations of 18
months and 15 months, respectively); conversely, the
survival times of patients with other cancers were relatively
short.
Many studies have reported that patients with single bone
lesions in the absence of metastases in other organs have a
longer survival duration relative to those with multiple bone
metastases (15-17). In a study of 42 patients with solitary
bone metastases, Hoshi et al. (15) reported a median survival
duration of 30 months and a 1-year survival rate of 76.5%. In
the present study, the 11 patients with single bone lesions
had a median survival duration of 32 months and a 1-year
survival rate of 68%. The survival durations were shorter

mass was significantly more frequent among osteolytic


lesions (62%). The frequencies of bone metastasis with a
tumor mass were low among patients with breast or prostate
cancer and similar among those with other types of cancer.
Specifically, 2536% of patients with other types of cancer
(non-breast or prostatic) had bone lesions with tumor
masses.
Bone metastases are associated with a particular set of
complications, and the frequency of these complications
varies depending on the features of the metastatic lesions.
For example, pathologic fractures and spinal cord compression are encountered more frequently with osteolytic lesions,
as these lesions cause bone destruction (2,11). It is rational to
expect that bone metastases with tumor masses would
present more complications; indeed, spinal cord compression
was more frequent among cases of bone metastasis with a
tumor mass in the current study. However, an elevated
serum ALP level was more frequently observed in cases of
bone metastasis without a tumor mass. In terms of
pathologic fractures, serum calcium levels, surgical intervention, pain severity, and responses to radiotherapy, no
differences were observed between patients with bone
metastasis with a tumor mass and those with other types
of bone metastases.

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CLINICS 2015;70(8):535-540

Table 3 - Independent prognostic factors affecting the duration of survival after the development of bone metastasis, as determined
by multivariate analysis.
Overall survival
95% CI2

p-value

1
1.62

1.111.76

*0.011

1
0.45

0.310.64

o0.001

1
1.39

1.021.90

0.034

0.200.57

o0.001

0.300.67

0.001

HR
Bone metastasis with a tumor mass
No
Yes
Gender
Male
Female
Weight loss
No
Yes
Primary disease
Lung
Breast
Lung
Prostate
Type of bone metastasis
Osteolytic
Osteoblastic
Osteolytic
Mixed
Serum ALP3 level
p129 U/L
4129 U/L
Serum calcium level
p10.6 mg/dL
410.6 mg/dL

1
0.32
1
0.45
1
0.56
1
0.56

0.390.81

0.002

0.380.83

0.004

1
1.34

1.032.00

0.030

1
2.22

1.034.81

0.042

Abbreviations: 1HR, hazard ratio; 2CI, confidence interval; 3ALP, alkaline phosphatase

among the patients with osteolytic lesions compared with


patients with osteoblastic or mixed lesions. Moreover,
patients with bone metastases with tumor masses had
significantly shorter survival durations compared with those
with bone metastases without tumor masses (median
survival durations of 3 months and 11 months, respectively;
1-year survival rates of 28% and 50%, respectively).
Two major limitations of the present study were its
retrospective design and its heterogeneous study population.
We believe that studies of more specific groups would yield
more significant results.
The presence of bone metastasis with a tumor mass
appeared to be a strong negative prognostic factor and was
associated with a higher incidence of spinal cord compression.

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AUTHOR CONTRIBUTIONS
Ycel B designed the research and analyzed the data. Ycel B, Celasun
MG, ztoprak B, Hasbek Z, Bahar S, Kacan T, Bahceci A, and Seker MM
performed the research. Kacan T and Seker MM contributed analytical
tools. Ycel B and ztoprak B wrote the paper. The authors have no
nancial disclosures to declare, no conicts of interest to report, and have
no commercial or proprietary interest.

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3. Mundy GR. Metastasis to bone: causes, consequences and therapeutic
opportunities. Nat Rev Cancer. 2002;2(8):584-93, http://dx.doi.org/
10.1038/nrc867

540

CLINICAL SCIENCE

Ocular risk management in patients undergoing general


anesthesia: an analysis of 39,431 surgeries
Newton Kara-Junior,I,II Rodrigo Franca de Espindola,II ,* Joao Valverde Filho,I Christiane Pellegrino Rosa,I
Andre Ottoboni,I Enis Donizete SilvaI
I

Srio-Libanes Hospital, Sao Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sao Paulo, Ophthalmology Department, Sao Paulo/SP, Brazil.

OBJECTIVE: This study sought to describe and analyze ocular findings associated with nonocular surgery in
patients who underwent general anesthesia.
METHODS: The authors retrospectively collected a series of 39,431 surgeries using standardized data forms.
RESULTS: Ocular findings were reported in 9 cases (2.3:10,000), which involved patients with a mean age of 58.9
19.5 years. These cases involved patients classified as ASA I (33%), ASA II (55%) or ASA III (11%). General
anesthesia with propofol and remifentanil was used in 4 cases, balanced general anesthesia was used in 4 cases,
and regional block was used in combination with balanced general anesthesia in one case. Five patients (55%)
underwent surgery in the supine position, one patient (11%) underwent surgery in the lithotomy position, two
patients (22%) underwent surgery in the prone position, and one patient (11%) underwent surgery in the
lateral position. Ocular hyperemia was detected in most (77%) of the 9 cases with ocular findings; pain/burning
of the eyes, visual impairment, eye discharge and photophobia were observed in 55%, 11%, 11% and 11%,
respectively, of these 9 cases. No cases involved permanent ocular injury or vision loss.
CONCLUSION: Ophthalmological findings after surgeries were uncommon, and most of the included patients
were relatively healthy. Minor complications, such as dehydration or superficial ocular trauma, should be
prevented by following systematic protocols that provide appropriate ocular occlusion with a lubricating
ointment and protect the eye with an acrylic occluder. These procedures will refine the quality of anesthesia
services and avoid discomfort among patients, surgeons and anesthesia staff.
KEYWORDS: Blindness; Anesthesia; Eye Injuries.
Kara-Junior N, Espindola RF, Valverde Filho J, Rosa CP, Andre Ottoboni, Silva ED. Ocular risk management in patients undergoing general
anesthesia: an analysis of 39,431 surgeries. Clinics. 2015;70(8):541-543
Received for publication on January 22, 2015; First review completed on April 7, 2015; Accepted for publication on May 12, 2015
E-mail: rodrigo166@uol.com.br
*Corresponding author

INTRODUCTION

Because the frequency of ocular complications is very low,


few peer-reviewed studies have analyzed ocular symptoms
and vision loss after surgeries under general anesthesia. The
aim of this retrospective study was to contribute to the
prevention of ocular complications during anesthesia by
determining and analyzing the ocular findings from a large
series of cases involving general anesthesia.

Postoperative visual loss (POVL) following general surgery is a relatively uncommon but devastating complication
that is most frequently associated with cardiac, spine, head
and neck operations. Estimates have indicated that POVL
occurs in up to 0.2% (1) and 4.5% (2) of spine and cardiac
surgeries, respectively.
Although studies of 65,000 and 400,000 patients who
underwent anesthesia for all types of surgery at two large
academic institutions suggested a low prevalence of perioperative vision loss in surgeries other than cardiac and spinal
fusion procedures, the actual prevalences of perioperative
vision loss for the most common types of operations remain
unknown (3,4).

MATERIALS AND METHODS


This retrospective study included 39,431 nonocular surgeries. We began the study by reviewing the documented
cases of ocular findings after surgical procedures performed
at our institution between January 2007 and December 2010.
The preoperative variables included age, sex, American
Society of Anesthesiologists (ASA) physical status classification, urgency of surgery (emergency or elective), duration of
the procedure, ocular findings (signs and symptoms) and
surgical position during surgery. Other variables included
the use of ocular lubricant during anesthesia, the required
treatment and the final diagnosis.

Copyright & 2015 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.
DOI: 10.6061/clinics/2015(08)02

541

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CLINICS 2015;70(8):541-543

patients received ocular lubricant (in the form of eye drops,


serum or gel). These findings indicated the precautions
implemented by the staff to prevent ocular injury. However,
these actions cannot avoid ocular complications in all cases.
The most common diagnoses found in our study were
direct trauma and dry eye. Preventive strategies are the only
option to reduce the effect of ocular complications during
general anesthesia. Ocular occlusion and the use of eyelubricating ointment are important strategies to prevent
dehydration of the ocular surface during long surgeries. In
these situations, the mechanisms of aggravation can include
not only corneal exposure if the eyelids remain open but also
decreased tear secretion induced by the anesthesia. After the
surgical procedure, the maintenance of the patients ocular
occlusion is recommended during the postanesthetic period,
when the blink reflex is poor and the patient remains sleepy.
The main symptoms of dry eye are pain, redness and tearing.
With respect to the treatment of corneal deepithelialization
due to exposure keratopathy, eye lubrication with artificial
tears and occlusion with ocular lubricating ointment are
frequently recommended in severe cases. The ocular administration of saline should be avoided due to the risk of further
dehydration of the cornea. Frequent review of dry eye cases
by an ophthalmologist is necessary due to the risk of
progression to ulceration of the cornea, which can lead to
permanent vision loss.
Direct trauma to the eye is generally caused by pressure
exerted by the surgeons arm or hand on the patients eye
during surgery or by direct corneal injury with instruments
or components of the surgical field. During intubation, the
anesthetist himself may cause trauma to the patients eye.
Ocular trauma can be prevented by the systematic use of
acrylic eye protection similar to the postoperative protection
used after eye surgery to prevent patients from exerting
pressure on their eyes; the use of such protection may be
particularly important for patients whose surgical sites are
near the head (20).
In the present study, there were no cases of permanent
vision loss. Many strategies can be used to prevent blindness,
particularly PION-related blindness. Maneuvers to keep the
head at or above heart level to reduce venous congestion in
the head have been recommended in the ASA practice
advisory for perioperative visual loss associated with spine
surgery (21). Minimizing the duration of time in the prone
position and maximizing hemostasis may also be beneficial.
In summary, an understanding of the risk factors and
characteristics that promote the occurrence of perioperative
ocular lesions is extremely important for the development of
prevention strategies. Despite the low incidence of these
complications, the potential for serious and permanent visual

All demographic variables were analyzed using descriptive statistics; in particular, means and SDs were determined
for continuous variables, and frequencies (in percentages)
were calculated for categorical variables.

RESULTS
This retrospective study included 39,431 nonocular surgeries.
Ocular findings were reported in 9 cases (2.3:10,000), which
involved patients with a mean age of 58.919.5 years. Table 1
presents the characteristics of all 9 cases. Examinations of
individual variables revealed that male patients (66%), ASA II
status (55%), elective surgery (88%) and the supine position
(66%) were each involved in the majority of these cases.
For patients with ocular findings who were subjected to
general anesthesia (9 cases), pain (55%) and photophobia
(22%) were the main symptoms, and hyperemia (77%) was
the main sign (Table 2). Only one patient presented with
blurred vision (11%).
The main diagnoses in these cases were direct trauma
(44%) and dry eye (33%) (Table 2). All 9 patients experienced
ocular occlusion during surgery, and 5 patients (55%) also
received lubricant. No patient exhibited permanent ocular
injury or significant visual loss.

DISCUSSION
Perioperative ischemic optic neuropathy (PION) has been
reported after spine (5-7), orthopedic (8), neck (9-13), heart,
and abdominal surgeries (14,15). Intraoperative variables
that reportedly play roles in the pathogenesis of PION
include hypotension, anemia, and elevated intraocular
pressure associated with the prone position during spinal
surgery (16). Vascular risk factors, such as diabetes, coronary
artery disease, and hypertension, are present in many
patients who experience PION (17,18), although vision loss
has been reported in children and healthy adults who exhibit
none of these factors (6).
Given that the mechanisms and risk factors for PION are
poorly understood, the risks of vision loss should be considered
in preoperative discussions with patients who expect to
undergo spine surgery or surgery requiring cardiopulmonary
bypass because such procedures are associated with the highest
incidences of this rare complication (19).
In the present study, the incidence of ocular findings was
2.3:10.000. No patient experienced permanent ocular injury
or significant visual loss. However, certain of the observed
symptoms/signs could significantly impact eye health.
All 9 of the patients with ocular findings experienced
ocular occlusion during their procedures, and 55% of these
Table 1 - Patient characteristics.
Patient
1
2
3
4
5
6
7
8
9

Sex

Age

Duration (min)

ASA

Surgery

male
male
female
male
female
male
female
male
male

69
21
69
77
34
62
60
58
80

105
450
345
150
255
275
345
135
135

I
I
I
II
II
II
II
II
III

elective
elective
elective
elective
emergency
elective
elective
elective
elective

542

Anesthesia
balanced general anesthesia
general anesthesia propofol
balanced general anesthesia
general anesthesia propofol
general anesthesia propofol
general anesthesia propofol
balanced general anesthesia
balanced general anesthesia
general anesthesia propofol

Position

and remifentanil
and remifentanil
and remifentanil
and remifentanil

and remifentanil

supine
supine
supine
prone
supine
lithotomy
prone
supine
supine

Ocular risk in general anesthesia


Kara-Junior N et al.

CLINICS 2015;70(8):541-543

Table 2 - Description of ocular findings (signs and symptoms), treatments and final diagnoses for patients subjected to general
anesthesia.
Patient

Use of ocular
lubricant

Type of
lubricant

yes

eye drops

yes

3
4

no
yes

serum
physiological
solution
gel

no

6
7

Sign(s)

Symptom(s)

Treatment(s)

Diagnosis

hyperemia,
edema
hyperemia

pain

antibiotics

direct trauma

pain

antibiotics, mydriatic drugs,


corticosteroids

direct trauma/
dehydration by serum

discharge
hyperemia

pain

serum physiological solution


lubrication, antibiotics

yes
no

gel
-

yes

ointment

hyperemia
hyperemia,
palpebral edema
hyperemia

photophobia
pain; photophobia;
blurred vision
pain

no

hyperemia

cold compress, lubrication,


corticosteroids
lubrication
eye anesthetics, antibiotics,
lubrication, cold compress
eye anesthetics, lubrication, cold
compress
corticosteroids

dry eye
corneal
deepithelialization
dry eye

injuries such as retinal ischemia and PION justify appropriate care and the active pursuit of high-quality anesthesia
services. Since 2010, a protocol involving ocular occlusion
with the instillation of lubricant eye drops during relatively
complex procedures has been systematically adopted by the
anesthesia services of Srio Libans Hospital. Beginning in
2014, guided by the results and insights of this study, which
was conducted and analyzed in collaboration with ophthalmologists, lubricating ointment and ocular occlusion with an
acrylic occluder for eye protection have been used for all
surgeries involving general anesthesia. It is recommended
that these procedures, which have been implemented to
achieve the objective of further improving patient safety
during surgery, should be followed from the induction of
anesthesia to the complete awakening of the patient in the
postanesthesia recovery room.
Minor complications, such as dehydration or superficial
ocular trauma, which can generally be rapidly resolved
during the postoperative period, should be prevented by
following systematic protocols that include appropriate
ocular occlusion with lubricating ointment and protection
of the eye with an acrylic occluder. These protocols will
refine the quality of anesthesia services and avoid discomfort
among patients, surgeons and anesthesia staff.

exposure keratopathy
direct trauma
direct trauma
dry eye

3. Roth S, Thisted RA, Erickson JP, Black S, Schreider BD. Eye injuries after
non ocular surgery: a study of 60,965 anesthetics from 1988 to 1992.
Anesthesiology 1996;85(5):10207.
4. Warner ME, Warner MA, Garrity JA, MacKenzie RA, Warner DO. The
frequency of perioperative vision loss. Anesth Analg. 2001;93(6):141721.
5. Katz DM, Trobe JD, Cornblath WT, Kline LB: Ischemic optic neuropathy
after lumbar spine surgery. Arch Ophthalmol. 1994;112(7):92531.
6. Alexandrakis G, Lam BL. Bilateral posterior ischemic optic neuropathy
after spinal surgery. Am J Ophthalmol. 1999;127(3):3545.
7. Cheng MA, Sigurdson W, Tempelhoff R, Lauryssen C. Visual loss after
spine surgery: A survey Neurosurgery. 2000;46(3):62531.
8. Roth S, Nunez R, Schreider BD. Unexplained visual loss after lumbar
spinal fusion. J Neurosurg Anesthesiol. 1997;9(4):3468.
9. Bhatti MT, Enneking FK. Visual loss and ophthalmoplegia after shoulder
surgery. Anesth Analg. 2003;96(3):899902.
10. Marks SC, Jaques DA, Hirata RM, Saunders JR Jr. Blindness following
bilateral radical neck dissection. Head Neck. 1990;12(4):3425.
11. Nawa Y, Jaques JD, Miller NR, Palermo RA, Green WR. Bilateral posterior
optic neuropathy after bilateral radical neck dissection and hypotension.
Graefes Arch Clin Exp Ophthalmol. 1992;230(4):3018.
12. Schobel GA, Schmidbauer M, Millesi W, Undt G. Posterior ischemic optic
neu- ropathy following bilateral radical neck dissection. Int J Oral
Maxillofac Surg. 1995;24(4):2837.
13. Worrell L, Rowe M, Petti G. Amaurosis: A complication of bilateral radical
neck dissection. Am J Otolaryngol. 2002;23(1):569.
14. Pazos GA, Leonard DW, Blice J, Thompson DH. Blindness after bilateral
neck dissection: Case report and review. Am J Otolaryngol. 1999;
20(5):3405.
15. Asensio JA, Forno W, Castillo GA, Gambaro E, Petrone P. Posterior
ischemic optic neuropathy related to profound shock after penetrating
thoracoabdominal trauma. South Med J. 2002;95(9):10537.
16. Johnson MW, Kincaid MC, Trobe JD. Bilateral retrobulbar optic nerve
infarctions after blood loss and hypotension. A clinicopathologic case
study. Ophthalmology. 1987; 94(12):157784.
17. Cheng MA, Todorov A, Tempelhoff R, McHugh T, Crowder CM,
Lauryssen C. The effect of prone positioning on intraocular pressure in
anesthetized patients. Anesthesiology. 2001;95(6):13515.
18. Kim JW, Hills WL, Rizzo JF, Egan RA, Lessell S. Ischemic optic neuropathy following spine surgery in a 16-year-old patient and a ten-year-old
patient. J Neuroophthalmol. 2006;26(1):303.
19. Holy SE, Tsai JH, McAllister RK, Smith KH. Perioperative Ischemic Optic
Neuropathy. A Case Control Analysis of 126,666 Surgical Procedures at a
Single Institution. Anesthesiology. 2009; 110(2):24653.
20. Carvalho RS, Kara-Jose N, Temporini ER, Kara-Junior N, Noma RK. Selfmedication: the first attempt in patients seen in an ophthalmologic
emergency room. Clinics. 2009;64(8):73541.
21. American Society of Anesthesiologists Task Force on Perioperative
Blindness: Practice advisory for perioperative visual loss associated with
spine surgery: A report by the American Society of Anesthesiologists Task
Force on Perioperative Blindness. Anesthesiology. 2006;104(6):131928.

AUTHOR CONTRIBUTIONS
Kara-Junior N: study conception and design; drafting of the manuscript;
and critical revision. Espindola RF: drafting of the manuscript; critical
revision; and analysis and interpretation of study data. Valverde Filho J,
Rosa CP, Ottoboni A, and Silva ED: study conception and design; data
acquisition; and analysis and interpretation of study data.

REFERENCES
1. Stevens WR, Glazer PA, Kelley SD, Lietman TM, Bradford DS. Ophthalmic complications after spinal surgery. Spine. 1997;22(12):131924.
2. Shaw PJ, Bates D, Cartlidge NE, Heaviside D, French JM, Julian DG, et al.
Neuro-ophthalmological complications of coronary artery bypass graft
surgery. Acta Neurol Scand. 1987;76(1):17.

543

CLINICAL SCIENCE

Flow-through anastomosis using a T-shaped vascular


pedicle for gracilis functioning free muscle transplantation in brachial plexus injury
Yi Hou, Jiantao Yang, Yi Yang, Bengang Qin, Guo Fu, Xiangming Li, Liqiang Gu*, Xiaolin Liu,
Qingtang Zhu, Jian Qi
The First Affiliated Hospital of Sun Yat-sen University, Department of Microsurgery, Orthopedic Trauma, and Hand Surgery, Guangzhou, China

OBJECTIVE: In gracilis functioning free muscle transplantation, the limited caliber of the dominant vascular
pedicle increases the complexity of the anastomosis and the risk of vascular compromise. The purpose of this
study was to characterize the results of using a T-shaped vascular pedicle for flow-through anastomosis in
gracilis functioning free muscle transplantation for brachial plexus injury.
METHODS: The outcomes of patients with brachial plexus injury who received gracilis functioning free muscle
transplantation with either conventional end-to-end anastomosis or flow-through anastomosis from 2005 to 2013
were retrospectively compared. In the flow-through group, the pedicle comprised a segment of the profunda
femoris and the nutrient artery of the gracilis. The recipient artery was interposed by the T-shaped pedicle.
RESULTS: A total of 46 patients received flow-through anastomosis, and 25 patients received conventional endto-end anastomosis. The surgical time was similar between the groups. The diameter of the arterial anastomosis
in the flow-through group was significantly larger than that in the end-to-end group (3.87 mm vs. 2.06 mm,
respectively, po0.001), and there were significantly fewer cases of vascular compromise in the flow-through
group (2 [4.35%] vs. 6 [24%], respectively, p=0.019). All flaps in the flow-through group survived, whereas 2 in
the end-to-end group failed. Minimal donor-site morbidity was noted in both groups.
CONCLUSIONS: Flow-through anastomosis in gracilis functioning free muscle transplantation for brachial plexus
injury can decrease the complexity of anastomosis, reduce the risk of flap loss, and allow for more variation in
muscle placement.
KEYWORDS: Brachial plexus injury; Functioning free muscle transplantation; Flow-through anastomosis;
Gracilis muscle; T-shaped pedicle.
Hou Y, Yang J, Yang Y, Qin B, Fu G, Li X, et al. Flow-through anastomosis using a T-shaped vascular pedicle for gracilis functioning free muscle
transplantation in brachial plexus injury. Clinics. 2015;70(8):544-549
Received for publication on February 10, 2015; First review completed on March 30, 2015; Accepted for publication on May 12, 2015
E-mail: guliqiang1963@aliyun.com
*Corresponding author

INTRODUCTION

FFMT consists of the surgical transplantation of normal


muscle with neurovascular anastomosis to replace a
destroyed or denervated muscle. FFMT has been used to
restore upper limb function following traumatic BPI (12),
muscle loss or denervation (13), Volkmanns ischemic
contracture (14), and tumor resection (15). In contrast to
common free cutaneous or musculocutaneous flap transfer,
FFMT aims to restore function rather than to provide simple
wound coverage and soft tissue repair. In essence, FFMT is a
vascularized free tissue transfer, and many factors can
adversely affect the outcomes of flaps, especially including
failure of the vascular anastomosis (16). Therefore, reliable
anastomosis of blood vessels is one of the most important
prerequisites for successful FFMT.
The gracilis muscle is the donor muscle most widely
used in FFMT, and the limited caliber of the vascular
pedicle is problematic. In particular, the diameter of the
gracilis artery is only 1.0 mm to 2.5 mm (17-19), which

Traumatic brachial plexus injury (BPI) is a severe and


devastating condition observed in up to 4.2% of multitrauma
victims (1). Although transfer of multiple nerves has shown
satisfactory results for traumatic BPI (2-5), the management of
total BPI, or brachial plexus avulsion, is challenging. Functioning free muscle transplantation (FFMT) with or without transfer
of multiple nerves has been increasingly accepted as an
important option for achieving functional reconstruction (6-11).

Copyright & 2015 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.
No potential conflict of interest was reported.
DOI: 10.6061/clinics/2015(08)03

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CLINICS 2015;70(8):544-549

harvest time was defined as the time from incision to


complete isolation of the muscle. The study was approved by
the Institutional Review Board of the hospital, and all
patients provided written informed consent for the surgical
procedure performed and for their images and data to be
used for research purposes.

causes difficulty in finding matched-size recipient vessels


and which also limits blood flow at the site of anastomosis.
Thus, FFMT using the gracilis muscle is susceptible to
vascular compromise. Moreover, BPI associated with
vascular injury is not uncommon. The concomitant
vascular damage, although insidious, often involves the
commonly used arterial branches in the recipient limb,
leading to less-than-optimal vascular anastomosis and an
increased failure risk in FFMT. An anastomosis method
other than the conventional end-to-end or side-to-side
method is thus needed to decrease the risk of gracilis flap
failure in FFMT.
Flow-through anastomosis uses a T-shaped vascular
pedicle to bridge the recipient blood vessels. Flaps with a
flow-through design were originally used in complex
trauma surgery and malignant tumor resection, which
require simultaneous wound coverage and blood vessel
repair. By using a T-shaped vascular pedicle, injured or
deficient vessels at the recipient site can be repaired via
circulation through the flap while the flap is simultaneously
revascularized by the recipient blood vessels. Foucher et al.
(20) first reported a flow-through flap in 1984, and Costa et
al. (21) reported 1-stage coverage and revascularization of
traumatized limbs using a flow-through flap in 1991. Since
that time, the technique has been widely applied in trauma
surgery to achieve wound coverage and vascular repair in
one stage (22-24). In addition, due to its versatility, the flowthrough technique has been used in various free flap
transfers to increase blood inflow, decrease overall resistance, and capture more variant perforators (25-30). However, there are few reports of this techniques use with
FFMT.
Thus, the purpose of this study was to compare the results
of flow-through anastomosis with those of traditional end-toend anastomosis in gracilis FFMT used for the repair of
traumatic BPI.

Surgical technique
All of the FFMTs were performed by a single senior
professor and his team, including a resident and an attending
physician. Each patient was placed in the supine position
with hip joint flexion, abduction, and external rotation and
knee joint flexion. In this position, the adductor longus could
be palpated in the medial thigh. Additionally, a line was
drawn along the prominence from the pubic tubercle to the
medial knee to indicate the anterior boarder of the gracilis
muscle, and the skin flap overlying the gracilis muscle was
marked (Figure 1).
An incision was made along the anterior boarder of the
skin flap, and dissection between the adductor longus and
the gracilis was performed, preserving the fascia surrounding
the gracilis. The dominant pedicle and motor nerve were
identified beneath the adductor longus (Figure 2A); however,
the neurovascular pedicle was not dissected at this time.
Next, the incision was extended to the insertion of the gracilis,
and the posterior border of the skin flap was incised. The
gracilis was then isolated from adductor longus anterolaterally and from the adductor magus posterolaterally.
With the adductor longus retracted anteriorly, the vascular
pedicle of the gracilis was exposed and dissected, and the
branches to the adductor longus, brevis, and magnus were
ligated. The nerve branches of the muscle were also identified
(Figure 2B). The adductor longus was also retracted posteromedially so that the artery pedicle could be traced to its
origin at the profunda femoris. A 2 cm to 3 cm segment of
the profunda femoris was then isolated and cut (Figure 2C).
A T-shaped artery pedicle comprising the profunda femoris
and the nutrient artery of the gracilis was harvested, and the
venae comitantes of the nutrient artery were also harvested
from their origin. The profunda femoris arterial segment had
an obviously larger caliber (Figure 2D). After dividing the
motor nerve, the gracilis was harvested.
Incisions in the upper limb were performed based on the
aim of reconstruction. The recipient artery, which was usually
the brachial artery, axillary artery, or radial artery, was divided
and interposed with the T-shaped pedicle, with both ends
anastomosed (Figure 3). The venae comitantes of the gracilis
were also anastomosed with the matched recipient veins in an
end-to-end fashion. If there were 2 venae comitantes, they
were anastomosed with the superficial and deep venous
systems, respectively (Figure 3C). When only a single vena
comitans was present, only one anastomosis was performed.
In the end-to-end anastomosis group, a single arterial end-toend anastomosis was performed. The spinal accessory nerve
was the first choice for nerve innervation in both groups. For
patients whose spinal accessory nerves were unavailable,
intercostal nerves, the medial brachial cutaneous nerve, and a
bundle of ulnar nerves reinnervated by CC7 were utilized.

