world journal of · 551 tyms/kras/braf molecular profiling predicts survival following adjuvant...

World Journal ofGastrointestinal Oncology

World J Gastrointest Oncol 2019 July 15; 11(7): 509-566

ISSN 1948-5204 (online)

Published by Baishideng Publishing Group Inc

W J G OWorld Journal ofGastrointestinalOncology

Contents Monthly Volume 11 Number 7 July 15, 2019

MINIREVIEWS509 Utilizing gastric cancer organoids to assess tumor biology and personalize medicine

Lin M, Gao M, Cavnar MJ, Kim J

518 Recent progress of chemotherapy and biomarkers for gastroesophageal cancerMaeda O, Ando Y

527 Sarcopenia in pancreatic cancer – effects on surgical outcomes and chemotherapyChan MY, Chok KSH

ORIGINAL ARTICLE

Retrospective Cohort Study

538 Intraoperative intraperitoneal chemotherapy increases the incidence of anastomotic leakage after anterior

resection of rectal tumorsWang ZJ, Tao JH, Chen JN, Mei SW, Shen HY, Zhao FQ, Liu Q

Retrospective Study

551 TYMS/KRAS/BRAF molecular profiling predicts survival following adjuvant chemotherapy in colorectal

cancerNtavatzikos A, Spathis A, Patapis P, Machairas N, Vourli G, Peros G, Papadopoulos I, Panayiotides I, Koumarianou A

WJGO https://www.wjgnet.com July 15, 2019 Volume 11 Issue 7I

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ContentsWorld Journal of Gastrointestinal Oncology

Volume 11 Number 7 July 15, 2019

ABOUT COVER Editorial Board Member of World Journal of Gastrointestinal Oncology, WaelM Abdel-Rahman, MD, PhD, Professor, Department of Medical LabSciences, University of Sharjah, Sharjah 27272, Sharjah, United ArabEmirates

AIMS AND SCOPE World Journal of Gastrointestinal Oncology (World J Gastrointest Oncol, WJGO,online ISSN 1948-5204, DOI: 10.4251) is a peer-reviewed open accessacademic journal that aims to guide clinical practice and improve diagnosticand therapeutic skills of clinicians. The WJGO covers topics concerning carcinogenesis, tumorigenesis,metastasis, diagnosis, prevention, prognosis, clinical manifestations,nutritional support, etc. The current columns of WJGO include editorial,frontier, field of vision, review, original articles, case report. We encourage authors to submit their manuscripts to WJGO. We will givepriority to manuscripts that are supported by major national andinternational foundations and those that are of great clinical significance.

INDEXING/ABSTRACTING The WJGO is now indexed in Science Citation Index Expanded (also known as

SciSearch®), PubMed, and PubMed Central. The 2019 edition of Journal Citation

Reports® cites the 2018 impact factor for WJGO as 2.758 (5-year impact factor: 3.220),

ranking WJGO as 52 among 84 journals in gastroenterology and hepatology (quartile in

category Q3), and 131 among 229 journals in oncology (quartile in category Q3).

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NAME OF JOURNALWorld Journal of Gastrointestinal Oncology

ISSNISSN 1948-5204 (online)

LAUNCH DATEFebruary 15, 2009

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PUBLICATION DATEJuly 15, 2019

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Submit a Manuscript: https://www.f6publishing.com World J Gastrointest Oncol 2019 July 15; 11(7): 509-517

DOI: 10.4251/wjgo.v11.i7.509 ISSN 1948-5204 (online)

MINIREVIEWS

Utilizing gastric cancer organoids to assess tumor biology andpersonalize medicine

Miranda Lin, Mei Gao, Michael J Cavnar, Joseph Kim

ORCID number: Miranda Lin(0000-0002-1250-1254); Mei Gao(0000-0001-8960-8749); Michael JCavnar (0000-0002-6197-7945);Joseph Kim (0000-0001-6313-1516).

Author contributions: All authorscontributed equally to conceptionand design of the study, drafting ormaking critical revisions tointellectual content in themanuscript, and final approval ofthe final version of the article.

Conflict-of-interest statement: Nopotential conflicts of interest.

Open-Access: This article is anopen-access article which wasselected by an in-house editor andfully peer-reviewed by externalreviewers. It is distributed inaccordance with the CreativeCommons Attribution NonCommercial (CC BY-NC 4.0)license, which permits others todistribute, remix, adapt, buildupon this work non-commercially,and license their derivative workson different terms, provided theoriginal work is properly cited andthe use is non-commercial. See:http://creativecommons.org/licenses/by-nc/4.0/

Manuscript source: Invitedmanuscript

Received: February 23, 2019Peer-review started: February 23,2019First decision: April 15, 2019Revised: April 25, 2019Accepted: June 12, 2019Article in press: June 13, 2019Published online: July 15, 2019

Miranda Lin, Mei Gao, Michael J Cavnar, Joseph Kim, Department of Surgery, University ofKentucky, Lexington, KY 40536, United States

Corresponding author: Joseph Kim, FACS, MD, Surgical Oncologist, Department of Surgery,University of Kentucky, 800 Rose St., Lexington, KY 40536, United [email protected]: +1-859-3238920Fax: +1-859-3236840

AbstractWhile the incidence and mortality of gastric cancer (GC) have declined due topublic health programs, it remains the third deadliest cancer worldwide. Forpatients with early disease, innovative endoscopic and complex surgicaltechniques have improved survival. However, for patients with advanceddisease, there are limited treatment options and survival remains poor. Therefore,there is an urgent need for more effective therapies. Since novel therapies requireextensive preclinical testing prior to human trials, it is important to identifymethods to expedite this process. Traditional cancer models are restricted by theinability to accurately recapitulate the primary human tumor, exorbitant costs,and the requirement for extended periods of development time. An emerging invitro model to study human disease is the patient-derived organoid, which is athree-dimensional system created from fresh surgical or biopsy tissues of apatient’s gastric tumor. Organoids are cultured in plastic wells and suspended ina gelatinous matrix, providing a substrate for extension and growth in alldimensions. They are rapid-growing and highly representative of the molecularlandscape, histology, and morphology of the various subtypes of GC. Organoidsuniquely model tumor initiation and growth, including steps taken by normalstomach cells to transform into invasive, intestinal-type tumor cells. Additionally,they provide ample material for biobanking and screening novel therapies.Lastly, organoids are a promising model for personalized therapy and warrantfurther investigation in drug sensitivity studies for GC patients.

Key words: Organoids; Gastric cancer; Cancer models; Drug sensitivity; Drug screening;Personalized medicine

©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.

Core tip: Patient-derived organoids are three-dimensional models of human cancer useful

WJGO https://www.wjgnet.com July 15, 2019 Volume 11 Issue 7509


https://dx.doi.org/10.4251/wjgo.v11.i7.509

http://orcid.org/0000-0002-1250-1254

http://orcid.org/0000-0001-8960-8749

http://orcid.org/0000-0002-6197-7945

http://orcid.org/0000-0001-6313-1516

http://creativecommons.org/licenses/by-nc/4.0/



P-Reviewer: Dang SC, Wu JMS-Editor: Ji FFL-Editor: AE-Editor: Xing YX

for investigating tumor biology and drug discovery. There are now many reports on theutility of organoids in cancer research and personalized therapy. However, none focus onthe use of organoid technology in improving outcomes for patients with gastric cancer(GC), which is one of the deadliest cancers worldwide. Our objective is to report thecurrent progress in GC organoid technology in comparison to traditional cancer modelsand evaluate their potential role in informing personalized clinical decision making forpatients with GC.

Citation: Lin M, Gao M, Cavnar MJ, Kim J. Utilizing gastric cancer organoids to assess tumorbiology and personalize medicine. World J Gastrointest Oncol 2019; 11(7): 509-517URL: https://www.wjgnet.com/1948-5204/full/v11/i7/509.htmDOI: https://dx.doi.org/10.4251/wjgo.v11.i7.509

INTRODUCTIONThe incidence of gastric cancer (GC) has declined due to global improvements insanitation, hygiene, and food preservation, and there has been a correspondingdecrease in mortality with the implementation of early detection strategies[1,2].However, GC remains the third leading cause of cancer-related deaths worldwide,with a low 28% 5-year survival rate[1,3]. Although surgical techniques such as D2lymph node dissection have improved survival for patients with early or localizeddisease, there is still an urgent need to increase the number of effective therapiesavailable to patients with advanced GC[4].

A variety of cancer models have been developed to bring new therapies to theclinic. The traditional in vitro cancer model used to screen novel therapies is the cancercell line (CCL)[5]. CCLs are rapid-growing cancer models that are derived from asingle patient and immortalized[5]. CCLs have the advantage of quick developmentwhile producing abundant biological material for testing novel therapies, howeverdue to serial passaging and wide distribution over many generations, they developextensive chromosomal changes and lose the ability to accurately model the originalprimary tumor[6]. Additional drawbacks include the two-dimensional nature of thiscancer model and lack of structural modeling. Thus, CCLs do not meet the need for acancer model that represents the molecular profile and three-dimensional architectureof the primary tumor.

After screening of potential new therapies in CCLs, drugs that show the mostpromise typically undergo testing in vivo. Patient-derived tumor xenograft (PDTX)models are the gold standard in vivo model, created from either subcutaneous ororthotopic implantation of CCLs or patient tumor tissues into animals[7]. PDTXs arebeneficial in that they accurately represent genotypic and phenotypic characteristicsof the primary tumor as well as interactions with the microenvironment. However,they are quite costly and require a lengthy amount of time to create, limiting theirability to provide immediate clinically actionable data[7].

Recent studies have demonstrated the potential of using next generationsequencing to predict optimal therapies for individual patients through identificationof driver mutations and use of targeted therapies[8]. However, sequencing can berestricted by tumor tissue availability from the patient, cost, time, and the lack oftargeted drugs for cancers that do not harbor driver mutations, or when thecorresponding targeted drug has not been developed[9]. Thus, there is an importantneed for an appropriate cancer model that is able to address these limitations andidentify the most effective therapy for individual patients with currently availabledrugs in a timely manner.

Patient-derived organoids are three-dimensional in vitro human model systems thatrecapitulate disease development[10]. The architecture of a typical GI organoidresembles the organ from which it has been derived and maintains its genomicprofile[10]. Organoids present an accurate model that provide quicker turnover forexperimental procedures and yield results in a shorter period of time than traditionalhuman cancer models. The first gastric organoid culture was developed in 2010 tomodel the self-renewing and proliferative capacity of Lgr5+ve stem cells in the gastricpylorus[11]. Since then, organoids have also been successfully created from GCspecimens to study oncogenic events and drug sensitivities[12-15]. GC organoidsprovide the potential to aid development of new therapies and more importantly totest and predict which therapies may work best in GC patients.


Lin M et al. Personalizing medicine with gastric cancer organoids

510

In this review, we provide a detailed description of current strides and benefits ofGC organoid technology. A brief description of GC organoid culture will be followedby analysis of organoid representation of primary tumor histology, morphology, andheterogeneity, as well as organoid modeling of the subtypes of GC andcarcinogenesis. This review will also discuss GC organoids as a vehicle for drugsensitivity testing and biobanking, and finally, describe investigations utilizing GCorganoid technology for personalizing therapy and the promise of clinical trials usingGC organoids to predict therapies for individual patients.

ORGANOID CULTUREGC organoids can be propagated from surgically resected specimens or biopsies, andvarious methodologies have been proposed for their successful creation[12-16]. In brief,tumor specimens are washed and centrifuged, and then minced and digested intosmall pieces (2-5 mm2)[16]. From our experience, surgical specimens require extensivemanual mincing with a scalpel and enzymatic digestion from muscle layers, whilebiopsies require no enzymatic digestion and only light applied pressure on amicroscope slide (Figure 1)[12]. Once the cells are isolated, they are then suspended inmatrigel, a basement membrane matrix extracted from Englebreth-Holm Swarmmouse tumors and containing the extracellular matrix components laminin, type IVcollagen, and enactin[17]. Matrigel provides a gelatinous matrix for organoids toelongate and proliferate three-dimensionally. GC organoids are overlaid withorganoid medium, which consists of Advanced Dulbecco’s Modified Eagle’sMedium/F12, Glutamax, HEPES, and penicillin/streptomycin (Figure 2) [18].Additionally, specific factors important for gastric epithelial cell proliferation andmaturation are added to promote growth of organoids, including Wnt3A, R-spondin,Noggin, hEGF, hFGF10, and gastrin (Figure 2)[10]. Once plated in wells and enriched inmedium, organoids are incubated at 37 oC and 5% CO2

[16].Initially, GC organoids appear as clumped cells in the three-dimensional matrix. In

3-4 days, they develop into a spherical cystic body surrounded by differentiated cells,including stem cells, mucinous cells, gastric pit cells, and endocrine cells (Figure 3)[10].If the culture is healthy, GC organoids produce buds and replicate (Figure 4). Oncehighly confluent, organoids are passaged, i.e., they are digested and resuspended inmatrigel into additional wells. Passaging allows cultures to grow and cultivate formany months, producing substantial biological material for testing or freezing forlater use[10,14,15].

ACCURATE MODELS OF GC

Retention of primary tumor characteristics and representation of tumor morphologyThe current gold standard in vitro model of GC has been the CCL[5]. However, CCLcultures consist of clones of a single cell that have been distributed and passagedmany times, altering chromosomal characteristics to where they no longer representthe original tumor[5]. In a study of four HeLa cell line cultures, it was found that eachculture had a unique genomic profile and activated different pathways in response toa hypoxic environment, demonstrating the effect of different culture conditions andserial passaging on the genomic integrity of CCLs[19]. In contrast, GC organoids retainalterations found in the primary tumor even after serial passaging as evidenced byour own experience showing GC organoids retained copy number alterations aftermultiple passages[12] and a study showing six GC organoids maintained a stabletranscriptome even after six months in culture[14].

Since faithful chromosomal segregation during mitosis requires intact tissuearchitecture and extracellular cues, CCLs may not represent the degree ofchromosomal stability found in the primary tumor from which it was derived[20]. Incontrast, organoids contain a three-dimensional architecture and preserve structuralintegrity found in the primary tumor. They are composed of multiple cell types andrepresent the diversity of cell populations found in tumors (Table 1)[21]. They varymorphologically depending on the original tumor’s location in the stomach andtumor histology[15]. GC organoids derived from intestinal-type tumor specimensresemble typical GI cancer organoids in that they are glandular and form a singlelumen lined by a single layer of epithelial cells[13]. Moderately differentiated intestinaltype tumors develop buds, while poorly differentiated intestinal-type tumors formsolid clusters[14]. GC organoids developed from diffuse-type tumor specimens formeither loosely cohesive isolated cells or solid cell clusters that do not surround alumen[13,14]. Mixed-type tumors develop into organoids containing all morphological



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Figure 1

Figure 1 Creation of gastric cancer organoids. Endoscopic biopsy and surgical specimens are obtained from the patient and washed and centrifuged extensively.Endoscopic biopsy specimens are then placed on a microscope slide and pressure is applied by a coverslip onto the tissues to release gastric glands. Surgicalspecimens are enzymatically digested and minced with a scalpel for gland isolation. Once isolated, glands are suspended in matrigel and plated in drops in pre-warmed wells. Organoids are overlaid with medium.

patterns[13,14].

Representation of histological and molecular subtypesRecently, therapeutic research efforts have been enhanced by classification systemsthat delineate molecular features of certain GC subtypes. The classification systemproposed by The Cancer Genome Atlas (TCGA) identifies four gastric tumorsubtypes: Epstein-Barr Virus-positive (EBV, 9%), those with microsatellite instability(MSI, 22%), genomically stable (20%), and tumors with chromosomal instability (CIN,50%)[22]. The Lauren classification identifies two histological subtypes of GC based onpathology, epidemiology, and etiology: Intestinal and diffuse type adenocarcinoma.Each GC subtype contains distinct features and biomarkers that may be targeted byspecific therapies. Organoids are unique in that they represent the entire genomiclandscape and display distinguishable genetic properties depending on the GCsubtype outlined by TCGA and Lauren classifications of the primary tumor, makingthem useful models for drug discovery[22,23]. Chromosomal stability is maintained inMSI and EBV organoids[14]. Diffuse-type organoids encompass both CIN andgenomically stable subtypes, while intestinal- and mixed-type organoids displayextensive chromosomal instability[14,15]. GC organoids established from a genomicallystable tumor contain typical CDH1 gene alterations and organoids established fromMSI tumor tissues display characteristic CpG island methylator phenotype and MLH1hypermethylation[13-15]. Furthermore, EBV organoid lines retain the viral genome aswell as characteristic ARID1A and PIK3CA mutations[14]. In one study, a diffuse-typeGC organoid line contained the CLDN18-ARHGAP6 fusion gene, which is found ingenomically stable tumors. This mutation is not represented in any GC CCLs, furthersupporting the advantage of organoids to represent all types of GC compared toCCLs[14].