METHODS
Patients
The cases of consecutive patients with traumatic BPI
treated with gracilis FFMT at our center from 2005 to 2013
were retrospectively reviewed. The criteria for inclusion in
this study were patients with traumatic BPI who received
FFMT using the gracilis muscle as the donor muscle. Patients
with traumatic muscle loss and those who received FFMT
using muscles other than the gracilis as the donor muscle
were excluded. At the beginning of the study period, we
used traditional end-to-end vascular anastomosis when
performing FFMT, whereas in the later part of the study
period, we used the flow-through technique; thus, patients
were divided into a traditional anastomosis group and a
flow-through anastomosis group.
Data regarding patient age and gender, the etiology of the
injury, the harvest time, the total operation time, vascular
compromise and other postoperative complications, and
donor-site complications were collected from the medical
records. The data were specifically collected from the
medical records by a physician who was not involved in
the study and who was not aware of which type of
anastomosis was performed. As is routine in our department,
two groups of surgeons began preparation of the donor site
and recipient site simultaneously. The operation time was
defined as the time from incision to wound closure, and the

Postoperative care and follow-up


Postoperatively, the patients were administered antibiotics
to prevent infection and were also given anticoagulation
and anticonvulsant treatments. Plaster splints were applied for

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CLINICS 2015;70(8):544-549

Figure 1 - Design of the gracilis musculocutaneous flap

Donor-site discomfort and dysfunction were evaluated 1


year after the FFMT. Subjective donor-site discomfort was
specifically evaluated using a questionnaire adapted from

6 weeks after surgery, and passive functional exercises were


begun immediately after surgery. The rehabilitation protocol
comprised acupuncture, moxibustion, and electrical stimulation.

Figure 2 - Intraoperative images - A) Exposure of the dominant vascular pedicle. To avoid injury during the operation, the pedicle was
not dissected at first. B) The neurovascular pedicle of the gracilis. Note that the sensory nerve branch ($) must be resected to ensure
enough motor nerve fiber regeneration (a, b). C) Exposure of the profunda femoris. A segment of the profunda femoris was prepared.
It is unnecessary to perform a long dissection. D) The T-shaped arterial pedicle of the gracilis musculocutaneous flap (flap placed with
the skin paddle downward).

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Figure 3 - Flow-through anastomosis of the T-shaped pedicle - A) The diameter of the profunda femoris segment is obviously larger
than that of the nutrient artery of the gracilis. B) The brachial artery was resected, and the diameters of the segment profunda femoris
and brachial artery were well matched. C) Interposed anastomosis to bridge the brachial artery. Two veins were anastomosed in direct
end-to-end fashion.

32 brachial arteries, five axillary arteries, and nine radial


arteries. In the end-to-end group, the recipient vessels
included four circumflex humeral arteries, six brachial artery
branches, six axillary artery branches, three deep brachial
artery branches, and two thoracoacromial arteries.
As shown in Table 1, the harvest time (p=0.167), total
operation time (p=0.721), and donor-site score (p=0.288) were
similar between the two groups. The diameter of the arterial
anastomosis in the flow-through group was significantly
larger than that in the end-to-end group (3.870.42 mm vs.
2.060.44 mm, respectively, po0.001), and there were
significantly fewer cases of vascular compromise in the
flow-through group (2 [4.35%] vs. 6 [24%], respectively,
p=0.019). Moreover, in the flow-through group, there were
two cases of reversible venous spasms, and the flaps were
salvaged after re-exploration. In contrast, in the end-to-end
group, there were six cases of vascular compromise,
including one case of venous spasm, four of venous
thrombosis, and one of arterial thrombosis; four flaps were
salvaged, whereas two failed.
In addition, one patient in the flow-through group and
two in the end-to-end group developed donor-site hematomas that resolved with local care. However, no adduction
muscle strength loss was noted at 1 year after surgery in
either group, as determined by a manual muscle strength
test. The donor-site scores were similar between the two
groups (Table 1), and no scores of 3 or more were noted in
either group. Discomfort included itching, numbness, and
hyperesthesia around the scar. However, all discomfort was
easily bearable and did not affect the activities of daily life.

that of Carr et al. (31). Briefly, donor-site symptoms were


classified into 11 grades, with 0 defined as no discomfort; 1 to
9, as increasing degrees of discomfort; and 10, as unbearable
discomfort.

Statistical analysis

Continuous variables are presented as the meanstandard deviation (SD) and were compared using independentsample t-tests. Categorical variables are expressed as a
number and percentage and were compared using Fishers
exact test. A two-tailed po0.05 was considered statistically
significant. All analyses were performed using SPSS Version
20 (SPSS Statistics V20, IBM Corporation, Somers, New
York).

RESULTS
A total of 71 patients treated with FFMT for traumatic BPI
due to a motorcycle accident, a machine injury, or a crush
injury were included in the analysis. A total of 46 patients
received flow-through anastomosis, and 25 patients received
conventional end-to-end anastomosis; these 2 groups were
comparable with respect to age and sex (both p40.05;
Table 1). The purpose of reconstruction for the patients in the
flow-through group was to restore elbow flexion and finger
extension (n=33), to restore elbow flexion and finger flexion
(n=12), or to restore elbow extension (n=1). In the end-to-end
group, the goal of reconstruction was to restore elbow flexion
and finger extension (n=21) or to restore elbow flexion (n=4).
In the flow-through group, the recipient vessels included

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CLINICS 2015;70(8):544-549

Table 1 - Patient characteristics and outcomes

Age (y)
Gender, male
Harvest time (min)
Total operation time (min)
Diameter of arterial anastomosis* (mm)
Vascular compromise
Donor-site score

Flow-through group (n=46)

End-to-end anastomosis group (n=25)

27.858.80
44 (95.6)
98.679.16
400.1173.14
3.870.42
2 (4.35)
1.090.69

26.447.94
24 (96)
101.526.02
408.2098.74
2.060.44
6 (24)
0.880.67

p-value
0.508
0.718
0.167
0.721
o0.001
0.019
0.228

* The diameter was measured once the vessels were mobilized during surgery.
The data are expressed as a number (percentage) or the meanstandard deviation.

DISCUSSION

femoris and the medial circumflex femoral artery can be


easily identified and well matched with a recipient artery.
The most important advantage of a T-shaped vascular
pedicle is the large caliber, and there are unique advantages
to using this technique in FFMT. Miyamoto et al. (35)
revealed that a flow-through flap allowed greater blood flow
into the flap through the anastomotic site than end-to-end
and end-to-side anastomoses did. For FFMT, increased blood
flow is particularly advantageous for intramuscular blood
circulation during the early stage after surgery. Extra
dissection to identify an anonymous recipient artery is
unnecessary because of matching with the brachial, axillary,
or radial artery. This approach successfully avoids problems
due to previous vascular injury and also allows for variation
of the insertion point of the transplanted muscle, without
restriction due to inconvenient recipient vessel positioning.
Additionally, the vascular system of the recipient site can be
kept intact, which reduces the risk of additional ischemia if
recipient vessels must be sacrificed.
There are, however, several unresolved issues with the
application of a T-shaped venous pedicle. Ichinose et al. (36)
noted that dual venous anastomosis of separate venous
systems is conducive to reducing the risk of flap failure and
affords protection against venous catastrophe through a selfcompensating mechanism. There are one or two venae
comitantes in the gracilis vascular pedicle, and with flowthrough venous anastomosis, drainage occurs through a
single venous system. Therefore, when there are two venae
comitantes, it is recommended that they be respectively
anastomosed with branches from the deep and superficial
venous systems. Conversely, if only one vena comitans is
present, flow-through venous anastomosis is recommended
due to the advantages of the large caliber.
In the present study, less vascular compromise was
observed with flow-through anastomosis; the increased
inflow not only supplies oxygen-rich blood but also
improves the outflow of venous blood (35). This is likely
the reason that significantly less vascular crisis occurred in
the flow-through group, even though only the artery
underwent flow-through anastomosis. Due to abundant
communication in the femoral arterial system in the thigh,
ischemia of the lower limb after sacrifice of the profunda
femoris is rare. In our study, no ischemic necrosis or
contracture of the muscles at the donor site occurred, and
no obvious decrease in muscle strength was observed.
There are no specific contraindications for the use of flowthrough anastomosis with a T-shaped arterial pedicle in
FFMT. However, caution must be exercised in patients with a
history of vascular injury to the femoral artery. Computed
tomography angiography (CTA) may be useful for assessing

We have performed FFMT for brachial plexus avulsion


since 2005, and end-to-end vascular anastomosis was initially
used. As the technique evolved, we adopted flow-through
vascular anastomosis for the procedure. In the present study,
we compared the results of gracilis FFMT for traumatic BPI
using flow-through or end-to-end anastomosis. The results
showed that the diameter of the arterial anastomosis in the
flow-through group was significantly larger than that in the
end-to-end group. Additionally, there were significantly fewer
cases of vascular compromise and less flap failure in the flowthrough anastomosis group, whereas the operation time and
donor-site morbidity were similar between the groups.
The treatment of BPI is challenging, especially if there is
complete avulsion of the brachial plexus. Limited donor
nerve for nerve transfer and long distances to the target
muscle are the main obstacles. For certain patients, FFMT is
the only choice for the restoration of limb function. In these
cases, FFMT failure would be catastrophic. The reported
success rate of free tissue transplantation ranges from 91% to
99%, with the majority of failures being due to technical
errors with vessel anastomosis, such as a mismatch of vessels
(16). Generally, small vessels are more vulnerable to
thrombosis than large-diameter vessels are, and a T-shaped
vascular pedicle can afford a larger vessel for anastomosis.
Although primarily used in trauma with main artery injury,
application of the flow-through technique has been extended
to various situations without vascular injury. For example,
Haffey et al. (25) reported a flow-through anterolateral thigh
free flap that could capture vascular perforators from separate
sources, regardless of the vascular branching pattern of
the pedicle. Moreover, Kawamura et al. (27) reported the
feasibility of harvesting a flow-through flap from the scapular
region, and Koshima et al. (28) reported a flow-through free
anterolateral or anteromedial thigh flap with a wide but short
vascular pedicle to avoid problems associated with variation
and to shorten the operation time, with good results. Thus, the
versatility of the technique has been clearly shown, laying
the foundation for its application in FFMT.
The gracilis muscle has a type II blood supply system (32).
The dominant pedicle enters the muscle 6 to 10 cm inferior to
the pubic tubercle, with one or two minor pedicles entering
the muscle approximately 20 cm inferior to the pubis. The
muscle can survive by relying only on the dominant vascular
pedicle, and the caliber of the dominant pedicle is 1 to 2.5 mm
(17). The dominant artery generally originates from the
profunda femoris (33), although certain authors have
reported that it originates from the medial circumflex femoral
artery (34). Regardless of the origin, both the profunda

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CLINICS 2015;70(8):544-549

blood flow in the femoral artery system preoperatively. In


addition, the method is not recommended if the ipsilateral
gracilis is chosen as the donor muscle; due to the relative
locations of the donor and recipient vessels, folding and
twisting of the vascular pedicle is likely to occur.
The primary limitations of this report are the relatively small
number of patients and the retrospective nature of the study.
Traumatic muscle loss is also an important indication for FFMT;
however, we limited this study to patients without muscle loss
to reduce the influence of this variable on the results. Lastly, we
did not include data on the long-term outcomes of the surgery
because the purpose of this report was to examine the effect of
the flow-through technique on flap survival.
The use of flow-through anastomosis in gracilis FFMT for
BPI can decrease the complexity of anastomosis and reduce
the risk of flap loss. The technique allows for a recipient
artery to be chosen such that muscle placement can be
performed in accordance with the aims of reconstruction and
also allows for preservation of the original vessels in the
recipient limb. Thus, we believe that flow-through anastomosis should be considered when performing FFMT.

13. Lin SH, Chuang DC, Hattori Y, Chen HC. Traumatic major muscle loss in
the upper extremity: reconstruction using functioning free muscle transplantation. J Reconstr Microsurg. 2004;20(3):227-35, http://dx.doi.org/
10.1055/s-2004-823110
14. Chuang DC, Carver N, Wei FC. A new strategy to prevent the sequelae of
severe Volkmanns ischemia. Plast Reconstr Surg. 1996;98(6):1023-31,
http://dx.doi.org/10.1097/00006534-199611000-00015
15. Ihara K, Shigetomi M, Kawai S, Doi K, Yamamoto M. Functioning muscle
transplantation after wide excision of sarcomas in the extremity. Clin
Orthop Relat Res. 1999;(358):140-8.
16. Gardiner MD, Nanchahal J. Strategies to ensure success of microvascular
free tissue transfer. J Plast Reconstr Aesthet Surg. 2010;63(9):e665-73,
http://dx.doi.org/10.1016/j.bjps.2010.06.011
17. Macchi V, Vigato E, Porzionato A, Tiengo C, Stecco C, Parenti A, et al. The
gracilis muscle and its use in clinical reconstruction: an anatomical,
embryological, and radiological study. Clin Anat. 2008;21(7):696-704,
http://dx.doi.org/10.1002/ca.v21:7
18. Juricic M, Vaysse P, Guitard J, Moscovici J, Becue J, Juskiewenski S.
Anatomic basis for use of a gracilis muscle flap. Surg Radiol Anat. 1993;15
(3):163-8, http://dx.doi.org/10.1007/BF01627695
19. Giordano PA, Abbes M, Pequignot JP. Gracilis blood supply: anatomical
and clinical re-evaluation. Br J Plast Surg. 1990;43(3):266-72, http://dx.
doi.org/10.1016/0007-1226(90)90071-7
20. Foucher G, van Genechten F, Merle N, Michon J. A compound radial
artery forearm flap in hand surgery: an original modification of the
Chinese forearm flap. Br J Plast Surg. 1984;37(2):139-48, http://dx.doi.
org/10.1016/0007-1226(84)90001-8
21. Costa H, Guimares I, Cardoso A, Malta A, Amarante J, Guimares F.
One-staged coverage and revascularisation of traumatised limbs by a
flow-through radial mid-forearm free flap. Br J Plast Surg. 1991;44(7):
533-7, http://dx.doi.org/10.1016/0007-1226(91)90012-9
22. Kells AF, Broyles JM, Simoa AF, Lewis VO, Sacks JM. Anterolateral thigh
flow-through flap in hand salvage. Eplasty. 2013;13:e19.
23. Yokota K, Sunagawa T, Suzuki O, Nakanishi M, Ochi M. Short interposed
pedicle of flow-through anterolateral thigh flap for reliable reconstruction
of damaged upper extremity. J Reconstr Microsurg. 2011;27(2):109-14,
http://dx.doi.org/10.1055/s-0030-1268209
24. Kasten SJ, Chung KC, Tong L. Simultaneous revascularization and soft
tissue coverage in the traumatized upper extremity with a flow-through
radial forearm free flap. J Trauma. 1999;47(2):416-9, http://dx.doi.org/
10.1097/00005373-199908000-00042
25. Haffey TM, Lamarre ED, Fritz MA. Auto flow-through technique for
anterolateral thigh flaps. JAMA Facial Plast Surg. 2014;16(2):147-50,
http://dx.doi.org/10.1001/jamafacial.2013.2263
26. Parr JM, Adams BM, Wagels M. Flow-through flap for salvage of fibula
osseocutaneous vascular variations: a surgical approach and proposed
modification of its classification. J Oral Maxillofac Surg. 2014;72(6):
1197-202, http://dx.doi.org/10.1016/j.joms.2013.12.011
27. Kawamura K, Yajima H, Kobata Y, Shigematsu K, Takakura Y. Anatomy of
Y-shaped configurations in the subscapular arterial system and clinical
application to harvesting flow-through flaps. Plast Reconstr Surg. 2005;116
(4):1082-9, http://dx.doi.org/10.1097/01.prs.0000178791.85118.ca
28. Koshima I, Fujitsu M, Ushio S, Sugiyama N, Yamashita S. Flow-through
anterior thigh flaps with a short pedicle for reconstruction of lower leg
and foot defects. Plast Reconstr Surg. 2005;115(1):155-62.
29. Brooks D, Buntic RF, Nguyen N. Salvage of a radial forearm flap transferred onto a by-pass graft with conversion from a high-to-low-resistance
circulatory pattern: case report. J Reconstr Microsurg. 2005;21(6):355-7,
http://dx.doi.org/10.1055/s-2005-915201
30. Rozen WM, Leong J. Arterialized venous flow-through flaps with dual
discontiguous venous drainage: a new modification to improve flap
survival. Plast Reconstr Surg. 2012;130(1):229e-31e, http://dx.doi.org/
10.1097/PRS.0b013e3182550260
31. Carr MM, ManktelowRT, Zuker RM. Gracilis donor site morbidity. Microsurgery. 1995;16(9):598-600, http://dx.doi.org/10.1002/(ISSN)1098-2752
32. MathesSJ, Nahai F. Classification of the vascular anatomy of muscles:
experimental and clinical correlation. Plast Reconstr Surg. 1981;67(2):
177-87, http://dx.doi.org/10.1097/00006534-198167020-00007
33. Morris SF, Yang D. Gracilis muscle: arterial and neural basis for subdivision. Ann Plast Surg. 1999;42(6):630-3, http://dx.doi.org/10.1097/
00000637-199906000-00008
34. Kappler UA, Constantinescu MA, Buchler U, Vogelin E. Anatomy of the
proximal cutaneous perforator vessels of the gracilis muscle. Br J Plast
Surg. 2005;58(4):445-8, http://dx.doi.org/10.1016/j.bjps.2004.11.021
35. Miyamoto S, Okazaki M, Ohura N, Shiraishi T, Takushima A, Harii K.
Comparative study of different combinations of microvascular anastomoses in a rat model: end-to-end, end-to-side, and flow-through anastomosis. Plast Reconstr Surg. 2008;122(2):449-55, http://dx.doi.org/
10.1097/PRS.0b013e31817d62c5
36. Ichinose A, Terashi H, Nakahara M, Sugimoto I, Hashikawa K, Nomura T,
et al. Do multiple venous anastomoses reduce risk of thrombosis in free-flap
transfer? Efficacy of dual anastomoses of separate venous systems. Ann Plast
Surg. 2004;52(1):61-3, http://dx.doi.org/10.1097/01.sap.0000096425.18223.60

ACKNOWLEDGMENTS
This study was supported by the NHFPC Special Fund for Health Scientic
Research in the Public Welfare (Number 201402016).

AUTHOR CONTRIBUTIONS
We declare that all of the listed authors have participated actively in the
study and meet the requirements for authorship. Hou Y and Gu L designed
the study and wrote the protocol. Hou Y and Yang J performed the
research/study. Liu X and Zhu Q contributed constructive suggestions
about writing the article. Yang Y and Qin B managed the literature
searches and analyses. Fu G and Li X performed the statistical analysis.
Hou Y wrote the rst draft of the manuscript.

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2. Gu YD, WU MM, Zhen YL, Zhao JA, Zhang GM, Chen DS, et al. Phrenic
nerve transferfor treatment of root avulsion of the brachial plexus. Chin
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3. Mcguiness CN, Kay SP. The prespinal route in contralateral C7 nerve root
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5. Terzis JK, Kostopoulos VK. The surgical treatment of brachial plexus
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org/10.1097/01.prs.0000254859.51903.97
6. Terzis JK, Kostopoulos VK. Free muscle transfer in posttraumatic plexopathies: part 1: the shoulder. Ann Plast Surg. 2010;65(3):12-7, http://dx.
doi.org/10.1097/SAP.0b013e3181cbfe9d
7. Terzis JK, KostopoulosVK. Free muscle transfer in posttraumatic plexopathies part II: the elbow. Hand (N Y). 2010;5(2):160-70.
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org/10.1055/s-0030-1253242
10. Songcharoen P. Management of brachial plexus injury in adults. Scand J
Surg. 2008;97(4):317-23.
11. Chuang DC. Nerve transfer with functioning free muscle transplantation.
Hand Clin. 2008;24(4):377-88, http://dx.doi.org/10.1016/j.hcl.2008.03.012
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549

CLINICAL SCIENCE

Treatment with dasatinib or nilotinib in chronic myeloid


leukemia patients who failed to respond to two
previously administered tyrosine kinase inhibitors
a single center experience
Beatriz Felicio Ribeiro, Eliana C.M. Miranda, Dulcineia Martins de Albuquerque, Marcia T. Delamain,
Gislaine Oliveira-Duarte, Maria Helena Almeida, Bruna Verglio, Rosana Antunes da Silveira,
Vagner Oliveira-Duarte, Irene Lorand-Metze, Carmino A. De Souza, Katia B.B. Pagnano*
Universidade de Campinas (Unicamp), Centro de Hematologia e Hemoterapia Campinas/SP, Brazil.

OBJECTIVE: To evaluate hematological, cytogenetic and molecular responses as well as the overall, progressionfree and event-free survivals of chronic myeloid leukemia patients treated with a third tyrosine kinase inhibitor
after failing to respond to imatinib and nilotinib/dasatinib.
METHODS: Bone marrow karyotyping and real-time quantitative polymerase chain reaction were performed at
baseline and at 3, 6, 12 and 18 months after the initiation of treatment with a third tyrosine kinase inhibitor.
Hematologic, cytogenetic and molecular responses were defined according to the European LeukemiaNet
recommendations. BCR-ABL1 mutations were analyzed by Sanger sequencing.
RESULTS: We evaluated 25 chronic myeloid leukemia patients who had been previously treated with imatinib and a
second tyrosine kinase inhibitor. Nine patients were switched to dasatinib, and 16 patients were switched to nilotinib
as a third-line therapy. Of the chronic phase patients (n=18), 89% achieved a complete hematologic response, 13%
achieved a complete cytogenetic response and 24% achieved a major molecular response. The following BCR-ABL1
mutations were detected in 6/14 (43%) chronic phase patients: E255V, Y253H, M244V, F317L (2) and F359V. M351T
mutation was found in one patient in the accelerated phase of the disease. The five-year overall, progression-free and
event-free survivals were 86, 54 and 22% ( po0.0001), respectively, for chronic phase patients and 66%, 66% and 0%
( po0.0001), respectively, for accelerated phase patients. All blast crisis patients died within 6 months of treatment.
Fifty-six percent of the chronic phase patients lost their hematologic response within a median of 23 months.
CONCLUSIONS: Although the responses achieved by the third tyrosine kinase inhibitor were not sustainable, a
third tyrosine kinase inhibitor may be an option for improving patient status until a donor becomes available
for transplant. Because the long-term outcome for these patients is poor, the development of new therapies for
resistant chronic myeloid leukemia patients is necessary.
KEYWORDS: CML; Dasatinib; Nilotinib; Third-line TKI treatment.
Ribeiro BF, Miranda EC, Martins de Albuquerque D, Delamain MT, Oliveira-Duarte G, Almeida MH, et al. Treatment with dasatinib or nilotinib in
chronic myeloid leukemia patients who failed to respond to two previously administered tyrosine kinase inhibitors a single
center experience. Clinics. 2015;70(8):550-555
Received for publication on February 25, 2015; First review completed on March 30, 2015; Accepted for publication on May 21, 2015
E-mail: kborgia@unicamp.br
*Corresponding author

INTRODUCTION

have failed to respond to imatinib as a first-line therapy.


Indeed, approximately 50% of chronic phase (CP) patients
achieve a complete cytogenetic response (CCyR) when
treated with second-generation TKIs (1,2). Nevertheless,
approximately 52% of patients must discontinue secondline TKI therapy, most often due to resistance or intolerance (3).
Allogeneic transplantation is the treatment of choice for
patients who fail to respond to at least one second-generation
TKI (2nd TKI). However, transplantation is not feasible for
many older patients, for patients with poor performance
status and for patients who do not have an available donor.

Second-generation tyrosine kinase inhibitors (TKIs),


such as nilotinib and dasatinib, are effective therapeutic
options for chronic myeloid leukemia (CML) patients who

Copyright & 2015 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.
No potential conflict of interest was reported.
DOI: 10.6061/clinics/2015(08)04

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CLINICS 2015;70(8):550-555

scale (IS). Major molecular response (MMR) was defined as a


transcript level p0.1% (IS).

These patients may be switched to a different TKI that was


not used previously or switched to other drugs, such as
interferon (INF) or hydroxyurea (HU) (4). Patients treated
with a 3rd TKI should be closely monitored because novel
mutations can occur with sequential TKI therapy, increasing
the risk of resistance (5,6).
In Brazil, fewer treatment options are available for
resistant cases as other TKIs, such as bosutinib and
ponatinib, are not available. In this study, we present our
experience with CML patients treated with dasatinib or
nilotinib as a third-line (3rd TKI) therapy and emphasize the
importance of developing other therapeutic options for these
patients.

Detection of BCR-ABL1 kinase domain mutations


Mutations were detected by direct sequencing of DNA
from peripheral blood samples collected from TKI-resistant
CML patients who failed or displayed a sub-optimal
response to IM or a 2nd TKI, according to methods that
were described previously (13,14). Briefly, total RNA was
transcribed to cDNA and then was amplified using Taq
platinum high fidelity and primers; the forward primer
annealed to BCR exon 2, and the reverse primer annealed to
ABL exon 10. The PCR product was amplified in a seminested reaction, resulting in a 863-base pair fragment that
was sequenced in both directions. The sample nucleotide
sequences were compared to the GenBank accession
no. X16416.

PATIENTS AND METHODS


Between July 2008 and December 2014, 213 CML patients
were treated at the Hematology and Hemotherapy Center at
the University of Campinas according to the 2006 and 2009
European LeukemiaNet recommendations (7,8). The firstline treatment for CML in Brazil is imatinib. The second-line
TKI is chosen based on clinical factors, BCR-ABL1 mutation
status and drug availability. Dasatinib was approved in 2008
and nilotinib was approved in 2009 in Brazil; before 2008,
these drugs were available only through clinical trials. A
total of 25 consecutive adult CML patients, 18 (72%) of
whom were in the CP stage, 3 (12%) of whom were in the AP
stage and 4 (16%) of whom were in the BC stage, who were
resistant (n=23) or intolerant (n=2) to two prior TKIs and
were switched to a 3rd TKI, were included in our analysis.
Most of the patients were treated at our center since their
initial diagnosis; however, three patients were referred from
other treatment centers at the time of initiation of the 2nd TKI
treatment and were followed at our center after discontinuation of the 2nd TKI. Patients were treated with 100-140 mg
dasatinib daily (n=9) (after failure with imatinib and
nilotinib) or 400-800 mg nilotinib daily (n=16) (after failure
with imatinib and dasatinib). Doses were adjusted according
to tolerance. Hematologic, cytogenetic and molecular
responses as well as the CML phases were defined according
to the European LeukemiaNet recommendations (8,9). Bone
marrow karyotyping was performed using the GiemsaTrypsin-Wright stain banding technique at baseline and at
3, 6, 12 and 18 months after the initiation of therapy with the
3rd TKI. Twenty metaphase cells were analyzed for each
sample (10).

Statistical methods
Probabilities of overall survival (OS), progression-free
survival (PFS) and event-free survival (EFS) were calculated
using the Kaplan-Meier method. OS was calculated at the
initiation of therapy with the 3rd TKI until the final follow-up
or death for any reason. PFS was defined as survival without
transformation to the accelerated or blastic phase after
starting the 3rd TKI and was judged based on an event of
progression or death. EFS was defined as loss of complete
hematological response (CHR), CCyR, MMR, progression to
advanced phases, death or 3rd TKI discontinuation for any
reason (toxicity, resistance, transplant or patient lost to
follow-up). Po0.05 was considered statistically significant.
The cut-off for the data analysis was March 2015.

Ethics
The study protocol was approved and was conducted in
accordance with the ethical standards of the local Research
Ethics Committee on human experimentation and the
Helsinki Declaration of 1975, which was revised in 1983.
Patients provided written informed consent for their
participation.

RESULTS
Clinical and laboratory characteristics of the 25 CML
patients at the time of diagnosis and before the initiation of
the 3rd TKI are presented in Tables 1 and 2, respectively.
Chronic-phase CML patients (CP-CML) (n=18) were
analyzed separately. Thirteen CP-CML patients were resistant to imatinib (72%), and 5 were intolerant to imatinib
(28%). Five patients were treated with dasatinib (28%), and
13 patients were treated with nilotinib (72%). Sixteen patients
(89%) were resistant to the 2nd TKI, and 2 patients (11%) were
intolerant to the 2nd TKI. The resistant patients never
achieved a previous CCyR with imatinib or with the 2nd
TKI. The median follow-up duration was 52 (7-75) months,
and 16/18 patients (89%) achieved or maintained a complete
hematologic response during this period. Of 15 patients who
were subjected to cytogenetic analysis, 2 (13%) achieved
CCyR. Of 17 CP-CML patients with available molecular
analysis data, 4 (24%) achieved a major molecular response
(MMR), and 2 achieved a complete molecular response
(CMR). For CP-CML patients, the frequencies of the transcript
levels at baseline and at 3 and 6 months after the initiation of
the 3rd TKI are shown in Table 3.

Detection of BCR-ABL1 transcripts


BCR-ABL1 transcripts were measured in the peripheral
blood by real-time quantitative polymerase chain reaction
(RQ-PCR) at baseline and then every 3 months using
procedures described elsewhere with some modifications
(11). First, cDNA was amplified using the ABI 7300 sequence
detection system (Applied Biosystems) and TAQMAN
Universal Master Mix in a final reaction volume of 25 mL
according to the instructions recommended by the manufacturer. ABL1 was used for normalization. BCR-ABL1
transcripts were measured in duplicate. The copy numbers
were calculated by comparison with a standard curve
generated from serial dilutions (4-6 dilutions) of a linearized
plasmid containing a BCR-ABL1 insert, which has been
described previously (12). The results were reported as BCRABL1/ABL1 ratio (%) after conversion to the international

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CLINICS 2015;70(8):550-555

Table 1 - Characteristics of chronic myeloid leukemia patients at diagnosis (n=25).


Variables

n.

Median age (range) years


Gender: male
Sokal risk group
Low
Intermediate
High
Missing
Additional chromosomal abnormalities*
Splenomegaly
Spleen size 410 cm below the costal margin
White cell count x 109/L (median, range)
Platelet count x 109/L (median, range)
Hemoglobin, g/L (median, range)
Blasts PB, % (median, range)
Basophils PB, % (median, range)

45 (14-72)
13

52

5
1
9
10
01/09
11/16
06/11
137.10 (17.1 494.4)
352.0 (141.0 - 2,901.0)
10.2 (5.1 13.7)
3.5 (0 - 17)
4 (0 - 34)

20
4
36
40
11.1
68.7
54.4

* 47, XX, t (9;22) (q34;q11), +der(22)

Mutation analysis

F317L mutation. A second HSCT was performed, which


resulted in the achievement of a complete molecular response,
but the patient died due to graft-versus-host disease.

BCR-ABL1 mutations were evaluated in 14 of 18 CP-CML


patients, and mutations were detected in 6/14 patients
(43%). One patient in the AP stage presented with the
mutation M351T. The mutation F317L was found in 3
patients before the initiation of the 3rd TKI (during secondline dasatinib therapy), and the mutation F359V was found
in one patient, who displayed imatinib resistance, before the
initiation of dasatinib as a 2nd-line therapy. Five mutations
were found during 3rd-line TKI therapy: E255V (dasatinib),
Y253H (dasatinib), M244V (dasatinib), and F317L (nilotinib).
The patient with the F359V mutation presented with a long
history of disease and had been treated previously for
12 years at another center with busulfan and hydrea before
imatinib treatment. The F359V mutation was detected for the
first time when a patient developed imatinib resistance, but
at the time there were no 2nd-line inhibitors available in
Brazil. The patient underwent hematopoietic stem cell
transplantation (HSCT) and relapsed one and a half years
later with persistence of the F359V mutation. The patient was
treated with dasatinib and achieved CHR but never achieved
a major cytogenetic response. After 4 years of dasatinib
treatment, the patient progressed to the AP stage. At this
time, a new mutation analysis was performed, which
revealed no evidence of the F359V mutation, but a new
mutation, F317L, was identified. The patient was treated
with nilotinib and achieved CHR but relapsed after
5 months; at the time of relapse, the patient maintained the

Survival analysis
One patient in the CP stage died during 3rd TKI therapy.
CP-CML patients had 5-year OS, PFS and EFS values of 86,
54 and 22% (po0.0001), respectively, whereas AP-CML
patients had 5-year OS, PFS and EFS values of 66, 66 and
0%, respectively (po0.0001). BC-CML patients showed no
response in the first year after treatment (Figures 1, 2 and 3).

Long-term outcome
During treatment, 9/16 (56%) CP-CML patients lost CHR
within a median of 23 (3-37) months. Two patients lost CCyR
after 12 and 13 months. One patient lost MMR after
7 months. Six (34%) patients are currently taking their 3rd
TKI, although 3 of these patients lost their response (1 MMR,
1 CCyR and 1 CHR). Three CP-CML patients (17%)
progressed to the BC (blast crisis) stage, and 2 CP-CML
patients subsequently died. Discontinuation of the 3rd TKI
occurred in 16 (89%) cases due to resistance (8); intolerance
(3); loss to follow-up (3); and death (2) during the treatment.
Three AP-CML patients reached CHR, but one of these
patients lost their response. Only one patient achieved CCyR
and MMR, but those responses were lost. One patient
discontinued treatment due to intolerance in the 4th month.

Table 3 - Molecular responses of chronic phase-chronic myeloid


leukemia patients treated with a 3rd tyrosine kinase inhibitor.

Table 2 - Clinical and laboratory characteristics of chronic


rd

myeloid leukemia patients at the initiation of the 3


kinase inhibitor (n=25).

tyrosine
Time

Variables
Median age (range) years
Median time of imatinib therapy (range) months
Achievement of CCyR with imatinib treatment n (%)
Interval diagnosis 3rd TKI (range) months
Treated with dasatinib 100-140 mg once daily n (%)
Treated with nilotinib 400 mg BID n (%)
Disease status before 3rd TKI n (%)
CP
AP
BC

Baseline

n= 25
56
30
3
98
16
09

(22-75)
(1-66)
(12%)
(12-404)
(64%)
(36%)

3 months

6 months
18 (72%)
03 (12%)
04 (16%)

552

RQ-PCR (IS)%

410
1 10
0.1 1o
p0.1
410
1 10
0.1 1o
p0.1
410
1 10
0.1 1o
p0.1

11/18
04/18
03/18
0
09/12
02/12
0
1/12
04/08
01/08
01/08
02/08

61
22
17
0
75
17
0
08
50
12.5
12.5
25

CML patients treated with a third TKI


Ribeiro BF et al.

CLINICS 2015;70(8):550-555

Figure 1 - Kaplan-Meier survival analysis. Five-year OS of chronic myeloid leukemia patients treated with a 3rd tyrosine kinase inhibitor
according to disease phase.

Figure 2 - Kaplan-Meier survival analysis. Five-year PFS of chronic myeloid leukemia patients treated with a 3rd tyrosine kinase inhibitor
according to disease phase.

553

CLINICS 2015;70(8):550-555

CML patients treated with a third TKI


Ribeiro BF et al.

Figure 3 - Kaplan-Meier survival analysis. Five-year event-free survival of chronic myeloid leukemia patients treated with a 3rd tyrosine
kinase inhibitor according to disease phase.

Regarding molecular responses, most of our patients had


BCR-ABL1 transcript levels 410% at 3 months (75%) and
41% at 6 months (62.5%). The achievement of early
responses to first- and second-line therapies, such as BCRABL1 transcript levels o10% at 3 months and o1% at
6 months, has been associated with long-term cytogenetic
and molecular responses and better clinical outcomes
(3,1722). Only one patient in the CP stage achieved the
optimal response criteria within 3 months and 6 months,
respectively.
In our study, the EFS was 44% at 27 months for CP-CML
patients, which is similar to the findings reported by Ibrahim
et al. (23), where 26 CP-CML patients who failed to respond
to two prior TKIs had 45.7% EFS at 30 months after the
initiation of a 3rd TKI. These results show that although
patients can achieve hematological and cytogenetic
responses with a 3rd TKI, those responses are not sustainable.
Similar observations were made by Garg et al.(24). The
authors evaluated 48 CML patients, 25 of whom were in
the CP stage, treated sequentially with three TKIs. Three
patients in the CP stage and one in the AP stage achieved
CCyR; the median duration of the response was 16.3 months.
The median failure-free survival was 20 months for patients
in the CP stage, 5 months for patients in the AP stage, and
3 months for patients in the BP stage.
Patients treated with sequential TKIs also have a higher
risk of developing resistance and novel mutations (5,6). In
fact, 76% of our patients discontinued the 3rd TKI, and 42%
of those discontinuations were due to resistance. We
found 5 BCR-ABL1 mutations in 14 CP patients during the
3rd TKI therapy. One patient harbored a F359V mutation
and responded to dasatinib, however, another mutation

Four BC-CML patients did not reach hematological or


cytogenetic responses and died within four months of the
initiation of the 3rd TKI.
Regarding other treatments after discontinuation of the 3rd
TKI, 14 patients were treated with the following drugs:
hydrea (8), hydrea followed by HSCT (2), hydrea followed
by low dose ARA-C and imatinib (1), interferon followed by
hydrea (1), imatinib (1), and conventional chemotherapy
followed by hydrea (1).

DISCUSSION
Our data show that only 22% of patients in the CP stage
showed long-term benefits from the administration of a 3rd
TKI after imatinib and a 2nd TKI failure. We found that 89%
of our patients in the CP stage achieved CHR, 13% achieved
CCyR, and 24% achieved MMR; however, 50% of those
patients lost CHR within a median of 23 months. All patients
with CCyR lost their response after 12 months, and 25% of
patients lost MMR after 7 months.
Our results are in agreement with prior reports. QuintasCardama et al. (15) performed a study on 23 CML patients
treated with dasatinib after imatinib and nilotinib failure and
found that 43% of these patients achieved CHR and 30%
achieved a cytogenetic response. Giles et al. (16), performed a
study analyzing 60 patients treated with nilotinib after
imatinib and dasatinib failure and found that 70% of CPCML patients achieved CHR and 43% of CP-CML patients
achieved a major cytogenetic response (MCyR). The authors
also found that after 18 months, 59% of CP-CML patients
were progression-free, and their estimated survival was 86%.

554

CML patients treated with a third TKI


Ribeiro BF et al.

CLINICS 2015;70(8):550-555

was selected in this patient when the disease progressed


(F317L).
Although the responses to 3rd-line TKI therapy are not
sustainable, 3rd-line TKIs may be an alternative for patients
with CML who failed to respond to imatinib and a second
generation TKI and are not eligible for HSCT (4). A 3rd-line
TKI can improve the patients condition until an alternative
transplant donor is available. Nevertheless, because the longterm outcome of these patients is poor, it is important to
emphasize the importance of developing new therapies for
CML resistant patients.

9. Cortes JE, Talpaz M, OBrien S, Faderl S, Garcia-Manero G, Ferrajoli A,


et al. Staging of chronic myeloid leukemia in the imatinib era: an
evaluation of the World Health Organization proposal. Cancer. 2006: 106
(6):1306-15, http://dx.doi.org/10.1002/(ISSN)1097-0142.
10. Testoni N, Marzocchi G, Luatti S, Amabile M, Baldazzi C, Stacchini M,
et al. Chronic myeloid leukemia: a prospective comparison of interphase
fluorescence in situ hybridization and chromosome banding analysis for
the definition of complete cytogenetic response: a study of the GIMEMA
CML WP. Blood. 2009;114(24):493943, http://dx.doi.org/10.1182/blood2009-07-229864.
11. Machado MP, Tomaz JP, Lorand-Metze I, Souza CA De, Vigorito AC,
Delamain MT, et al. Monitoring of BCR-ABL levels in chronic myeloid
leukemia patients treated with imatinib in the chronic phase the
importance of a major molecular response. Rev Bras Hematol Hemoter.
2011;33(3):2115, http://dx.doi.org/10.5581/1516-8484.20110056.
12. Cross NC, Feng L, Chase A, Bungey J, Hughes TP, Goldman JM. Competitive polymerase chain reaction to estimate the number of BCR-ABL
transcripts in chronic myeloid leukemia patients after bone marrow
transplantation. Blood. 1993;82(6):192936.
13. Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J, et al.
Monitoring CML patients responding to treatment with tyrosine kinase
inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations
and for expressing results. Blood. 2006;108(1):2837, http://dx.doi.org/
10.1182/blood-2006-01-0092.
14. Branford S, Rudzki Z, Walsh S, Parkinson I, Grigg A, Szer J, et al.
Detection of BCR-ABL mutations in patients with CML treated with
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mutations in the ATP phosphate-binding loop (P-loop) are associated
with a poor prognosis. Blood. 2003;102(1):27683, http://dx.doi.org/
10.1182/blood-2002-09-2896.
15. Quintas-Cardama A, Kantarjian H, Jones D, Nicaise C, OBrien S, Giles F,
et al. Dasatinib (BMS-354825) is active in Philadelphia chromosomepositive chronic myelogenous leukemia after imatinib and nilotinib
(AMN107) therapy failure. Blood. 2007;109(2):4979, http://dx.doi.org/
10.1182/blood-2006-07-035493.
16. Giles FJ, Abruzzese E, Rosti G, Kim D-W, Bhatia R, Bosly A, et al. Nilotinib is active in chronic and accelerated phase chronic myeloid leukemia
following failure of imatinib and dasatinib therapy. Leukemia. 2010;24
(7):1299301, http://dx.doi.org/10.1038/leu.2010.110.
17. Marin D, Ibrahim AR, Lucas C, Gerrard G, Wang L, Szydlo RM, et al.
Assessment of BCR-ABL1 transcript levels at 3 months is the only
requirement for predicting outcome for patients with chronic myeloid
leukemia treated with tyrosine kinase inhibitors. J Clin Oncol. 2012;
30(3):2328, http://dx.doi.org/10.1200/JCO.2011.38.6565.
18. Hughes TP, Hochhaus A, Branford S, Mller MC, Kaeda JS, Foroni L, et al.
Long-term prognostic significance of early molecular response to imatinib
in newly diagnosed chronic myeloid leukemia: an analysis from the
International Randomized Study of Interferon and STI571 (IRIS). Blood.
2010;116(19):375865, http://dx.doi.org/10.1182/blood-2010-03-273979.
19. Hanfstein B, Mller MC, Hehlmann R, Erben P, Lauseker M, Fabarius A,
et al. Early molecular and cytogenetic response is predictive for long-term
progression-free and overall survival in chronic myeloid leukemia (CML).
Leukemia. 2012;26(9):2096102, http://dx.doi.org/10.1038/leu.2012.85.
20. Branford S, Kim D-W, Soverini S, Haque A, Shou Y, Woodman RC, et al.
Initial molecular response at 3 months may predict both response and
event-free survival at 24 months in imatinib-resistant or -intolerant
patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase treated with nilotinib. J Clin Oncol. 2012;30
(35):43239, http://dx.doi.org/10.1200/JCO.2011.40.5217.
21. Shah NP, Guilhot F, Cortes JE, Schiffer C a, Le Coutre P, Brmmendorf TH,
et al. Long-term outcome with dasatinib after imatinib failure in chronicphase chronic myeloid leukemia: follow-up of phase 3 study. Blood.
2014;123(15):2317-24, http://dx.doi.org/10.1182/blood-2013-10-532341.
22. Quints-Cardama A, Kantarjian H, Jones D, Shan J, Borthakur G, Thomas
D, et al. Delayed achievement of cytogenetic and molecular response is
associated with increased risk of progression among patients with chronic
myeloid leukemia in early chronic phase receiving high-dose or standarddose imatinib therapy. Blood. 2009;113(25):631521, http://dx.doi.org/
10.1182/blood-2008-07-166694.
23. Ibrahim AR, Paliompeis C, Bua M, Milojkovic D, Szydlo R, Khorashad JS,
et al. Efficacy of tyrosine kinase inhibitors (TKIs) as third-line therapy in
patients with chronic myeloid leukemia in chronic phase who have failed
2 prior lines of TKI therapy. Blood. 2010;116(25):5497500, http://dx.doi.
org/10.1182/blood-2010-06-291922.
24. Garg RJ, Kantarjian H, OBrien S, Quints-Cardama A, Faderl S,
Estrov Z, et al. The use of nilotinib or dasatinib after failure to 2 prior
tyrosine kinase inhibitors: long-term follow-up. Blood. 2009;114(20):
43618, http://dx.doi.org/10.1182/blood-2009-05-221531.

ACKNOWLEDGEMENTS
Beatriz Felicio Ribeiro received a scholarship from FAPESP and Katia
Pagnano received nancial support from FAPESP. The authors thank the
Universidade de Campinas (UNICAMP), Centro de Hematologia e
Hemotherapia, Campinas/SP, Brazil for supporting this study.

AUTHOR CONTRIBUTIONS
Ribeiro BF and Pagnano KB conceived and designed the study. Ribeiro
BF, Duarte VO, Miranda EC, Almeida MH, and Pagnano KB performed
the data collection. Delamain MT, Oliveira-Duarte G, and Pagnano KB
treated the patients. Lorand-Metze I, Souza CA, Pagnano KB, Ribeiro BF,
Verglio B, Silveira RA, and Albuquerque DM performed the BCR-ABL1
mutation analysis and quantitative PCR experiments. Miranda ECM
managed, analyzed, and interpreted the data. Ribeiro BF, Miranda ECM,
Albuquerque DM, Delamain MT, Oliveira- Duarte G, Almeida MH,
Verglio B, Silveira RA, Oliveira-Duarte V, Lorand-Metze I, Souza CA,
and Pagnano KB approved the nal manuscript. All authors contributed to
the collection, analysis and interpretation of the data and contributed to the
critical revision of the article for intellectual content.

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555

CLINICAL SCIENCE

The effect of elemene on lung adenocarcinoma A549


cell radiosensitivity and elucidation of its mechanism
Kun Zou, Caigang Liu, Zhuo Zhang*, Lijuan Zou*
2nd Affiliated Hospital of Dalian Medical University, Radiotherapy Department, Dalian/Liaoning, China.

OBJECTIVE: To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular
mechanism.
METHODS: Apoptosis of A549 cells was detected by flow cytometry and fluorescence microscopy. The effect of
double-strand break (DSB) damage repair in A549 cells was evaluated using the neutral comet assay. Protein
expression levels were detected using western blotting, and the correlation between protein levels was
analyzed.
RESULTS: Elemene exhibited a radiosensitizing effect on A549 cells. The level of apoptosis induced by elemene
combined with radiation was significantly greater (po0.01) than that elicited by either radiation or elemene
alone. Following radiation and subsequent repair for 24 h, the tail intensity of A549 cells treated with a
combination of elemene and radiation was greater than that of cells treated with either elemene or radiation
alone (po0.01). This result indicates that elemene inhibits cellular DSB repair. Both elemene combined with
radiation and radiation alone decreased the protein expression of DNA-PKcs and Bcl-2 compared to elemene
alone (po0.01), while p53 protein expression was increased (po0.01). A negative correlation was observed
between DNA-PKcs and p53 expression (r=-0.569, p=0.040), while a positive correlation was found between
DNA-PKcs and Bcl-2 expression (r=0.755, p=0.012).
CONCLUSIONS: Elemene exhibits a radiosensitizing effect on A549 cells, and its underlying molecular
mechanism of action may be related to the downregulation of DNA-PKcs gene expression.
KEYWORDS: Elemene; Radiosensitivity; A549 cells; DNA-PKcs; Bcl-2; p53.
Zou K, Liu C, Zhang Z, Zou L. The effect of elemene on lung adenocarcinoma A549 cell radiosensitivity and elucidation of its mechanism.
Clinics. 2015;70(8):556-562
Received for publication on January 6, 2015; First review completed on February 27, 2015; Accepted for publication June 1, 2015
E-mail: 799832582@qq.com, zoulijuanfl@163.com
*Corresponding authors

INTRODUCTION

damage repair-related proteins (5). Thus, inhibition of DNAPKcs gene expression can block DNA double-strand break
(DSB) repair and improve cellular radiosensitivity.
Cellular apoptosis is the core characteristic of radiotherapy, and regulation of this process thus plays an
important role in cellular radiosensitivity (6,7). Previous
studies have shown that apoptosis-related genes, such as
phosphoprotein (p53), p16, B-cell lymphoma-2 (Bcl-2), and
erythroblastic leukemia viral oncogene homolog 2 (erbB-2),
are associated with tumor radiosensitivity (8,9), especially
p53 and Bcl-2. It has also been reported that elemene
interacts with the frontier orbitals of DNA bases to form
complexes between DNA molecules. Specifically, Jiang et al.
(10) showed that elemene increases the radiosensitivity of
A549 cells, the mechanism for which may be related to the
upregulation of p53, downregulation of Bcl-2, and induction
of cellular apoptosis.
Elemene, which is extracted from Zingiberaceae plants
(Curcuma aromatica Salisb.), is a non-cytotoxic antitumor
compound that can improve the radiosensitivity of tumor
cells (11). Results of an in vitro study showed that elemene
increased the radiosensitivity of renal carcinoma cells,
tongue squamous cancer cells, and non-small cell lung

Lung cancer is one of the most common malignant tumors,


and radiotherapy is the prevailing method of choice for
treatment (1), especially for middle- to late-stage lung cancer.
Radiation works by damaging the DNA of cancerous cells
and altering apoptosis-related genes or proteins, leading to
cell death. Improving the radiosensitivity of tumor cells is a
significant factor that would improve the efficacy of
radiotherapy.
DNA-dependent protein kinase (DNA-PK) is an important
enzyme that participates in DNA damage repair and has
become the main target of radiation sensitivity interventions
(2-4). The catalytic subunit (cs) of DNA-PK affects cellular
radiosensitivity by regulating the phosphorylation of DNA

Copyright & 2015 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.
No potential conflict of interest was reported.
DOI: 10.6061/clinics/2015(08)05

556

Effect of elemene on lung cancer


Zou K et al.

CLINICS 2015;70(8):556-562

the survival fraction (SF) of each group was calculated as


follows:

cancer cells (10,12,13). Animal experiments further showed


that elemene exhibited radiotherapy-sensitizing effects in
many types of tumor cells, such as transplanted murine U14
tumors, kidney cancer GRC-1 cells, and tongue squamous
carcinoma Tca-8113 cells (13-15). In addition, beta elemene
enhances A549 cell radiosensitivity through enhancement of
DNA damage and suppression of DNA repair (16).
In the current study, A549 cells were irradiated following
elemene treatment, and the changes in expression of the
apoptosis-related genes Bcl-2 and p53 as well as the doublestranded DNA damage repair-related gene DNA-PKcs were
evaluated. These experiments were conducted to further
understand elemenes molecular mechanism of action in
enhancing radiation sensitivity of A549 cells.

SER=control group (D0,

Dq)/experimental

group (D0,

Dq).

Morphological assessment of apoptosis


The cells were treated as follows: the control group
received RPMI 1640 medium; the radiation group received
a radiation dose of 4 Gy; the drug group was treated with 10
or 20 mg/mL elemene; and the drug plus radiation group
was treated with 10 or 20 mg/mL elemene followed by a
radiation dose of 4 Gy. Following incubation with elemene,
the exponentially growing cells were irradiated as described
above. Samples of 3  105 cells were then collected from
each group, treated with pancreatic enzyme digesting cells,
rinsed twice with PBS, and centrifuged at 1,000 rpm for
5 min. The SF was determined using the following equation:

MATERIALS AND METHODS


Cell culture
The human lung adenocarcinoma A549 cell line was
purchased from the Chinese Academy of Medical Sciences
(CAMS) Cell Center and passaged at the Second Affiliated
Hospital of Dalian Medical University Center Laboratory.
The cells were cultured in RPMI 1640 medium containing
10% inactivated fetal bovine serum (FBS) at 37 C under an
atmosphere of 5% CO2 and saturated humidity. The cells
were subcultured when they reached the exponential phase.

SF=colony number/(plating cell number  PE).


The dose survival curve was fitted using the linearquadratic (LQ) function model S=e-(ad+bd2) (17) for calculating radiobiological parameters, including the sensitivity
enhancement ratio (SER), SER of the mean lethal dose (D0)
SERDq, and SER of the quasi-threshold dose (Dq) SERD0.
Nuclear morphology was examined using fluorescence
microscopy following Hoechst 33342 staining (final concentration 8 mg/mL) for 15 min at 37 C. Imaging was
performed using an Olympus BX-51 fluorescent microscope
with appropriate filter cubes. The excitation and emission
wavelengths were 350 nm 460 nm, respectively.

Reagents and instruments


Elemene (0.1 g/20 mL), which was obtained from DaLian
JinGang Pharmaceutical Co. Ltd. (China), was dissolved in
RPMI 1640 medium to final working concentrations of 10
and 20 mg/mL before use. RPMI 1640 medium was obtained
from Gibco (USA); FBS was obtained from TianJin TBD
Biotechnology Company (China); p53 and Bcl-2 antibodies
were obtained from Santa Cruz (USA, 1:1,000); and DNAPKcs antibody (1:2,000), anti-human b-actin mouse monoclonal antibody and histone H1 internuclear internal
reference antibodies (1:200) were obtained from Neomarker
(USA). The Jim-X half-dry transfer electrophoresis apparatus
was obtained from DaLian JingMai Biotechnology Co. Ltd.
(China). The flow cytometer was purchased from the Gene
Company (USA). The CK2 type inverted microscope was
obtained from Olympus (Japan). The BX51 type fluorescent
microscope was also obtained from Olympus (Japan).

Apoptosis standard
Normal cells demonstrated uniform dispersion of lowdensity fluorescence, while apoptotic cells showed highdensity fluorescence, characterized by a bright blue hue.

Assessment of apoptosis
The cells used were grouped and treated as specified
above. Then, samples of 3  105 cells were collected from
each group, treated with pancreatic enzyme digesting cells,
rinsed twice with PBS, and centrifuged at 1,000 rpm for
5 min. The cells were treated with 100 mL 2% Triton X-100 for
20 min, rinsed twice with PBS, and centrifuged at 1,000 rpm
for 5 min. Next, 200 mL of DNA-Prep LPR reagent (BeckmanCoulter Ltd) was added for 20 min, and the cells were rinsed
twice with PBS, followed by centrifugation at 1,000 rpm for
5 min. The cells were resuspended in PBS and 50 mg/mL
propidium iodide (PI) reagent containing 480 mL of PBS, 5 mL
of PI (5 mg /mL), and 5 mL of RNase (10 mg /mL), and
10 mL of Triton X-100 (10%) was added 30 s later. Single-cell
suspensions were analyzed by flow cytometry to determine
the cellular apoptosis rate.

Irradiation conditions
Cell irradiation was performed using the Varian 2300C/D
medical linear accelerator (Varian Companies, USA) with a
coverage field of 20 cm  20 cm. The culture dish was
placed in the radiation field above 1.5 cm of organic glass.
Cells with irradiated with 6 MV X-ray irradiation at a dosage
rate of 300 cGy/min, a rack angle of 180 , and source-tosurface distance (SSD) of 100 cm.

Clonogenic assay
Neutral comet assay

Logarithmic-growth phase cells were inoculated in a


60-mm culture dish. After adherence, the cells in the drug
and combined irradiation groups were cultured in the
presence of 10 or 20 mg/mL elemene and seeded in culture
plates at 100 cells/well for 24 h. The cells were exposed to 0,
2, 4, 6, 8, and 10 Gy of irradiation and cultured for another 14
days. The number of cell clones viewed under a low
magnification microscope was 50. The plating efficiency
(PE) was calculated relative to the control group (0 Gy), and

A549 cells were irradiated in the absence or presence of


elemene (10 or 20 mg/mL) and assayed immediately after
radiation or returned to the incubator for 24 h to permit
repair. The comet assays were performed immediately after
incubation with elemene, irradiation or combination treatment. Tumor cells were grown on glass microscope slides
using a standard protocol (18). The slides were submerged in
lysing solution containing 30 mM ethylenediaminetetraacetic

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Table 1 - Survival fraction (%).


Group
Control
10 mg/ml
20 mg/ml

0 Gy

2 Gy

4 Gy

6 Gy

8 Gy

10 Gy

100
97.720.2
95.418.8

84.113.2
76.210.4
63.67.5

70.210.5
49.56.4
30.43.0

66.31.9
22.52.3
5.71.1

47.32.5
7.51.8
2.70.6

26.01.3
3.40.2
0.70.1

Cells were incubated with 10 or 20 mg/mL concentrations of elemene for 24 h and were then irradiated with 0, 2, 4, 6, 8, and 10 Gy and cultured for
another 14 days. The number of cells forming more than 50 clones was counted under the inverted microscope to calculate the cloning efficiency (CE):
CE (%)=(Clone formation average of treatment group/inoculated cell number)  100%. Survival fraction (SF)=(irritated group CE/non-irradiated group
PE)  100%. The experiment was repeated 3 times to calculate the average.