TUMORIGENESIS WITH GC ORGANOIDS

Models of tumor formation, carcinogenesis, and metastasisThe initiation of GC has been studied extensively to target specific genes andmolecular events leading to tumor development. Organoids have been used toaccurately model this progression. Nanki et al[13] induced diffuse GC-like morphologyin normal gastric organoids by knocking out the tumor suppressor CDH1, whichplays a role in cell-cell adhesion[13,24]. Further knockout of the oncogene RHOA, a RhoGTPase belonging to the Ras superfamily, in CDH1-KO organoids resulted inreversion back to normal morphology[13,25]. This suggests malignant phenotype may beinduced in normal organoids and organoids can also model reversion back to normalmorphology, making them useful for studying initiating events in tumorigenesis.Organoids therefore, may serve as a valuable tool to study phenotypic consequencesleading to tumor development after activation or inhibition of specific genes.

Unlike CCLs, organoids have the unique ability to model the development ofcancer. Normal gastric organoids and organoids derived from a lesion with intestinalmetaplasia from the same patient were not morphologically distinct, but the intestinalmetaplasia-derived organoids contained the marker CDX2 while normal organoidsdid not[13]. Similarly, organoids derived from dysplasia and invasive tumor from the



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Figure 2

Figure 2 Complete list of ingredients for gastric cancer organoid medium. Organoid medium consists mainly of advanced DMEM/F12, Glutamax, HEPES, andpenicillin/streptomycin and further supplemented with growth factors and enzymes to promote organoid growth.

same patient both expressed representative early GC mutations however, haddistinctive chromosomal patterns and unique gene mutations, illustrating the abilityof organoids to document the important transition from dysplasia to adenocarci-noma[14]. Altogether, these studies reveal organoids created from pre-malignantlesions are accurate surrogates of the sequence of events leading to cancer, which arenot possible with cell lines, and therefore serve as a useful tool for identifying andtargeting key mutations of tumor development.

Lastly, organoids created from the same patient’s primary tumor and lymph nodemetastases show varying degrees of differences, which is believed to model niche-specific changes during metastasis[14]. Thus, the ability of organoids to portray thegenotypic and phenotypic events during cancer progression and metastasis providesa unique platform to study important oncological events not accessible through othermodels.

DRUG SENSITIVITY TESTING

Current limitations of bringing drugs to clinicThe costs associated with supporting a cancer drug from preclinical development toapproval by the United States Food and Drug Administration averages about $719.8million[26]. The high costs and lengthy amount of time, approximately 5 to 15 years, tobring newly developed drugs into the clinic limit the ability to provide new therapiesto patients. One specific delay is the constraint associated with creating PDTX models,which are costly and require 4-8 mo prior to drug testing[7]. From our experience,organoids can be utilized for drug sensitivity testing within two weeks and are lessexpensive than animal models (Table 1)[12]. While organoids lack the ability to modelangiogenesis, they provide ample biological material to rapidly test drugs prior toanimal studies.

Investigation of drug sensitivity testingOrganoids have the ability to accurately discriminate between different drugtreatments. Previously, we created and treated organoids from a patient’s gastrictumor biopsies with the cytotoxic chemotherapies 5-fluorouracil, cisplatin, oxaliplatin,and irinotecan[12]. We observed varying levels of organoid sensitivity to thechemotherapies that correlated with varying IC50 values. Similarly, Vlachogiannis etal[27] concluded that organoids are useful for modeling patient drug response. AnErbB2-amplified GC organoid line was treated with lapatinib, an antibody that targetsthe EGFR and HER2 tyrosine kinases that is currently used in combination withcapecitabine to treat metastatic breast cancer patients [28]. While the ErbB2-overexpressing GC organoids exhibited strong sensitivity to lapatinib, a GC organoidline with ErbB2 wild-type genotype did not respond. In addition, a GC organoid linewith amplified AKT1 was the only organoid line in their cohort to respondsignificantly to two anti-AKT antibodies, MK-2206 and GSK690693[27]. As such, GCorganoids have potential for drug sensitivity screening.

Creation of GC organoid biobanks for novel drug screening



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Figure 3

Figure 3 Image of gastric cancer organoids. A: Image of passage 2 gastric cancer organoids created from biopsy. Scale bar 200 μm; B: High magnification imageof passage 2 gastric cancer organoid. Scale bar 100 μm.

Organoid biobanks have been created for various cancer types[14,29-32]. They portray thediverse molecular landscape and corresponding phenotypic characteristics foundwithin a single cancer type and provide abundant biological material for drugsensitivity screening of newly synthesized drugs undergoing preclinical development.In one particular study, 9 GC organoids created from 7 patients were selected for drugscreening[14]. Thirty-seven cancer therapies were tested in the organoids, whichshowed parallel response to that of clinical response with resistance to 5-fluorouraciland cisplatin and sensitivity to oxaliplatin, epirubicin, and paclitaxel. The GCorganoids also responded to recently FDA-approved therapies, napabucasin andabemaciclib, and therapies currently undergoing clinical trials, vistusertib and VE-822[14]. These data highlight the potential utility of an organoid biobank to screennovel therapies for efficacy prior to in vivo trials, bypassing the additional cost andtime constraints associated with screening therapies in mouse models[33]. GC organoidbiobanks serve as a useful tool for drug screening by bridging the gap between ex vivoand in vivo models by accurately portraying the genetic profile of cancers withdecreased time and cost constraints.

PERSONALIZED MEDICINE

Limitations of personalized medicineEven within GC subtypes, every patient’s tumor is vastly different and therefore,could benefit from a personalized approach. Currently, after biopsy or surgicalresection of the primary tumor, a patient’s preserved tumor specimen may be sent forgenomic sequencing, such as that offered by Foundation Medicine[8], to provideinformation about key driver mutations to guide treatment. While genomic testingprovides invaluable biomarker and gene mutation information to help inform certaintherapies and clinical trials, there are a few limitations. Optimally, a tumor percentageof 30% is required per specimen for Foundation Medicine[8] and a minimum thresholdof 60% tumor percentage has historically been required for large next generationsequencing studies[22]. In contrast, organoid establishment is not limited by tumorpercentage and furthermore, organoids are created from minimal fresh tissue andexpanded in culture, even when tumor tissue is limited[27]. Finally, and perhaps mostimportantly, next generation sequencing testing has been criticized for lack of benefitto the majority of patients who have somatic alterations in their cancers that have nocorresponding targeted therapies available in the clinic[9]. These patients may benefitfrom functional assays using organoids to test a variety of drugs to determine the bestavailable treatment.

Yielding drug treatment results in a clinically actionable time periodGC organoids established directly from patients may be used to test standard of carecytotoxic and targeted drug regimens, providing great potential for personalizedmedicine. However, GC organoids are mostly created from surgically resected tumorspecimens. Surgery provides abundant tissue and serves as a good source for creatingorganoid biobanks for drug discovery and signaling studies. However, creatingorganoids from surgical specimens does not benefit the patient undergoingneoadjuvant therapies. Additionally, many patients with GC have metastatic diseaseand are not candidates to undergo surgery. Therefore, in order to personalizemedicine for individual patients, a new avenue for organoid creation must be



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Figure 4

Figure 4 Gastric cancer organoid with budding. High magnification image of passage 2 gastric cancer organoidwith budding. Scale bar 100 μm.

explored. We recently published a novel technique for successfully establishing GCorganoids from esophagogastroduodenoscopy (EGD) (Figure 1)[12]. Many patientssuspected to have GC will undergo EGD for diagnosis and staging. Therefore,creating organoids from EGD biopsies for drug sensitivity testing may directly yieldclinically relevant results for patients who are ineligible for surgical intervention. Ourtechnique has yielded abundant EGD derived organoids for testing multiple standardof care drug regimens and combination therapies within two weeks of organoidestablishment (unpublished data), providing a valuable model for clinical predictionof the best therapies for individual patients. Furthermore, since almost every patientsuspected of GC will undergo EGD, creating organoids from biopsies may serve as anadditional source of abundant human biological material for biobanking.

Clinical trials using GC organoids to personalize therapyWith the ability to quickly develop large numbers of organoids that accuratelyportray an individual patient’s tumor, GC organoids show high clinical utility forpredicting drug sensitivities in patients. This unique advantage is currently beingassessed in a human clinical trial where GC organoids created from patients withresectable cancer will be treated with the same neoadjuvant systemic therapy receivedby the patient, and in vitro cytotoxicity will be correlated to in vivo response(OPPOSITE trial: NCT03429816)[34]. The future of personalized medicine in GCincludes human trials implementing GC organoids to test various drugs and predictthe best treatment for individual patients with advanced GC.

CONCLUSIONOrganoid technology has provided the ability to model GC with more accuracy thantraditional in vitro models. They retain GC biomarkers and display varyingmorphology depending on the histology and molecular subtype of the primary tumorfrom which they are derived. GC organoids are unique in that they recapitulateimportant molecular events leading to carcinogenesis and metastasis unlike CCLS.They are less costly and less time-consuming to create than in vivo PDTX models,making them an appropriate vehicle for biobanking and comprehensive drugscreening. While GC organoids present a unique, in vitro model, there are a fewlimitations. Unlike PDTX models, organoids are unable to mimick the in vivo tumormicroenvironment in that they lack blood vessels for studying cancer angiogenesisand metastasis. In addition, they are grown in optimal conditions and growth-promoting media. Despite these restrictions, GC organoids overcome currentlimitations in precision medicine that require minimum tumor percentage and yet,organoids provide a more relevant predictor of drug response than next generationsequencing methods. With the ability to create GC organoids from diagnostic biopsyprocedures, multiple drug regimens can be tested at once to yield actionable results ina clinically relevant time interval, providing avenues for personalized medicine forGC patients.



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Table 1 Comparison of patient-derived cancer models

Cancer cell lines Xenografts Organoids

Model system In vitro In vivo In vitro

Shape 2D 3D 3D

Time prior to drug testing 1-2 d 4-8 mo 10-14 d

Cellularity Clones of single cell Tumor in microenvironment Multiple cell types

Biological material produced Abundant Scarce Abundant

Cost Low High Medium

Tumor architecture None Represented Represented

Primary tumor represented No Yes Yes

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MINIREVIEWS

Recent progress of chemotherapy and biomarkers forgastroesophageal cancer

Osamu Maeda, Yuichi Ando

ORCID number: Osamu Maeda(0000-0003-4700-6541); Yuichi Ando(0000-0002-6849-2297).

Author contributions: Maeda Owrote the manuscript; Ando Ysupervised the review; and allauthors read and approved thefinal manuscript.

Conflict-of-interest statement: Nopotential conflict of interest.



Received: February 20, 2019Peer-review started: February 22,2019First decision: April 16, 2019Revised: April 17, 2019Accepted: May 28, 2019Article in press: May 29, 2019Published online: July 15, 2019

P-Reviewer: Munoz M, Pekgoz MS-Editor: Ji FFL-Editor: A

Osamu Maeda, Yuichi Ando, Department of Clinical Oncology and Chemotherapy, NagoyaUniversity Hospital, Nagoya 466-8560, Japan

Corresponding author: Osamu Maeda, MD, PhD, Lecturer, Department of Clinical Oncologyand Chemotherapy, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan. [email protected]: +81-52-7441903Fax: +81-52-7441903

AbstractKey cytotoxic drugs of chemotherapy for gastroesophageal cancer includefluoropyrimidine, platinum, taxanes and irinotecan. Concurrentchemoradiotherapy is one of the main treatment strategies, especially foresophageal cancer. As molecular target agents, the anti-HER2 antibodytrastuzumab for HER2-positive gastric cancer and the anti-angiogenesis agentramucirumab combined with paclitaxel have been proven to improve thesurvival of gastric cancer patients. Recently, anti-PD-1 antibodies have becomeavailable as second- or later-line chemotherapy. Microsatellite instability is alsouseful as a biomarker to select patients suitable for immunotherapy.Furthermore, genome-wide analysis has improved our understanding of thebiological features and molecular mechanisms of gastroesophageal cancer andwill provide optimized treatment selection.

Key words: Gastroesophageal cancer; Chemotherapy; Biomarker; HER2


Core tip: This article reviewed the current status and recent developments ofgastroesophageal cancer and its related biomarkers for treatment selection. Platinum,fluoropyrimidines, taxanes, irinotecan, trastuzumab and ramucirumab are key drugs.Recently, anti-PD-1 antibodies have become available. PD-L1 expression andmicrosatellite instability are used to predict the effectiveness of immunotherapy.Genome-wide analysis will provide a better understanding of the biology ingastroesophageal cancer.

Citation: Maeda O, Ando Y. Recent progress of chemotherapy and biomarkers forgastroesophageal cancer. World J Gastrointest Oncol 2019; 11(7): 518-526




http://orcid.org/0000-0003-4700-6541

http://orcid.org/0000-0002-6849-2297




E-Editor: Xing YX URL: https://www.wjgnet.com/1948-5204/full/v11/i7/518.htmDOI: https://dx.doi.org/10.4251/wjgo.v11.i7.518

INTRODUCTIONGastroesophageal cancer is one of the main causes of death worldwide. According toa report by the World Health Organization, gastric cancer and esophageal cancer havethe 3rd and 6th highest mortality rates, respectively. The best way to cure gastroe-sophageal cancer is the complete removal of cancer by surgical resection.Chemotherapy and radiation also contribute to improving the prognosis. Drugs usedfor systemic chemotherapy include cytotoxic agents and molecular target drugs.Recently, immune checkpoint inhibitors have also become available. Althoughmultiple options can be used as a treatment strategy, the effectiveness and side effectsare different depending on individual patients. Therefore, biomarkers to predict theeffectiveness for the optimization of treatment selection and individualization aredesired. In the present review, current chemotherapy options for gastroesophagealcancer and biomarkers are discussed.

FIRST-LINE CHEMOTHERAPY FOR GASTRIC CANCER

Doublet regimens for gastric cancerAs first-line chemotherapy for gastroesophageal cancer, a combination of platinumand fluoropyrimidine is essential. Doublet or triplet platinum/fluoropyrimidinecombinations are recommended for fit patients with advanced gastric cancer[1]. Sinceoral fluoropyrimidines have the advantage of simplicity, many studies using S-1 orcapecitabine have been performed. In the SPIRITS trial, the combination of oralfluoropyrimidine S-1 and cisplatin (SP) improved the survival of advanced gastriccancer patients compared with S-1 alone (median survival time: 13.0 mo vs 11.0 mo, P= 0.04)[2]. Fluorouracil, leucovorin plus oxaliplatin or cisplatin[3] was also effective. Thecombination of another oral fluoropyrimidine, capecitabine, with cisplatin showedsignificant noninferiority for progression-free survival vs fluorouracil plus cisplatin(FP)[4]. Cisplatin induces severe nausea and vomiting, i.e., it is highly emetogenic, andit also has strong nephrotoxicity, in which a large amount infusion is necessary toprevent renal impairment. In contrast, oxaliplatin is moderately emetogenic and lessnephrotoxic than cisplatin. A study comparing two platinum and two fluoro-pyrimidine drugs showed that capecitabine and oxaliplatin are as effective asfluorouracil and cisplatin[5]. For oral fluoropyrimidine, the equality of S-1 andcapecitabine in effectiveness was evaluated. A comparison between S-1 plusoxaliplatin and capecitabin plus oxaliplatin[6] showed equal efficacy. In a comparisonwith S-1 plus cisplatin and S-1 plus oxaliplatin (SOX) (G-SOX trial), both showedequivalent efficacy[7]. According to a meta-analysis comparing fluoropyrimidines,toxicity profiles were different, but a lower frequency of relevant adverse events wasobserved with S-1. This report concluded that choosing fluoropyrimidines should bebased on their individual toxicity profiles because their efficacies was similar[8].

Triplet regimens for gastric cancerTo strengthen the efficacy of first-line chemotherapy, triplet regimens includingfluorouracil, platinum and taxane have been investigated. In the V325 study,combination with docetaxel, cisplatin and fluorouracil was superior in survivalcompared with FP[9,10] (median survival time: 9.2 mo vs 8.6 mo, P = 0.02). However,severe adverse events, including grade 3 or 4 neutropenia and complicatedneutropenia, were observed in 82% and 29% of the patients, respectively. In tripletregimens, capecitabine and S-1 were also used as fluoropyrimidines, and oxaliplatinwas used as platinum.

The combination of docetaxel, cisplatin and capecitabine (DCX)[11] for advancedcancer achieved a median overall survival of 14.4 mo, but 62.5% of patientsexperienced grade 3 or 4 neutropenia. DCX was reported to be used as neoadjuvantchemotherapy, which was administered before surgery for resectable diseases[12], and63% of the patients achieved R0 resection. To maintain effectiveness and avoid severeadverse events, a modified DCX regimen in which docetaxel was reduced wasreported[13]. With this regimen, three out of eight patients underwent conversiongastrectomy and achieved long-term survival.


Maeda O et al. Chemotherapy and biomarkers for gastroesophageal cancer

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The combination with docetaxel, cisplatin and S-1 for unresectable gastric cancer(KDOG 0601) was also reported[14]. In this study, the objective response rate was 81%,and the median survival time was 18.5 mo. In a study with docetaxel, cisplatin and S-1 (DCS) as neoadjuvant chemotherapy (JCOG1002), the response rate was 57.7%, andR0 resection was achieved in 84.6% of patients[15]. In another study (JCOG1002),neoadjuvant DCS achieved a 90% R0 resection rate[16].

HER2-positive gastric cancerApproximately 20% of gastric cancer is HER2 positive, and the anti-HER2 antibodytrastuzumab is effective. In comparison between combination cisplatin pluscapecitabine with or without trastuzumab (ToGA trial), the median overall survivalwas 13.8 mo with trastuzumab and 11.1 mo without trastuzumab[17]. For HER2-positive breast cancer, lapatinib, trastuzumab emtansine and pertuzumab areavailable as anti-HER2 agents. However, for HER2-positive gastric cancer, the trials oflapatinib[18,19], trastuzumab emtansine (T-DM1)[20] and pertuzumab[21] did not show asurvival benefit and were not used for gastric cancer. Therefore, only trastuzumab asan anti-HER2 agent is available for HER2-positive gastric cancer.

SECOND OR LATER-LINE FOR GASTRIC CANCERSeveral phase III trials revealed evidence of a survival benefit in second-linechemotherapy. For example, the COUGAR-02[22] trial showed the benefit of docetaxel,an improvement of survival with irinotecan[23] was proven by ArbeitsgemeinschaftInternistische Onkologie, and a Korean study revealed the effectiveness of docetaxelor irinotecan[24]. The WJOG 4007 study compared paclitaxel and irinotecan andshowed an equivalent effectiveness of both agents[25]. Ramucirumab is a moleculartarget agent that binds to vascular endothelial growth factor receptor-2 (VEGFR2) andinhibits VEGFR-mediated angiogenesis. In the REGARD trial, ramucirumabmonotherapy showed a longer survival rate compared with the placebo[26]. TheRAINBOW trial revealed the benefit of the addition of ramucirumab to paclitaxel[27].Apatinib is a tyrosine kinase inhibitor that selectively binds to and strongly inhibitsVEGFR-2, with a decrease in VEGF-mediated endothelial cell migration, proliferation,and tumor microvascular density. Apatinib significantly improved the survival ofpatients for whom two or more prior lines of chemotherapy had failed[28].

Trifluridine/tipiracil (TAS-102) is a cytotoxic chemotherapy consisting of athymidine-based nucleoside analogue, trifluridine, and a thymidine phosphorylaseinhibitor, tipiracil. Trifluridine is incorporated into DNA, resulting in DNAdysfunction, and tipiracil blocks trifluridine degradation by thymidine phos-phorylase, increasing trifluridine bioavailability. Trifluridine/tipiracil improved theoverall survival compared with the placebo in patients who had previously receivedtwo or more regimens[29].

Recently, the effectiveness of immune checkpoint inhibitors has been shown invarious cancers. Nivolumab is an anti-PD-1 antibody, and its survival benefit wasproven as a third or later-line chemotherapy[30]. Pembrolizumab is also an anti-PD-1antibody and demonstrated promising activity and manageable safety in patientswith advanced gastric or gastroesophageal junction cancer who had previouslyreceived at least 2 lines of treatment[31]. In this study, durable responses were observedin patients with PD-L1–positive and PD-L1–negative tumors. In another study,second-line therapy with pembrolizumab and paclitaxel was compared for gastriccancer with a combined positive score (CPS) of 1 or higher of PD-L1. CPS is defined asthe number of PD-L1–positive cells (tumor cells, lymphocytes, macrophages) as aproportion of the total number of tumor cells multiplied by 100. Althoughpembrolizumab did not significantly improve overall survival compared withpaclitaxel as a second-line therapy for advanced gastric or gastroesophageal junctioncancer, pembrolizumab had a better safety profile than paclitaxel[32].

CHEMOTHERAPY AND CONCURRENTCHEMORADIOTHERAPY FOR ESOPHAGEAL CANCER

Chemotherapy for esophageal cancerFor esophageal cancer, fluoropyrimidines and platinum are essential, and in additionto taxanes, they are also useful in gastric cancer. Perioperative FP was evaluated forgastroesophageal adenocarcinoma, and superiority in overall survival was showncompared with surgery alone[33]. For squamous cell carcinoma, surgery followingadjuvant FP was proven to be superior to surgery alone (JCOG9204)[34]. In comparison



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between preoperative and postoperative FP for localized advanced squamous cellcarcinoma of the esophagus, preoperative FP was superior to postoperative FP(JCOG9907)[35].

Chemoradiotherapy for esophageal cancerChemoradiation with FP followed by surgery (CALGB 9781) showed superioritycompared with surgery alone[36]. In the FFCD 9102 trial, no benefit was shown for theaddition of surgery after chemoradiation with FP compared with the continuation ofadditional chemoradiation for squamous cell carcinoma in the esophagus[37].Preoperative chemoradiotherapy with a combination of carboplatin and paclitaxelwas superior to surgery alone[38]. For esophageal adenocarcinoma, squamous cell, oradenosquamous carcinoma, a comparison with FP and the combination of oxaliplatin,leucovorin and fluorouracil revealed that both regimens are effective as definitivechemoradiotherapy for patients unsuitable for surgery[39]. For stage II-III esophagealsquamous cell carcinoma, definitive chemoradiation with FP was effective. Themedian survival time was 29 mo, with 3- and 5-year survival rates of 44.7% and36.8%, respectively, which was comparable to surgery with adjuvant chemotherapy(JOG9906)[40]. For stage I esophageal squamous cell carcinoma, chemoradiation withFP achieved 80.5% of the four-year survival proportion and 68% of the 4-year majorrelapse-free survival (JCOG9708)[41]. For patients with advanced squamous cellcarcinoma of the thoracic esophagus having either T4 tumor or distant lymph nodemetastasis (M1 Lym), chemoradiation with FP was administered. The response ratewas 68.3%, the complete response was 15%, the median survival time was 305.5 days,and the 2-year survival rate was 31.5% (JCOG 9516)[42]. The optimal dose of radiationwas also studied. For definitive chemoradiation using FP, the INT 0123 (RadiationTherapy Oncology Group 94-05) phase III trial compared 64.8 Gy and 50.4 Gy[43]. Thehigher radiation dose did not increase survival or local/regional control, and theyconcluded that the standard radiation dose for patients treated with concurrent 5-FUand cisplatin chemotherapy is 50.4 Gy[43].

Taxanes for esophageal cancerAs for second-line chemotherapy, taxanes are often used. In a phase II trial usingdocetaxel in which the majority of patients had squamous cell carcinoma, theresponse rate was 20%, and the median survival time was 8.1 mo[44]. In the COUGAR-02 trial, docetaxel was effective for esophageal adenocarcinoma as well as gastricadenocarcinoma[22]. Paclitaxel was also effective. In a phase II trial for esophagealcancer mainly of squamous cell carcinoma, the response rate was 44.2%, and themedian survival time was 10.4 mo[45]. In another trial with the majority of patients inwhich the histological diagnosis was adenocarcinoma, paclitaxel was also effective[46].

Other molecular targeted agentsAs described above, the angiogenesis inhibitor ramucirumab and the anti-HER2antibody trastuzumab are effective for gastric cancer. Although other moleculartargeted agents were investigated in multiple clinical trials, the benefits of most ofthem have not been proven. Bevacizumab is an angiogenesis inhibitor that is widelyused for cancer, including colorectal cancer and lung cancer. For gastric cancer,bevacizumab was examined combined with cytotoxic chemotherapy, and the survivalbenefit was not proven (AVAGAST trial)[47]. An anti-EGFR antibody, panitumumab,for first-line chemotherapy did not improve survival[48]. The EGFR inhibitor erlotinibis active in patients with gastroesophageal junction adenocarcinomas but appearsinactive in gastric cancers (SWOG 0127)[49]. The anti-EGFR antibody cetuximab isuseful for head and neck cancers and colorectal cancer with the wild-type RAS gene.A trial of cetuximab for gastric cancer evaluated the effect of the addition tocapecitabin and cisplatin (EXPAND). They concluded that the addition of cetuximabto capecitabine-cisplatin provided no additional benefit to chemotherapy alone[50]. Inthe CALGB 80403 (Alliance)/E1206 trial, cetuximab was applied for esophagealcancer with multiple regimens[51]. However, a trial of chemoradiotherapy withcetuximab in patients with esophageal cancer (SCOPE1) showed a shortened survivaltime compared with chemoradiotherapy without cetuximab. Therefore, concurrentchemoradiotherapy with cetuximab is not recommended[52]. Another anti-EGFRantibody, panitumumab, was also evaluated for esophagogastric cancer (REAL3)[48].The addition of panitumumab does not increase overall survival and cannot berecommended.

Microsatellite instabilityDNA mismatch repair (MMR) is a mechanism for recognizing and repairing DNAreplication errors. Microsatellite instability (MSI) is the condition of having apredisposition to mutations that result from deficient MMR. A potential determinant



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of the response to immune checkpoint inhibitors is mutation-associated neoantigens(MANAs) that are encoded by cancers. MMR-deficient cancers are predicted to havemany MANAs that might be recognized by the immune system.

The effect of pembrolizumab on patients with advanced MMR–deficient cancersacross 12 different tumor types was evaluated[53]. Responses were durable, objectiveresponses were observed in 53% of patients, and complete responses were achieved in21% of patients. According to another report, the response of pembrolizumab forMMR–deficient colorectal cancers is much better than that for MMR-proficientcolorectal cancers. Furthermore, patients with MMR–deficient noncolorectal cancerhad responses similar to those of patients with MMR–deficient colorectal cancer[54].Therefore, MSI/MMR deficiency is useful as a biomarker to predict the effectivenessof immunotherapy for solid tumors, including gastroesophageal cancer.

CURRENT APPLICATION OF BIOMARKERS AND FUTUREPERSPECTIVESAs mentioned above, HER2 overexpression and amplification are applied to definethe indication of trastuzumab. The expression of PD-L1 might be useful in predictingthe effect of pembrolizumab in some situations. MSI is also useful for selectingpatients who are suitable to use pembrolizumab for gastroesophageal cancer as wellas other solid tumors.

The Cancer Genome Atlas (TCGA) project proposes a molecular classificationdividing gastric cancer into four subtypes[55]: Tumors positive for Epstein–Barr virus,which display PIK3CA mutations, DNA hypermethylation, and an amplification ofJAK2, PD-L1 and PD-L2; microsatellite unstable tumors, which show elevatedmutation rates; genomically stable tumors, which are enriched for the diffusehistological variant and mutations of RhoA or fusions involving RHO-family GTPase-activating proteins; and tumors with chromosomal instability, which showaneuploidy and the focal amplification of receptor tyrosine kinases. TCGA alsoshowed the biological features of esophageal cancer[56]. According to this information,esophageal squamous cell carcinomas resembled squamous carcinomas of otherorgans more than they did esophageal adenocarcinomas. In contrast, esophagealadenocarcinomas strongly resembled the chromosomally unstable variant of gastricadenocarcinoma, suggesting that these cancers could be considered a single diseaseentity.

A study of perioperative chemotherapy for gastric cancer extracted the expressionof seven genes (CDH1, ELOVL5, EGFR, PIP5K1B, FGF1, CD44v8.10 and TBCEL) asbiomarkers to predict the prognosis of the patient[57]. In another study, the DNAmethylation profile was potentially related to a resistance to chemotherapy for gastriccancer[58]. The exploration of new antitumor agents accompanied by the developmentof a molecular diagnosis with will optimize the selection of therapy for individualpatients.

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MINIREVIEWS

Sarcopenia in pancreatic cancer – effects on surgical outcomes andchemotherapy

Miu Yee Chan, Kenneth Siu Ho Chok

ORCID number: Miu Yee Chan(0000-0001-5527-1471); Kenneth SiuHo Chok (0000-0001-7921-3807).

Author contributions: Chan MYperformed the literature reviewand drafted the manuscript; ChokKSH was responsible for theconcept and supervision of thestudy and final approval of themanuscript.

Conflict-of-interest statement: Theauthors declare that they have noconflict of interest.

Open-Access: This article is anopen-access article that wasselected by an in-house editor andfully peer-reviewed by externalreviewers. It is distributed inaccordance with the CreativeCommons Attribution NonCommercial (CC BY-NC 4.0)license, which permits others todistribute, remix, adapt, buildupon this work non-commercially,and license their derivative workson different terms, provided theoriginal work is properly cited andthe use is non-commercial. See:http://creativecommons.org/licenses/by-nc/4.0/


Received: January 26, 2019Peer-review started: January 28,2019First decision: April 15, 2019Revised: April 23, 2019Accepted: May 21, 2019Article in press: May 22, 2019Published online: July 15, 2019

Miu Yee Chan, Department of Surgery, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong,China

Kenneth Siu Ho Chok, Department of Surgery and State Key Laboratory for Liver Research,The University of Hong Kong, Hong Kong, China

Corresponding author: Kenneth Siu Ho Chok, FRCS (Ed), Associate Professor, Department ofSurgery and State Key Laboratory for Liver Research, The University of Hong Kong, 102 PokFu Lam Road, Hong Kong, China. [email protected]: +852-22553025

AbstractSarcopenia is found in up to 65% of pancreatic cancer patients. The definition anddiagnostic methods for sarcopenia have changed over the years, and themeasurement of skeletal muscle mass with cross-sectional imaging has becomethe most popular way of assessment, although the parameters measured varyamong different studies. It is still debatable that there is an association betweensarcopenia and postoperative pancreatic fistula, but most studies showed ahigher risk in patients with sarcopenic obesity. Long-term survival is worse insarcopenic patients, as shown by meta-analysis. Sarcopenia is also associatedwith decreased survival and higher toxicity in patients receiving chemotherapy,and chemotherapy also tends to potentiate sarcopenia. Treatment for sarcopeniastill remains an area for research, although oral supplements, nutritionalmodifications and exercise training have been shown to improve sarcopenia.

Key words: Sarcopenia; Pancreatic cancer; Clinical outcomes; Surgical outcomes;Chemotherapy; Radiotherapy


Core tip: Sarcopenia is a common condition found in pancreatic cancer patients. There isgrowing evidence showing that sarcopenia is associated with worse survival outcomes.This article summarizes the current evidence for the definition and diagnosis ofsarcopenia, as well as its relationship with surgical outcomes, survival andchemotherapy.

Citation: Chan MY, Chok KSH. Sarcopenia in pancreatic cancer – effects on surgical




http://orcid.org/0000-0001-5527-1471

http://orcid.org/0000-0001-7921-3807




P-Reviewer: Huang L, KaramouzisMVS-Editor: Ji FFL-Editor: FilipodiaE-Editor: Xing YX

outcomes and chemotherapy. World J Gastrointest Oncol 2019; 11(7): 527-537URL: https://www.wjgnet.com/1948-5204/full/v11/i7/527.htmDOI: https://dx.doi.org/10.4251/wjgo.v11.i7.527

INTRODUCTIONThe topic of sarcopenia in pancreatic cancer has come under the spotlight in the pastdecade. With the updates on several consensus statements, including those from theAsian Working Group for Sarcopenia in 2016[1] and European Working Group onSarcopenia in Older People (EWGSOP) in 2018[2], it is now known that sarcopenia is acondition that not only relates to age but is also affected by multiple factors, such assystemic inflammation, physical inactivity, and inadequate intake. The relationshipbetween pancreatic cancer and cachexia has long been recognized, but it is only in thelast decade that researchers have started to understand the importance of sarcopenia.

Pancreatic cancer is one of the most deadly malignancies worldwide, with a 5-yearsurvival of only about 5%, despite numerous efforts to improve various therapeuticstrategies over the decades[3]. It has become the third leading cause of cancer-relateddeaths in the United States and is projected to become the second by 2030[4]. Amongpancreatic cancer patients, those who have undergone resection have much bettersurvival rates than those who are unresectable[5]. Unfortunately, less than one-fifth ofpatients with this malignancy are considered resectable[3]. The low resection rate isdue to unfavorable tumor stage and location and also to comorbidities and poorfunctional performance of patients[6]. In pancreatic cancer patients, poor oral intake,altered metabolism due to malignancy, and malabsorption because of obstruction orexocrine insufficiency can all come into play at the same time and contribute to bothcachexia and sarcopenia[7]. These in turn worsen the patients’ performance status andtheir suitability for surgery.