Statistical analysis

acid (EDTA) and 0.5% sodium dodecyl sulfate (SDS, pH 8.3)


for 1.5 h at 37 C. Following lysis, the slides were rinsed three
times in Tris-borate-EDTA (TBE) buffer consisting of 90 mM
Tris, 90 mM boric acid, and 2 mM EDTA, pH 8.5, and stored
overnight in TBE buffer at 4 C. Slides were transferred to an
electrophoresis unit with TBE buffer and electrophoresed at
1 V/cm for 20 min. Following electrophoresis, the slides
were neutralized with 0.4 M Tris buffer (pH 7.5) and stained
with ethidium bromide (20 mg/mL). Finally, the slides were
viewed using an Olympus BX-51 fluorescent microscope
(excitation filter 549 nm, barrier filter 590 nm). Images of 50
randomly selected cells from each slide were analyzed with
Comet Assay Software Project casp-1.2.2 (University of
Wroclaw, Poland). The tail moment was used as a parameter
to assess DNA damage. The assay was completed three
separate times, and 50 cells were evaluated per experiment.

Data were analyzed using the statistical package for the


social sciences (SPSS) v13.0 software. Data are expressed as
the mean standard deviation (SD). The statistical
significance of differences between groups was determined
by one-way analysis of variance (ANOVA), followed by post
hoc analysis using the least significant difference (LSD) for
multiple comparisons. The Spearman test was used for the
correlation analysis of the relationships between the expression levels of genes. The level of significance was set at
po0.05 and po0.01 for all statistical analysis.

RESULTS
Effects of elemene on cell radiosensitivity
The survival fraction of A549 cells decreased following
treatment with different doses of radiation and the same
concentration of elemene. Conversely, the A549 cell
survival fraction decreased in the groups treated with the
same dose of radiation in combination with increasing
concentrations of elemene (Table 1). Following treatment
with 10 or 20 mg/mL elemene, the A549 cell survival curve
shifted to the left, the shoulder area was diminished, and
the steepness of the curve increased (Figure 1). Based on
the cell survival curve, the radiobiological parameters and
radiosensitization ratio were obtained and are listed in
Table 1. These data show that, compared with the control
group, the SERD0 and SERDq values for the 10 and 20 mg/mL
elemene treatment groups were greater than 1. Furthermore,
the ratio gradually increased with an increasing drug
concentration (Table 2).

Western blot assay


Western blotting was performed to detect the expression
levels of DNA-PKcs, p53, and Bcl-2. The cells were grouped
and treated as specified above. After 24 h, western blot
analysis was performed using cytosolic fractions as previously described (19). Equal amounts of cytosolic protein
were separated on 812% SDS polyacrylamide denaturing
gels and transferred to nitrocellulose membranes. The
membranes were blocked in TBS-Tween (TBS-T, 10 mM
Tris-HCl, pH 7.4; 150 mM NaCl; and 0.1% Tween-20) with
5% non-fat milk for 2 h and incubated with specific primary
antibodies overnight at 4 C. Finally, the membranes were
incubated with horseradish peroxidase (HRP)-conjugated
secondary antibodies at 37 C for 2 h and assayed using an
enhanced chemiluminescence plus detection system.

Effect of elemene on A549 cell apoptosis


Fluorescence microscopy showed that compared with the
control group, the groups treated with radiation alone and
elemene alone contained more apoptotic cells (Figure 2).
Furthermore, significantly higher apoptotic levels were
observed in the groups treated with radiation and elemene
at 10 or 20 mg/mL (Figure 2A). Flow cytometry revealed that
Table 2 - Radiation parameters for a single-hit multi-target
model.
Group

D0 (Gy)

Dq (Gy)

SF2 (%)

SERD0

SERDq

Control
2.540.24 2.680.25 84.620.9
10 mg/mL 1.640.15 1.870.22 56.314.9 1.540.20 1.430.15
20 mg/mL 1.550.13 1.530.11 43.210.7 1.630.32 1.750.19
SPSS17.0 was used to calculate survival parameters including SF2
(surviving fraction of 2 Gy), D0 (mean lethal dose or final slope), and
Dq (quasi-thres-hold dose). SER (sensitization enhancement ratio). SER D0
(Dq)=control group D0 (Dq)/drug group D0 (Dq) . Three parallel samples
were set at each radiation dosage.

Figure 1 - Cell survival curves of A549 cells following treatment


with 0, 10 or 20 mg/mL elemene and exposure to x-ray doses from
0 to 10 Gy.

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CLINICS 2015;70(8):556-562

Figure 2 - Elemene induces apoptosis in A549 cells. Cells were incubated with 10 or 20 mg/mL elemene for 24 h, followed by irradiation with
4 Gy X-rays and washing with PBS. (A) Cells were imaged with an Olympus BX-51 fluorescent microscope using appropriate filter cubes. (B)
Chromatin condensation was analyzed by fluorescence microscopy after DNA staining with Hoechst 3334. The results are expressed as the
mean SD of three independent experiments, n=3, **po0.01 compared to control, ##po0.01 compared to radiation alone.

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CLINICS 2015;70(8):556-562

Figure 3 - Influence of elemene on radiation-induced DSB. A549 cells were irradiated with 4 Gy X-rays in the absence or presence of
elemene (10 or 20 mg/mL) and assayed immediately after radiation or returned to the incubator for 24 h to permit repair. Comet assays
were performed immediately after incubation with elemene treatment, irradiation, or combination treatment. Columns represent the
means of the tail moments from three independent experiments, and the bars represent the SD, **po0.01 vs. control, ##po0.01
compared to radiation alone.

Protein expression correlation analysis

compared with the control group, the apoptosis rate of the


group treated with radiation alone appeared to increase, but
this was not found to be statistically significant (p40.05). The
apoptosis rate of the group treated with elemene alone did
not change (p40.05), while the apoptosis rate of the elemene
plus radiation group increased significantly (po0.01). The
rate of apoptosis increased with increasing concentrations of
elemene (Figure 2B).

Spearman correlation analysis showed that DNA-PKcs


and p53 protein expression was negatively correlated
(r=-0.569, po0.05), while DNA-PKcs expression was positively correlated with Bcl-2 protein expression (r=0.755,
po0.05).

DISCUSSION
Basic research in radiation biology has shown that
radiation therapy works mainly by damaging tumor cell
DNA and altering the expression of apoptosis-related genes
and proteins. The radiosensitivity of tumor cells relates to
their capacity to repair DSB via the related genes DNAPKcs, Ku70/80, and ataxia telangiectasia mutated (ATM).
Other genes known to be involved in radiosensitivity and
responsible for apoptosis regulation include p53, Bcl-2,
c-myc proto-oncogene (c-myc), and survivin (9). Beta
elemene, which is the active component of elemene, has
recently been demonstrated to enhance the radiosensitivity
of human cancer cell lines in vitro and in an animal tumor
model in vivo (16,20). In particular, beta elemene was found
to enhance radiosensitivity by influencing the cell cycle
distribution of gastric cancer MKN28 cells, and the
mechanisms responsible for this effect include the induction
of G2/M phase arrest, inhibition of sublethal damage repair,
and induction of cell apoptosis, which enhances the killing
effects of radioactive rays (21). The results of the current
study show that the SERD0 and SERDq values of A549 cells
exposed to a low concentration of cytotoxic elemene were
greater than 1. In addition, elemene enhanced the sensitivity

Effect of elemene on DSB repair in A549 cells


Figure 3 (0 h) shows that both elemene and irradiation
alone increased the tail intensity in A549 cells, and the
degree of DSB was augmented as the elemene concentration
increased. In particular, the results showed a higher number
of DSB in the combination group than the irradiation alone
and elemene alone groups (po0.01). As shown in Figure 3
(24 h), following incubation for 24 h, the tail intensity in the
irradiation alone group returned to background levels,
while there was a significant increase in the amount of
remaining tail intensity in the combination group compared
with the irradiation alone and elemene alone groups
(po0.01).

Effects of elemene on DNA-PKcs, Bcl-2, and p53


protein expression in A549 cells
The results shown in Figure 4A revealed that in the 10 and
20 mg/mL combined treatment groups, a significant decrease in
the protein expression of DNA-PKcs (po0.01, Figure 4B) and
Bcl-2 (po0.01, Figure 4C) was observed, while the p53 protein
expression level was significantly increased (po0.01, Figure 4C).

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p53 and induce A549 cell apoptosis, thereby increasing cell


radiosensitivity.
Interestingly, when Bcl-2 and p53 gene expression was
significantly altered, DNA-PKcs protein expression was
significantly decreased in the combined treatment group.
This result indicates that elemene is also involved in
regulating DNA damage repair pathways. Protein kinase
activation leads to the phosphorylation of downstream
DNA repair proteins, which initiate DNA chain fracture
repair (27), and the relationship between DNA-PKcs and
radiotherapy sensitivity has been a topic of significant
research in recent years. It is well established that
inhibiting tumor cell expression of DNA-PKcs increases
radiation sensitivity. Panet al. (28) studied the relationship
between DNA-PKcs expression and radiation sensitivity in
non-small cell lung cancer cell lines, and in adenocarcinomas and large cell carcinomas, DNA-PKcs was shown to
be an important component regulating cellular radiosensitivity. This result indicates that DNA-PKcs may be
predictive of non-small cell lung cancer cell radiosensitivity. Zou et al. (29) silenced the DNA-PKcs gene of human
mammary epithelial cells (MCF10F) using small interfering RNA (siRNA) technology. Simultaneously, the expression of DNA repair-related proteins, such as DNA-PKcs,
Ku80, ATM, and p53, was decreased in these cells, while
their sensitivity increased with low doses of radiation.
Small molecule inhibitors of DNA-PKcs were also shown
to enhance radiation sensitivity of cervical cancer
cells (30). Our experimental results showed that elemene
inhibited DNA-PKcs expression in A549 cells,
reduced DNA damage repair, and increased cellular
radiosensitivity.
DNA-PKcs is a protein with a wide range of functions and
is involved in DNA damage repair, apoptosis, and V(D)J
recombination (31). Yu et al. (32) found that in non-small cell
lung cancer, high expression of DNA-PKcs increased the
activity of the DNA damage repair system. In addition,
apoptosis inhibition caused by mutant p53 and Bcl-2
expression exhibited a combined effect, which may explain
the development of resistance to radiotherapy in small-cell
lung cancer. Daido et al. (33) indicated that following
exposure to low doses of radiation, human malignant glioma
M059J cells that lack DNA-PKcs underwent massive autophagic cell death that was significantly increased after
exposure to DNA-PKcs inhibitors. Furthermore, DNA-PKcs
inhibitors exert radiotherapy-sensitizing effects on glioma
cells by enhancing type II programmed cell death. Lee et al.
(34) found that p53-inducible gene 3 (PIG3) is involved in
apoptosis caused by p53 activation and that this molecule
can regulate DNA-PKcs expression. Moreover, knockdown
of PIG3 was shown to decrease the level of DNA-PKcs in
cells. Our study further addressed the correlation between
DNA-PKcs, Bcl-2, and p53 expression, and the results
showed that DNA-PKcs expression was significantly positively correlated with that of Bcl-2 (r=0.755, po0.05) and
significantly negatively correlated with p53 (r=0.569,
po0.05). We further showed that DNA-PKcs was closely
related to apoptosis and that elemene increased apoptosis of
A549 cells and strengthened cellular radiosensitivity by
inhibiting DNA-PKcs expression.
In summary, elemene exhibits radiotherapy-sensitizing
effects on lung adenocarcinoma A549 cells, and its mechanism of action involves the upregulation of p53 and down-

Figure 4 - Western blot analysis of the protein levels of Bcl-2, p53


and DNA-PKcs. A549 cells were irradiated with 4 Gy X-rays
following treatment with 10 or 20 mg/mL elemene for 24 h.
Proteins were extracted and separated by SDS-PAGE. (A) Levels of
Bcl-2, p53 and DNA-PKcs were quantitated by densitometry, and
the ratios of the three proteins are displayed. Values represent
the mean SD, n=3, **po0.01 compared to the control,
##
po0.01 compared to radiation alone. Elemene inhibited the
protein expression of (B) DNA-PKcs and (C) Bcl-2 and promoted
the protein expression of p53. Key: 1, Control; 2, irradiation; 3,
10 mg/mL; 4, 20 mg/mL; 5, 10 mg/mL + irradiation; 6, 20 mg/mL +
irradiation.

of A549 cells to radiotherapy. Cellular apoptosis is fundamental to radiotherapy, and its regulatory mechanism plays
an important role in cellular radiosensitivity. Apoptosisrelated genes such as p53 and Bcl-2 have important
regulatory functions in the progression of rapid apoptosis
induced by radiation therapy. For instance, a previous study
showed that the levels of the antiapoptotic genes Bcl-2 and
Bcl-xl in A549 cells decreased, while p53 expression and the
production of exosomes increased, following elemene
treatment (22). This result demonstrates that both p53 and
Bcl-2 have important regulatory actions in cervical cancer
cell apoptosis induced by radiation. A number of experimental studies have further shown that elemene is involved
in regulating the expression of Bax, c-myc, p53, poly (ADPribose) polymerase (PARP), survivin, and livin as well as
inducing tumor cell apoptosis (23-26). Our results showed
that, compared with the exposure alone group, the group
that received elemene combined with irradiation exhibited
increased p53 gene expression and significantly decreased
Bcl-2 gene expression, and the expression of both genes was
significantly correlated. Furthermore, elemene was shown to
regulate expression of the apoptosis-related genes Bcl-2 and

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CLINICS 2015;70(8):556-562

regulation of Bcl-2 to promote cell apoptosis, as well as the


downregulation of DNA-PKcs to inhibit DSB repair. However, the specific mechanism of action of elemene requires
further elucidation.

15. She JJ, Wang ZM, Che XM, Pan CE. Radiosensitization of beta-elemene on
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0284186X.2014.925582.
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ACKNOWLEDGEMENT
This study was supported by grants from the National Natural Science
Foundation of China (No. 81473452).

AUTHOR CONTRIBUTIONS
Zou K, Zhang Z and Zou L designed the research study and wrote the
paper. Zou K and Liu C performed the research. Zou K and Zhang Z
analyzed the data.

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562

CLINICAL SCIENCE

Non-alcoholic steatohepatitis-related liver cirrhosis is


increasing in China: A ten-year retrospective study
Ji Xiong,I Jun Wang,II Juan Huang,I Wenjing Sun,I Jun Wang,I Dongfeng ChenI,*
I
Third Military Medical University, Daping Hospital, Institute of Surgery Research, Department of Gastroenterology, Chongqing, China. II Medical Team of
Chinese Peoples Armed Police Force, Xinjiang, China.

OBJECTIVE: Little is known about metabolic factors in cirrhotic patients in China. Therefore, we aimed to quantify
the prevalence of both metabolic factors and non-alcoholic steatohepatitis-related liver cirrhosis in China.
METHODS: The medical records of 1,582 patients diagnosed with liver cirrhosis from June 2003 to July 2013 at
Daping Hospital (Chongqing, China) were retrospectively reviewed through a computer-generated search.
RESULTS: Serum hepatitis B virus surface antigen was present in 1,083 (68.5%) patients, and hepatitis B was found
to be the only etiological factor in 938 (59.3%) of all patients. Obesity, diabetes mellitus, and arterial hypertension
were observed in 229 (14.5%), 159 (10.1%), and 129 (8.2%) patients, respectively. From 2012-2013, the proportion
of non-alcoholic steatohepatitis-related liver cirrhosis increased to 3.2%, whereas the average proportion of nonalcoholic steatohepatitis-related liver cirrhosis in the previous ten years was 1.9%. The incidence of hepatocellular
carcinoma was much higher in males than in females (6.3% vs. 3.7%, respectively, p=0.036). Obesity and diabetes
mellitus did not significantly increase the incidence of hepatocellular carcinoma in the whole cirrhotic group. The
presence of hepatitis B virus was the only risk factor for hepatocellular carcinoma in cirrhotic patients (po0.001).
CONCLUSIONS: Although hepatitis B virus remains the main etiology of liver cirrhosis in China, steatohepatitisrelated liver cirrhosis is increasing in frequency. Hepatitis B virus was the sole significant risk factor for
hepatocellular carcinoma in the whole cirrhotic group in the present study, in contrast to obesity and diabetes
mellitus, for which only a trend of increased hepatocellular carcinoma was found.
KEYWORDS: Non-Alcoholic Steatohepatitis; Non-Alcoholic Fatty Liver Disease; Liver Cirrhosis; Obesity; Metabolism.
Xiong J, Wang J, Huang J, Sun W, Wang J, Chen D. Non-alcoholic steatohepatitis-related liver cirrhosis is increasing in China: A ten-year
retrospective study. Clinics. 2015;70(8):563-568
Received for publication on January 29, 2015; First review completed on March 20, 2015; Accepted for publication on June 1, 2015
E-mail: chendongfeng1981@163.com
*Corresponding author

INTRODUCTION

histological characteristic features of non-alcoholic fatty liver


disease (NAFLD) such as fatty infiltration tend to be lost in
NASH-related cirrhosis (3). Third, there is a lack of strict
criteria for diagnosing NASH-related cirrhosis. According to
one nationwide Japanese survey, 2.1% of cirrhosis cases were
attributable to NASH-related cirrhosis based on the researchers own criteria, which mainly diagnosed NASH-related
cirrhosis as cryptogenic cirrhosis associated with obesity
(BMI over 25), DM or metabolic syndrome (Met-s) (4).
If appropriate criteria are used, NASH-related cirrhosis
may account for two thirds of cases currently labeled as
cryptogenic cirrhosis (3). Herein, we retrospectively analyzed
etiological factors in LC in patients admitted to our hospital
over the past 10 years to evaluate the proportion and time
trend of NASH-related LC, with special emphasis on the
factors favoring HCC development.

The main etiology of liver cirrhosis (LC) in China is


hepatitis B virus (HBV) infection (1). However, the changing
proportions of the various potential etiological factors over
the last 10 years remain poorly documented, especially the
presence of metabolic characteristics such as obesity (or
increased body mass index [BMI]), diabetes mellitus (DM),
hypertension and alcoholism. In Western countries, nonalcoholic steatohepatitis (NASH)-related cirrhosis is increasingly considered as a major causal factor of cirrhosis.
Moreover, hepatocellular carcinoma (HCC) is related to
NASH-related cirrhosis in Germany (2). For several possible
reasons, few data exist regarding NASH-related LC in China.
First, NASH-related cirrhosis is diagnosed by exclusion, and
ruling out all other possible causes is difficult. Second, the

PATIENTS AND METHODS


Copyright & 2015 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.

The medical records of patients diagnosed with LC from


June 2003 to July 2013 at Daping Hospital (Chongqing,
China) were retrospectively reviewed through a computergenerated search. The study included 1,582 patients.

DOI: 10.6061/clinics/2015(08)06

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NASH-related liver cirrhosis in China


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CLINICS 2015;70(8):563-568

Table 1 - Etiologies of liver cirrhosis from 2003-2013.

LC was diagnosed based on clinical symptoms, imaging


data, and/or histological findings (only 41 patients had a
liver biopsy in the present series). Classical criteria for each
etiology potentially involved in LC were applied to
categorize cirrhotic patients. For NASH-related LC, we
adopted the following criteria. First, all NASH-related LC
had to be cryptogenic LC and had to be coupled with alcohol
consumption of less than 20 grams per day. Second, the BMI
of each patient had to be over 25, with or without DM or
Met-s; this was slightly different from the Japanese criteria
(4), which did not include obesity as a necessary criterion. If
the above two criteria were met, then the patient was
clinically suspected of having NASH-related LC. Furthermore, if histological findings such as micronodular cirrhosis,
perisinusoidal fibrosis or fatty changes were found, then the
patient was histologically diagnosed with NASH-related LC.
Among the 30 patients diagnosed with NASH-related LC,
only one patient was histologically diagnosed with NASHrelated LC by liver biopsy.
Clinical information (age, gender, alcohol consumption,
virus infection, hepatic complications) was also collected for
all patients with LC. Metabolic risk factors such as obesity,
DM and arterial hypertension were systematically collected
based on the diagnostic history. The Child-Pugh criteria were
used to grade cirrhosis.

Category
HBV alone
HCV alone
HBV/HCV
HBV/HEV
Alcohol
HBV/Alcohol
HCV/Alcohol
AIH
PBC
AIH/PBC
Wilsons disease
NASH-LC
Cryptogenic
Other
Total

No. of patients (%)


938
15
8
2
158
135
5
32
69
4
6
30
167
13
1,582

(59.3)
(1.0)
(0.5)
(0.1)
(9.9)
(8.5)
(0.3)
(2.0)
(4.4)
(0.3)
(0.4)
(1.9)
(10.6)
(0.8)
(100)

HBV alone: hepatitis B virus was the only etiological factor for cirrhosis;
HCV alone: hepatitis C virus was the only etiological factor for cirrhosis;
HBV/HCV: hepatitis B virus combined with hepatitis C virus; HBV/HEV: hepatitis B virus combined with hepatitis E virus; HBV/Alcohol: hepatitis B virus
combined with alcohol abuse; HCV/Alcohol: hepatitis C virus combined
with alcohol abuse; AIH: autoimmune hepatitis; PBC: primary biliary cirrhosis; AIH/PBC: autoimmune hepatitis combined with primary biliary
cirrhosis.

The remaining 167 patients had cryptogenic LC, as defined by


the absence of identified causes, including NASH-related LC
criteria.

Statistical analysis
The dichotomous data and continuous data were analyzed
by the chi-square test and Students t test, respectively, using
Statistical Package for the Social Sciences (SPSS, version 15.0;
Chicago, IL, USA). Logistic regression was used to analyze
the selected risk factors for HCC. When examining the
differences between males and females, Fishers exact test
and the Mann-Whitney U test were also used. A p-value of
o0.05 was considered statistically significant.

Etiological differences between males and females


HBV was clearly the main etiology in both males and
females (59.8% vs. 57.9%, respectively, p40.05) (Figure 1).
The percentages of patients with alcohol abuse, HBV plus
alcohol abuse, and HCV plus alcohol abuse were higher in
males vs. females (11.2% vs. 1.0%, po0.01; 12.2% vs. 0.2%,
po0.01; and 0.5% vs. 0%, po0.01, respectively). However,
the percentages of AIH and PBC were much higher in
females vs. males (4.9% vs. 0.7%, po0.01; 12.6% vs. 0.7%,
po0.01, respectively). No difference between males and
females existed for NASH-related LC (1.7% vs. 2.3%,
respectively, p=0.47).

RESULTS
Etiology of LC
A total of 1,582 patients diagnosed with LC from 20032013 were included in this retrospective study. A total of
1,097 (69.4%) patients were male, 485 (30.6%) were female,
and 87 (5.5%) were diagnosed with HCC during
hospitalization.
In our study, 14 etiological categories were set for LC
(Table 1). The most common etiology was HBV; hepatitis B
surface antigen (HBs Ag) was present in the sera of 1,083
(68.5%) patients. HBV was found to be the only etiological
factor in 938 (59.3%) patients. Hepatitis C virus (HCV)
accounted only for 1.8% of cases (1.0% of patients had HCV
alone, 0.5% had HCV combined with HBV, and 0.3% had
HCV combined with alcohol abuse). Hepatitis E virus
combined with HBV was found in only two patients. In
total, 298 (18.7%) patients had a history of alcohol abuse (135
of them also had HBV infection, and 5 patients also had HCV
infection; alcohol was the dominant factor in 158 patients).
There were 32 patients with autoimmune hepatitis (AIH), 69
patients with primary biliary cirrhosis (PBC), 4 patients
with AIH combined with PBC, and 6 patients with
Wilsons disease. For NASH-related LC, 30 patients (1.9%)
met our criteria. Thirteen patients had other identified etiologies, including intra-hepatic bile-duct stones
(9 patients), Budd-Chiari syndrome (2 patients), parasitic
infection (1 patient), and veno-occlusive disease (1 patient).

Time trend of metabolic factors and HCC in


cirrhotic patients
The metabolic information recorded during the periods
from 2003-2008 and 2008-2013 was compared (Table 2).
Whereas the total number of patients from 2008-2013 was
two times higher than the number in the period from 20032008 (1,091 and 491, respectively), no significant differences
were noted in terms of age, sex ratio, or BMI between the two
time periods. Trends of increased obesity, DM, hypertension
and HBs Ag positivity were observed in the second five-year
period. In contrast, the proportion of HCC significantly
decreased in the period from 2008-2013 (4.7% vs. 7.3%,
p=0.033) (Table 2).

NASH-related LC
Among the total of 197 patients with cryptogenic LC, 30
patients met our criteria for NASH-related LC. As shown in
Figure 2, the proportion of NASH-related LC increased in
the last two years of the study to 3.2%, compared with 1.9%
for the whole study period.

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CLINICS 2015;70(8):563-568

Figure 1 - Difference in etiologies between males and females.

DISCUSSION

Among the 30 patients with NASH-related LC, 19 patients


were men, and 11 were women (no statistical significance).
Ascites and upper gastrointestinal bleeding (UGB) were the
main complications in patients with NASH-related LC, and the
complication rate of both ascites and UGB was higher in women,
but the difference was not significant. For Child-Pugh grading of
NASH-related LC, the rate of Child A was lower in women than
in men, but the difference was not significant. HCC was not
found in women or men with NASH-related LC. (Table 3).

In our study, HBV remained the main etiological factor


(68.5%) for LC in China, whereas HCV was by far the most
frequent cause of cirrhosis (60.9%) according to a nationwide
survey in Japan (4). HCV accounted for only 1.8% of our
cirrhotic patients. Alcoholic liver disease and hepatitis C are
the most common causes of cirrhosis in the Western world,
whereas hepatitis B is the prevailing cause in most parts of
Asia and sub-Saharan Africa (5). For example, in the United
Kingdom (6), a total of 3,360 incident cases of cirrhosis in
patients aged 25 or older were diagnosed with LC between
1992 and 2001, and 35.9% of these cases were induced by
alcohol, whereas only 5.4% were induced by viral hepatitis.
In the USA, 7% of cirrhosis cases were attributable to HBV,
and 27% were to HCV from 1989-2000(7). Globally, 57% of
cirrhosis cases were attributable to either HBV (30%) or HCV
(27%) (7). In China, as reported in our study, 59.3% of
cirrhosis cases were only related to HBV infection. In a large
community-based study in eastern China (8), a total of
149,175 individuals were investigated in 60 communities in
three counties in Jiangsu province from 2011-2012. Of these
subjects, 1,175 (0.79%, 95% confidence interval (CI) 0.74-0.83)
were anti-HCV antibody positive. The prevalence was even
lower in children (0.09%, 95% CI 0.04-0.17). A seroepidemiological survey of HCV conducted in 2007 revealed a
lower rate of anti-HCV antibody positivity (0.80%) in
Chongqing, Southwest China than in the general population
(9). Thus, the prevalence of hepatitis C as a cause of LC is low
in Chongqing, China.
The difference between Asia or sub-Saharan Africa and the
Western world may be related to the wide implementation of
HBV programs, which has not been financially possible in
numerous developing countries. In 1992, the World Health
Organization (WHO) recommended that all countries
include a hepatitis B vaccine in their routine infant
immunization programs. As of 2003, the WHO/UNICEF
estimated 42% hepatitis B vaccination coverage in the global
birth cohort (7). In China, the HBV infection prevalence
decreased from 9.7% (in 1992) to 7.2% (in 2006) after the
introduction of a universal HBV vaccination program in 1988
(10). However, in our study, we did not observe the
theoretically expected decrease in the development of HBVinduced LC, possibly because HBV infection was present

Selected risk factors for HCC


Among the 87 patients with HCC, 69 patients were men,
and 18 patients were women. As shown in Table 4, only the
sex ratio (male to female) and HBs Ag positivity rate were
significantly increased in HCC patients compared with nonHCC patients (po0.05) by univariate analysis. Metabolic
factors, which included obesity, DM, and hypertension, were
not significantly different between men and women. HBs Ag
positivity was the sole risk factor for HCC in cirrhotic
patients (po0.001) in a multivariate analysis. Obesity and
DM did not increase the HCC incidence in the whole
cirrhotic group (5.7% vs. 5.5%, respectively, p=0.862; 5.0% vs.
5.6%, respectively, p=0.849). However, among patients with
cirrhosis induced only by HBV, a trend of increased rates of
HCC in obese and DM patients was found (8.4% vs. 6.5%,
respectively, p=0.436; 7.1% vs. 6.7%, respectively, p=0.589).
Table 2 - Clinical characteristics of cirrhotic patients.