In various studies, the prevalence of sarcopenia in pancreatic cancer patients rangesfrom 30% to 65%[8-10]. The wide variation is likely due to the heterogeneous groups ofpatients, difference in disease stage, and different methods of measuring sarco-penia[1,7,11]. Despite these variations, it has been repeatedly shown that sarcopeniapatients are more likely to have poorer outcomes[12-14]. This article aims to examine thecurrent evidence on sarcopenia, as well as its impact on the management of patientswith pancreatic ductal adenocarcinoma.

DEFINITION OF SARCOPENIASince the term “sarcopenia” was coined by Rosenberg[15] in 1997, remarkable progresshas been made in understanding this condition and its relationship with malignanciesand surgery. Instead of merely detecting the decline in muscle mass, EWGSOPredefined the condition in 2010 as the syndrome characterized by progressive andgeneralized loss of both skeletal muscle mass and quality (strength or performance)with a risk of adverse outcomes[16]. In the latest consensus by EWGSOP in 2018[2],muscle strength has come to the forefront in the diagnosis. From the evolution of thedefinition, it is clear that more emphasis has been put on muscle quality over quantityover the years. Similar definitions have been put forward by other groups, includingthe International Working Group on Sarcopenia[17], the European Society for ClinicalNutrition and Metabolism (ESPEN) Special Interest Group[18], the Society ofSarcopenia, Cachexia and Wasting Disorders[19], and the Asian Working Group forSarcopenia[20]. According to these definitions, the assessment of both muscle quantityand muscle quality is required when diagnosing sarcopenia (Table 1).

ASSESSMENT OF SARCOPENIA IN PANCREATIC CANCERPATIENTSDespite the relatively unified definition from different consensus groups, there is awide array of assessment tools for sarcopenia. Each tool differs in applicability inresearch settings, clinical settings and primary care settings. Since different studiesutilized different tools for assessment and there is no unified cut-off value, theinterpretation and comparison of results across different studies is particularlydifficult.


Chan MY et al. Sarcopenia in pancreatic cancer

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Table 1 Diagnostic criteria for sarcopenia by various working groups

Criteria Remarks

European Working Group on Sarcopenia inOlder People, 2010[16]

1 Low muscle strength Probable sarcopenia is identified by Criterion 1

2 Low muscle quantity or quality Diagnosis is confirmed by additionaldocumentation of Criterion 2

3 Low physical performance Sarcopenia is considered severe if all 3 criteria aremet

ESPEN Special Interest Group, 2010[18] 1 Low muscle mass Cut-off point should be more than 2 standarddeviations below mean value of referencepopulation using young adults of the same sexand ethnic background

2 Walking speed < 0.8 m/s in the 4-min test orreduced performance in functional test

Functional test can be any test used forcomprehensive geriatric assessment Both criteriashould be present

International Working Group on Sarcopenia,2011[17]

1 Gait speed < 1 m/s 2 Lean mass less than the20th percentile of values for healthy young adults

Both criteria should be present

European Working Group on Sarcopenia inOlder People, 2018 [2]

1 Low muscle mass Cut-off point should be more than 2 standarddeviations below mean value of referencepopulation using healthy young adults of thesame ethnic background

2 Low muscle strengt Diagnosis is based on documentation of Criterion1 plus Criterion 2 or Criterion 33 Low physical performance

Society of Sarcopenia, Cachexia and WastingDisorders, 2011[19]

1 Walking speed ≤ 1 m/s or < 400 m during 6-minwalk

Both criteria should be present

2 Lean appendicular mass corrected for heightsquared of more than 2 standard deviations belowhealthy adults of 20–30 years old of the sameethnic group

The traditional way to determine appendicular lean muscle mass is dual-energy X-ray absorptiometry[21-23]. However, it is less sensitive in evaluating intramuscular fat,which can make up 5%-15% of muscle mass in obese people[24]. Other methods such asbioimpedance analysis and urinary metabolites have also been mentioned in theliterature[25] but are subject to error. As most patients diagnosed with pancreaticcancer would have had cross-sectional imaging such as computed tomography ormagnetic resonance imaging, most of the studies used these scanning methods todiagnose sarcopenia. Both computed tomography and magnetic resonance imaginghave been shown to be more sensitive to small changes in muscle area than dual-energy X-ray absorptiometry[23,26] and are now considered to be the gold standard forevaluating muscle mass[27].

There are a number of measurements that can be taken from cross-sectionalimaging. The areas of fat, fat-free and lean muscle can be calculated with the specificHounsfield unit[27] and then converted into whole-body fat mass, fat-free mass andlean muscle mass[28]. The most commonly used landmark is the cross-sectional area ofmuscle at the L3 vertebra, and there are studies showing that the measurement at thislevel significantly correlates with whole-body muscle mass[28,29]. There are also othermeasurements such as the cross-sectional area of the psoas muscle[30] and the volumeof the psoas muscle[31], but some researchers opined that these measurements mightnot be representative enough to be a surrogate marker, as the psoas muscle is a minormuscle[32]. It is important to examine which measurement was used in a study, as wellas whether the results were adjusted for height, weight or body mass index. Assuggested by EWGSOP[2] and the ESPEN Special Interest Group[18], the cut-off pointfor the measurements should be more than two standard deviations below the meanreference value of healthy young adults of the same sex and same ethnicity.

As mentioned above, sarcopenia is not only defined by a decrease in muscle mass.As in osteoporosis, where an increase in bone mass does not necessarily translate intoa lower fracture risk, an increase in muscle mass does not translate into better physicalperformance. Physical performance is a combination of many aspects, and musclequantity is only a small part of it. Other aspects, including muscle quality, strength,power, motor control and coordination all play a part. Therefore, a decline in musclestrength or power should be documented. There are simple methods to assess musclestrength and power, such as handgrip strength with dynamometry and sit-to-standtime[2,33]. According to EWGSOP in 2018[2], physical performance should also beassessed by a test such as gait speed, 400-meter walk test, or the short physical



529

performance battery[34]. Although there may be certain limitations in these tests, like inpatients with mobility problems due to orthopedic or neurological problems, attemptsshould be made to include these parameters when discussing sarcopenia. However, inthe current available studies on pancreatic cancer patients, these parameters wererarely included (Table 1). Therefore, the true prevalence of sarcopenia in the studypopulations may still be unknown.

IMPACT OF SARCOPENIASurgical resection remains as the only potentially curative treatment for pancreaticcancer. The evolvement of operative techniques and perioperative care has loweredthe perioperative mortality rate to 3%-5% at high-volume centers and the morbidityrate to about 40%[35]. Despite the advances in surgery and the combination ofchemotherapy and radiotherapy, the median survival after resection andchemotherapy is only around 30 mo, with a 5-year survival rate of around 30%[36,37].Therefore, there has been ongoing research trying to identify the risk factors for suchpoor outcomes, and sarcopenia is a factor being investigated.

SurgeryPerioperative outcomes: A study by Peng et al[14] in 2012 is one of the earliest studiesreporting the relationship between sarcopenia and surgical outcomes of pancreaticcancer. The study included 557 patients who underwent pancreatic surgery forpancreatic cancer, and 139 of them (25.0%) were found to be sarcopenic aftermeasurement of their total psoas area. Sarcopenic and non-sarcopenic patients had nostatistically significant difference in hospital stay, intensive care unit stay, or overallmorbidity rate. Sarcopenia was not associated with increased hazard of 90-d mortality[hazard ratio [HR] 2.31, 95% confidence interval (CI): 0.78–6.77; P = 0.13].

Such discrepancy in results was likely partially due to the different assessmentparameters used. It is important to bear this in mind when interpreting results fromdifferent studies. For example, Pecorelli et al[38] reported that sarcopenia, as defined byPrado et al[39] using total abdominal muscle area (TAMA), was not a significantprognostic factor for 60-d postoperative mortality (P = 0.224). However, the ratio ofvisceral fat area (VFA) to TAMA was found to be a significant predictor for 60-dmortality when the ratio was > 3.2 in multivariable analysis [odds ratio (OR) 6.76,95%CI: 2.41-18.99; P < 0.001]. Similarly in another study by Amini et al[31], total psoasvolume was used instead of total psoas area in patients who underwent curativesurgery. With a different assessment tool, they were able to show that sarcopenia wasassociated with adverse short-term outcomes. While sarcopenia based on total psoasarea was not associated with morbidity after operation (OR 1.06, 95%CI: 0.77-1.47; P =0.72), sarcopenia based on total psoas volume was found to be associated with asignificantly higher complication risk (OR 1.79, 95%CI: 1.25-2.56; P = 0.002) andsignificantly longer intensive care unit stay (P = 0.002).

Meta-analysis by Ratnayake et al[40] reported that there was no statistical differencein the incidence of delayed gastric emptying (sarcopenic 19% vs non-sarcopenic 17%,95%CI: 0.80-1.29; P = 0.895), postoperative bile leakage (sarcopenic 7% vs non-sarcopenic 7%, 95%CI: 0.61-1.71; P = 0.933), surgical site infection (sarcopenic 17% vsnon-sarcopenic 22%, 95%CI: 0.75-1.16; P = 0.518), or morbidity of Clavien-Dindograde 3 or above (sarcopenic 30% vs non-sarcopenic 24%, 95%CI: 0.86-1.14; P = 0.869).The only significant difference was in postoperative hospital stay, which was longerin the sarcopenic group (mean difference 0.73 d, 95%CI: 0.06-1.40; P = 0.033).However, some studies in this meta-analysis included patients receiving pancreaticsurgery for both benign and malignant conditions, and not all studies used the sameparameters to diagnose sarcopenia. Overall, the impact of sarcopenia on short-termsurgical outcomes did not seem significant, but further research in this area is neededto have a more definitive answer.

Postoperative pancreatic fistula: Postoperative pancreatic fistula (POPF) is one of themost concerning complications in patients undergoing pancreatic surgery. There werea number of studies that examined the relationship between sarcopenia andpancreatic fistula. Nishida et al[41] measured the skeletal muscle index [skeletal musclearea at L3/(body height)2] of 266 patients who underwent pancreatoduodenectomy. Atotal of 61.3% of patients had pancreatic malignancy. The authors reported asignificantly higher rate of major complications (Clavien-Dindo grade 3 and above)and, specifically, a higher rate of POPF (sarcopenic 22.0% vs non-sarcopenic 10.4%; P= 0.011) in sarcopenia patients. Sarcopenia was also a significant independent riskfactor for clinically relevant POPF (OR 2.869, 95%CI: 1.329-6.197; P = 0.007) inmultivariate analysis taking into account factors including body mass index, presence



530

of pancreatic tumor, portal vein or superior mesenteric vein resection, diameter of thepancreatic duct, and consistency of the pancreas.

In the study by Pecorelli et al [38 ] in 2016, 202 patients who underwentpancreatoduodenectomy were included. The VFA and TAMA at L3 on computedtomography were measured. A high VFA-to-TAMA ratio was associated with 60-dmortality by multivariate analysis (OR 6.76, 95%CI: 2.41-18.99; P < 0.001). Only a largeVFA, but not TAMA or VFA-to-TAMA ratio, was associated with POPF (OR 4.05,95%CI: 1.85-8.84; P < 0.001). Although a relationship between TAMA and POPF couldnot be identified, a VFA-to-TAMA ratio > 3.2 was shown to be predictive of a highermortality risk (OR 6.33, 95%CI: 1.37-29.21; P = 0.018) in the subgroup of patients withmajor complications.

In the meta-analysis by Ratnayake et al[40], which included 13 studies involving 3608patients, six studies reported on POPF. There was no difference in the incidence ofPOPF between the sarcopenic and non-sarcopenic groups [risk ratio (RR) 1.05, 95%CI:0.68-1.61; P = 0.843]. Two of these studies reported on patients with sarcopenicobesity. Yamane et al[42] analyzed the ratio of visceral adipose tissue area to skeletalmuscle index of 99 patients who underwent pancreaticoduodenectomy. Multivariateanalysis showed that a ratio ≥ 2.0 was one of the independent risk factors associatedwith clinically significant POPF (grade B or C). In another study by Sandini et al[43], theVFA-to-TAMA ratio was measured in 124 patients. It was reported that the rate ofPOPF was slightly higher in patients with sarcopenic obesity after pan-creaticoduodenectomy, but it did not reach statistical significance (46.7% vs 32.3%; P =0.103). This may imply that sarcopenia alone is not associated with POPF, but patientswith sarcopenic obesity may have a higher risk of POPF.

Long-term survival: In the study by Peng et al[14] in 2012 cited above, the 3-yearsurvival rates of men (non-sarcopenic 39.2% vs sarcopenic 20.3%; P < 0.05) andwomen (non-sarcopenic 40.8% vs sarcopenic 26.1%; P < 0.05) were both significantlylower in the sarcopenic group. Sarcopenia was found to be associated with 3-yearmortality in both univariate (HR 1.68, 95%CI: 1.34-2.11; P < 0.001) and multivariateanalyses (HR 1.63, 95%CI: 1.28-2.07; P < 0.001)[44].

Table 2 is a summary of long-term survival outcomes in sarcopenic patients fromeight studies. In the study by Amini et al[31], a low total psoas volume was found to beassociated with worse survival (HR 1.72, 95%CI: 1.36-2.19; P < 0.001). Similar resultswere obtained by Okumura et al[45], who used the total psoas index at umbilical levelrather than at L3. Overall survival and disease-free survival were both significantlylower in the sarcopenic group (median overall survival: sarcopenic 17.7 mo vs non-sarcopenic 33.2 mo; P < 0.001; actual median disease-free survival not available; P <0.001). There were also studies that did not find any significant difference betweensarcopenic and non-sarcopenic patients, such as the studies by Joglekar et al[46] andVan Dijk et al[47].

Most of the studies in Table 2 used measurements from total psoas area or totalpsoas index for comparison. However, the cut-off points for sarcopenia varied widely.Some studies, such as the one by Van Dijk et al[47], did not find any significant resultswith more commonly used parameters (TAMA) but had significant findings usingvalues derived from computed tomography (radiation attenuation of skeletal muscle).Whether this indicates a low sensitivity of the initial parameter requires furtherinvestigation.

Mintziras et al[48] conducted a meta-analysis including 11 studies of pancreaticcancer and sarcopenia and concluded that the hazard of death was 1.4 times higher insarcopenic patients (summary adjusted HR 1.35, 95%CI: 1.18-1.54), and the hazardwas even higher for patients with sarcopenic obesity (summary adjusted HR 2.01,95%CI: 1.55-2.61). Nevertheless, studies on both palliative and curative surgeries wereincluded in this meta-analysis. Some studies also included pathologies other thanpancreatic cancer.

The vicious cycle of sarcopenia and chemotherapyMost of the available studies on chemotherapy for pancreatic cancer reported a poorerresponse and worse survival in sarcopenic patients[49,50]. In the study by Dalal et al[9],patients with inoperable locally advanced pancreatic cancer received bevacizumab incombination with capecitabine and radiation. An increased loss in skeletal muscleindex of more than 3.8% was found to be associated with poorer survival (P = 0.02).The effect on survival was especially obvious in sarcopenic obesity. Pretreatmentsarcopenic obesity was significantly associated with overall survival (P = 0.04) in thestudy by Cooper et al[51]. Patients with sarcopenia or obesity alone also had a shortermedian survival, but the difference did not reach statistical significance. In theretrospective study by Kays et al[49], six out of 53 patients with advanced pancreaticcancer treated with FOLFIRINOX were found to have sarcopenic obesity. This group



531

Table 2 Summary of long-term survival outcomes in sarcopenic patients in eight studies

Ref. No. ofpatients Indication Operation Assessment

of sarcopenia

Cut-off pointsforsarcopenia

Outcomes P value

Peng et al[14],2012

557 Pancreaticcancer

PD and DP Total psoasindex

Lowest quartileof the studycohort

3-yr survival,male

Sarcopenic:20.3% Non-sarcopenic:39.2%

< 0.05

3-yr survival,female

Sarcopenic:26.1% Non-sarcopenic:40.8%

< 0.05

Amini et al[31],2015

763 Pancreaticadenocarci-noma

PD, DP and TP Total psoasvolume(adjusted forheight), totalpsoas index

Cut-off valuefrom Peng etal[14]

OS Sarcopenia asindependentrisk factor

< 0.001

UV: HR 1.72,95%CI:1.36–2.19 MV:HR 1.11,95%CI:1.11–1.91

0.006

Joglekar etal[46], 2015


PD and DP Total psoasindex

Lowest quartileof the studycohort

OS No significantdifference

0.44

Okumura etal[45], 2015


PD, DP and TP Total psoasindex(measured atumbilical level)

Calculatedfrom receiver-operatingcharacteristiccurves

Median OS Sarcopenic: 17.7mo Non-sarcopenic: 33.2mo

< 0.001

DFS Significantlyshorter survivalin sarcopenicgroup

< 0.001

Onesti et al[66],2016

270 Both benignand malignantconditions

PD, DP, centraland TP

Total psoasarea

Lowest tertileof the studycohort

OS Significantlyworse survivalfor sarcopenicgroup infemales only

0.005

Ninomiya etal[67], 2017


PD, DP and TP Totalabdominalmuscle area(adjusted forheight)

Cut-off valuefrom Prado etal[39]

Median OS Sarcopenic: 23.7mo Non-sarcopenic: 25.8mo

0.185

Van Dijk etal[47], 2017

199 Cancer ofpancreatichead, ampulla,distal bile ductor duodenum

PD Totalabdominalmuscle area(adjusted forheight),radiationattenuation ofskeletal muscleat L3

Lowest tertileof the studycohort

Median OS No differencewhen totalabdominalmuscle areawas compared

Not reported

Significantlyshorter survivalin patients withlow radiationattenuation

0.008

Sugimoto etal[68], 2018


PD, DP and TP Totalabdominalmuscle area(adjusted forheight)

Cut-off valuefrom Fearon etal[29]


0.412

DFS No significantdifference

0.390

Lowest quartilefrom studycohort


0.075

DFS No significantdifference

0.172

PD: Pancreaticoduodenectomy; DP: Distal pancreatectomy; TP: Total pancreatectomy; OS: Overall survival; DFS: Disease-free survival; UV: Univariateanalysis; HR: Hazard ratio; CI: Confidence interval; MV: Multivariate analysis.

of patients had a significantly shorter median overall survival when compared withthe rest of the cohort (10.4 mo vs 16.1 mo; P = 0.04).