Age (years)
Female
Male
Male/female
BMI (Mean SD)
Obesity (%)
DM (%)
Hypertension (%)
HBs Ag+ (%)
HCC (%)
Number of patients

Total

20032008

20082013

p-value#

52.812.8
485
1,097
2.26
22.13.3
229 (14.5)
159 (10.1)
129 (8.2)
1,083 (68.5)
87 (5.5)
1,582

52.312.8
147
344
2.34
22.13.5
71 (14.5)
44 (8.9)
33 (6.7)
325 (66.2)
36 (7.3)
491

52.712.7
338
753
2.23
22.13.2
158 (14.5)
115 (10.5)
96 (8.8)
758 (69.5)
51 (4.7)
1,091

0.519
0.678

0.894
0.991
0.334
0.162
0.193
0.033

BMI: body mass index; DM: diabetes mellitus; HBS Ag+: hepatitis B virus
surface antigen positive; HCC: hepatocellular carcinoma.
#
p-value comparing 20032008 with 20082013.

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CLINICS 2015;70(8):563-568

study, with the difference that a BMI higher than 25 was


necessary but not the sole criterion to consider cryptogenic
cirrhosis as NASH-related cirrhosis because abnormal
glucose metabolism, arterial hypertension and high triglyceride levels in the patients were also considered. Although
NASH can be found in lean patients, the identification of
metabolic features or performance of a liver biopsy is
required for an accurate diagnosis. Due to the retrospective
nature of our study, we could not document the number of
lean patients; thus, we adopted a BMI higher than 25 as a
necessary criterion. Notably, a liver biopsy may lack relevant
metabolic features when cirrhosis is present and therefore
may not always contribute to determining the etiology.
Although we considered obesity and DM in our study
when diagnosing NASH-related LC, we did not find
significant differences in the proportions of obesity, DM
and hypertension when comparing the 2003-2008 and 20082013 time periods.
Several studies have reported that Met-s (obesity or DM)
not only promotes the development of LC but also increases
the risk of HCC (17-19). In China, obesity has become an
important health problem because the prevalence of obesity
in adults, which was 7.1% in 2002, has increased by 97.2%
since 1992 (20,21). In children, a dramatic increase in obesity
has been observed, with a two- to three-fold increase in
Beijing and Shanghai between 1985 and 1995. In addition,
the prevalence of overweight and obese children approached
29% in 2000 in 7- to 12-year-old boys and 15-17% in girls (21).
A 2013 study from Tianjing, China, also found that the
prevalence of obesity in children under 7 years old
approached 8.2% (22). These data illustrate the rapid
development of obesity in both adults and children. In the
USA, data from the 2009-2010 National Health and Nutrition
Examination Survey reveal that 36% of Americans are obese
and that 30% of the general adult population may have
NAFLD (23). In the USA, the yearly cumulative HCC
incidence in NASH-related LC was 2.6%, compared with
4.0% among patients with HCV-related cirrhosis, over an
average follow-up period of 3.2 years (24). Therefore, the
rapid development of obesity in China could be contributing
to an increase in NAFLD, leading to the increased prevalence
of NASH-related LC shown in our study. The importance of
obesity in the natural history of NASH-related LC explains

Figure 2 - Time trend of NASH-related LC over the ten year period


from 2003-2013.

prior to the start of the vaccination campaign; the development of cirrhosis usually requires a long period of time (11).
Nevertheless, the efficacy of the Chinese HBV immunization
program is indicated by the decreased prevalence of HBV
carrier status to 2.1% among all children and to 1.0% among
those born after 1999, together with the presence of anti-HBs
antibodies (1,12). Therefore, it is likely that HBV-related
etiology of Chinese LC will be significantly reduced in the
future.
In Western countries, NAFLD is now considered to be the
most common cause of chronic liver disease, with 20-30% of
the total population presenting with NAFLD (13,14). In
China, NAFLD affects approximately 15% of adults and is
present in 68.5% of obese children (15, 16). The proportion of
NASH-related LC (1.9%) in our study was similar to that in
Japan (2.1%) (4). However, our study was retrospective and
may not represent the incidence of LC in the general
population. In our study, an increasing frequency was noted
over the ten years covered by the study; this trend is likely to
result in a significant increase in patients with NASH-related
LC in the future.
A limitation of the current study, especially in the context
of a retrospective study, is that diagnosing LC related to
NASH requires a BMI higher than 25. However, our criteria
were based on those adopted by the Japanese nationwide
Table 3 - Clinical characteristics of NASH-related LC.

Number of patients
Age (years)
BMI (Kg/m2)
Arterial hypertension (%)
DM (%)
Complications
Ascites (%)
HCC (%)
UGB (%)
HRS (%)
HE (%)
Child-Pugh grading
A (%)
B (%)
C (%)

Total

Female (F)

Male (M)

p-value (F vs. M)

30
57.714.1
27.22.1
11 (36.7)
8 (26.7)

11
60.612.4
27.22.2
6 (54.6)
1 (9.1)

19
55.915.0
27.22.0
5 (26.3)
7 (36.8)

0.388a
0.965a
0.238b
0.199b

(40.0)
(0)
(20.0)
(0)
(0)

5 (45.5)
0 (0)
3 (27.3)
0 (0)
0 (0)

7 (36.8)
0 (0)
3 (15.8)
0 (0)
0 (0)

16 (53.3)
12 (40.0)
2 (6.7)

5 (45.5)
4 (36.4)
2 (18.2)

11 (57.9)
8 (42.1)
0 (0)

12
0
6
0
0

0.712b
0.641b

0.308c

BMI: body mass index; DM: diabetes mellitus; HCC: hepatocellular carcinoma; UGB: upper gastrointestinal bleeding; HRS: hepatorenal syndrome; HE: hepatic
encephalopathy.
p-value determined by a: Students t test; b: Fishers exact test; c: the Mann-Whitney U test.

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CLINICS 2015;70(8):563-568

Table 4 - Selected risk factors for HCC.


Variable

Age
o65
X65
Sex
Female
Male
Obesity
Obese
Non-obese
Hypertension
Hypertensive
Non-hypertensive
DM
Diabetic
Non-diabetic
HBs Ag
HBs Ag positive
HBs Ag negative
Hepatitis B*
HBV+obesity
HBV+non-obesity
HBV+DM
HBV+non-DM
Alcohol#
HBV+alcohol abuse
HBV+no alcohol abuse

Control

HCC

N=1,495 (%)

N=87 (%)

OR (95% CI)

p-value

1,227 (94.6)
268 (94.0)

70 (5.4)
17 (5.9)

0.93 (0.52-1.64)

0.796

468 (96.3)
1,027 (93.7)

18 (3.7)
69 (6.3)

1.81 (1.04-3.14)

0.036

216 (94.3)
1,279 (94.5)

13 (5.7)
74 (5.5)

1.06 (0.58-1.96)

0.862

123 (95.3)
1,362 (94.4)

6 (4.6)
81 (5.6)

0.90 (0.37-2.21)

0.825

151 (95.0)
1,344 (94.5)

8 (5.0)
79 (5.6)

0.93 (0.43-1.99)

0.849

1,008 (93.1)
487 (98.6)

75 (6.9)
7 (1.4)

2.64 (1.01-6.92)

0.049

11
52
5
58

(8.4)
(6.5)
(7.1)
(6.7)

1.31 (0.66-2.58)

0.436

1.27 (0.53-3.07)

0.589

12 (8.9)
63 (6.7)

0.74 (0.39-1.42)

0.364

121
754
65
810

(91.7)
(93.6)
(92.8)
(93.3)

123 (91.1)
885 (93.4)

Univariate analysis

Multivariate analysis
OR (95% CI)

p-value

3.35 (1.76-6.36)

0.000

* Cirrhotic patients with chronic liver disease caused only by HBV infection (938 patients).
Alcohol abuse in cirrhotic patients with HBs Ag positivity (1,083 patients).

nationwide study (4). A study from England found that


NAFLD accounted for 41 (34.8%) cases of HCC in 2010 (30).
Another study on HCC from Germany also found an
increasing proportion of NASH in HCC patients (2). With
the promotion of an HBV vaccination program and the
improvement of economic development, this type of time
trend may also occur in China. Therefore, we should expand
our attention from patients with cirrhosis caused by chronic
viral hepatitis to those with cirrhosis induced by NASH in
the future. Additionally, patients with NASH-related LC
must be followed in the coming decades, paying special
attention to the effects in obese children, as the vaccination
program has protected them from HBV.
For NASH-related LC, we found that female patients were
older than males and that the rates of complications and
Child-Pugh grade C tended to be higher in women, which is
in accordance with the Japanese nationwide study (4).
However, the difference in our study was not significant,
possibly due to the small sample size. The epidemiological
study in Japan observed a higher NAFLD prevalence in men
than in women. However, in contrast to men, NAFLD
increased with age in women, showing a higher prevalence
among women aged 40-49 years and after menopause (31).
Aging is considered as a risk factor for NAFLD in premenopausal women (32). Our study showed an average age
of 60 years for women who developed NASH-related LC,
indicating that they were menopausal women. Because
estradiol has been reported to attenuate hepatic stellate cell
activation and therefore decrease the fibrotic process (33),
estradiol diminution after menopause might contribute to
the development of NASH-related cirrhosis.
Our study has several limitations. First, because the study
was retrospective, it was not possible to collect certain

why we decided to integrate this risk factor as a necessary


criterion for diagnosing NASH-related LC in our study.
Although the increases in NASH-related LC and obesity
may lead to a higher incidence of HCC, we unexpectedly
found that the HCC incidence was significantly lower in the
time period from 2008-2013 than in the period from 20032005. Another recent study, which used data from the Cancer
Registration Database records in Shanghai, China, also noted
a decreasing HCC incidence (25). This study additionally
found that the age-standardized incidence rates for each year
and the age-standardized mortality rates decreased from
2006-2008 and that the incidence and mortality rates
increased with age. The incidence and mortality rates of
primary liver cancer were relatively higher in the suburbs
than in urban areas and were also higher in males than in
females. Additionally, a study from Taiwan found that male
gender was a risk factor for HCC, which is in accordance
with our results based on univariate analysis. However, DM
and obesity were not associated with HCC in areas where
either HBV or HCV was endemic (26). In our study, obesity
and DM were not associated with HCC in the whole cirrhotic
population, in accordance with the Taiwanese study. However, when considering only the HBV-related cirrhotic
subgroup, we observed a statistically insignificant trend of
increased HCC in patients who were obese or diabetic. The
prevalence of NAFLD-related HCC is rising worldwide:
4-22% of HCC cases in Western countries are now attributed
to NAFLD (13). Additionally, 10-75% of cases of NAFLDrelated HCC occur in patients without cirrhosis (27,28).
A higher BMI in childhood increases the risk of primary liver
cancer in adults (29). In our study, HCC was not found in
patients with NASH-related LC, whereas HCC was present
in 31.5% of NASH-related LC cases in the Japanese

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CLINICS 2015;70(8):563-568

important metabolic parameters such as blood glucose for all


cirrhotic patients, which may have led to underestimation of
the rate of DM in LC. Second, histological data were rarely
obtained for our patients, which confined the diagnosis of
NASH-related cirrhosis to clinical, biochemical, and imaging
data. However, it should be recalled that when cirrhosis is
histologically present, the etiological features may disappear,
so a liver biopsy may not be able to ascertain NASH-related
cirrhosis. Third, the relatively small number of NASHrelated LC cases might explain the absence of significant
differences when considering important risk factors reported
in other studies. Finally, the history of NAFLD and the
duration of obesity or DM could not be obtained from the
database.
In conclusion, HBV remains the main etiology of LC in
China. However, NASH-related LC has clearly increased in
frequency in recent years. Further studies are warranted to
evaluate the exact importance of obesity and diabetes in
HCC development in the overall cirrhotic population.

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Debasish Das. Hepatocellular cancer: The impact of obesity, type 2 diabetes and a multidisciplinary team. Journal of hepatology. 2014;60:110-7,
http://dx.doi.org/10.1016/j.jhep.2013.08.011.
31. Rodrigues MH, Bruno AS, Nahas-Neto J, Santos ME, Nahas EA. Nonalcoholic fatty liver disease and metabolic syndrome in postmenopausal
women. Gynecol Endocrinol. 2014;30(5):325-9, http://dx.doi.org/10.3109/
09513590.2013.875992.
32. Hamaguchi M, Kojima T, Ohbora A, Takeda N, Fukui M, Kato T. Aging is
a risk factor of nonalcoholic fatty liver disease in premenopausal women.
World J Gastroenterol. 2012;18(3):237-43, http://dx.doi.org/10.3748/wjg.
v18.i3.237.
33. Shimizu I, Ito S. Protection of estrogens against the progression of chronic
liver disease. Hepatol Res. 2007;37(4):239-47, http://dx.doi.org/10.1111/
hep.2007.37.issue-4.

ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of
China (Grant No. 81170382 and 81200297).

AUTHOR CONTRIBUTIONS
Xiong J designed the questionnaire for data collection from the database,
was responsible for the data entry and statistical analysis, and wrote the rst
version of the manuscript. Wang J collected data from the database and
participated in data entry. Huang J collected data from the database and
participated in data entry. Sun W checked the data and collected references
for this manuscript. Wang J revised the manuscript. Chen D participated in
the overall process of writing and revising the manuscript and served as the
corresponding author.

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568

CLINICAL SCIENCE

Glutamine treatment attenuates hyperglycemia-induced


mitochondrial stress and apoptosis in umbilical vein
endothelial cells
Sher Zaman Safi,I,* Kalaivani Batumalaie,I Marzida Mansor,II Karuthan Chinna,III Syam Mohan,IV
Hamed Karimian,IV Rajes Qvist,I Muhammad Aqeel Ashraf,V Garcie Ong Siok YanII
I

University of Malaya, Faculty of Medicine, Department of Medicine, Kuala Lumpur, Malaysia. II University of Malaya, Faculty of Medicine, Department of
Anesthesiology, Kuala Lumpur, Malaysia. III University of Malaya, Faculty of Medicine, Department of Social and Preventive Medicine, Kuala Lumpur,
Malaysia. IV University of Malaya, Faculty of Medicine, Department of Pharmacy, Kuala Lumpur, Malaysia. V University of Malaya, Faculty of Science,
Department of Geology, Kuala Lumpur, Malaysia.

OBJECTIVE: The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis,
mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical
vein endothelial cells.
MATERIALS AND METHODS: Human umbilical vein endothelial cells were grown and subjected to glutamine
and insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and the
production of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicity
assays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay.
RESULTS: Glutamine maintains the integrity of the mitochondria by reducing the cell permeability and
cytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level was
significantly (po0.005) reduced when the cells were treated with glutamine. An apoptosis assay revealed
significantly reduced apoptosis (po0.005) in the glutamine-treated cells. Moreover, glutamine alone or in
combination with insulin modulated inflammatory cytokine levels. Interleukin-10, interleukin-6, and vascular
endothelial growth factor were up-regulated while tumor necrosis factor-a was down-regulated after
treatment with glutamine.
CONCLUSION: Glutamine, either alone or in combination with insulin, can positively modulate the mitochondrial
stress and cell permeability in umbilical vein endothelial cells. Glutamine regulates the expression of
inflammatory cytokines and maintains the balance of the mitochondria in a cytoprotective manner.
KEYWORDS: Hyperglycemia; Sepsis; Apoptosis; Cytokine; Glutamine.
Safi SZ, Batumalaie K, Mansor M, Chinna K, Mohan S, Karimian H, et al. Glutamine treatment attenuates hyperglycemia-induced mitochondrial
stress and apoptosis in umbilical vein endothelial cells. Clinics. 2015;70(8):569-576
Received for publication on April 29, 2015; First review completed on June 9, 2015; Accepted for publication on June 9, 2015
E-mail: safi.nust@yahoo.com
*Corresponding author

INTRODUCTION

been observed in the vascular cells, myocardium, and nerves


of diabetic experimental animals and human subjects,
although whether it contributes to or is a marker for
complications in these tissues is unclear. Vascular diseases
are the major cause of morbidity and mortality in diabetic
patients. Several studies suggest that the endothelium is the
initial site where these vascular complications develop (4).
During critical illness, hyperglycemia alone or hyperglycemia coupled with a relative insulin deficiency may directly or
indirectly yield a predisposition to a spectrum of complications, such as multi-organ failure and death (5-7). Morphological correlates of these functional abnormalities were not
initially identified; however, several later studies showed an
increase in apoptosis in several organs affected by diabetes
and sepsis, including the eye (8,9), heart, and vascular
endothelium (10,11).

Hyperglycemia is the abnormality underlying diabetes


and several other complications. Chronic hyperglycemia
condition initiates a wide range of complications, including
cardiovascular disease, which is the most frequent cause of
death in the diabetic population (1,2). Hyperglycemia also
induces the production of reactive oxygen species (ROS) and,
ultimately, DNA damage and apoptosis (3). Apoptosis has

Copyright & 2015 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.
DOI: 10.6061/clinics/2015(08)07

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divided into three groups. In the first group, 40 mM of glutamine was added. In the second group, 1.0 x 10-6 units/ml of
insulin was added. In the third group, glutamine (40 mM)
and insulin (1.0 x 10-6 units/ml) were added. The cells were
then incubated for the required length of time (24 hours). For
the cytokine and TUNEL analyses, 0.7x106 cells were grown
in T25 flasks using the same treatment groups. The cells were
harvested and frozen until required for analysis.

Vascular endothelial cells are among the first cells in the


body to interact with bacterial endotoxins during sepsis.
Sepsis is a very complex and heterogeneous clinical
condition that is associated with hyperglycemia and insulin
resistance (12). Vascular endothelial cells possess mechanisms that recognize structural patterns of bacterial constituents and initiate the expression of proinflammatory and
anti-inflammatory pathways, which are tightly controlled
to maintain homeostatic balance (13). However, in severe
sepsis, external stimuli, such as a severe infection, can
activate the immune cells and unleash a systemic inflammatory response that is expressed through various pathways
(14). There is also increasing evidence to suggest that cell
death by apoptosis plays an important role in the pathogenesis of severe sepsis/septic shock (15).
Several studies have shown that endothelial cells can
undergo apoptosis in response to sepsis-related factors such
as Lipopolysaccharide (LPS) and Tumor necrosis factor alpha
(TNF-a) (16-18). In addition, studies using in vitro models of
infection have demonstrated that certain organisms are
capable of inducing endothelial apoptosis (19). Along with
the interaction of inflammation with apoptosis in sepsis,
mitochondrial dysfunction seems to have a major impact in
sepsis patients because it has been closely linked to programmed cell death. Alterations in mitochondrial function
have been described in the muscle and liver mitochondria
from septic rats and primates. Furthermore, mitochondrial
dysfunction has been suggested as a potential mechanism to
explain tissue hypoxia despite normal oxygen availability
during sepsis (20,21). Pro-inflammatory cytokines, such as
interleukin 6 (IL-6), TNF-a, and other molecules, are released
during acute inflammation and result in endothelial activation
and a significant increase in the expression of endothelial
leukocyte adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1),
and vascular endothelial growth factor (VEGF). These proteins
promote leukocyte rolling, adherence, and migration, which
initiate inflammation in the endothelium and other cells
(22,23). We included IL-10 as an anti-inflammatory marker.
Therefore, the aim of this study was to determine the
mechanism of endothelial cell apoptosis and the expression
of inflammatory cytokines under hyperglycemic conditions
and to examine the effects of glutamine and insulin.

Western blotting
The endothelial cells were first lysed in cold lysis buffer
containing 20 mmol/l of TRIS HCl, 140 mmol/l of NaCl, 1
mmol/l of EDTA and complete miniprotease inhibitor cocktail, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1
mmol/l NaF, and 1 mmol orthovanadate. The proteins (30 mg)
were then loaded on 10% SDS polyacrylamide gels and
transferred to activated nitrocellulose membranes. The membranes were blocked with Tris-buffered saline (TBS) containing 5% nonfat milk and incubated overnight with the primary
antibodies to IL-10 and TNF-a, obtained from Santa Cruz, at
4 C. Beta-actin was used as a loading control. After extensive
washes in TBS, the membranes were incubated for one hour at
room temperature with the appropriate horseradish peroxidase-conjugated secondary antibodies, and the proteins were
visualized using a chemiluminescence substrate according to
the manufacturers instructions (Amersham Life Sciences).

Multiple cytotoxicity assays


The Cellomics Multiparameter Cytotoxicity 3kit was used
as previously reported in detail by Cheah et al. (24). The
Multiparameter Cytotoxicity 3kit enables parallel measurements of six independent parameters that monitor cell
health, namely, changes in cell permeability, cell loss and
nuclear size; changes in mitochondrial membrane potential;
cytochrome c release; and morphological changes. Briefly, the
cells were plated at 1x104 cells per well for 24 hours. Glucose
(5 or 20 mM), glutamine (40 mM), and insulin (1.0 x 10-6
units/ml) were added in different combinations as described
in the cell treatment section, and the incubation was
continued for 24 hours. The MMP dye and the cell
permeability dye were added to the live cells, and the cells
were incubated for 1 hour. The cells were fixed, permeabilized, and blocked with 1  blocking buffer before they were
incubated with the primary cytochrome c antibody and
conjugated secondary antibody for 1 hour. The cells were
rinsed three times with wash buffer II, and the plates were
analyzed using the Array Scan HCS high content system
(Cellomics, PA, USA).

MATERIALS AND METHODS


Cell culture
Endothelial cells were obtained from VEC Technologies
(New York, USA). The cells were thawed at 37 C and
cultured in T25 flasks coated with 50 mg/ml of fibronectin.
The cells were immersed in 5 ml of complete medium
(MCDB-B-131), supplemented with 10% FBS, 1% penicillinstreptomycin, and epidermal growth factor (EGF, 10 ng/ml).
The cells were incubated at 37 C with 5% CO2. Trypsin/
EDTA (1 ml for each flask) was used to detach the cells upon
confluency. All the experiments were performed at passages
2-5.

Measurement of transmembrane mitochondrial


potential
The mitochondrial transmembrane potential results from
the asymmetric distribution of protons and other ions on the
two sides of the inner mitochondrial membrane, which gives
rise to the chemical, pH, and electric gradients that are
essential for mitochondrial function. The inner side of the
inner mitochondrial membrane is negatively charged. Consequently, cationic lipophilic fluorochromes are distributed
within the mitochondrial matrix as a function of the Nernst
equation describing the transmembrane mitochondrial
potential (c). Using a cytofluorometer, these dyes were
employed to measure variations in the transmembrane
potential (c) on a per-mitochondrion or per-cell basis.

Cell treatment
The cells were seeded at 1x104 cells in each well and
incubated for 24 hours. Various concentrations of glucose,
ranging from a normal value (5 mM) to a hyperglycemic
level (20 mM), were added to the individual wells. The
hyperglycemic cells (glucose concentration 20 mM) were

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Terminal deoxynucleotidyl transferase dUTP nick


end-labeling (TUNEL) assay

Bio-Plex Cytokine Assay (Bio-Rad Laboratories) was conducted according to the manufacturers protocol. The
calculated concentrations for each cytokine were averaged,
and the standard deviations were determined. Statistical
significance was determined using the t-test, where po0.05
designated increased/decreased cytokine production in the
presence of glutamine or glutamine in combination with
insulin.

DNA damage was investigated using a 96well colorimetric apoptosis detection kit (R&D System) according to the
manufacturers instructions. Umbilical vein endothelial cells
were cultured and transferred to a 96-well plate (1x105 cells/
well). The cells were then fixed with 3.7% buffered
formaldehyde for 5 minutes, followed by washing with
PBS. The washing was followed by permeabilization of the
cells with 100% methanol for 20 minutes and another wash
with PBS. Following the manufacturers protocol, the cells
were then subjected to the labeling procedure, and the
reaction was stopped with 0.2 N HCl after 30 minutes. The
cells were treated with NucleaseTM to generate DNA breaks
and to confirm the permeabilization and labeling reactions.
An unlabeled control was included to indicate the level of
background labeling associated with non-specific binding of
the Strep-HRP. The absorbance at 450 nm was measured
using a microplate reader.

Statistical analysis
Each experiment was performed at least two times.
Statistical analysis was performed using one-way analysis
of variance (ANOVA).

RESULTS
Glutamine reduces the cytochrome C levels and
apoptosis in hyperglycemic human umbilical vein
endothelial cells
Umbilical vein endothelial cells were stained with Hoechst
in the presence of glucose (20 mM) alone, 20 mM glucose +
40 mM glutamine, 20 mM glucose + 10-6 M insulin, or 20
mM glucose + 40 mM glutamine + 10-6 M insulin, and the
staining intensity was determined. As shown in Figures 1
and 2, glucose alone (20 mM) reduced the number of cells,
possibly by apoptosis, as well as reduced the level of

Cytokine measurements
The cytokines TNF-a, IL-6, and IL-10 were measured in
triplicate using the Protein Bio-Plex Cytokine Assay (Bio-Rad
Laboratories). T25 flasks containing 0.7x106 cells were
cultured, and the lysate was filter-sterilized (0.22-mm pore
size). The protein concentrations were determined, and the

Figure 1 - Representative images of endothelial cells treated with medium alone (control), glucose alone (20mM), glucose (20mM) +
insulin (10-6 M), glucose (20mm) + glutamine (40mM) and glucose (20mM) + glutamine (40mM) + insulin (10-6 M). Cells were stained
with Hoechst for nuclear, cell permeability dye, mitochondrial membrane potential dye and cytochrome c. The image from each row
was obtained from the same field of the same treatment sample (magnification 20 xs).

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Glutamine restores the loss of mitochondrial


membrane potential (MMP) in Human Umbilical
Endothelial Cells (HUVEC)

cytochrome c. There was a significant change (po0.005) in


the fluorescence intensity in the cells that were treated with
glucose along with glutamine. This result indicates that these
cells were more viable. In addition, the cytochrome c levels in
the endothelial cells treated with glucose (20 mM) +
glutamine (40 mM) were significantly (po0.005) lower than
the levels in the cells treated with glucose (20 mM) alone. The
cytochrome c levels in the cells treated with glucose (20 mM)
+ insulin (10-6 M) and glucose (20 mM) + insulin (10-6 M) +
glutamine (40 mM) also changed significantly (po0.05)
compared with the levels in the cells treated with glucose
(20 mM) alone (Figures 1 and 2B). Similarly, the TUNEL
assay revealed a significantly reduced level of apoptosis
when the hyperglycemic cells were treated with insulin
(po0.05), glutamine (po0.005), or glutamine + insulin
(po0.005) (Figure 2A).

According to previous studies, a reduction in the


mitochondrial membrane potential occurs when cells
undergo oxidative stress and hyperglycemia in the
presence of other co-factors. The cells showed a significant change in the MMP when treated with glutamine
and a considerable, but not significant, change in the
MMP when they were treated with glucose + glutamine
+ insulin compared with 20 mM glucose only. Glucose
(20 mM) + insulin (10 -6 M) and glucose (20 mM) + insulin
(10-6 M) + glutamine (40 mM) also yielded an increase in the
MMP (DCm) compared with the control. These results show
that the MMP was reduced after the cells had been treated

Figure 2 - Shows (A) TUNEL assay which revealed a significantly reduced apoptosis when hyperglycemic cells were treated with insulin
(po0.05) glutamine (po0.005) and glutamine + insulin (po0.005). (B) Cytochrome c intensity for the endothelial cells treated with
glucose (20 mM) + glutamine (40 mM) significantly changes when compare to glucose (20 mM) alone. Cytochrome c intensity for the
cells treated with glucose (20 mM) + insulin (10-6 M) and glucose (20 mM) + insulin (10-6 M) + glutamine (40 mM) changes
insignificantly as compare to glucose (20 mM) alone. (C) and (D) show mitochondrial membrane potential and cell permeability
respectively.

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Glutamine and inflammatory cytokines

with 20 mM glucose alone; however, glucose (20 mM) in


combination with glutamine (40 mM) was shown to be more
protective against the fall in the MMP and oxidative stressinduced cell death (Figure 2C).

When glucose (20 mM) and insulin (10-6 M) were added to


the culture, there was no difference in the expression of IL-6.
However, glucose + glutamine significantly increased the
expression of IL-6. The combined treatment with glucose
(20 mM), glutamine (40 mM), and insulin (10-6 M) also
significantly increased the expression of IL-6 (po0.005).
Similarly, treatment with insulin alone did not alter the
expression of IL-10, but insulin in combination with
glutamine increased the expression of IL-10 in the endothelial cells. A small increase in the expression of VEGF was
noted while a reduced expression of TNF-a was observed
when the cells were treated with the same concentration of
glutamine alone or in combination with insulin (Figure 3A,
3B, 3C, and 3D). Detailed values of all cytokines are given in
Table 1. IL-10 is the most important anti-inflammatory

Effect of glutamine on cell permeability


When the MMP decline reaches a certain point, the
opening of the permeability transition pore (PTP) starts to
cause extensive cell damage and, consequently, cell death.
The hyperglycemia-induced permeability was reversed
when we treated the HUVECs with glucose + glutamine
or glucose + glutamine + insulin compared with glucose
(20 mM) alone. However, there was a slight increase in the
cell permeability when the cells were treated with glucose (20
mM) + insulin (10-6 M) (Figure 2D).

Figure 3 - (A) shows that insulin did not alter the expression of IL10 when treated alone but insulin in combination with glutamine
increased IL10 in endothelial cells (po0.05). (B) Shows a significantly reduced TNFa when cells were treated with the same
concentration of glutamine in combination with insulin. (C) IL6 expression which is significantly increased when treated with
glutamine in combination with insulin (po0.005). (D) A mild increase in the expression of VEGF was noted when treated with
glutamine or glutamine in combination with insulin.

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Table 1 - Cytokine analysis comparing 20 mM glucose, 20 mM

carried out Western blotting for IL-10 and TNF-a (Figure 4).
The levels of both IL-10 and TNF-a changed in the same
manner, as shown in Figure 3.

glucose + Ins, 20 mM glucose + Gln, and 20 mM glucose + Ins


+ Gln.
Variable
Hu IL-6

Hu IL-10

Hu TNF-a

Hu VEGF (45)

Group
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20

mM
mM+Ins
mM+Gln
mM+Ins+Gln
mM
mM+Ins
mM+Gln
mM+Ins+Gln
mM
mM+Ins
mM+Gln
mM+Ins+Gln
mM
mM+Ins
mM+Gln
mM+Ins+Gln

Mean

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3

55.5
56.2
73.7
91.0
26.3
27.5
32.2
34.8
13.3
12.8
10.7
9.7
22.0
20.7
22.5
24.7

p-value

DISCUSSION

NS
0.035
0.004

Our results show that glutamine, either alone or in


combination with insulin, disrupts mitochondrial stress and
improves cell viability. Anti-inflammatory cytokines were highly
expressed in the glutamine-treated cells. With respect to
cytochrome C, there was a significant change in the fluorescence
intensity in the cytosol in the cells treated with glutamine alone,
indicating an increase in the viability of the cells (Figure 2B) on
the basis that increased cytochrome c levels in the cells trigger
programmed cell death through apoptosis (25,26). Similarly,
there was an increase in the mitochondrial potential (Figure 2C).
Our results demonstrate that the cytochrome c levels in cells
are increased under hyperglycemic conditions. This protein is
known for its function in the mitochondria as a key participant
in the life-supporting function of ATP. Our data also support the
hypothesis that hyperglycemia, through the production of
oxidative stress, could be an apoptotic stimulus that triggers
the release of cytochrome c into the cytosol, thereby activating
the mitochondrial pathway that leads to the permeabilization of
the outer mitochondrial membrane and increasing the level of
cytochrome c (27,28). In the cytosol, cytochrome c engages
apoptotic protease activating factor-1 (APAF1) and forms the
apoptosome. The cell then dies via the apoptotic pathway or
necrosis due to the collapse of electron transport, generation of
oxygen free radicals, and production of ATP (29,30). As shown
in Figure 2B, in our study, the level of cytochrome c in cells

NS
NS
0.011
NS
NS
0.006
NS
NS
NS

cytokine found within the human response. It is a potent


inhibitor of Th1 cytokines and a potent activator of
monocyte/macrophage proinflammatory cytokine synthesis.
In addition, IL-10 attenuates the surface expression of TNF
receptors and promotes the shedding of the TNF receptors
into the systemic circulation. Furthermore, there is an
interesting relationship between IL-10 and the soluble TNFa receptor. Specifically, when IL-10 increases, it causes an
increase in the levels of the soluble TNF-a receptor, which
results in decreased TNF-a levels. To confirm this effect, we

Figure 4 - (A) shows the effect of insulin and glutamine on the expression of IL10 and TNFa in endothelial cell. (B) shows significantly
reduced IL10 and (C) shows TNFa in graphical form.

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determined the effect of insulin and glutamine on the


expression of IL-6, a cytokine with antiinflammatory and
proinflammatory functions. When insulin was added to the
cultures, there was no difference in the expression of
IL-6 (Figure 3), but glutamine had an additive effect with
insulin, and the combination of glutamine and insulin
significantly increased the expression of IL-6. Like many
other cytokines, IL-6 has both proinflammatory and antiinflammatory properties. Recent evidence generated using
IL-6 knockout mice has demonstrated that IL-6, like other
members of the gp130 receptor ligand family, acts predominantly as an antiinflammatory cytokine. IL-6 down-regulates the synthesis of IL-1 and TNF-a and attenuates the
synthesis of proinflammatory cytokines. Simultaneously,
IL-6 inhibits the production of proinflammatory cytokines,
including GMCSF, IFNg, and MIP2. Interestingly, IL-6 may
down-regulate TNF-a and IL-b production and may be
important in limiting the inflammatory response. Our results
demonstrate the same limiting response of IL-6 on TNF-a;
however, VEGF was unexpectedly up-regulated by the
glutamine treatment. The net results of these immunologic
effects place IL-6 among the anti-inflammatory group (35).
The expression of IL-10 (Figure 3) was increased by
treatment with glutamine and insulin, with the addition of
insulin having an additive effect. IL-10 is the most important
anti-inflammatory cytokine in humans. It is a potent
inhibitor of the Th1 cytokines and a potent deactivator of
monocyte/macrophage pro-inflammatory cytokine synthesis. In addition, IL-10 attenuates the surface expression of
TNF receptors and promotes the shedding of the TNF
receptors into the systemic circulation (35-37).
The above cytokines have both anti-inflammatory and pro
inflammatory functions (38). Therefore, even during an
inflammatory disease such as sepsis, it is important to maintain
both the inflammatory and anti-inflammatory cytokines, and
this balance seems to be achievable by the effect of glutamine.
According to previous reports, glutamine supplementation has
been shown to maintain the Tlymphocyte population in the
spleen and to significantly enhance the mRNA expression
levels of Th1 and Th2 cytokines and TNF-a in the spleens of
rats with septic peritonitis (39,40).
Pharmacological supplementation with glutamine helps to
maintain the intestinal barrier, modulate cytokine production,
and prevent organ injury during sepsis. However, the exact
protective mechanism remains to be explored. It has already
been demonstrated that glutamine significantly attenuates the
plasma levels of cytokines produced by macrophages and
endothelial cell necrosis after cecal ligation and puncture in rats
(41,42). Recently, it was reported that glutamine treatment
directly augmented macrophage TNF-a production in vitro but
decreased TNF-a release in vivo, even though the expression of
HSP72 was increased in both cases (43). Another report
suggests that dietary glutamine administration results in higher
inflammatory cytokine production and greater neutrophil
recruitment during the early stage of acute lung injury (44).
In conclusion, our data suggest that glutamine alone or in
combination with insulin can modulate the production of
inflammatory and anti-inflammatory cytokines and maintain
the cytokine balance under hyperglycemic conditions,
producing a cytoprotective effect. At the same time, our
data indicate that glutamine maintains the integrity of the
mitochondria, the dysfunction of which in hyperglycemic
endothelial cells may reflect the degree of systemic injury in
severe sepsis.

cultured in 20 mM glucose (hyperglycemic conditions) was


significantly reduced by the addition of glutamine (40 mM), and
the viability of the cells was thus increased compared with that
of cells incubated with insulin alone or insulin plus glutamine.
Cells induced to undergo apoptosis show an early
reduction in the incorporation of c-sensitive dyes, which
indicates a decrease in the transmembrane potential. This
transmembrane potential disruption can be detected in many
different cell types, irrespective of the apoptosis-inducing
stimulus. The transmembrane potential disruption occurs
before the cells exhibit nuclear DNA fragmentation, indicating that the membrane potential change constitutes an early
common event of the apoptotic cascade. Purified cells with a
low transmembrane potential rapidly proceed to DNA
fragmentation. In our study, there was a decrease in the
mitochondrial potential under the hyperglycemic condition,
suggesting that hyperglycemia could act as an apoptosisinducing stimulus. The decrease in the mitochondrial
potential was reduced in the presence of glutamine or
glutamine plus insulin (Figure 2C), although this reduction
did not reach significance in the case of glutamine and
insulin combined. An intact transmembrane potential (c) is
indispensable for normal mitochondrial function because
cells undergoing apoptosis cease mitochondrial biogenesis at
both the translational and transcriptional level (31). Moreover, during apoptosis, mitochondrial inner membrane
proteins, including cytochrome c, leak out into the cytosol.
Mitochondria play a central role in the apoptotic process,
in which the dissipation of MMP, increased mitochondrial
oxidant production, and release of apoptogenic proteins
(e.g., apoptosis-inducing factor and cytochrome c) caused
by opening of the permeability transition pore are observed.
In our study, the cell permeability was increased under
hyperglycemic conditions, but the permeability was reduced
in the cells treated with glutamine (Figure 2D).
Recent evidence (32) has implicated a general dysregulation of the endothelium, with apoptosis and necrosis as the
final pathway of endothelial dysfunction and with mitochondrial dysfunction caused by the central disruption of
cellular oxidative function. We therefore hypothesized that
mitochondrial dysfunction may also be present in endothelial cells during hyperglycemia and may reflect the degree of
systemic injury in patients with severe sepsis and hyperglycemia. Stress-induced hyperglycemia and insulin resistance
are exceedingly common in critically ill patients, particularly
in those with sepsis. Multiple pathogenic mechanisms are
responsible for this metabolic syndrome, with the increased
release of proinflammatory mediators and counter-regulatory hormones playing a pivotal role (33). Recent data
suggest that hyperglycemia may potentiate the proinflammatory response, while insulin has the opposite effect.
To investigate the possibility that hyperglycemia plays a
key role in the development of the inflammatory response in
sepsis, we assessed the patterns of IL-6, TNF-a, VEGF, and
IL-10 that are associated with severe sepsis. In an exploratory
analysis, one study (34) demonstrated that by using multiple
cytokine assays, distinct cytokine profiles were found to be
associated with the severity of sepsis, the development of
organ failure, and death. The inflammatory cytokines IL-1b,
IL-6, IL-8, IL-10, and TNF-a have been shown to be
associated with the various stages of severe sepsis. To
determine whether the same group of cytokines was
expressed under hyperglycemic conditions, we assessed the
expression of IL-6, TNF-a, VEGF, and IL-10. In our study, we

575

Glutamine treatment for mitochondrial stress


Safi SZ et al.

CLINICS 2015;70(8):569-576

ACKNOWLEDGEMENTS

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supplementation on lung injury induced by lipopolysaccharide administration. Am J Physiol Lung Cell Mol Physiol. 2009;296(3):L288-95,
http://dx.doi.org/10.1152/ajplung.90479.2008.

This work was supported in part by grants (No. RG074/09AFR, and RG52813HTM (UMRG)) from the University of Malaya. We thank Arokiasamy
Vinsent Rayappan (Department of Medicine, Faculty of Medicine, UM) for
helping in the cell culture work. We declare there is no conict of interest.

AUTHOR CONTRIBUTIONS
Sa SZ performed the basic work and wrote the manuscript. Batumalaie K
helped with the lab work. Karimian H helped with the reagents. Mansor M,
Mohan S, Qvist R, and Yan GOS designed the study and reviewed
the manuscript several times. Chinna K and Ahraf MA helped with the
statistical analysis.

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576

BASIC RESEARCH

Effect of hypertonic saline treatment on the


inflammatory response after hydrochloric acid-induced
lung injury in pigs
Carla Augusto Holms,I Denise Aya Otsuki,I,* Marcia Kahvegian,I Cristina Oliveira Massoco,II
Denise Tabacchi Fantoni,III Paulo Sampaio Gutierrez,IV Jose Otavio Costa Auler JuniorI
I

Faculdade de Medicina da Universidade de Sao Paulo, Laboratory of Anesthesiology (LIM-08), Sao Paulo/SP, Brazil. II Faculdade de Medicina Veterinaria e
Zootecnia da Universidade de Sao Paulo, Department of Pathology. III Department of Surgery, Sao Paulo/SP, Brazil. IV Instituto do Coracao do Hospital das
Clnicas da Faculdade de Medicina da Universidade de Sao Paulo, Laboratory of Pathology, Sao Paulo/SP, Brazil.

OBJECTIVES: Hypertonic saline has been proposed to modulate the inflammatory cascade in certain
experimental conditions, including pulmonary inflammation caused by inhaled gastric contents. The present
study aimed to assess the potential anti-inflammatory effects of administering a single intravenous dose of
7.5% hypertonic saline in an experimental model of acute lung injury induced by hydrochloric acid.
METHODS: Thirty-two pigs were anesthetized and randomly allocated into the following four groups: Sham,
which received anesthesia and were observed; HS, which received intravenous 7.5% hypertonic saline solution
(4 ml/kg); acute lung injury, which were subjected to acute lung injury with intratracheal hydrochloric acid; and
acute lung injury + hypertonic saline, which were subjected to acute lung injury with hydrochloric acid and
treated with hypertonic saline. Hemodynamic and ventilatory parameters were recorded over four hours.
Subsequently, bronchoalveolar lavage samples were collected at the end of the observation period to measure
cytokine levels using an oxidative burst analysis, and lung tissue was collected for a histological analysis.
RESULTS: Hydrochloric acid instillation caused marked changes in respiratory mechanics as well as blood gas and
lung parenchyma parameters. Despite the absence of a significant difference between the acute lung injury and
acute lung injury + hypertonic saline groups, the acute lung injury animals presented higher neutrophil and
tumor necrosis factor alpha (TNF-a), interleukin (IL)-6 and IL-8 levels in the bronchoalveolar lavage analysis. The
histopathological analysis revealed pulmonary edema, congestion and alveolar collapse in both groups;
however, the differences between groups were not significant. Despite the lower cytokine and neutrophil levels
observed in the acute lung injury + hypertonic saline group, significant differences were not observed among
the treated and non-treated groups.
CONCLUSIONS: Hypertonic saline infusion after intratracheal hydrochloric acid instillation does not have an
effect on inflammatory biomarkers or respiratory gas exchange.
KEYWORDS: Acute lung injury; Hypertonic saline; Pigs; Hydrochloric acid; Inflammation.
Holms CA, Otsuki DA, Kahvegian M, Massoco CO, Fantoni DT, Gutierrez OS, et al. Effect of hypertonic saline treatment on the inflammatory
response after hydrochloric acid-induced lung injury in pigs. Clinics. 2015;70(8):577-583
Received for publication on May 5, 2015; First review completed on May 19, 2015; Accepted for publication on May 19, 2015
E-mail: lim08@usp.br
*Corresponding author

INTRODUCTION

compromise and is frequently observed among patients


who have depressed airway protective reflexes, such as
traumatized patients (3,4). In addition to the potential risk
of developing ARDS (5), the inhalation of gastric contents
is a common cause of donor lung rejection (6).
Hyperosmolar solutions, such as hypertonic saline, have been
tested in an attempt to reduce inflammation in ALI (7-9).
However, previous results are contradictory and indicate that
the effects of hypertonic saline depend on the type and agent of
lung injury and timing of administration (10,11).
The ALI model used in the current study involves the
intratracheal instillation of hydrochloric acid (HCl) and
attempts to mimic the clinical scenario of gastric content
aspiration, wherein a pulmonary lesion is caused by the action

The aspiration of acid gastric contents is the second most


common cause of direct acute lung injury (ALI) (1), and it
accounts for 10% of all acute respiratory distress syndrome
(ARDS) cases (2). Pulmonary inhalation of gastric contents
may provoke ALI with different degrees of respiratory

Copyright & 2015 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.
DOI: 10.6061/clinics/2015(08)08

577

CLINICS 2015;70(8):577-583

Hypertonic saline in lung injury


Holms CA et al.

Figure 1 - Study design.

of HCl. The acid causes chemical lesions in the lungs, resulting


in diffuse injury to the alveoli, release of inflammatory
mediators, recruitment of polymorphonuclear cells and promotion of severe edema, and these complications cause a
significant reduction in pulmonary compliance and compromise gas exchange (12-14). We hypothesized that hypertonic
saline would modulate HCl-induced lung injury. The purpose
of this investigation was to test whether a 7.5% hypertonic
saline dose could attenuate the inflammatory response evoked
by HCl instillation.

conventional formulae, following indexes were derived:


cardiac index (CI), pulmonary vascular resistance index
(PVRI) and systemic vascular resistance index (SVRI).
The inspiratory peak pressure, plateau inspiratory pressure, static pulmonary compliance, respiratory frequency
and tidal volume were obtained directly from the ventilator
monitor. Arterial blood samples were collected at each time
point and were immediately analyzed (ABL 555; Radiometer,
Copenhagen, Denmark), and the PaO2/FiO2 ratio was
calculated.

METHODS

Study design
After preparation, all of the animals were submitted to
three series of recruitment maneuvers consisting of 30 s of
sustained inflation with 20 cmH2O pressure followed by 30 s
of regular ventilation. The animals were allowed to stabilize
for 30 min and then randomly assigned to four groups: Sham
(n=8), hypertonic saline (HS; n=8), acute lung injury (ALI;
n=8) and acute lung injury + hypertonic saline (ALI+HS;
n=8). ALI was induced in the ALI and ALI+HS groups via
intratracheal instillation of HCl through the distal port of a
bronchoscope. The ALI+HS animals were treated with 7.5%
hypertonic saline (4 mg/kg) 15 min after HCl instillation.
The Sham and HS groups served as controls. The animals
from the HS group were administered 7.5% hypertonic saline
(4 ml/kg) 15 min after the baseline measurement.
The experimental protocol is outlined in Figure 1.

This study was approved by the Ethics and Animal


Investigation Committee of the affiliated institution (CAPPesq
0363/08) and was performed in accordance with the Guide for
the Care and Use of Laboratory Animals (15). Thirty-two
young female Landrace pigs weighing between 27 and 33 kg
(292.9) were included in this study. Animals were submitted
to 12-hour fasting with free access to water, pre-medicated
intramuscularly with ketamine (5 mg/kg) and midazolam
(0.5 mg/kg), and catheterized using a marginal ear vein for
drug and fluid administration. After sedation, the animals were
anesthetized with an intravenous dose of propofol (3-5 mg/
kg), orally intubated (6.5 mm internal diameter cuffed endotracheal tube) and placed in the supine position. The lungs
were mechanically ventilated (Primus, Drger, Germany) using
pressure-controlled ventilation (FiO2 50%, tidal volume of
8 ml/kg, inspiratory:expiratory (I:E) of 1:2 and positive endexpiratory pressure (PEEP) of 5 cmH2O), with the respiratory frequency adjusted to maintain end-tidal CO2 between
35 and 40 mmHg. To achieve patient-ventilator synchrony,
pancuronium bromide (0.3 mg/kg/h) was administered
through continuous venous infusion. Anesthesia was maintained with 1.5% isoflurane administered through a calibrated vaporizer (Vapor 2000, Draeger, Lubeck, Germany).
The body temperature was maintained between 37 C and
38 C using a circulating water mattress.

Collecting points
Following a 30-min stabilization period, baseline (BL)
measurements were performed. ALI was induced immediately after BL measurements in the ALI and ALI+HS
groups. A new series of measurements was collected 30 min
after the administration of HCl (T30) and hourly thereafter
(T90, T150, T210, T270).
Blood samples were collected at the above time points, and
bronchoalveolar lavage (BAL) sampling was performed with
3 x 20 ml phosphate buffered saline (PBS) at T270 through
bronchoscopy of the upper right lobe of the lung.

Monitoring
An arterial catheter was inserted into the right femoral
artery, and a 7.5 French pulmonary artery catheter that
measured continuous cardiac output (Edwards Lifesciences
Corp., Irvine, CA) was inserted into the right jugular vein.
The heart rate (HR), mean arterial pressure (MAP), mean
pulmonary artery pressure (MPAP), pulmonary artery
wedge pressure (PAWP) and central venous pressure (CVP)
were obtained directly using a multiparametric monitor
(IntelliVue MP40, Phillips, Bblinger, Germany). Using

ALI induced by HCl


A 0.1 N HCl pH 1.0 solution was prepared by the
hospitals pharmacy department and instilled at a dose of
4 ml/kg body weight at the level of the carina over a 4 min
period through a bronchoscope (Pentax FB-15H, Montvale,
NJ). ALI was established when the PaO2/FiO2 ratio fell
below 300 mmHg, which was achieved approximately 10
min after hydrochloric acid inhalation.

578

Hypertonic saline in lung injury


Holms CA et al.

CLINICS 2015;70(8):577-583

Table 1 - Hemodynamics and respiratory variables in the control (SHAM and hypertonic saline) and acid lesion (acute lung injury and
acute lung injury + hypertonic saline) groups.
Variable
2

CI (L/min/m )

HR (bpm)

MAP (mmHg)

MPAP (mmHg)

CVP (mmHg)

PVRI (dyne.sec.cm

SVRI (dyne.sec.cm

PPlat (cmH2O)

Compl (cmH2O)

.m2)

.m2)

Group

BL

T30

T90

T150

T210

T270

SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS

3.60.6
3.70.3
3.90.6
3.50.3
948
10415
10613
9212
547
673
708
663
142
162
162
143
93
102
101
91
13120
12815
15231
13735
1317188
1185124
1228152
1266175
131
141
141
141
314.1
294.5
284.2
284.2

3.70.5
4.10.4
4.20.9
4.30.4*
9711
10511
12415
11413
596*
724*
8016*
8317*
162
161
253*w#
265*w#
112
111
9 1
111
12918
11930
331114*w#
364127*w#
1249138
1178140
1343335
1349291
141*
141,8
231.3*w#
231.6*w#
304.5
284.6
141.4*w#
132*w#

4.00.5
4.90.4*w
4.40.9
4.80.5*
10119
11515
11219
12021
6910*
8410*
808*
8311*
153
191*
193*w#
213*w#
103
111
101
91
12116
13040
22551*w#
20757*w#
1348158
1157202
1317354
1222254
141.7*
141.8*
211.5*w#
211.7*w#
294
263.8*
162*w#
151.6*w#

4.30.5
4.90.6*
4.40.8
4.80.4*
10415
12918
12231
12112
6612*
8112*
849*
8511*
153
166
223*w#
243*w#
103*
101*
101*
101*
13130
13027
26677*w#
26653*w#
1302236
1115200
1372320
1247280
141.6*
151.6*
211.7*w#
211.6*w#
294.3*
263.8*
161.9*w#
151.7*w#

4.30.7
4.60.7*
4.20.7
4.80.4*
10721
12218
11930
12417
619*
769*
828*
8512*
163
182**
213*w#
244*w#
102*
102*
101*
101*
13228
13231
23058*w#
26666*w#
1362202
1116228
1419352
1251274
141.6*
151.9*
211.8*w#
211.2*w#
284*
254.4*
161.5*w#
151.7*w#

4.30.7
4.50.7*
4.20.5
4.60.5*
10828
11710
11921
12119
618*
777*
815*
828*
152
192*
224*w#
233*w#
102
102
101
101
13130
14928*
29051*w#
26358*w#
1274111
1172272
1375187
1241269
141.6*
152*
211.7*w#
211.2*w#
283.6*
244.3*
162.5*w#
152*w#

Data are expressed as the meanstandard deviation. CI: cardiac index; HR: heart rate; MAP: mean arterial pressure; MPAP: mean pulmonary arterial
pressure; CVP: central venous pressure; PVRI: pulmonary vascular resistance index; SVRI: systemic vascular resistance index; PPlat: plateau pressure; Compl:
pulmonary compliance; * po0.05 compared with the baseline; w po0.01 compared with the Sham group; # po0.05 compared with the HS group.

Neutrophil count and oxidative burst analysis

containing pig-specific monoclonal antibodies, according to the


manufacturers instructions (R&D Systems, Minneapolis, MN,
USA). The obtained concentrations were transformed into
pg/ml values using a nonlinear regression curve.

The BAL samples were pooled, and a 0.2 ml aliquot was


transferred to an Eppendorf tube, stained with 0.2% trypan
blue solution and counted using a Neubauer chamber.
As previously described (16,17), oxidative burst analysis
was performed using a flow cytometer (Becton Dickinson
Immunocytometry System, San Jose, CA, USA) connected to
a computer (Apple, Fremont, CA, USA). BAL cells (2 x 105
cell) were incubated in a 37 C shaking water bath for 30 min
with 200 ml of dichloro-dihydro-fluorescein diacetate
(DCFH-DA; 0.3 mM, Molecular Probes, Invitrogen, Carlsbad,
CA, USA), 200 ml of DCFH-DA and 100 ml of phorbol-12myristate-13-acetate (PMA; 1 ng/1 ml, Calbiochem, Gibbstown, NJ, USA). In total, 10,000 cellular events were
analyzed by Cell Quest Software, and the results were
expressed as the geometric mean fluorescence intensity
(GMFI).

Lung histology
At the end of the study, the trachea was clamped at the end of
the inspiratory cycle, and the animals thorax was opened. Four
samples were collected from the middle area of the left apical
(Wests zone 2) and diaphragmatic lung lobes as well as from
the middle of the right apical and diaphragmatic lung lobes. The
samples were fixed in a 10% formaldehyde solution for
subsequent histologic analysis. The tissue samples were
embedded in paraffin, and 5 mm histological sections were
stained with hematoxylin and eosin. Two pathologists who
were blinded to the study groups performed the histological
analyses simultaneously. Examinations included testing for the
presence of edema, intra-alveolar and interstitial hemorrhages
and polymorphonuclear and mononuclear cell infiltration.
Each assessed histological characteristic was attributed a
score from 0 to 3 according to the level observed in the tissue
(absent (0), mild (1), moderate (2) or severe (3)). The final score
for the animal was determined according to the sum of the
scores from each lobe (maximum score 12).

Cytokine measurements
BAL samples (10 ml) were centrifuged (2,000 rpm, 10 min,
4 C), and the supernatant was stored at -80 C for subsequent
analysis. BAL cytokine levels (interleukin (IL)-1, IL-8, IL-10, and
tumor necrosis factor alpha (TNF-a)) were assessed using
commercially available immunoenzymatic assay (ELISA) kits

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Statistical analysis

were found in the ALI+HS group, followed by the ALI group


(Figure 3).
The ALI and ALI+HS groups (34.43.5 and 30.27.7
neutrophils x 103/ml, respectively) presented higher BAL
neutrophil counts relative to the Sham and HS groups
(18.71.6 and 17.91.5 neutrophils x 103/ml, respectively).
Additionally, the ALI and ALI+HS groups presented
significantly higher activity (244.8130 and 232.6122
GMFI, respectively) in BAL neutrophils in the PMA-induced
burst responses compared with that of the Sham and HS
groups (51.722 and 5720.2 GMFI, respectively).

The results were expressed as the meanstandard


deviation (SD). Statistical analyses were performed using a
repeated measures analysis of variance (ANOVA) followed
by Tukeys test to analyze the group effect on the investigated
parameters. A one-way ANOVA was used to analyze the
histopathological results. Significance was established at the
level of 5% (po0.05).

RESULTS
The body weights of the animals did not differ between
groups. In addition, the administered amount of infused
7.5% hypertonic saline (HS: 17116 ml; ALI+ HS: 11211 ml)
and HCl (ALI: 11912 ml; ALI + HS: 11212 ml) were also
similar among the groups.

Histological analysis
The score for histological injury was significantly higher in
the ALI and ALI+HS groups compared with the Sham and
HS groups (Figure 4).
The pattern of lung injury observed in the ALI and ALI+
HS groups was heterogeneous and more evident in the
diaphragmatic lobes. Examination of these lung tissues
revealed large areas of alveolar architecture destruction,
hemorrhage, edema and inflammatory polymorphonuclear
and mononuclear cell infiltration (Figure 5). However,
significant differences were not observed between the scores
exhibited by animals in the ALI and ALI+HS groups.