It has been well reported that chemotherapy for other cancers affects the bodycomposition throughout the treatment course[52-54]. It was estimated that patients



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undergoing chemotherapy for pancreatic cancer experienced a relative muscle loss of2.9% every 100 d (95%CI: -5.2--0.8; P = 0.01)[55]. This rate of muscle loss is much greaterthan that in a healthy adult, who generally loses muscle at a rate of 1%–1.4% peryear[56,57]. The muscle-loss effect is especially prominent in the case of neoadjuvantchemotherapy. It was reported that the relative mean difference in loss of musclemass was 4.5% more in patients receiving neoadjuvant chemotherapy than in thosehaving palliative chemotherapy[55]. From this, one may postulate that the effect ofmuscle loss is not from disease progress alone, but from the chemotherapy as well.

Not only does chemotherapy potentiate sarcopenia, sarcopenia also increases thetoxicity of chemotherapy[58,59]. This is likely due to the fact that the dosage ofchemotherapy is largely dependent on the patient’s height and weight (i.e. bodysurface area), with the change in body composition factored out[60-62]. Patients withsarcopenia tend to receive a higher dose of chemotherapeutic agent for a relativelysmall lean muscle mass and are thus more likely to suffer toxicity. Such a relationshipis not limited to a specific tumor type or chemotherapy. In a phase 1 trial by Cousin etal[63], a low skeletal muscle index was the only factor associated with dose-limitingtoxicity, regardless of cancer type. With a higher incidence of toxicity, there is also ahigher incidence of treatment termination and hospitalization. This implies that thecurrent method of dosage calculation still has room for improvement. The optimalway of adjustment for sarcopenia when prescribing chemotherapeutic agents is stillan area for further research.

DISCUSSIONAssessment of nutritional status of cancer patients has evolved from a simple“eyeballing test” at bedside to sophisticated tests, such as bioelectrical impedanceanalysis and lean muscle mass calculation from various imaging studies. In order toidentify patients with sarcopenia and provide timely intervention, a more proactiveapproach should be employed. Proper assessment of sarcopenia should beincorporated into the management of pancreatic cancer. Ideally, all patients receivingimaging studies can be screened for sarcopenia, but this requires special software andtrained personnel. Even without those sophisticated measures, measurements fromsimple tests, such as hand grip strength, gait speed and bioelectrical impedance, canbe obtained relatively easily in clinical settings.

In spite of all the knowledge of sarcopenia and its relationship with oncology, thereis still no optimal treatment to reverse sarcopenia. On the one hand, cancer patientsneed adequate amounts of protein intake for anabolism, but on the other hand,excessive energy intake may potentiate obesity[64]. Sarcopenic obesity has been shownto have a more deleterious effect on outcomes. The endocrine activity of visceraladipose tissue may work synergistically with cancer hormone-like mechanisms andprotein wasting[65]. Therefore, a careful balance of nutrition intake is crucial in themanagement of sarcopenia and sarcopenic obesity.

In additional to nutritional modification, exercise intervention is also beneficial inreversing sarcopenia. Resistance training intervention and compound exerciseintervention (a blend of aerobic, resistance, flexibility and balance training) have beenshown to improve muscle mass and/or physical performance[11]. However, thesetraining programs were mainly conducted in community-dwelling elderly people.They would be challenging for cancer patients due to various reasons, includingfatigue and cancer-related pain.

With a better understanding of sarcopenia, clinical strategies should be revo-lutionized to identify and combat the condition once a patient is diagnosed withpancreatic cancer. Screening for sarcopenia in this group of patients should be made aroutine practice. They should be referred to respective allied health professionals forearly optimization, with reassessment at regular intervals if surgery is pending. Adedicated multidisciplinary team consisting of surgeons, oncologists, nurses,dietitians and physiotherapists will be needed.

To conclude, sarcopenia is prevalent in pancreatic cancer patients and is associatedwith worse survival outcomes after surgical resection and chemotherapy. Inparticular, sarcopenic obesity has higher morbidity and mortality risks, including therisk of POPF. The relationship between sarcopenia and other short-term surgicaloutcomes still remain unclear, as different studies used different cut-off values anddiagnostic methods. With the latest guidelines and consensus, it is hoped that morestandardized reporting can be used in upcoming studies so that good quality level 1studies can be conducted.



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ORIGINAL ARTICLE

Retrospective Cohort Study

Intraoperative intraperitoneal chemotherapy increases the incidenceof anastomotic leakage after anterior resection of rectal tumors

Zhi-Jie Wang, Jin-Hua Tao, Jia-Nan Chen, Shi-Wen Mei, Hai-Yu Shen, Fu-Qiang Zhao, Qian Liu

ORCID number: Zhi-Jie Wang(0000-0003-2930-4668); Jin-Hua Tao(0000-0003-4703-9271); Jia-Nan Chen(0000-0002-6673-6884); Shi-Wen Mei(0000-0002-9735-3261); Hai-Yu Shen(0000-0002-2961-5098); Fu-QiangZhao (0000-0003-0676-8371); QianLiu (0000-0003-2510-3113).

Author contributions: Wang ZJ andTao JH designed the research;Chen JN, Mei SW, Shen HY, andZhao FQ collected and analyzedthe data; Wang ZJ drafted thearticle; Liu Q revised the paper.

Supported by Medicine and HealthTechnology Innovation Project ofChinese Academy of MedicalSciences, No. 2017-12M-1-006.

Institutional review boardstatement: Our investigationreceived approval from the ethicscommittee of the National CancerCenter/Cancer Hospital, ChineseAcademy of Medical Sciences andPeking Union College.

Informed consent statement: Allpatients have signed an informedconsent form before the study.

Conflict-of-interest statement: Theauthors declare that there is noconflict of interest in regard to thisresearch.

STROBE statement: The authorshave carefully read the STROBEStatement checklist of items andprepared the manuscript based onthe requirements of STROBEStatement checklist of items.

Open-Access: This article is anopen-access article which was

Zhi-Jie Wang, Jin-Hua Tao, Jia-Nan Chen, Shi-Wen Mei, Hai-Yu Shen, Fu-Qiang Zhao, Qian Liu,Department of Colorectal Surgery, National Cancer Center/Cancer Hospital, Chinese Academyof Medical Sciences and Peking Union College, Beijing 100021, China

Corresponding author: Qian Liu, MD, Professor, Department of Colorectal Surgery, NationalCancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking UnionCollege, No. 17, Panjiayuan Nanli, Chaoyang District, Beijing 100021, [email protected]: +86-10-87787110Fax: +86-10-87787110

AbstractBACKGROUNDIntraoperative intraperitoneal chemotherapy is an emerging treatment modalityfor locally advanced rectal neoplasms. However, its impacts on postoperativecomplications remain unknown. Anastomotic leakage (AL) is one of the mostcommon and serious complications associated with the anterior resection of rectaltumors. Therefore, we designed this study to determine the effects ofintraoperative intraperitoneal chemotherapy on AL.

AIMTo investigate whether intraoperative intraperitoneal chemotherapy increases theincidence of AL after the anterior resection of rectal neoplasms.

METHODSThis retrospective cohort study collected information from 477 consecutivepatients who underwent an anterior resection of rectal carcinoma using thedouble stapling technique at our institution from September 2016 to September2017. Based on the administration of intraoperative intraperitoneal chemotherapyor not, the patients were divided into a chemotherapy group (171 cases withintraperitoneal implantation of chemotherapy agents during the operation) or acontrol group (306 cases without intraoperative intraperitoneal chemotherapy).Clinicopathologic features, intraoperative treatment, and postoperativecomplications were recorded and analyzed to determine the effects ofintraoperative intraperitoneal chemotherapy on the incidence of AL. The clinicaloutcomes of the two groups were also compared through survival analysis.

RESULTSThe univariate analysis showed a significantly higher incidence of AL in the




http://orcid.org/0000-0003-2930-4668

http://orcid.org/0000-0003-4703-9271

http://orcid.org/0000-0002-6673-6884

http://orcid.org/0000-0002-9735-3261

http://orcid.org/0000-0002-2961-5098

http://orcid.org/0000-0003-0676-8371

http://orcid.org/0000-0003-2510-3113


selected by an in-house editor andfully peer-reviewed by externalreviewers. It is distributed inaccordance with the CreativeCommons Attribution NonCommercial (CC BY-NC 4.0)license, which permits others todistribute, remix, adapt, buildupon this work non-commercially,and license their derivative workson different terms, provided theoriginal work is properly cited andthe use is non-commercial. See:http://creativecommons.org/licenses/by-nc/4.0/

Manuscript source: Unsolicitedmanuscript

Received: January 27, 2019Peer-review started: January 28,2019First decision: April 15, 2019Revised: May 1, 2019Accepted: May 28, 2019Article in press: May 29, 2019Published online: July 15, 2019

P-Reviewer: Abdel-Rahman WM,Kuo SH, Senchukova MS-Editor: Ji FFL-Editor: Wang TQE-Editor: Xing YX

patients who received intraoperative intraperitoneal chemotherapy, with 13(7.6%) cases in the chemotherapy group and 5 (1.6%) cases in the control group (P= 0.001). As for the severity of AL, the AL patients who underwent intraoperativeintraperitoneal chemotherapy tended to be more severe cases, and 12 (92.3%) outof 13 AL patients in the chemotherapy group and 2 (40.0%) out of 5 AL patientsin the control group required a secondary operation (P = 0.044). A multivariateanalysis was subsequently performed to adjust for the confounding factors andalso showed that intraoperative intraperitoneal chemotherapy increased theincidence of AL (odds ratio = 5.386; 95%CI: 1.808-16.042; P = 0.002). However, thesurvival analysis demonstrated that intraoperative intraperitoneal chemotherapycould also improve the disease-free survival rates for patients with locallyadvanced rectal cancer.

CONCLUSIONIntraoperative intraperitoneal chemotherapy can improve the prognosis ofpatients with locally advanced rectal carcinoma, but it also increases the risk ofAL following the anterior resection of rectal neoplasms.

Key words: Anastomotic leakage; Rectal neoplasms; Lobaplatin; Fluorouracil implants;Postoperative complications; Intraoperative intraperitoneal chemotherapy


Core tip: Intraoperative intraperitoneal chemotherapy is gradually administered duringoperations for locally advanced rectal cancer patients. It is believed that this treatmentcan improve the oncological outcomes and survival rates. However, surgicalcomplications related to intraoperative chemotherapy have also been reported. Weconducted this study to determine the relationship between intraperitoneal chemotherapyand anastomotic leakage to help surgeons weigh the benefits and risks of intraoperativechemotherapy.

Citation: Wang ZJ, Tao JH, Chen JN, Mei SW, Shen HY, Zhao FQ, Liu Q. Intraoperativeintraperitoneal chemotherapy increases the incidence of anastomotic leakage after anteriorresection of rectal tumors. World J Gastrointest Oncol 2019; 11(7): 538-550URL: https://www.wjgnet.com/1948-5204/full/v11/i7/538.htmDOI: https://dx.doi.org/10.4251/wjgo.v11.i7.538

INTRODUCTIONTumor recurrence can lead to an unfavorable prognosis for patients with locallyadvanced rectal carcinoma. The peritoneum is a common recurrence site for patientswho undergo a radical resection of rectal carcinoma. In a retrospective study, it wasreported that of the 1354 patients with rectal cancer who were included in the report,5.4% went on to develop peritoneal recurrence after radical surgery[1]. Furthermore, Tstage and N stage are independent risk factors that affect the incidence of peritonealcarcinomatosis[2]. Therefore, it is recommended that patients with locally advancedrectal cancer of stage T3/T4 or N1/N2 receive preoperative chemoradiotherapy todecrease the risk of recurrence. Previous randomized trials have shown that patientswho undergo preoperative chemoradiotherapy have significantly reduced localrecurrence rates and improved rates of disease-free survival (DFS)[3]. However, for asubset of patients who have already suffered from peritoneal metastases,cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) havebeen widely used to improve their oncological prognosis[4]. Few reports have focusedon the use of intraoperative intraperitoneal chemotherapy without hyperthermia toprevent peritoneal carcinomatosis in rectal cancer patients. Intraoperativeintraperitoneal chemotherapy is an emerging modality that can improve theprognosis of rectal cancer patients with a high risk of cancer recurrence, andrepresents a distinct approach from both preoperative chemoradiotherapy and HIPECthat can be easily administrated in the clinic[5,6].

Tumor recurrence results from residual tumor cells that are present after theremoval of a primary tumor. Previous studies have shown that residual tumor cells


Wang ZJ et al. Intraoperative chemotherapy in rectum surgery

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can exhibit a transient period of increased growth rate and become more vulnerable tochemotherapeutic agents during the first 3 d following the resection of a primarytumor, which provides the rationale for the administration of intraoperativeintraperitoneal chemotherapy to decrease the incidence of peritoneal recurrence[7,8].Over the past few years, intraoperative intraperitoneal chemotherapy has beengradually incorporated into the treatment for rectal carcinoma patients in Easterncountries[5,6]. Locally advanced rectal carcinoma has a higher risk of peritonealrecurrence, and as a result, clinical stages T3/T4 and N1/N2 of the disease are oftenregarded as indications for the use of intraoperative intraperitoneal chemotherapy. Inthis procedure, chemotherapy agents are placed into the pelvic cavity after neoplasmresection and digestive reconstruction to inhibit the proliferation and dissemination ofthe remaining tumor cells. However, there are no uniform guidelines for the type anddose of chemotherapeutic agent and most decisions are determined by the surgeon’srecommendations and the patient’s economic conditions. Common drug optionsinclude fluorouracil implants, lobaplatin, and raltitrexed. Reports have shown thatintraoperative intraperitoneal chemotherapy can reduce locoregional recurrence ratesand increase long-term survival rates[5]. Nevertheless, the effects of intraoperativeintraperitoneal chemotherapy on postoperative complications have rarely beenexplored, which raises concerns about the safety and feasibility of this new treatmentmodality. Given that anastomotic leakage (AL) is the most common and seriousoperation-associated complication after rectal surgery, we aimed to evaluate the roleof intraoperative intraperitoneal chemotherapy in the occurrence of AL.

AL is a common and severe postoperative complication that can develop after ananterior resection of rectal neoplasms and has a high incidence that ranges from 6.1%to 11.9%[9-11]. AL prolongs hospitalization times and increases short-term morbidityand mortality. Moreover, several studies have shown that AL contributes to the risk oflocal recurrence and decreased overall survival[12-14]. Previous reports have indicatedthat male sex, history of smoking and ischemic heart disease, tumor location and size,malnutrition, and intersections of staple lines are possible factors that can lead to ALin rectal tumor patients[10,15-18]. Prophylactic ileostomy, transanal tube placement, andintracorporeal reinforcing sutures may decrease the incidence of AL[19,20]. Additionally,some assay indexes and prediction models have been established to evaluate thepossibility of AL[21-23]. Exploring the association between intraoperative intraperitonealchemotherapy and AL can improve our understanding of the indications andcontradictions of this new treatment modality.