Hemodynamic and respiratory data


The hemodynamic measurements were similar for each of
the investigated groups throughout the period of observation, and significant changes were not observed in the MAP,
CVP, or SVRI values. However, the pulmonary arterial
pressure and PVRI values exhibited significant increases
after T30 and remained high throughout the observation
period in the ALI and ALI+HS groups (Table 1). Following
HCl instillation, both lung injury groups exhibited significant
reductions in static pulmonary compliance and increases in
Pplat (plateau pressure). The PaO2/FiO2 ratio exhibited a
significant reduction at T30 in injured animals, or those in the
ALI and ALI+HS groups (24040 and 24451, respectively, po0.001), compared with those in the control animals,
or the Sham and HS groups (46737 and 44557,
respectively), and this ratio remained low throughout the
observation period (Figure 2).

DISCUSSION
In the present study, we demonstrated that hypertonic
saline infused after intratracheal HCl instillation attenuated
increases in BAL neutrophil counts and inflammatory
cytokine concentrations. HCl instillation alone induced a
severe direct lung injury as evidenced by an intense
inflammatory reaction observed in the lung histology, BAL
cytokine levels and oxidative burst. Lung function was also
adversely affected, which was indicated by decreased gas
exchange and reduced lung compliance.
Previous studies have attributed beneficial effects to hypertonic saline in a number of ALI models, such as oleic acid and
ischemia/reperfusion-induced lung injury (7,18,19). The use of
hypertonic saline solution has also demonstrated potential antiinflammatory effects related to neutrophil activation (20) in cell
cultures as well as in experimental models of sepsis and
hemorrhagic shock (21-23). Hypertonic saline solution acts on
polymorphonuclear A2 adenosine receptors and causes a
feedback mechanism that stimulates cAMP and PKA release,
thus blocking neutrophil activation (21-23).
It is believed that hyperosmolar solutions can also
decrease pulmonary vascular permeability and leukocyte
adhesion molecule expression, especially P-selectin and
L-selectin. This expression hinders neutrophil adhesion to the
endothelium and may result in reduced lung injury (24,25).
Contrary to the results obtained in studies performed in a
HCl-induced lung injury model (8) and an experimental oleic
acid-induced lung injury model (7), which demonstrated that
pulmonary edema decreased in rats treated with 7.5%
hypertonic saline, our histopathological results did not show
a significant differences between the ALI and ALI+HS
groups with regard to the investigated parameters. However,
the ALI+HS group tended to show lower histopathological
scores relative to the ALI group, although this difference was
not significant. Regarding the control groups, significant
differences were not observed between the HS and Sham
groups, which demonstrates that the administration of 7.5%

BAL cytokines, neutrophil count and oxidative


burst analysis
ALI animals showed higher TNF and IL-8 levels relative to
the Sham, HS and ALI+HS groups. Higher levels of IL-1B

Figure 2 - Changes in the PaO2/FiO2 ratio in the control (Sham


and HS) and acid-lesion (acute lung injury and AI + hypertonic
saline) groups. *: po0.05 compared with TB; w: po0.05
compared with the Sham group; #: po0.05 compared with the
hypertonic saline group.

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CLINICS 2015;70(8):577-583

Figure 3 - Bronchoalveolar lavage cytokines. The values are expressed in pg/ml. Data are presented as the group meanSD. w: po0.05
compared with the Sham group; #: po0.05 compared with the hypertonic saline group; y: po0.05 compared with the acute lung injury group.

Therefore, our findings of alveolar and interstitial edema and


polymorphonuclear and mononuclear cells sequestration in
response to injury are consistent with the data reported in the
literature.
Although significant differences were not observed
between the ALI and ALI+HS groups, higher concentrations
of TNF-a and IL-8 in the ALI-group BAL were observed.

hypertonic saline as an isolated agent did not result in


worsening lung injury scores.
Alveolar-capillary barrier destruction and increased microvascular permeability are known to trigger the process of
lung injury via acid aspiration, which leads to the activation
of leukocytes and their migration to pulmonary tissue as well
as the production of numerous inflammatory mediators (26).

Figure 4 - Scores for histological injury of the lungs. w: po0.05 compared with the Sham group; #: po0.05 compared with the
hypertonic saline group.

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Figure 5 - Representative photomicrographs with hematoxylin and eosin (H&E) staining (x200) of the lungs of pigs submitted to acute
lung injury. A) Sham group. B) hypertonic saline group. C) acute lung injury group. D) acute lung injury + hypertonic saline group.
Extensive alveolar and interstitial inflammatory infiltration was seen in both injury groups.

not observed in pulmonary compliance, which remained lower


for the duration of the study. Vascular occlusion and hypoxic
vasoconstriction may shift blood from non-aerated to aerated
lung areas, thus contributing to oxygenation improvement.
In addition, to minimize secondary lung injury caused by
the ventilator, we limited the pressure-controlled ventilation
to a volume of 8 ml/kg and 5 cmH2O PEEP. We also
maintained Pplat values below 30 cmH2O throughout the
study, which is similar to the standard defined for patients
with ARDS [30]. We observed a significant Pplat increase in
animals after HCl instillation, which was positively correlated with increases in the mPAP and PVRI values. Moreover, the ALI and ALI+HS groups also showed significantly
reduced pulmonary compliance after HCl instillation compared with the control groups. These changes in ventilatory
parameters remained throughout the study period and
corroborate the results of other studies using HCl- or oleic
acid-induced lung injury (29,31).
This ALI model was capable of inducing increased
changes in ventilatory parameters, and it also induced the
release of inflammatory cytokines in the bronchoalveolar
lavage samples in the ALI and ALI+HS groups. The
histopathological analysis identified areas of heterogeneous
injury characterized by polymorphonuclear infiltrates, alveolar hemorrhage, and edema in the groups subjected to ALI.
Although the use of 7.5% hypertonic saline was shown to
be safe in our study, the benefits of its use remain uncertain
because we did not observe improvements in any of the
investigated parameters among animals in the injury group.

The concentration of IL-10, an anti-inflammatory cytokine,


was significantly lower in the ALI-HS group, which may
have been caused by the lower TNF-a values in this group
because IL-10 expression is closely related to TNF-a
expression (27). However, BAL samples were collected
270 min after acid administration, and the IL-10 concentration may subsequently increase.
The observed increases in MPAP and PVRI suggest that
pulmonary hypertension occurred as a result of the initial
lung injury triggered by HCl. The two main causes were
chemical injury, which led to the destruction of the alveolarcapillary barrier and an increase of pulmonary permeability
(28), and pulmonary blood flow redistribution triggered by a
physiological process known as hypoxic pulmonary vasoconstriction (HPV), which optimizes the ventilation/perfusion ratio and blood oxygenation by gas exchange.
Regarding the gas exchange, we also found a statistically
significant decrease in the PaO2/FiO2 ratio, which was
approximately 50% lower in the ALI and ALI+HS groups
compared with the other groups. This finding is consistent
with the results obtained by Inci et al. (29) in rats after HCl
instillation. The PaO2 values, however, were only significantly different among the injury and control groups at the
T30 time point and returned to BL values at subsequent time
points. Although the PaO2/FiO2 ratio gradually increased
throughout the study, these values remained lower in the
treated groups compared with the non-injured groups.
Although the PaO2 value and PaO2/FiO2 ratio gradually
increased over the observation period, a similar response was

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ACKNOWLEDGMENTS

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Junior JO, Tabacchi Fantoni D. Modulation of inflammation during acute
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Hypertonic saline infusion for pulmonary injury due to ischemia-reperfusion.
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WG. Hypertonic saline resuscitation reduces neutrophil margination by
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resuscitation. Shock. 2011;35(2):178-83, http://dx.doi.org/10.1097/SHK.
0b013e3181f221fb.
22. Pascual JL, Ferri LE, Seely AJ, Campisi G, Chaudhury P, Giannias B, et al.
Hypertonic saline resuscitation of hemorrhagic shock diminishes neutrophil rolling and adherence to endothelium and reduces in vivo vascular leakage. Ann Surg. 2002;236(5):634-42, http://dx.doi.org/10.1097/
00000658-200211000-00014.
23. Pascual JL, Khwaja KA, Ferri LE, Giannias B, Evans DC, Razek T, et al.
Hypertonic saline resuscitation attenuates neutrophil lung sequestration and
transmigration by diminishing leukocyte-endothelial interactions in a two-hit
model of hemorrhagic shock and infection. J Trauma. 2003;54(1):121-30;
discussion 30-2, http://dx.doi.org/10.1097/00005373-200301000-00015.
24. Safdar Z, Wang P, Ichimura H, Issekutz AC, Quadri S, Bhattacharya J.
Hyperosmolarity enhances the lung capillary barrier. J Clin Invest.
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25. Coimbra R, Hoyt DB, Junger WG, Angle N, Wolf P, Loomis W, et al.
Hypertonic saline resuscitation decreases susceptibility to sepsis
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http://dx.doi.org/10.1097/00005373-199704000-00004.
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T, et al. Quantitative computed tomography in porcine lung injury with
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10.1097/CCM.0b013e3182186d09.
27. Park WY, Goodman RB, Steinberg KP, Ruzinski JT, Radella F, 2nd, Park
DR, et al.Cytokine balance in the lungs of patients with acute respiratory
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Analg. 1989;69(1):87-92.
29. Inci I, Ampollini L, Arni S, Jungraithmayr W, Inci D, Hillinger S, et al. Ex
vivo reconditioning of marginal donor lungs injured by acid aspiration.
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j.healun.2008.07.027.
30. Silversides JA, Ferguson ND. Clinical review: Acute respiratory distress
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31. Richard JC, Bregeon F, Leray V, Le Bars D, Costes N, Tourvieille C, et al.
Effect of activated protein C on pulmonary blood flow and cytokine production in experimental acute lung injury. Intensive Care Med. 2007;33(12):
2199-206, http://dx.doi.org/10.1007/s00134-007-0782-0.

This work was supported by grants from Fundaco de Amparo e Pesquisa


do Estado de So Paulo (FAPESP 08/55376-7 and 08/56732-4) and LIM
08 (Medical Investigation Laboratories Institute, Hospital das Clinicas da
Faculdade de Medicina).

AUTHOR CONTRIBUTIONS
Holms CA conducted the study and data analysis. Otsuki DA helped design
the study, conduct the study, collect and analyze data and prepare the
manuscript. Kahvegian M helped conduct the study. Massoco CO helped
conduct the study and data analysis. Fantoni DT designed the study and
helped analyze the data. Gutierrez PS performed the histological analysis.
Auler Jr JO helped design the study and prepare the manuscript. All of the
authors read and approved the nal manuscript.
*Presented in part at the 30th International Symposium on Intensive Care
and Emergency Medicine, 2010, Brussels.

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583

REVIEW

Operative versus nonoperative treatment for displaced


midshaft clavicle fractures: a meta-analysis based on
current evidence
Xin-Hua Wang, Wei-Jun Guo, A-Bing Li, Guang-Jun Cheng, Tao Lei, You-Ming Zhao*
The Second Affiliated Hospital of Wenzhou Medical University, Department of Orthopedics Surgery, Wenzhou, China.

Literature searches of the Cochrane Library, PubMed, EMBASE, Web of Science, LILACS, China National Knowledge
Infrastructure, and Wanfang Data databases were performed from 1966 to September 2014. Only randomized
and quasi-randomized controlled clinical trials comparing operative and nonoperative treatments for displaced
midshaft clavicle fractures were included. Data collection and extraction, quality assessment, and data analyses
were performed according to the Cochrane standards. Thirteen studies were considered in the meta-analysis.
Constant scores and the Disabilities of the Arm, Shoulder and Hand scores were improved in the operative fixation
group at a follow up of one year or more. The nonunion and symptomatic malunion rates were significantly
lower in the operative group. Additionally, the nonoperative group had a higher likelihood of neurological
symptoms compared with the operative group. A significantly higher risk of complications was found in patients
treated conservatively than in those who underwent operative fixation. However, when patients with nonunion
and symptomatic malunion were excluded from the analysis, no significant differences in the complication rate
were found. We concluded that based on the current clinical reports, operative treatment is superior to
nonoperative treatment in the management of displaced midshaft clavicle fractures. However, we do not support
the routine use of primary operative fixation for all displaced midshaft clavicle fractures in adults.
KEYWORDS: Clavicle; Midshaft clavicles; Operative treatment; Nonoperative treatment; Meta-analysis.
Wang XH, Guo WJ, Li AB, Cheng GJ, Lei T, Zhao YM Operative versus nonoperative treatment for displaced midshaft clavicle fractures: a meta-analysis
based on current evidence. Clinics. 2015;70(8):584-592
Received for publication on February 04, 2015; First review completed on March 23, 2015; Accepted for publication on April 30, 2015
E-mail: wzmuorthopaedic@sina.com
*Corresponding author

INTRODUCTION

A few meta-analyses comparing operative versus nonoperative approaches for the treatment of midshaft clavicle
fractures have been published in recent years (11,12), but the
results were inconclusive due to the relatively small sample
size in each published study. However, because several
relevant studies have been published on this topic in recent
years, the present meta-analysis is more precise.
The purpose of the present systematic review and metaanalysis was to determine the effectiveness of operative
versus nonoperative treatment for displaced midshaft
clavicle fractures by comparing the clinical results reported
in all of the available related evidence.

Clavicle fractures, which account for approximately 2.6% of


total body fractures and 34-45% of shoulder girdle injuries in
adults, are among the most common bone injuries in the body
(1,2). Appproximately 69-81% of clavicle fractures are in the
middle one-third of the clavicle, which is the thinnest part and
contains the smallest amount of soft tissue; 17% of clavicle
fractures are in the lateral one-third, and 2% are in the medial
one-third (3). Conventionally, most acute displaced midshaft
clavicles fractures are treated nonoperatively with the expectationsof a high probability of fracture union, good functional
outcomes and a high level of patient satisfaction (4-8).
However, the outcomes of nonoperative treatment are not
as favorable as once thought, and the trend to surgically treat
these fractures has grown (9,10). Whether surgical treatment
is associated with improved outcomes remains unknown.

MATERIALS AND METHODS


Literature search
This study was performed with guidance from the
Cochrane Handbook for Systematic Reviews of Interventions
and the Preferred Reporting Items for Systematic Reviews
and Meta-Analyses statement (13,14). The PubMed,
Cochrane Library, EMBASE, Web of Science, LILACS, China
National Knowledge Infrastructure and Wanfang Data
databases were searched (from 1966 to September 2014).
Keywords combined with MeSH terms, including clavicle,

Copyright & 2015 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.
DOI: 10.6061/clinics/2015(08)09

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CLINICS 2015;70(8):584-592

Critical Appraisal

clavicular, and fractures, were used for searching; the search


was performed without language restrictions but was
limited to human subjects. Additionally, the reference lists
of identified studies were manually checked to include other
potentially eligible trials. This process was performed
iteratively until no additional articles could be identified.

All selected articles were critically appraised by two


reviewers independently, using the Jaded score (16). The
score for each article could range from 0 (lowest quality) to 5
(highest quality). Scores of 3 to 5 denote good to excellent
quality, and scores of 0 to 2 denote poor to low quality. All
disagreements between the authors were resolved by
consensus, and a third author was consulted if necessary.

Inclusion and exclusion criteria


The search results were screened based on the following
inclusion criteria: (i) the studies were randomized or quasirandomized controlled clinical trials on patients with
displaced midshaft clavicle fractures that had occurred
within less than two weeks; (ii) the studies compared
operative (including plate, intramedullary nail fixation) with
nonoperative treatment (including sling or a figure-eight
bandage), (iii) the patients were at least 16 years of age; and
(iv) the studies included comparisons of the functional
outcomes, measured with Disabilities of the Arm, Shoulder
and Hand (DASH) and Constant scores, nonunion, symptomatic malunion, and complications. The exclusion criteria
were as follows: (i) studies including patients with pathological fractures or preexisting shoulder abnormalities; (ii)
studies concerning adolescent fractures; (iii) studies concerning open fractures; (iv) review literature, repeated reports,
retrospective studies, or case reports; and (v) studies that did
not report outcomes of interest.

RESULTS
Characteristics of the eligible studies
Details of the literature search are presented in a flow
diagram (Figure 1). Thirteen studies with relatively low
quality were included in the final analysis. Among them, the
report by Smith et al. (17) was an abstract that met the
inclusion criteria, and the sample sizes of the studies ranged
from 40 to 178 patients. Information on the general
characteristics, participants, and methodological quality of
the 13 studies is summarized in Table 1. Of a total of 959
included patients, 507 were treated with operative
approaches, and the others were treated with conservative
approaches. Allocation concealment was reported in 8 trials
(18-25) and was not stated in the other trials. Blinding was
rarely used in the included studies; only one study by
Robinson et al. (23) was blinded in the functional assessment.

Nonunion and symptomatic malunion

Data extraction

All 13 studies reported nonunion incidences. The pooled


results of our primary outcome measure, nonunion incidence,
presented a significant difference favoring operative over
nonoperative treatment (RR, 0.16; 95%CI, 0.09-0.30;
po0.00001). Subgroup analysis concerning fixation methods
showed that plate fixation (RR, 0.15; 95%CI, 0.07-0.29;
po0.00001) but not intramedullary nailing fixation (RR, 0.23;
95% CI, 0.06-0.92; p=0.04) was associated with a reduced risk
compared with nonoperative treatment (Figure 2A). One
study reported by Bhme et al. (27), in which fractures were
reduced and fixed with both plates and nails in the operative
groups, was excluded from the subgroup analysis.
Information on the incidence of symptomatic malunion
was provided in 9 studies (17,18,20-22,24-27). Using the fixed
effects model, the rate of symptomatic malunion was
significantly lower in the operative group compared with
that in the nonoperative group (RR 0.13, 95%CI 0.070.24,
po0.00001) (Figure 2B). No significant heterogeneity was
detected among these studies (Chi2=10.89, df=12, I2=0%,
p=0.74 and Chi2=2.46, df=8, I2=0%, p=0.96, respectively).

Two reviewers independently extracted the following data


from each included study: first author, year of publication,
number of patients, number of patients lost to follow up,
type of interventions, functional outcomes, and rates of
nonunion, symptomatic malunion, neurological symptoms
and total complications.

Outcomes for analysis


The primary outcome was the incidence of nonunion and
symptomatic malunion; the secondary outcomes were
clinical function measured by the DASH and Constant
Shoulder scores, complications and subgroup analyses
(neurological symptoms and complications without nonunion or symptomatic malunion).

Statistical analysis
Estimates of the treatment effect were expressed as risk
ratios (RRs) for dichotomous outcomes and weighted mean
differences (WMDs) for continuous outcomes, both with 95%
confidence intervals (CIs). For studies that did not present
standard deviations, the standard deviations were calculated
from the P-value or CI following the guidance of the
Cochrane Handbook for Systematic Reviews of Interventions
(13). Homogeneity across the studies was assessed with a
chi-square analysis, considering po0.10 significant. A fixed
effects model was used when the heterogeneity was not
significant, and a random effects model was adopted if
significant heterogeneity was present. A sensitivity analysis
was performed by omitting one study each time to explore
potential sources of heterogeneity and to test the stability of
pooled results. Publication bias was observed with the funnel
plot. Review Manager (RevMan) software (Version 5.3.5. The
Nordic Cochrane Centre, Copenhagen, Denmark) (15),
provided by The Cochrane Collaboration, was used for
graphical representation of the pooled data.

Functional outcomes
Nine studies (18,10-23,25,27-29) reported Constant scores
(eight at a follow up of one year or more and one at a follow
up of 6 months); the Constant scores of the operative group
were higher than those of the nonoperative group. Three
(27-29) of the nine studies were excluded from the analysis
due to a lack of data on the standard deviation or to
insufficient follow-up. The test for heterogeneity was
significant (Chi2=14.13, df=5, I2=65%, p=0.01). Using the
random effects model, the aggregated results suggested that
the Constant score was significantly higher in the operative
group compared with the nonoperative group (WMD 4.74,
95%CI, 2.457.03, po0.0001) (Figure 3A). Subsequently, we
performed a sensitivity analysis to explore potential sources
of heterogeneity. Exclusion of the trial conducted by

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Figure 1 - Flow chart showing article selection.

Mirzatolooei (21) reduced the heterogeneity (Chi2=2.47, df=4,


I2=0%, p=0.65) but did not materially alter the pooled results
(WMD 3.74, 95%CI, 2.39 5.08, po0.00001).
DASH scores were used in eight studies (18, 20-23,25,28,29);
the DASH scores of the operative group were lower than those
of the nonoperative group at a follow up of one year
or more, but the actual standard deviations were only included
in five studies (18,21-23,25). Pooled data showed that the
DASH score in the operative group was significantly lower
than that in the nonoperative group (WMD -6.34,
95%CI -11.28 -1.39, p=0.01) (Figure 3B). Significant heterogeneity was detected among these studies (Chi2=33.93, df=4,
I2=88%, po0.00001). Similarly, exclusion of the trial conducted by Mirzatolooei (21) resolved the heterogeneity
(Chi2=2.77, df=3, I2=0%, p=0.43) without materially altering
the pooled results (WMD -3.64, 95%CI -5.58 -1.69,
p=0.0002).

Complications
Because the definition of complications varied in all of the
studies, we defined complications as all adverse events that
were reported in those trials: nonunion (usually defined as no
evidence of healing at fifty-two weeks after injury), delayed
union (no evidence of healing at twenty-four weeks after
injury), symptomatic malunion, infection, hardware removal,
neurological symptoms, and refracture, among others.
In an overall analysis of the 13 selected studies, significant heterogeneity (Chi2=22.50, df=12, I2=47%, p=0.03) was
detected among these studies. Sensitivity analysis found that
the study reported by Judd et al. (19) was the source of
heterogeneity, probably owing to a high rate of hardwarerelated complications associated with the use of Hagie pins
in this study. Thus, the random effects model was applied. A
significantly higher risk of complications was found in
patients treated conservatively than in those who underwent

Table 1 - Characteristics and methodological quality of the included studies.


Study

Smith (2001)
Jubel (2005)
COTS (2007)
Figueiredo (2008)
Judd (2009)
Smekal (2009)
Bohme (2011)
Chen (2011)
Mirzatolooei (2011)
Kulshrestha (2011)
Virtanen (2012)
Robinson (2013)
Mohsen (2014)

Design

No. of Patients Assessed


(O/N)

Range of Ages
(years)

Follow-up
(months)

Internal
Fixation

Nonoperative
Treatment

Jadad
Score

RCT
QRCT
RCT
RCT
RCT
RCT
QRCT
RCT
RCT
QRCT
RCT
RCT
QRCT

30/35
26/27
62/49
24/16
29/28
30/30
58/38
30/30
26/24
45/28
26/25
86/92
35/30

Adults
Adults
1660
18-58
1740
1865
18-70
18-63
1865
20-50
1870
1660
18-60

12
6
12
12
12
24
8
15
12
18
12
12
6

Plate
Nail
Plate
Plate
Nail
Nail
Plate/Nail
Nail
Plate
Plate
Plate
Plate
Plate

Sling
Bandage
Sling
Sling
Sling
Sling
Bandage
Sling
Sling
Sling
Sling
CollarCuff
Bandage

3
1
3
3
3
3
1
3
3
1
3
4
1

O/N: operative group/nonoperative group, RCT: randomized controlled trial, QRCT: quasi-randomized controlled trial.

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CLINICS 2015;70(8):584-592

Figure 2 - Forest plot showing comparison of nonunion rate (A) and symptomatic malunion rate (B) between operative (experimental)
and nonoperative (control) groups.

operative fixation (RR 0.57, 95%CI 0.46-0.72, po0.00001)


(Figure 4A). Furthermore, we also performed a subgroup
analysis of the complications without nonunion and symptomatic malunion. The aggregated results suggested that there
were no significant differences between groups in the rates of
complications (RR 1.30, 95%CI 0.881.92, p=0.19) (Figure 4B).
Significant heterogeneity was detected among these studies
(Chi2=25.81, df=12, I2=54%, p=0.01). We then performed
sensitivity analysis and found that the study reported by
Mohsen et al. (29) was source of heterogeneity (Chi2=12.95,
df=11, I2=15%, p=0.30). Although heterogeneity was found,
statistically similar results to those of the overall analysis
were obtained in the sensitivity analysis. The predominant

complications in the nonoperative group were nonunion,


neurological symptoms (including brachial plexus irritation
and compression) and symptomatic malunion. The operative
complications tended to be hardware related (including plate
irritation, pin protrusion and removal).

Neurologic symptoms
Nine studies reported neurological symptoms (17-22,25,
27,28). Pooled data showed that the operative group had a
significantly lower likelihood of developing neurological
symptoms compared with the nonoperative group (RR 0.40,
95%CI 0.230.70, p=0.001). No significant heterogeneity was

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CLINICS 2015;70(8):584-592

Figure 3 - Forest plot showing comparison of Constant scores (A) and DASH scores (B) between operative (experimental) and
nonoperative (control) groups.

detected among these studies (Chi2=8.07, df=8, I2=1%, p=0.43)


(Figure 4C).

specific conclusions as to which patients were most likely to


suffer from one of these significant complications; we also do
not support the routine use of primary operative fixation for
all displaced clavicle fractures in adults because an unacceptably high number of patients would be exposed to the
risks of surgery (23).
Although significant differences were found between the
two treatment groups in terms of functional outcomes, the
reasons for functional outcome differences are probably
multifactorial because most individuals who developed
nonunion or symptomatic malunion had significantly lower
outcome scores (i.e., a mean of sixteen points worse on the
DASH score in nonunion patients in the study by Virtanen
et al. (22)). Additionally, Robinson et al. (23) showed that the
development of nonunion was the only independent
predictor of functional outcomes. When patients with
nonunions were excluded, there was only a trend toward
better functional outcomes in the operative group, with no
significantly different scores at any time. Therefore, we
thought that the improved outcomes may have resulted from
the prevention of nonunion and symptomatic malunion by
operative fixation. Unfortunately, without sufficient original
data, we cannot perform a subgroup analysis of patients
with overall healed fractures.
Overall complication and neurological symptom rates
were higher in the nonoperative group than in the operative
group. The subgroup analysis of the complications without

Publication bias
Publication bias was assessed by comparing the WMDs of
nonunion; no evidence of publication bias was detected
(Figure 5).

DISCUSSION
Our meta-analysis revealed that primary operative fixation
could effectively reduce the rates of nonunion, symptomatic
malunion, neurological symptoms and overall complications. In addition, DASH and Constant scores were
significantly improved after operative fixation compared
with nonoperative treatment after a follow up of one year or
more. Based on current clinical reports, we conclude that
operative treatment is superior to nonoperative treatment in
the management of displaced midshaft clavicle fractures.
Pooled data showed that 14% of 452 patients in the
nonoperative group developed a nonunion, which is
significantly higher (p=0.00001) than the 1.7% rate of
nonunion in the 507 patients of the operative group.
Symptomatic malunion was also significantly more common
in the nonoperative group (20% in the nonoperative group
versus 1.8% in the operative group, po0.00001). However,
with the data available, we were not able to draw any

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CLINICS 2015;70(8):584-592

Figure 4 - Forest plot showing comparison of complications rates (A and B) and neurologic symptoms rates (C) between operative
(experimental) and nonoperative (control) groups.

implants or improved surgical techniques. The predominant complications in the nonoperative group were nonunion, neurological symptoms (including brachial plexus
irritation and compression) and symptomatic malunion;
however, most of those complications require operative
intervention.

nonunion and symptomatic malunion suggested that no


significant between-group differences existed in the complication rates (p=0.23). The most common complications in
the operative group were hardware related (including plate
irritation, pin protrusion and removal). Theoretically, these
complications could be reduced by using less prominent

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CLINICS 2015;70(8):584-592

Figure 5 - Funnel plot of detection of publication bias.