MATERIALS AND METHODS

PatientsOur investigation received approval from the ethics committee of our center and wasperformed in accordance with the Helsinki Declaration of World Medical Association.Every patient signed an informed consent form before participation in the study. Weextracted data from 477 consecutive patients who underwent anterior resection ofrectal cancer at the National Cancer Center/Cancer Hospital, Chinese Academy ofMedical Sciences and Peking Union Medical College from September 2016 toSeptember 2017. Information regarding the intraoperative use of chemotherapyagents was carefully collected from medical records. Follow-up data were acquired byoutpatient reexamination and telephones.

The inclusion criteria were defined as follows: (1) All patients were definitivelydiagnosed with rectal cancer through abdominal and pelvic enhanced computedtomography (CT) scans, rectal magnetic resonance imaging (MRI), colonoscopy, tissuebiopsy, and pathological examination; (2) All patients were confirmed to have TNMstage II-III rectal cancer through rectal MRI at the time of diagnosis; (3) The distalborder of the tumor from the anal verge was less than 15 cm; and (4) All patientsunderwent an anterior resection surgery using the double stapling technique.

The exclusion criteria were defined as follows: (1) Patients who were considered tohave TNM stage I or IV rectal cancer at the time of diagnosis; (2) Patients whoreceived hand suture anastomosis, Hartmann’s surgery, intersphincteric resection, orabdominal perineal resection; and (3) Patients whose information was not clearly andaccurately presented in the medical records.

AL was diagnosed through clinical symptoms and signs of fever, abdominal pain,peritonitis, and discharge of intestinal contents from pelvic drainage. Pelvic CT scansand rectoscopy can be used to provide additional confirmation of AL. Furthermore,hydrops and pneumatosis in the pelvic cavity in CT images or anastomotic defects inendoscopy images can also imply the existence of AL (Figure 1). We classified all ALpatients as grade A, B, or C according to the proposal from the International Study



540

Group of Rectal Cancer in 2010. Grade A AL patients require no active medicalintervention whereas grade B AL patients require only a conservative treatment. Incontrast, grade C AL patients require a secondary operation[24]. Given the limitednumber of grade A AL individuals due to its low rate of clinical manifestation and thelack of a need for active medical intervention, only grade B and C patients wereanalyzed in our study.

Surgical procedureAll patients underwent bowel preparation by taking oral sulfate-free polyethyleneglycol electrolyte powder the day before surgery. A standardized anterior resection ofrectal neoplasms was then performed for each patient by surgeons specialized incolorectal tumors. Both laparotomy and laparoscopic surgeries were performed at ourinstitution. A double stapling technique was used to form an end-to-end anastomoticstoma when the surgeons reconstructed the intestinal tract. Peritoneal lavage wasroutinely performed after the intestinal anastomosis. One to two pelvic drainage tubeswere then placed around the anastomotic stoma. Based on the patient’s condition, atransanal tube was selectively placed. The resected specimens were delivered toprofessional pathologists to determine the tumor stage.

Lobaplatin or fluorouracil implants were utilized for the patients who receivedintraoperative intraperitoneal chemotherapy. The dosage of lobaplatin was 60 mg,and it was dissolved in 500 mL of glucose solution at a concentration of 0.05 g/mL.The solution was then poured into the pelvic cavity through the drainage tube afterthe abdominal incision was closed. The tubes were occluded for 4 to 6 h to preventdrainage of the agents. Fluorouracil implants were placed directly into the pelviccavity before the incision was closed and remained permanently inside the body. Thecommon dosage ranged from 500 to 1000 mg.

Analyzed factorsTo evaluate the comparability between the chemotherapy group and the controlgroup, and to decrease any confounding bias, a total of 32 variables were included inour investigation. All factors can be roughly divided into demographic characteristics,comorbidities, preoperative oncological therapies, operative treatments, and tumorstaging. All of these data were described in detail in the medical records. We carefullyexamined the accuracy of our data to reduce bias from data collection.

Statistical analysisOur study was statistically reviewed by a biomedical statistician from our institution.All data were analyzed using the Statistical Package for the Social Sciences (SPSSversion 24.0; IBM Corp., Armonk, NY). Since we excluded patients whose informationwas not clearly and accurately presented in their medical records, there were nomissing data in this study. Quantitative data that were normally distributed arepresented as the mean ± SD and were further analyzed using a t-test. Quantitativedata that were not normally distributed are presented as the median (range) and werecompared using Mann-Whitney U tests. Qualitative data are expressed as the numberof cases and percentage and were further compared using Pearson’s χ2 test or Fisher’sexact test. Ordinal data are also presented as cases and percentage but were furtherexamined using Mann-Whitney U tests. To control confounding biases, factors thatwere regarded to be clinically associated with AL and imbalanced factors between thetwo groups with a P-value < 0.05 were included in the multivariate logistic regressionanalysis and stratification analysis to determine the independent risk factors for AL.Overall survival rates and DFS rates were calculated by the Kaplan-Meier method andfurther compared by a log-rank test. All tests were two-sided, and a P-value < 0.05was regarded as statistically significant.

RESULTS

Patient characteristicsOur investigation included 477 patients with an average age of 58.7 ± 10.9 years. Ofthese patients, 301 (63.1%) were male and 176 (36.9%) were female. A total of 171patients received intraoperative intraperitoneal chemotherapy, including 8 treatedwith lobaplatin alone, 157 treated with fluorouracil implants alone, and 6 treated withboth. The remaining 306 patients did not receive intraoperative intraperitonealchemotherapy. Patient-related factors are presented in Table 1. Patient demographics,habits, comorbidities, preoperative therapy, nutritional status, and American Societyof Anesthesiologists grade were comparable between the chemotherapy group andthe control group. Surgery-related factors are presented in Table 2. Most patientsreceived laparoscopic surgery in our study, including 167 (97.7%) in the chemo-



541

Figure 1

Figure 1 Images of anastomotic leakage. A: Pelvic computed tomography image showing hydrops andpneumatosis around the anastomotic stoma (the circle with high density in the figure); B: Endoscopic image showingthe defect of anastomotic stoma.

therapy group and 300 (98.0%) in the control group (P = 0.751). Natural orificespecimen extraction (NOSE) surgery is a surgical method that emerged in the lastdecade in which resected specimens are obtained from the vagina or anus instead ofan additional abdominal incision. More patients in the control group underwent theNOSE procedure (1.8% in the chemotherapy group vs 5.9% in the control group, P =0.035). The placement of a transanal tube was more common in patients who did notreceive intraoperative intraperitoneal chemotherapy (43.9% in the chemotherapygroup vs 55.6% in the control group, P = 0.014). Additionally, more patients from thecontrol group received more than 2 stapler firings during the digestive reconstruction(14.6% in the chemotherapy group vs 25.2% in the control group, P = 0.007). Noobvious differences were observed between the two groups for operation time,reinforcing suture, defunctioning stoma, blood loss, perioperative transfusion, orpreservation of the left colic artery. Tumor-related variables are detailed in Table 3.All patients enrolled in our study presented with TNM stage II or III disease at thedate of diagnosis prior to treatment. However, of the 97 patients who receivedneoadjuvant therapy, a total of 11 achieved complete pathological remission and 19regressed to TNM stage I through the postoperative pathological examination. Tumorlocation, pathological stage, and degree of differentiation were comparable betweenthe two groups.

ALThe details for the occurrence of AL are presented in Table 4. In total, 18 (3.8%)individuals developed AL in our study. A significantly higher incidence of AL wasobserved in the group that received intraoperative intraperitoneal chemotherapy thanin the control group (7.6% vs 1.6%, P = 0.001). Among the AL patients, none withgrade A were enrolled in our study. A total of 4 of the 18 AL patients were classifiedas grade B and received a conservative treatment, while 14 of the 18 patients wereclassified as grade C and received a secondary surgery according to the standardsfrom the International Study Group of Rectal Cancer released in 2010. The AL patientswho underwent intraoperative intraperitoneal chemotherapy tended to be moresevere cases and were more likely to receive a secondary operation (P = 0.044).Additionally, the majority of the cases developed AL within a week after surgery and1 case occurred two months after the procedure. No deaths were observed during theperioperative period.

Multivariate analysisDescriptive analysis identified significant imbalances between the chemotherapygroup and the control group for NOSE surgery (P = 0.035), placement of transanaltubes (P = 0.014), and the number of stapler firings (P = 0.007). These variables andother factors that have a confirmed association with AL from previous reports wereincluded in the subsequent multivariate analyses (Table 5). After adjusting forconfounding factors, intraoperative intraperitoneal chemotherapy was confirmed tosignificantly increase the incidence of AL [odds ratio (OR) = 5.386; 95% confidenceinterval (CI): 1.808-16.042; P = 0.002].

Stratification analysisStratification analysis was also performed to control for confounding biases. Theinfluence of intraoperative intraperitoneal chemotherapy on AL was further analyzed



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Table 1 Patient-related variable

Variable Intraoperative chemotherapy (+) (n = 171) Intraoperative chemotherapy(-) (n = 306) P-value

Age (yr, mean ± SD) 58.1 ± 10.1 59.1 ± 11.3 0.627

Sex, n (%) 0.829

Male 109 (63.7) 192 (62.7)

Female 62 (36.3) 114 (37.3)

BMI (kg/m2, mean ± SD) 24.4 ± 3.1 23.9 ± 3.4 0.123

Smoking, n (%) 59 (34.5) 87 (28.4) 0.168

Alcohol, n (%) 46 (26.9) 67 (21.9) 0.218

Hypertension, n (%) 46 (26.9) 75 (24.5) 0.565

Ischemic heart disease, n (%) 6 (3.5) 7 (2.3) 0.622

Diabetes, n (%) 22 (12.9) 39 (12.7) 0.970

Hepatitis, n (%) 14 (8.2) 16 (5.2) 0.202

History of malignancy, n (%) 9 (5.3) 12 (3.9) 0.493

Incomplete intestinal obstruction, n (%) 20 (11.7) 25 (8.2) 0.206

Preoperative chemotherapy, n (%) 32 (18.7) 65 (21.2) 0.511

Preoperative radiotherapy, n (%) 21 (12.3) 44 (14.4) 0.522

Preoperative hemoglobin, (g/L, mean ± SD) 136.8 ± 18.1 136.2 ± 18.0 0.813

Preoperative albumin (g/L, mean ± SD) 43.6 ± 3.5 44.2 ± 3.7 0.075

ASA grade, n (%) 0.987

1 6 (3.5) 14 (4.6)

2 154 (90.1) 269 (87.9)

3 11 (6.4) 23 (7.5)

SD: Standard deviation; BMI: Body mass index; ASA: American Society of Anesthesiologists.

in subgroups defined by sex, diabetes, incomplete intestinal obstruction, tumorlocation, NOSE surgery, consolidation suture, defunctioning stoma, transanal tube,and the number of stapler firings. Intraoperative intraperitoneal chemotherapysignificantly promotes the occurrence of AL in individuals who fell into thesubgroups of male, nondiabetic, without incomplete intestinal obstruction, withtumors located above the peritoneal reflection, without NOSE surgery, withoutconsolidation sutures, without defunctioning stoma, without transanal tubes, andboth who underwent 1 or 2 stapler firings and who underwent more than 2 staplerfirings. Although the OR values were different in different subgroups, the ORhomogeneity test through the Woolf method demonstrated that the data between thetwo subgroups were homogenous (P > 0.05). Therefore, we calculated the overall ORvalues through the Mantel-Haenszel method. The overall P-values and OR valuesshowed that intraoperative intraperitoneal chemotherapy remained significantlyassociated with AL even though the discussed variables caused weak confoundingeffects (Table 6).

Survival outcomesThe patients were followed for a median period of 24 mo (range: 1-31 mo). The 1-yearsurvival rate and 2-year survival rate were 98.2% and 96.2% in the chemotherapygroup and 99.3% and 96.1% in the control group, respectively. There were nosignificant differences in the survival rates between the two groups (P = 0.952).However, an increased DFS rate was confirmed in patients who receivedintraoperative intraperitoneal chemotherapy (P = 0.020). Both the 1-year DFS rate(92.8% in the chemotherapy group vs 88.4% in the control group) and the 2-year DFSrate (89.7% in the chemotherapy group vs 81.3% in the control group) were higher inthe chemotherapy group (Figure 2).

DISCUSSIONLocally advanced (T3/T4 or N1/N2) rectal cancer patients are at an increased risk oflocal recurrence and distant metastasis. Neoadjuvant therapy has been accepted as astandard treatment for locally advanced rectal carcinoma and can significantlyimprove the prognosis of these patients[25]. However, we observed that only 13.6% of



543

Table 2 Surgery-related variables


Approach, n (%) 0.751

Open 4 (2.3) 6 (2.0)

Laparoscopic 167 (97.7) 300 (98.0)

NOSE surgery, n (%) 3 (1.8) 18 (5.9) 0.035a

Operation time, (min, mean ± SD) 164.0 ± 54.7 172.0 ± 62.9 0.187

Consolidation suture, n (%) 27 (15.8) 37 (12.1) 0.256

Defunctioning stoma, n (%) 46 (26.9) 64 (20.9) 0.137

Estimated blood loss (mL, mean ± SD) 59.7 ± 54.4 67.1 ± 100.1 0.764

Transfusion, n (%) 4 (2.3) 14 (4.6) 0.219

Left colic artery preservation, n (%) 11 (6.4) 20 (6.5) 0.965

Transanal tube, n (%) 75 (43.9) 170 (55.6) 0.014a

Number of stapler firing, n (%) 0.007a

1 and 2 146 (85.4) 229 (74.8)

More than 2 25 (14.6) 77 (25.2)

aP < 0.05 vs Control. SD: Standard deviation; NOSE: Natural orifice specimen extraction.

patients had received preoperative radiotherapy and 20.3% of patients had receivedpreoperative chemotherapy. This might be because many patients were reluctant toreceive neoadjuvant treatment due to their poor economic conditions and fear of sideeffects. However, based on the postoperative pathological stage, most of the patientsin our study who did not receive neoadjuvant therapy were recommended to receivepostoperative radiotherapy or chemotherapy. Intraoperative intraperitonealchemotherapy is a newly developed independent modality to treat locally advancedrectal tumor that is distinct from neoadjuvant therapy. Furthermore, patients whohave already received neoadjuvant treatment can still receive intraoperativeintraperitoneal chemotherapy.

Previous reports have demonstrated that recurrent rectal carcinoma derives fromresidual intraperitoneal tumor cells after radical resection of the primary tumor site.These residual tumor cells tend to be more sensitive to chemotherapeutic agents sincethey show a transiently increased growth rate after surgery. However, systematicchemotherapy is not viable due to the poor physical condition of postoperativepatients. Intraperitoneal chemotherapy can reach a higher concentration inside theabdominal cavity while maintaining a lower concentration in blood, which canimprove the efficacy of killing residual tumor while decreasing systematic side effects.Based on these mechanisms and the high recurrence rate in locally advance rectalcancer patients, intraoperative intraperitoneal chemotherapy has been increasinglyused for these patients over the last decade[5,26]. This treatment is administered at theend of the surgery by placing the chemotherapeutic agents into the pelvic cavity andaims to kill the exfoliated tumor cells. A prospective randomized clinical trialconfirmed that patients treated by intraoperative implantation of fluorouracilimplants experienced improved oncological and survival outcomes[5]. In our study,there were no overall improvements in the survival rate in the chemotherapy grouprelative to the control group. This might be due to our much shorter follow-up periodwhen compared to previous reports. The longest follow-up time was only 31 months,and only 6 of 171 patients in the chemotherapy group and 10 of the 306 patients in thecontrol group died from the tumor recurrence. However, there was a significantincrease in DFS rates for the chemotherapy group, which is consistent with thefindings of previous studies.

Although it has been confirmed that intraoperative intraperitoneal chemotherapyimproves clinical outcomes in locally advanced rectal cancer patients, its impacts onpostoperative complications remain controversial. Considering that AL is one of themost common complications associated with the anterior resection of rectalcarcinomas, we evaluated the safety of intraoperative intraperitoneal chemotherapythrough the incidence of AL. AL is an extremely severe complication for rectal cancerpatients, and most symptomatic AL patients require a secondary operation. Previousstudies have identified risk factors for AL after an anterior resection of rectalcarcinoma, but the connection between intraoperative intraperitoneal chemotherapyand AL has not been thoroughly examined.

We found that AL occurred more frequently in patients who underwent



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Table 3 Tumor related variables

Variable Intraoperative chemotherapy (+)(n = 171)

Intraoperative chemotherapy(-)(n = 306) P-value

Distance of tumor from anal verge(cm, mean ± SD)

7.9 ± 3.0 8.3 ± 2.9 0.092

Tumor location, n (%) 0.621

Above peritoneal reflection 121 (70.8) 223 (72.9)

Below peritoneal reflection 50 (29.2) 83 (27.1)

Pathological T stage, n (%) 0.865

T1, T2, and no tumor residual afterpreoperative therapy

25 (14.6) 43 (14.1)

T3 and T4 146 (85.4) 263 (85.9)

Pathological N stage, n (%) 0.337

N0 and no tumor residual afterpreoperative therapy

81 (47.4) 131 (42.8)

N1 and N2 90 (52.6) 175 (57.2)

TNM stage, n (%) 0.374

No tumor residual afterpreoperative therapy

3 (1.8) 8 (2.6)

I 8 (4.7) 11 (3.6)

II 70 (40.9) 112 (36.6)

III 90 (52.6) 175 (57.2)

Degree of differentiation, n (%) 0.628

Low and low-middle grades 58 (30.2) 94 (30.8)

Middle, high-middle, and highgrades and no tumor residual aftertherapy

134 (69.8) 211 (69.2)

SD: Standard deviation.

intraperitoneal chemotherapy, and this trend was verified both in the univariate andmultivariate analyses. Lobaplatin and fluorouracil implants were used in ourprocedures. Lobaplatin was dissolved in solution and poured into the pelvic cavity,which led to the anastomotic stoma being immersed in the lobaplatin solution. Incontrast, the fluorouracil implants are solid microcapsules that are placed primarilyaround the anastomotic stoma in the pelvic wall. Therefore, the chemotherapy drugscan have a direct impact on anastomotic stoma. Both agents are cytotoxic drugs thatcan inhibit cell proliferation and induce apoptosis, particularly for cells with rapidproliferation[27,28]. Given that the healing process of the rectal anastomotic stomarequires the rapid proliferation of regenerative cells, we hypothesize thatintraoperative intraperitoneal chemotherapy increases the incidence of AL byinhibiting cell proliferation.

Previous reports have demonstrated that systematic chemotherapy could delay andimpair the healing process of wound. Moreover, treatment for this problem is reallydifficult as the proliferation of cells in the wound is inhibited[29-31]. Similarly, the sideeffects of chemotherapy on intestinal anastomosis healing were also observed inexperimental studies. AL tended to develop more often in rats receiving intra-operative administration of antineoplastics[32,33]. The anastomotic strength is generallyevaluated through bursting pressure, which increases slowly in the first fourpostoperative days but quickly thereafter[34]. However, the bursting pressure wassignificantly lower in rats receiving intraoperative intraperitoneal chemotherapycompared to those not. Decreased fibroblast activity and collagen deposition werefound in further histological examinations, which contributed to the mechanicalstrength of the anastomoses[35]. In addition, a series of animal experiments have alsoindicated that intraoperative intraperitoneal chemotherapy could bring detrimentaleffects on the intestinal anastomosis by increasing the inflammatory reaction,promoting oxidative stress, and reducing neoangiogenesis at the anastomotic site.These effects were observed in almost all the antitumor agents commonly used inintrapertoneal chemotherapy, including mitomycin C, cisplatin, oxaliplatin, 5-fluorouracil, iritotecan, and doxorubicin[32,36,37]. Moreover, the combination use ofdifferent chemotherapeutic agents showed enhanced negative effects on the healingprocess of intestinal anastomoses compared to those receiving only one type of



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Table 4 Incidence of anastomotic leakage


AL patients 13 (7.6) 5 (1.6) 0.001a

Grade, n (%) 0.044a

A 0 0

B 1 (0.6) 3 (1.0)

C 12 (7.0) 2 (0.7)

Occurrence time of AL 0.278

Early AL 13 (7.6) 4 (1.3)

Delayed AL 0 1 (0.3)

aP < 0.05 vs Control. AL: Anastomotic leakage.

agent [ 3 8 ]. However, previous clinical studies on the relationship betweenintraoperative intraperitoneal chemotherapy and AL are very limited. In our study, asignificantly higher incidence of AL was observed in the chemotherapy group, whichis in line with previous experimental studies. In addition, intraperitoneal usage ofantitumor agents appeared to be associated with the severity of AL. AL patients in thechemotherapy group were at higher risks of undergoing a secondary operation.Finally, we recognize that the sample size of the cases treated with lobaplatin was farless than the number of cases treated with fluorouracil implants, which mightintroduce bias. Further investigations with larger and more sufficient sample sizes areneeded to determine the association between the respective types of chemo-therapeutic agents and AL.

Our study has several limitations. First, since this is a retrospective cohort studyand the patients were divided into chemotherapy and control groups, there is the riskof selection bias, information bias, and confounding bias, although we tried to collectas many variables as possible and incorporated them into the multivariate analysisand stratification analysis. Additional large multicenter cohort studies or randomizedcontrolled trials are still required to assess the safety of intraoperative intraperitonealchemotherapy. Second, the incidence of AL at our center is relatively low whencompared with most other reports. There were only 18 AL patients observed in thisstudy, which made it difficult to perform dose-response relationship analyses tocontrol for bias.

In conclusion, this study determined that intraoperative intraperitonealchemotherapy increased the incidence of postoperative AL after the anterior resectionof rectal carcinoma, but it also improved the DFS rates in patients with locallyadvanced rectal carcinoma. Surgeons should carefully weigh the short-term risks ofpostoperative AL with the long-term benefits of improved oncological outcomesbefore choosing to utilize intraoperative intraperitoneal chemotherapy.



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Table 5 Multivariate logistic regression analysis

Variable P-value OR 95%CI

Intraoperative intraperitoneal chemotherapy 0.002 5.386 1.808-16.042

Gender 0.077 3.235 0.882-11.859

Diabetes 0.985 1.015 0.213-4.843

Incomplete intestinal obstruction 0.752 1.261 0.301-5.287

Distance of tumor from anal verge 0.869 0.985 0.824-1.178

NOSE surgery 0.388 2.696 0.283-25.675

Consolidation suture 0.319 0.340 0.041-2.835

Defunctioning stoma 0.157 0.312 0.062-1.565

Transanal tube 0.518 0.708 0.248-2.019

Number of stapler firing 0.733 0.811 0.244-2.698

OR: Odds ratio; CI: Confidence interval; NOSE: Natural orifice specimen extraction.

Table 6 Stratification analysis

Variable P-value OR 95%CI Woolf homogeneity test

Gender P = 0.805

Male 0.002 5.276 1.637-17.000

Female 0.589 3.767 0.335-42.393

Overall 0.003 4.962 1.733-14.206

Diabetes P = 0.441

Yes 1.000 1.810 0.108-30.436

No 0.001 5.759 1.823-18.193

Overall 0.002 4.941 1.732-14.093

Incomplete intestinal obstruction P = 0.305

Yes 0.161 2.471 1.712-3.565

No 0.009 3.915 1.313-11.673

Overall 0.003 4.936 1.714-14.217

Tumor location P = 0.964

Above peritoneal reflection 0.009 4.932 1.513-16.081

Below peritoneal reflection 0.296 5.234 0.529-51.758

Overall 0.002 4.997 1.748-14.286

NOSE surgery P = 0.209

Yes 0.143 10.000 2.685-37.239

No 0.003 4.354 1.506-12.585

Overall 0.002 4.855 1.727-13.649

Consolidation suture P = 0.588

Yes 0.422 2.423 1.805-3.253

No 0.002 4.800 1.656-13.910

Overall 0.002 5.162 1.797-14.829

Defunctioning stoma P = 0.302

Yes 1.000 1.400 0.085-22.978

No <0.001 6.319 1.994-20.026

Overall 0.002 5.092 1.793-14.461

Transanal tube P = 0.468

Yes 0.259 3.136 0.684-14.375

No 0.013 6.931 1.463-32.844

Overall 0.004 4.894 1.682-14.241

Number of stapler firings P = 0.480

1 and 2 0.011 4.136 1.272-13.446

More than 2 0.045 10.364 1.026-104.656



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Overall 0.002 4.942 1.733-14.096

OR: Odds ratio; CI: Confidence interval; NOSE: Natural orifice specimen extraction.

Figure 2

Figure 2 Overall survival and disease-free survival rates in the chemotherapy group and control group.

ARTICLE HIGHLIGHTSResearch backgroundTumor recurrence is common for patients with locally advanced rectal carcinoma after radicalresection surgery. Over the past few years, intraoperative intraperitoneal chemotherapy has beengradually incorporated into the treatment for rectal carcinoma patients to decrease therecurrence rate and showed improved clinical outcomes. Nevertheless, the effects ofintraoperative intraperitoneal chemotherapy on postoperative complications have rarely beenexplored. We conducted this research to determine the effects of intraoperative intraperitonealchemotherapy on the incidence of anastomotic leakage (AL), which would be meaningful topromote our knowledge about the safety and feasibility of this emerging therapy modality.

Research motivationOur study explored the safety of intraoperative intraperitoneal chemotherapy for patientsreceiving the anterior resection of rectal carcinoma. This is significant for surgeons to weigh thebenefits and risks of this treatment technique.

Research objectivesOur research aimed to evaluate the role of intraoperative intraperitoneal chemotherapy in theoccurrence of AL. Meanwhile, the prognosis of patients receiving this therapy was also analyzed.

Research methodsWe performed a retrospective cohort study and patients were divided into a chemotherapygroup and a control group. Important demographic variables and confounding factors werecollected and analyzed through univariate analysis, stratification analysis, and multivariateanalysis to control confounding bias. The oncological outcomes of the two groups werecompared through the Kaplan-Meier method and log rank test.

Research resultsWe found that intraoperative intrapertitoneal chemotherapy increased the incidence of AL inpatients receiving the anterior resection of rectal carcinoma, but this treatment also contributedto improved disease-free survival rate. This finding can help surgeons to weigh the benefits andrisks of this emerging treatment method. Moreover, the mechanisms of intraoperativeintraperitoneal chemotherapy leading to AL need to be further investigated in more basicstudies. The effects of different types of chemotherapeautic agents on AL can also be explored.

Research conclusionsIntraoperative intraperitoneal chemotherapy can improve the prognosis of patients with locallyadvanced rectal cancer, but it also increases the risks of AL in patients receiving anteriorresection of rectal carcinoma. Patients who have other risks of postoperative AL may not besuitable to receive this therapy.

Research prospectiveSurgeons need to think deeply about the indications and contraindications of intraoperativeintraperitoneal chemotherapy so that better clinical outcomes can be achieved in patients withrectal carcinoma. Moreover, our research is a retrospective study, and biases from data collectionand analysis may exist. More prospective randomized controlled trials need to be conducted to



548

explore the safety and feasibility of intraoperative intraperitoneal chemotherapy.

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ORIGINAL ARTICLE

Retrospective Study

TYMS/KRAS/BRAF molecular profiling predicts survival followingadjuvant chemotherapy in colorectal cancer

Anastasios Ntavatzikos, Aris Spathis, Paul Patapis, Nikolaos Machairas, Georgia Vourli, George Peros,Iordanis Papadopoulos, Ioannis Panayiotides, Anna Koumarianou

ORCID number: AnastasiosNtavatzikos (0000-0003-3343-3550);Aris Spathis (0000-0001-8867-3661);Paul Patapis (0000-0003-2349-769X);Nikolaos Machairas(0000-0003-3239-3905); GeorgiaVourli (0000-0002-9727-2808);George Peros (0000-0001-7401-2811);Iordanis Papadopoulos(0000-0002-0620-3584); IoannisPanayiotides (0000-0002-6394-117X);Anna Koumarianou(0000-0002-4159-2511).

Author contributions: NtavatzikosA, Spathis A, Panayiotides I andKoumarianou A designed theresearch; Ntavatzikos A, Patapis P,Spathis A and Koumarianou Acollected the data; Ntavatzikos A,Spathis A and Panayiotides Iperformed the research;Ntavatzikos A, Patapis P, SpathisA and Koumarianou A analyzedthe data; Ntavatzikos A andKoumarianou A wrote the paper;Panayiotides I and Papadopoulos Ioffered the technical or materialsupports; Ntavatzikos A, Spathis Aand Koumarianou A drafted themanuscript; all authors criticallyrevised the manuscript forimportant intellectual content.

Supported by Kapodistrias,National and KapodistrianUniversity of Athens, No.70/3/8006 (Pythagoras II, EPEAEKII, GSRT) and No. 70/3/9114;Spathis A was supported duringdata collection from No. 70/3/8462[PENED - European Social Fund(75%) and the Greek Ministry ofDevelopment-GSRT (25%)].

Anastasios Ntavatzikos, Anna Koumarianou, Hematology-Oncology Unit, 4th Department ofInternal Medicine, Medical School, National and Kapodistrian University of Athens,“ATTIKON” University Hospital, Athens 12462, Greece

Aris Spathis, Department of Cytopathology, National and Kapodistrian University of Athens,Medical School, “ATTIKON” University Hospital, Athens 12462, Greece

Paul Patapis, Nikolaos Machairas, 3rd Department of Surgery, Medical School, National andKapodistrian University of Athens, “ATTIKON” University Hospital, Athens 12462, Greece

Georgia Vourli, Department of Hygiene, Epidemiology and Medical Statistics, Medical School,National and Kapodistrian University of Athens, Athens 11527, Greece

George Peros, Iordanis Papadopoulos, Department of Surgery, Medical School, National andKapodistrian University of Athens, Evgenideio Therapeutirio S.A., “I AGIA TRIAS”, Athens11528, Greece

Ioannis Panayiotides, 2nd Department of Pathology, University of Athens, Medical School,“ATTIKON” University Hospital, Athens 12462, Greece

Corresponding author: Anastasios Ntavatzikos, MD, Research Scientist, Hematology-Oncology Unit, 4th Department of Internal Medicine, Medical School, National andKapodistrian University of Athens, “ATTIKON” University Hospital, Rimini 1, Haidari,Athens 12462, Greece. [email protected]: +30-210-5831687Fax: +30-210-5326446

AbstractBACKGROUNDPatients with stage II-III colorectal cancer (CRC) treated with adjuvantchemotherapy, gain a 25% survival benefit. In the context of personalizedmedicine, there is a need to identify patients with CRC who may benefit fromadjuvant chemotherapy. Molecular profiling could guide treatment decisions inthese patients. Thymidylate synthase (TYMS) gene polymorphisms, KRAS andBRAF could be included in the molecular profile under consideration.

AIMTo investigate the association of TYMS gene polymorphisms, KRAS and BRAFmutations with survival of CRC patients treated with chemotherapy.




http://orcid.org/0000-0003-3343-3550

http://orcid.org/0000-0001-8867-3661

http://orcid.org/0000-0003-2349-769X

http://orcid.org/0000-0003-3239-3905

http://orcid.org/0000-0002-9727-2808

http://orcid.org/0000-0001-7401-2811

http://orcid.org/0000-0002-0620-3584

http://orcid.org/0000-0002-6394-117X

http://orcid.org/0000-0002-4159-2511


Institutional review boardstatement: This study wasreviewed and approved by theInstitutional Review Board andEthical Committee of UniversityGeneral Hospital Attikon, Athens,Greece.

Informed consent statement:Patients were not required to giveinformed consent to the studybecause the analysis usedanonymous clinical data that wereobtained after each patient agreedto treatment by written consent.

Conflict-of-interest statement: Allauthors declare no conflicts-of-interest related to this article.



Received: March 14, 2019Peer-review started: March 15, 2019First decision: April 16, 2019Revised: April 30, 2019Accepted: June 12, 2019Article in press: June 13, 2019Published online: July 15, 2019

P-Reviewer: Arslan NC, Kasi PM,Watanapokasin RS-Editor: Ji FFL-Editor: AE-Editor: Xing YX

METHODSA retrospective study studied formalin-fixed paraffin-embedded tissues (FFPEs)of consecutive patients treated with adjuvant chemotherapy duringJanuary/2005-January/2007. FFPEs were analysed with PCR for the detection ofTYMS polymorphisms, mutated KRAS (mKRAS) and BRAF (mBRAF). Patientswere classified into three groups (high, medium and low risk) according to5’UTR TYMS polymorphisms Similarly, based on 3’UTR polymorphism ins/lossof heterozygosity (LOH) patients were allocated into two groups (high and lowrisk of relapse, respectively). Cox regression models examined the associated 5-year survival outcomes.

RESULTSOne hundred and thirty patients with early stage CRC (stage I-II: 55 patients;stage III 75 patients; colon: 70 patients; rectal: 60 patients) were treated withsurgery and chemotherapy. The 5-year disease free survival and overall survivalrate was 61.6% and 73.9% respectively. 5’UTR polymorphisms of intermediateTYMS polymorphisms (2RG/3RG, 2RG/LOH, 3RC/LOH) were associated withlower risk for relapse [hazard ratio (HR) 0.320, P = 0.02 and HR 0.343, P = 0.013respectively] and death (HR 0.368, P = 0.031 and HR 0.394, P = 0.029respectively). The 3’UTR polymorphism ins/LOH was independently associatedwith increased risk for disease recurrence (P = 0.001) and death (P = 0.005).mBRAF (3.8% of patients) was associated with increased risk of death (HR 4.500,P = 0.022) whereas mKRAS (39% of patients) not.