Although modern plate fixation techniques provide reliable healing rates, the optimal plate position and type remain
controversial. The clavicle contour and anatomy are curved
in multiple planes. The reconstruction plate is easier to
contour in all planes than the stiffer dynamic compression
plates (DCP), which allow bending only along the length of
the plate. For superior plating, a reconstruction plate or
precontoured plate can more precisely fit the S-shaped
anatomy. For anteroinferior plating, DCPs can be bent to
conform to the anatomy very well (39). Regarding stability,
two biomechanical studies have found greater stability with
compression plates compared with reconstruction plates
(31,32). In addition, a finite element study showed that
anteroinferior plating best resists the effects of most daily
living forces that act on the clavicle and can be considered
more mechanically physiological (33).
Will et al. (34) suggested that locked compression plates
(LCPs) provided more stiffness and less deflection than lowcontact dynamic compression plates (LC-DCPs). Using a
simulated segmental clavicle fracture model, another biomechanical study by Iannotti et al. (35) reported that LCDCPs offer significantly greater biomechanical stability than
reconstruction plates and DCPs and that clavicles plated at
the superior aspect exhibited significantly greater biomechanical stability than those plated at the anterior aspect.
However, most of the biomechanical studies must be
interpreted with caution because such testing can offer clean
comparisons of instrumentation and technique without
the confounding factors of patient and surgeon variations.
Nine of the 13 studies included in this review used plate
fixation; among them, three studies used reconstruction

plates (21,22,26), two studies used DCPs (24,29), and two


studies used LCPs (17,23). Another two studies that used a
mixture of plate implants (18,27) was not analyzed here. The
rate of nonunion was 6% in the DCP group, which was
higher than that in the reconstruction plate group (1%) and
LCP group (1%). Symptomatic malunion only occurred in the
reconstruction plate group, with a rate of 6%. In addition,
overall complication rates were higher than 20% in all of the
groups, which is consistent with previous literature (30). Recent
clinical studies have shown efficient healing, few complications,
and excellent return to function for anteroinferior plating
(36-38). The advantages of this technique include the avoidance
of potentially dangerous infraclavicular structures and the
reduction of patient complaints due to implant prominence (36).
A retrospective cohort study (39) with 156 midshaft clavicle
fractures concluded that anteroinferior clavicle fracture fixation
with DCPs results in excellent healing rates and lower removal
rates. Moreover, implant failure occurred more often with
reconstruction plates compared with dynamic compression
plates (p=0.029).
Considering these mechanical and clinical findings, the
plate type, precontouring, and position likely affect outcomes and implant-related complication rates. However,
these effects have yet to be fully examined and more
prospective trials are required to analyze the influence of
various plate types and positions on implant-related complications in the future.
Further subgroup analysis by type of surgery indicated
that plate fixation but not intramedullary nail fixation
was associated with a reduced risk of nonunion, symptomatic malunion and total complications compared with

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CLINICS 2015;70(8):584-592

There are some limitations of this study. First, the recruited


studies were not all randomized controlled trials. The lack of
inadequate allocation concealment and blinding, which can
lead to over-reporting of the treatment effect and selection or
allocation biases, likely affected the study results. Second, the
preoperative fracture pattern was found to be significantly
related to implant failure (49), but our meta-analysis could
not show fracture typespecific effects between operative
and nonoperative treatments because of the limited data of
the studies. Finally, despite our best efforts to use multiple
search methods, we may not have detected all eligible
existing trials with results that are applicable to our metaanalysis. Therefore, the results should be interpreted with
caution. Further research entailing high-quality randomized
controlled, multicenter trials with long-term functional outcomes and fracture typespecific designs is required to address
key clinical questions regarding the effects of operative
treatment versus nonoperative treatment in the management
of displaced midshaft clavicle fractures in adults.
In summary, we conclude that operative treatment is
superior to nonoperative treatment in the management of
displaced midshaft clavicle fractures, although we do not
support the routine use of primary operative fixation for all
displaced clavicle fractures in adults. Patients with a
completely displaced midshaft clavicle fracture should be
informed that they will be at a higher risk of sustaining
nonunion, symptomatic malunion and potential neurological
symptoms if the fracture is treated conservatively.

nonoperative treatment. No significant difference in the


functional outcomes between these two techniques was
observed. However, the pooled data showed that the
incidences of nonunion, symptomatic malunion and total
complications were comparable in the two operative groups
(2% versus 1%, 2% versus 0, 27% versus 27%, respectively).
Both the plating and intramedullary nail methods have
advantages and disadvantages. Biomechanically, plate fixation is superior to intramedullary fixation (40). Patients
treated with plate fixation can achieve full range of motion.
The disadvantages of plate fixation include the necessity for
increased exposure and soft tissue stripping, the increased
risk of damage to the supraclavicular nerve, slightly higher
infection rates, and the risk of refracture after plate removal
(18). A recent randomized clinical trial comparing locked
intramedullary nailing versus plating for displaced midshaft
clavicle fractures performed by Ferran et al. (41) showed no
significant differences between the two operative techniques.
Another two prospective comparative studies concluded that
both plating and intramedullary flexible nailing are equally
effective alternatives for the surgical fixation of displaced
midshaft clavicle fractures but that intramedullary techniques have potential advantages such as less soft tissue injury,
shorter operative times and hospital stays, less blood loss
and higher cosmetic satisfaction (42,43). The subgroup
analysis results of our review are partially consistent with
the results of the three trials; however, due to the limitations
of those studies such as their small sample sizes and single
center designs, we cannot provide any strong conclusions.
Studies with sound rationale and design are required to
accurately and definitively assess the differences in outcomes
between plate fixation and intramedullary fixation (44).
However, with a myriad of options available for both plate
fixation and intramedullary fixation, the question of which
form of fixation is superior remains. According to currently
available data, the superior surgical technique and implant
choice are those that the surgeon was originally trained to
perform and use.
Previous literature has addressed the issue of nails, yet the
difference between locked and unlocked nails has not been
considered. This study may provide additional interesting
clues for future research on this topic.
We identified six systematic reviews that approached the
comparison between surgical versus conservative interventions
to treat middle-third clavicle fracture in adults (11,12,45-48).
The results of our review are consistent with those of the eight
systematic reviews; however, the conclusions of those published reviews varied, which was partly in accordance with our
conclusions. The only Cochrane systematic review, reported by
Lenza et al, used more comprehensive statistical methods,
which were lacking in our present study. The most distinguishing characteristic of those published reviews was that analysis
was conducted with incomplete information. With the exception of one study by Lenza et al, the reviews did not include the
two RCTs (24,25). Additionally, new RCTs have been published
since then. Our review adds consistent information for current
clinical practice. We applied more specific subgroup analysis by
surgery and implant type and then discussed the results in
detail. Furthermore, we performed a broader literature search
that included non-English publications. Bias is inherent in
many analyses focusing on specific populations or geographic
areas. By including all of the available studies, including those
from multiple countries and reported in multiple languages, we
believe that our conclusions are applicable to most populations.

AUTHOR CONTRIBUTIONS
Wang XH conceived the study, collected the data, participated in the analysis
of samples, drafted the manuscript and performed the statistical analysis. Guo
WJ conceived the study and participated in its design, coordination and
drafting. Li AB participated in the analysis and interpretation of samples and
in the language translation of non-English studies. Cheng GJ participated in
the language translation of non-English studies. Lei T participated in the
revision of the manuscript. Zhao YM participated in the review, revision,
coordination and drafting of the manuscript and performed the analysis with
constructive discussions. The aim of this article was to identify the effects of
operative versus nonoperative treatment in the management of displaced
midshaft clavicle fractures in adults.

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592

REVIEW

The association between the rs11196218A/G polymorphism of the TCF7L2 gene and type 2 diabetes in
the Chinese Han population: a meta-analysis
Enting Ma,I Huili Wang,II Jing Guo,I Ruirui Tian,III Li WeiIV,*
I
General Hospital of Tianjin Medical University, Department of Pediatric Ward, Tianjin, China. II Xian International University, Department of Nursing,
Xian, China. III Tianjin Medical University, Department of Nursing, Tianjin, China. IV General Hospital of Tianjin Medical University, Department of Surgery,
Tianjin, China.

Transcription factor 7-like 2 has been shown to be associated with type 2 diabetes mellitus in multiple ethnic groups
in recent years. In the Chinese Han population in particular, numerous studies have evaluated the association
between the rs11196218A/G polymorphism of the transcription factor 7-like 2 gene and type 2 diabetes mellitus.
However, the results have been inconsistent, so we performed a meta-analysis to assess the association. Odds ratio
and 95% confidence interval values were calculated using a random-effects model or a fixed-effects model based on
heterogeneity analysis. The quality of the included studies was evaluated using the Newcastle-Ottawa Scale.
Subgroup analyses were conducted based on conformity with Hardy-Weinberg equilibrium in the control group as
well as on other variables, such as age, sex and body mass index. Sensitivity analysis was also performed to detect
heterogeneity and to assess the stability of the results. In total, 10 case-control studies comprising 7,491 cases and
12,968 controls were included in this meta-analysis. The combined analysis indicated that the rs11196218A/G
polymorphism was not associated with type 2 diabetes mellitus (G vs. A, OR = 1.04, 95% CI = 0.97-1.13, p = 0.28). The
subgroup analyses also did not show any association between the rs11196218A/G polymorphism and the risk of type
2 diabetes mellitus. Furthermore, the results of the subgroup analyses indicated that the absence of an association
was not influenced by age, sex or body mass index. The results of the sensitivity analysis verified the reliability and
stability of this meta-analysis. In conclusion, this study indicated that there is no significant association between the
rs11196218A/G polymorphism and the risk of type 2 diabetes mellitus in the Chinese Han population.
KEYWORDS: Type 2 diabetes mellitus (T2DM); Transcription factor 7-like 2 (TCF7L2); rs11196218A/G polymorphism;
Meta-analysis.
Ma E, Wang H, Guo J, Tian R, Wei L. The association between the rs11196218A/G polymorphism of the TCF7L2 gene and type 2 diabetes in the
Chinese Han population: a meta-analysis. Clinics. 2015;70(8):593-599
Received for publication on January 29, 2015; First review completed on March 20, 2015; Accepted for publication on May 12, 2015
E-mail: wlykdxzyy@126.com
*Corresponding author

INTRODUCTION

risk of T2DM in Icelandic (4), European (5-11), West African (12),


southern Asian (13), eastern Asian (14), and Chinese (15-19)
populations. The TCF7L2 gene is regarded as one of the most
important genes for determining genetic susceptibility to T2DM
that has been identified in humans so far.
With the growth of research efforts, studies on the association
between this variation and T2DM have been extensively
performed in China, but the results are disputable. Overall,
the most significant risk locus identified in Chinese individuals
is rs11196218. Therefore, only the rs11196218A/G polymorphism was considered in the present meta-analysis.
In contrast to a single study, meta-analyses are based on all
available studies, which has improved the statistical power
for exploring the above associations and has led to more
reliable conclusions in recent years (20). Therefore, in the
present study, a meta-analysis was performed to allow a
valuable conclusion to be drawn regarding the relationship
between the rs11196218A/G polymorphism and T2DM risk
in the Chinese Han population.

Type 2 diabetes mellitus (T2DM) is a complex metabolic


disease resulting from a combination of environmental and
genetic factors (1). With the development of society and the
improvement of peoples standard of living, the prevalence
of T2DM is increasing rapidly all around the world. T2DM is
very harmful to human health and life, and it also confers a
heavy burden on society.
The transcription factor 7-like 2 (TCF7L2) gene, located on
chromosome 10q25 (2), is part of the Wnt signaling pathway (3)
and has been shown to be strongly associated with an increased

Copyright & 2015 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.
DOI: 10.6061/clinics/2015(08)10

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CLINICS 2015;70(8):593-599

MATERIALS AND METHODS

Ethics statement

Our study adhered to the PRISMA Statement regarding


the reporting of systematic reviews and meta-analyses (21).

This article reports a meta-analysis of data obtained from


previous studies. All of the data were anonymized before
analysis. We also confirmed that none of the data involved
competing interests.

Search strategy
In this meta-analysis, we searched articles in PubMed,
Elsevier, SpringerLink, Embase, the Cochrane Library, ISI
Web of Science, Google Scholar and the China National
Knowledge Infrastructure (CNKI). The search languages
included English and Chinese. The following key words and
subject terms were used: TCF7L2, transcription factor 7-like
2, rs11196218, diabetes mellitus, type 2, type 2 diabetes
mellitus, T2DM, and T2D. The reference lists of eligible
studies and relevant review papers were additionally
identified via a manual search on this topic. The last research
update was performed on July 15, 2014.

Statistical analysis

In this meta-analysis, OR and 95% CI values were used to


assess the association between a polymorphism of the
TCF7L2 gene (rs11196218: G vs. A) and T2DM risk. In
addition, statistical significance was assessed using a Z-test,
and po0.05 indicated statistical significance for the association. The model selection was based on the heterogeneity
test; therefore, the w2-based Q-test was performed in this
study (23). When the Q-test yielded a p-value of more
than 0.10, a fixed-effects model was used (24); otherwise,
a random-effects model was applied (25). Heterogeneity
was also assessed using the I2 test. The I2 statistic was
specifically documented for the percentage of study
variability observed due to heterogeneity rather than
chance (I2 = 0-25%, no heterogeneity; I2 = 25-50%, moderate
heterogeneity; I2 = 50-75%, high heterogeneity; I2 = 75-100%,
extreme heterogeneity) (26).
Subgroup analyses were performed based on HWE.
Specifically, the p-values for HWE in the control group were
determined using the Pearson chi-square test, regardless of
whether the authors provided these values. HWE is the
principal law of population genetic studies: if p40.05,
the focus conforms to HWE, and the control samples are
representative. In Europeans, when excessive calories
are consumed, variation in TCF7L2 can cause impaired
b-cell function, indicating that body mass index (BMI) can
influence the effect size of TCF7L2 variants; hence, these two

Inclusion and exclusion criteria


The primary studies included in our meta-analysis had to
meet the following criteria: (1) the association between
the TCF7L2 polymorphism (rs11196218) and T2DM risk in
the Chinese Han population was clearly evaluated, (2) the
diagnosis of T2DM and the sources of the cases and controls
were clearly described, (3) a case-control study design was
employed, and (4) original data and sufficient information
were provided to estimate the odds ratio (OR) and the
corresponding 95% confidence interval (95% CI). The major
reasons for exclusion were as follows: (1) duplicate data were
presented; (2) the article consisted of an abstract, comment, or
review or focused on pathological mechanisms; and (3) more
than one article was published by the same author using the
same data series, in which case the most recent published
paper or the paper with the largest sample size was selected.

Quality assessment
The Newcastle-Ottawa Scale (NOS) (22) was used to assess
the quality of the studies included in our meta-analysis. The
NOS contains eight items and is categorized into three
dimensions: selection, comparability and exposure, for
case-control studies. In particular, the selection dimension
contains four items, the comparability dimension contains
one item, and the exposure dimension contains three items.
A star system is used to allow semi-quantitative assessment
of study quality, and a study can be awarded a maximum of
one star for each numbered item within the selection and
exposure categories. Meanwhile, a maximum of two stars
can be given for comparability. The NOS ranges from zero up
to nine stars, as follows: high-quality study: more than seven
stars; medium-quality study: between four and six stars;
poor-quality study: less than four stars.

Data extraction
For quality control, the data were extracted by two
reviewers using a standardized extraction form. If the
information on the genotype distribution was inadequate,
we tried to contact the authors by telephone or e-mail. The
following information was extracted from each article:
the last name of the first author, the year of publication,
the region, the numbers of cases and controls, the source of
the controls, the numbers of genotypes for cases and
controls, matching factors, and the Hardy-Weinberg equilibrium (HWE) in each control group. Disagreement was
resolved by consulting a third reviewer.

Figure 1 - Selection of the included studies.

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CLINICS 2015;70(8):593-599

Table 1 - The basic characteristics of the included studies addressing rs11196218.


First
author

Publication
year

Region

Ng

2007

Hong Kong

Zhang
Luo
Ma
Lin
Wen
Zhu
Zheng
Qiao
Zhai

2008
2009
2010
2010
2010
2011
2012
2012
2014

Hunan
Beijing
Shanghai
Chengdu
Shanghai
Anhui
Chongqing
Harbin
Henan

Source of
controls

Community-based and
hospital staff
Hospital-based
Hospital-based
Hospital-based
Hospital-based
N/D
Hospital-based
Hospital-based
Hospital-based
Community-based
and Hospital-based

Sample sizes

Cases

Controls

HWE

Comparability

Case

Control

433

419

676

190

599

239

Yes

Age*, sex*, BMI**

536
500
259
1529
1165
300
227
700
1842

475
500
200
1439
1136
300
152
560
7777

716
684
309
2171
1699
156
340
1025
2639

272
252
209
887
629
444
114
367
967

623
696
240
2015
1677
122
218
819
111287

221
240
168
863
595
478
86
295
4041

Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes

Age*, sex**, BMI*


Age**, sex**, BMI**
Age**, sex*, BMI**
Age**, sex*, BMI**
Age*, sex*, BMI*
Age*, sex*, BMI*
Age*, sex**, BMI**
Age*, sex*, BMI*
Age**, sex**, BMI**

Abbreviations: HWE, Hardy-Weinberg equilibrium; Yes, the genotype distribution conformed to HWE in the control group; No, the genotype distribution
not conform to HWE in the control group;
Hospital-based: subjects who were enrolled from health checks conducted at the hospital; community-based and hospital staff: subjects who were
enrolled from the community and hospital staff; N/D: no description; *: p40.05; **: po0.05.

allele G. Thus, a random-effects model was applied for the


meta-analysis. The results indicated that the rs11196218A/G
polymorphism was not associated with the risk of T2DM
(G vs. A: OR=1.04, 95% CI=0.97-1.13, p=0.28; heterogeneity
test w2 = 17.71, p = 0.04, I2 = 49%; Figure 2).
The subgroup meta-analysis of the studies that exhibited
consistency with HWE in the control group also showed that
there was no association between the rs11196218A/G
polymorphism and T2DM risk (G vs. A: OR = 1.08, 95%
CI = 0.98-1.19, p = 0.12; heterogeneity test w2 = 15.79, p = 0.03,
I2 = 56%; Figure 3).
The subgroup meta-analysis of the studies based on age
indicated that there was no significant association between
the rs11196218A/G polymorphism and T2DM risk (subgroup of age comparability: OR = 1.11, 95% CI = 0.96-1.28,
p = 0.18; heterogeneity test w2 = 14.81, p = 0.01, I2 = 66%; subgroup of age incomparability: OR = 1.00, 95% CI = 0.94-1.06,
p=0.93; heterogeneity test w2 = 1.45, p = 0.69, I2 = 0%).
The subgroup meta-analysis of the studies based on sex
demonstrated that there was no significant association
between the rs11196218A/G polymorphism and T2DM
risk (subgroup of sex comparability: OR = 1.10, 95% CI =
0.98-1.24, p = 0.12; heterogeneity test w2 = 13.04, p = 0.02,
I2 = 62%; subgroup of sex incomparability: OR = 0.98, 95%
CI = 0.91-1.05, p = 0.49; heterogeneity test w2 = 1.58, p = 0.66,
I2 = 0%).
Finally, the subgroup meta-analysis of the studies based on
BMI illustrated that there was no significant association
between the polymorphism and T2DM risk (subgroup of

factors will ultimately affect each other (27-29). This


interaction may also exist in Asians. For this reason, we
considered whether age, sex and BMI were matched between
cases and controls in our subgroup analyses. For example,
the studies showing a significant difference in age (po0.05)
between the cases and the controls were assigned to the
incomparability subgroup of age, whereas those exhibiting
p40.05 were assigned to the comparability subgroup.
However, due to the limited data in the studies, subgroup
analyses based on environment were not conducted.
Sensitivity analysis was also performed to search for
heterogeneity and to assess the stability of the results. One
case-control study was omitted each time to reflect the
influence of each dataset on the pooled OR.
Additionally, funnel plots were used to evaluate publication bias. All p-values were two tailed. Review Manager 5.0
(2011, The Cochrane Collaboration) software was used to
perform the meta-analysis.

RESULTS
Studies and data included in this meta-analysis
In total, 295 articles were relevant to our search terms, of
which 285 papers were excluded. Thus, 10 case-control
studies (15-19,30-34) comprising 7,491 cases of T2DM and
12,968 controls were ultimately included in this metaanalysis (Figure 1). All of these studies were published from
2007-2014.
The characteristics of these studies are summarized in
Table 1. The genotype frequency of the single nucleotide
polymorphism (SNP) was consistent with HWE in the
control group (p40.05) in all studies except for two (32,33).
The quality assessment of all included studies, evaluated
according to the NOS, is provided in Table 2. Most studies
were of high quality in terms of selection and exposure.
However, the quality of comparability was relativity low, as
only 3 studies (15,33,34) were comparable with the controls
regarding age, sex and BMI.

Table 2 - Quality assessment of all of the included studies.


First author
Ng
Zhang
Luo
Ma
Wen
Lin
Zhu
Zheng
Qiao
Zhai

Association between rs11196218A/G polymorphism


and T2DM risk
Using the heterogeneity test, we detected heterogeneity
among the studies in comparisons of the risk of carrying

595

Publication year

Selection

Comparability

Exposure

2007
2008
2009
2010
2010
2010
2011
2012
2012
2014

$$$
$$
$$
$$
$$
$$
$$$$
$$$$
$$$
$$

$
$
$
$
$$
$
$$
$
$$
$

$
$$
$$
$$
$$$
$$
$$
$$
$
$$

A meta-analysis
Ma E et al.

CLINICS 2015;70(8):593-599

Figure 2 - Meta-analysis of the association between the rs11196218A/G polymorphism and T2DM risk (G vs. A). n indicates the total
number of G alleles, and N indicates the total number of G alleles plus A alleles.

BMI comparability: OR = 1.03, 95% CI = 0.90-1.17, p = 0.72;


heterogeneity test w2 = 6.21, p = 0.10, I2 = 52%; subgroup of
BMI incomparability: OR = 1.06, 95% CI = 0.96-1.18, p = 0.26;
heterogeneity test w2 = 11.32, p = 0.05, I2 = 56%).

population, rs7903146 is very rare, and several studies have


indicated that rs11196218 is the most significant risk variant
(17). Nevertheless, the results regarding the rs11196218A/G
polymorphism and T2DM risk have been inconsistent for the
Chinese Han population. Certain studies (15-19) confirmed that
the G allele of the rs11196218A/G polymorphism was
significantly associated with T2DM risk, whereas studies by
Ma (30), Zheng et al. (31), Luo et al. (32), Wen et al. (33) and
Qiao et al. (34) did not find a significant association between
the rs11196218A/G polymorphism and T2DM risk in the
Chinese Han population. Therefore, to resolve the conflict
among these studies, we performed a meta-analysis to assess
the association between the rs11196218A/G polymorphism
and T2DM risk.
Previously, Luo et al. (32) conducted a meta-analysis to
evaluate the effect of TCF7L2 on genetic susceptibility to
T2DM in the East Asian population. However, only one
article analyzed included samples from the Chinese Han
population. In the current study, to increase the statistical
power of the analysis, a larger amount of data was collected
from the literature to perform an up-to-date meta-analysis
for the Chinese Han population.
Our meta-analysis, which included 7,491 T2DM cases and
12,968 controls from 10 case-control studies, explored the
association between rs11196218A/G and T2DM risk. Overall,

Sensitivity analysis
A single study included in the meta-analysis was omitted
each time to reflect the influence of each dataset on the
pooled OR values. We found that no single study could
change the pooled results (Table 3), which indicated that the
results were relatively reliable.

Publication bias
The shape of the funnel plots was symmetrical, suggesting
that there was no evidence of publication bias for the
rs11196218A/G polymorphism (Figure 4).

DISCUSSION
Since the initial discovery that TCF7L2 is strongly associated
with an increased risk of T2DM in Icelandic populations in
2006 (4), many replication studies have confirmed the role of
TCF7L2 in conferring susceptibility to T2DM in different
populations and ethnic groups (5,7,11,14,38,39), especially for
the rs7903146C/T polymorphism. However, in the Chinese

Figure 3 - Meta-analysis of the association between the rs11196218A/G polymorphism and T2DM risk (subgroup analyses for HWE in the
control group: G vs. A). n indicates the total number of G alleles, and N indicates the total number of G alleles plus A alleles.

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CLINICS 2015;70(8):593-599

Table 3 - The results of the sensitivity analysis with each rs1119

with other ethnic groups that have occurred in Hong Kong


may have resulted in different risk factors in different people
(40). Therefore, the genetic origins of risk may be relatively
different between this region and other areas. Pritchard et al.
(41) also suggested that low diversity may be sufficient to
lead to different results. However, although heterogeneity
was observed among studies, the results of the sensitivity
analysis verified the reliability and stability of the present
meta-analysis.
T2DM is a complex hereditary disease caused by
genetic and environmental factors (1). Other confounding factors, such as age, sex, BMI, the environment
and sophisticated gene-gene and gene-environment
interactions, may also affect the results of studies on
T2DM. Thus, the subgroup analyses in the present study
were also conducted based on variables such as age, sex
and BMI. However, the results indicated that the
association of the rs11196218A/G polymorphism with
T2DM was not influenced by age, sex or BMI in the
studied population. Due to the limited data available in
the studies analyzed, subgroup analyses based on the
environment and gene-gene and gene-environment interactions were not conducted.
There were certain advantages of our meta-analysis.
First, to the best of our knowledge, this is the most
comprehensive meta-analysis of the association between
the rs11196218A/G polymorphism of the TCF7L2 gene
and T2DM risk in the Chinese Han population conducted
to date, and our analysis had improved statistical power
for exploring this association. Second, the protocol for this
meta-analysis, using explicit methods and criteria for
study selection, data extraction, and data analysis, was
well designed before it was initiated. Third, a stringent
searching strategy based on computer-assisted and manual searches was applied to include as many eligible
studies as possible. Finally, the quality of the studies
included in our meta-analysis was satisfactory; in fact,
each article exhibited at least five stars. However, there
were still certain limitations of this meta-analysis. First, the
sample sizes of several of the studies included in our metaanalysis were relatively small. Second, due to the limited

6218A/G-focused study omitted.


Study omitted

OR

95% CI

p-value

Lin et al.
Luo et al.
Ma et al.
Ng et al.
Qiao et al.
Wen et al.
Zhai et al.
Zhang, Y.
Zheng et al.
Zhu, H.

1.01
1.03
1.02
1.00
1.02
1.03
1.04
1.03
1.02
1.01

0.96-1.07
0.98-1.08
0.97-1.07
0.95-1.05
0.97-1.07
0.98-1.09
0.98-1.11
0.98-1.08
0.97-1.07
0.96-1.06

0.62
0.32
0.44
0.90
0.41
0.26
0.16
0.32
0.50
0.69

Abbreviations: OR, odds ratio; CI, confidence interval.

we did not find that the rs11196218A/G polymorphism was


significantly associated with an increased risk of T2DM in
the Chinese Han population, which is consistent with the
findings of Ma (30), Zheng et al. (31), Luo et al. (32), Wen et
al.(33) and Qiao et al. (34). However, a previous metaanalysis performed by Luo et al. (32) found that rs11196218
showed a marginal association with T2DM risk (OR = 1.09,
95% CI = 1.00-1.19, p = 0.059). The most plausible explanation
for this discrepancy is the different genetic backgrounds
associated with different ethnic groups, areas and population
substructures.
In the present study, obvious heterogeneity was observed
in the comparisons among studies. Thus, a random-effects
model was applied for the meta-analysis, after which we
conducted a sensitivity analysis to identify the source of the
heterogeneity. After omitting the study by Ng et al. (17) from
the analysis, there was no heterogeneity among the
remaining studies. One plausible explanation for the
influential role of this study was that the populations
examined by Ng et al. (17) resided in the metropolis of Hong
Kong; as the residents of Hong Kong emigrated from
different areas of China, they have probably undergone
population admixture. Moreover, T2DM is a complex
hereditary disease, so the historical immigration, complex
ancestries, population movement, and recent intermarriages

Figure 4 - Funnel plot analysis to detect publication bias (G vs. A of the rs11196218A/G polymorphism). Each point represents an
independent study on the indicated association. The dark point represents two overlapping articles.

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CLINICS 2015;70(8):593-599

information on environment and lifestyle available the


included studies, it was not possible to perform a
subgroup meta-analysis or an interaction analysis based
on environment or lifestyle. Finally, similar to a casecontrol study, a meta-analysis is a retrospective study, and
recall bias might also exist.
In conclusion, we did not find any association between the
TCF7L2 gene rs11196218A/G polymorphism and T2DM risk
in the Chinese Han population. To achieve a better and more
comprehensive understanding of the association between
TCF7L2 gene polymorphisms and T2DM risk, we suggest
that studies including large samples and different ethnicities
and lifestyles and examining sophisticated gene-gene and
gene-environment interactions should also be considered in
future analyses.

14.

15.
16.
17.

18.

19.

AUTHOR CONTRIBUTIONS
Ma ET and Wei L developed the idea for the study and drafted the
manuscript. Ma ET, Wei L, Wang HL and Tian RR were responsible for
conducting the search, the data collection and the study quality assessment.
Ma ET, Wei L Wang HL and Guo J analyzed and interpreted the data. All
of the authors read and approved the nal version of the manuscript.

20.

21.

22.

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