CONCLUSIONProspective validating studies are required to confirm whether 2RG/3RG,2RG/LOH, 3RC/LOH, absence of ins/LOH and wild type BRAF may indicatepatients at lower risk of relapse following adjuvant chemotherapy.

Key words: Colorectal neoplasms; Thymidylate synthase; Untranslated regions;Fluorouracil; KRAS; BRAF; Prognosis


Core tip: There is a need to identify patients with colorectal cancer (CRC) who maybenefit from adjuvant chemotherapy. We investigated the survival in 130 patients withstage II-III CRC treated with adjuvant chemotherapy based on thymidylate synthase(TYMS) gene polymorphisms, KRAS and BRAF status. We found that TYMSpolymorphisms and BRAF status associate independently with the survival outcomes.Prospective validating studies are required.

Citation: Ntavatzikos A, Spathis A, Patapis P, Machairas N, Vourli G, Peros G, PapadopoulosI, Panayiotides I, Koumarianou A. TYMS/KRAS/BRAF molecular profiling predicts survivalfollowing adjuvant chemotherapy in colorectal cancer. World J Gastrointest Oncol 2019;11(7): 551-566URL: https://www.wjgnet.com/1948-5204/full/v11/i7/551.htmDOI: https://dx.doi.org/10.4251/wjgo.v11.i7.551

INTRODUCTIONColorectal cancer (CRC) is the third most common cancer in the United States ofAmerica while worldwide it is expected to increase by 60% to more than 2.2 millionnew cases and 1.1 million deaths by 2030[1,2]. In 2014, almost 153000 patients died fromCRC in the European Union, where it is the second leading cause of cancer death(Eurostat. Cancer statistics – specific cancers)[3]. At diagnosis, 74%-76% of patientshave a localized or regional CRC. Fluoropyrimidines remain the backbone of adjuvantchemotherapy for early stage CRC patients after curative surgery[4,5]. Fluoro-pyrimidines exert their action by different ways mainly by inhibiting the de novoformation of thymidylate (dTMP) from uridylate (dUMP)[6]. Other mechanisms ofaction are more complex than simply inhibition of TS expression, as they involveinhibition of DNA synthesis and function through misincorporation of FdUTP into


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cellular DNA and inhibition of RNA processing and mRNA translation through theincorporation of FUTP into cellular RNA[7]. The use of fluoropyrimidines is associatedwith reduction of recurrence in only 25% of patients with stage III CRC[8,9]. Only 3%-7% of patients with stage II CRC will benefit from adjuvant chemotherapy[10]. Thevariability of observed survival outcomes has been largely attributed to molecularheterogeneity and KRAS, BRAF and thymidylate synthase (TYMS) are beinginvestigated to this end[11]. KRAS belongs to the RAS subfamily of genes that encodes a21-kDa small-GTPase[12]. Activating mutations in RAS result in activation of majorsignaling pathways downstream of epidermal growth factor receptor (EGFR)stimulating cell proliferation and inhibiting apoptosis[13]. In the metastatic diseasesetting, KRAS mutations (mKRAS) is a predictor of resistance to EGFR inhibitors andis directly linked to poor patient survival, while its role in the adjuvant setting isunder investigation[14-16].

BRAF is an essential part of the RAS/RAF/MAP2K (MEK)-MAPK signalingcascade and its mutations have been likewise associated with inferior survival in CRCpatients after curative resection and adjuvant chemotherapy[17,18].

The TYMS gene (GeneID 7298) is located on the short arm of chromosome 18(18p11.32). There is conflicting evidence on the role of TYMS polymorphisms inpredicting response to 5FU–based chemotherapy[19-25]. The loss of heterozygosity(LOH) at the TYMS locus on chromosome 18 has been implicated as a factor affectingthe TYMS-related resistance to fluoropyrimidine-based therapy[26].

A TYMS polymorphism of the 5’ untranslated region (5’UTR) results by theinsertion of a 28 base-pair (bp) sequence (rs34743033)[19]. From the resulting alleles thatmay include two or three 28bp tandem repeats (2R or 3R respectively), the 3R allelewas associated with increased TYMS protein expression and TYMS enzymeactivity[27,28]. G->C single nucleotide polymorphism (SNP) in the tandem repeatsequence [rs2853542] was found to reduce the translational efficiency of a 3R to a2R[19,29]. Based on the presence of SNP polymorphisms (G or C) 3R are characterized as3RG and 3RC. In addition, the 3’UTR may contain a 6 bp polymorphism (rs34489327)affecting the TYMS mRNA stability, and resulting in increased intratumoral TYMSmRNA[19,30]. Depending on the presence of this 6 bp polymorphism, the three resultinggenotypes are ins/ins (homozygous for insertion of 6bp), del/del (homozygous fordeletion) and ins/del (heterozygous).

Based on all the above, the identification of potential markers that could elucidatewhich patients’ subgroups could benefit most from fluoropyrimidine-based therapyremains an unmet clinical need.

The present study aims to investigate the associations of TYMS polymorphisms,LOH, mKRAS and BRAF mutations (mBRAF) with clinicopathologic characteristicsand survival outcomes of patients with CRC treated with fluoropyrimidine-basedadjuvant chemotherapy.

MATERIALS AND METHODS

Patients and clinical dataThis was a retrospective study carried out by a single institution (University GeneralHospital “ATTIKON”). Formalin-fixed paraffin-embedded tissues (FFPE) and clinicaldata of consecutive patients with CRC referred for adjuvant chemotherapy fromJanuary 2005 to January 2007 were retrieved. Of these, only patients with histologiesreporting R0 surgical margins and treated with fluoropyrimidine-based adjuvantchemotherapy (and therefore with no redisual disease) were included in the analysis.In these cases, the integrity of mesocolon/mesorectum was preserved.

DNA extraction protocolDNA was extracted from 5 μm thick FFPE sections, containing at least 30% malignantcells, using a commercially available kit (Purelink Genomic DNA kit, Thermo FisherScientific, Germany). DNA was quantified by qPCR (Quant-iT™ PicoGreen® dsDNAAssay Kit, Thermo Fisher Scientific, Germany) and was diluted accordingly to achievea concentration of 10 ng/μL for TYMS polymorphisms and 4 ng/μL for mKRASdetection.

TYMS polymorphismsAnalysis was carried-out as previously described[31,32]. PCR was performed using 1U ofPlatinum® Taq DNA Polymerase (Thermo Fisher Scientific, Germany), 1.5 mmol/L ofMg and 200 nmol/L of dNTPs and primers. Althoug the same primers were used, 5’-UTR amplification was performed using a GC rich amplification kit (PCRX EnhancerSystem, Thermo Fisher Scientific, Germany) adding 1× of PRCx Enhancer.



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Genotyping for the 2R/3R polymorphism was performed by running 10 μL of thePCR product on a 1.5% agarose gel and staining with Ethidium Bromide as previouslydescribed (Ntavatzikos et al[31]). Similarly, for the 12G>C substitution, 10 μL of PCRproduct was digested with 1U of HaeIII restriction enzyme (Takara, Japan) at 37 oC for1 h and run on an 8% 19:1 polyacrylamide gel. Polyacrylamide gels were used for theanalysis of the 3’UTR. LOH analysis was achieved by analyzing the intensity of the5’UTR and 3’UTR bands of the pictures acquired using the GeneTools software(Syngene, United Kingdom). The sample was categorized as having LOH if one of thebands had an intensity score of < 50% of the other. Samples showing LOH weredefined as 2R/3RGLOH, 2RLOH/3RG, 2R/3RCLOH and 2RLOH/3RC indicating theallele that was partially lost. For quality control, selected products were sequenced toverify the sequence amplified. The amplified product was 242 bp for 3R and 214 bpfor 2R polymorphisms, as revealed by the blast of the sequenced products and thealignment with the latest human assemblies.

Mutational analysisDetection of mKRAS in codons 12 and 13 and BRAF activating mutation V600E wereperformed as previously described with a commercially available Real-Time PCR kit(Therascreen KRAS, DxS Diagnostics, United Kingdom) detecting 6 mutations ofcodon 12 (G12D, G12A, G12V, G12S, G12R, G12C) and 1 mutation of codon 13(G13D)[31,33]. A positive reaction mix for all mutations was included. To avoid falsenegative results caused by PCR inhibitors, a second exogenous reaction wassimultaneously taking place. If the sample’s ΔCt (Ct of control reaction-Ct mutationreaction) was lower than the value set by the manufacturer, then it was characterizedas bearing a mutation. BRAF activating mutation V600E was identified usingmolecular beacons as previously described[33]. One beacon for the wild type and onefor the mutant allele were added at a final concentration of 100 nmol/L in a 25 μLPCR reaction containing 1× PCR Buffer, 6 mmol/L MgCl2, 200 nmol/L dNTPs, 300nmol/L of each primer and 1U of Platinum® Taq. PCR profile applied was 95 oC 2min, followed by 40 cycles of 95 oC for 10 sec, 62 oC for 60 sec and 72 oC for 20 sec.DNA extracts from the series of melanoma cell lines SKMEL2 and SKMEL20 wereused as positive controls for both the wild type and mutant allele (CLS, Germany).The ABI 7500 Fast (Thermo Fisher Scientific, Germany) was used to perform all Real-Time PCR experiments.

TYMS-gene polymorphisms stratification modelBased on the predicted TYMS protein expression, 5’UTR polymorphisms wereassigned into low (2RG/2RG, 2RG/3RC, 3RC/3RC), medium (2RG/3RG,2RG/3RCLOH, 2RG/3RGLOH, 2RGLOH/3RC) and high TYMS protein expressiongroup (3RG/3RG, 3RG/3RC, 2RGLOH/3RG) [31 ]. The effect of each 3’UTRpolymorphism was examined against all the others by applying univariate analysisand it was found that only the ins/LOH polymorphism had a statistically significanteffect. Based on this finding, 3’UTR polymorphisms were allocated into two groupsdepending on the presence or not of ins/LOH. This classification is depicted in Table1.

Statistical analysisAssociation of TYMS polymorphisms with selected clinicopathological characteristicswas performed using the χ2 test with a 2-sided significance of 0.05. Time-to-eventdistributions were estimated using the Kaplan-Meier method. For all associations, thelevel of statistical significance was set at a = 0.05. Overall survival (OS) was defined asthe interval between initiation of adjuvant chemotherapy and death of any cause.Disease-free survival (DFS) was defined as the time from adjuvant chemotherapyinitiation to the first recurrence or death by any cause.

Surviving patients were censored at the date of last contact. Cox proportionalhazards model was used to estimate the relationship of clinicopathologicalparameters and TYMS polymorphisms with OS and DFS. The relationship of TYMSpolymorphisms and the groups to which classified with OS and DFS was assessed byunivariate Cox regression analysis. The final multivariate model was selected using abackward selection procedure, starting from an initial model that included allpotential risk factors and TYMS polymorphisms. Model selection was based onlikelihood ratio test, while the removal criterion was set at 0.10. All statistical analyseswere performed using the SPSS software version 24.0 (SPSS Inc, Chicago, IL, UnitedStates). The statistical methods of this study were reviewed by Georgia Vourli fromthe Department of Hygiene, Epidemiology and Medical Statistics, Medical SchoolUniversity of Athens.



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Table 1TYMS polymorphisms’ groups according to risk group and level of expressionrespectively

Groups Polymorphisms

3’UTR

A (low risk) del/del

del/LOH

ins/del

ins/ins

B (high risk) ins/LOH

5’UTR

A (low expression) 2RG

2RG/3RC

3RC

B (medium expression) 2RG/3RG

2RG/3RCLOH

2RG/3RGLOH

2RGLOH/3RC

C (high expression) 3RG

3RG/3RC

2RGLOH/3RG

UTR: Untranslated region; LOH: Loss of heterozygosity.

RESULTS

Patient characteristicsMedical records of 130 consecutive patients and their FFPE were retrieved foranalysis. Patients’ clinicopathologic data including age, gender, primary tumor site,histological grade, treatment and survival are shown in Table 2. With a medianfollow-up of 71.2 mo (range 0.5-157), 51 patients (39.2%) experienced diseaserecurrence while 45 patients (34.6%) died. The 5-year OS and DFS rate was 73.9% and61.6% respectively.

The frequency of TYMS polymorphisms involving G>C SNP and LOH arepresented in Table 3. Significant associations were found among patients’ tumorcharacteristics and polymorphisms as shown in Table 4.

Univariate survival analysisUnivariate Cox regression analysis of TYMS polymorphisms, mKRAS and mBRAF,LOH and selected clinicopathological patients’ characteristics are shown in Table 5.Univariate analysis indicated a trend for a better DFS and OS in the group of 5’UTRpolymorphisms with medium expression profile (group B), while ins/LOHpolymorphism of the 3’UTR were associated with a trend for worse DFS and OS. Theanalysis of mKRAS showed no significant effect on survival whereas BRAF V600Emutation was associated with increased risk of death. Clinical variables, close tostatistical significance, were age (< 65years old vs ≥ 65years old), primary site (rectalvs colon), histological grade (III-IV vs I-II) and stage (III vs Ι and II).

Multivariate survival analysisResults of the multivariate analysis including TYMS polymorphisms, mBRAF andselected clinicopathological characteristics are shown in Table 6. From the 5’UTRpolymorphisms, the group A (2RG/2RG, 2RG/3RC, 3RC/3RC) and group C(3RG/3RG, 3RG/LOH, 3RG/3RC) were associated with higher risk for diseaserecurrence and death as compared to group B (2RG/3RG, 2RG/LOH and 3RC/LOH).Similarly, group B of 3’UTR polymorphism (ins/LOH) was associated with increasedrisk of relapse and death as compared to group A.

Kaplan-Meier curves for DFS and OS according to TYMS 3’UTR and 5’UTRpolymorphisms groups are shown in Figure 1. Stage III increased independently therisk for relapse while the BRAF mutation increased independently the risk for death.Kaplan-Meier curves for OS according to mBRAF are shown in Figure 2.



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Table 2 Clinicopathologic data for colorectal cancer patients treated with adjuvantchemotherapy

Clinicopathologic data Total (n = 130)

Median age (range) 67 (37-88)

Male 79 (60.8)

Primary site

Rectum 60 (46.2)

Positive lymph nodes 76 (58.5)

Stage according to AJCC

I 1 (0.8)

II 54 (41.5)

III 75 (57.7)

Histological grade

I + II 83 (63.8)

III + IV 47 (36.2)

KRAS mutation 48 (36.9)

BRAF V600E mutation 5 (3.8)

TYMS LOH 34 (26.2)

Overall survival

Deaths n (%) 45 (34.6)

Mean time month (95%CI) 110.0 (99.5-120.5)

Disease-free survival

Events n (%) 51 (39.2)

Mean time month (95%CI) 100.1 (88.3-112.0)

Median follow up in months (range) 71.2 (0.5-156.8)

AJCC: American Joint Committee on Cancer 7th edition; TYMS: Thymidylate synthase gene; LOH: Loss ofheterozygosity; CI: Confidence interval.

DISCUSSIONThis is a retrospective study of 130 patients with CRC treated with surgery andadjuvant chemotherapy, studying for the first time the correlation of TYMSpolymorphisms, LOH, mKRAS and mBRAF with survival outcomes. We report thatthe 3’UTR and 5’UTR TYMS polymorphisms were independent factors associatedwith risk of disease relapse and death. In particular, ins/LOH increased risk ofdisease relapse and death, while the group of 5’UTR polymorphisms containing2RG/3RG, 2RG/LOH and 3RC/LOH decreased the risk of disease relapse and death.The study of mKRAS pointed out that it did not associate with disease relapse orrelated death, while the mBRAF increased independently the risk of death.

Since the early studies of adjuvant chemotherapy treatment with 5FU, 23 years ago,there have been two landmark advances in the field[34]. The first one involved theincorporation of oral capecitabine as an alternative to intravenously administered5FU[35]. The second was the addition of oxaliplatin to 5FU that lead to a 4.2% absoluteimprovement in OS of patients with T4 and N1 disease (stage III disease; MOSAICtrial) whereas stage II patients did not benefit[36,37]. As clinicopathologic parameters areimportant but not sufficiently useful in deciding which patients with stage II-III willbenefit from adjuvant chemotherapy, molecular markers are essential[38]. Severalstudies reported the association of TYMS polymorphisms, TYMS mRNA and TYMSprotein expression with survival in patients with CRC but with inconsistentfindings[20-22,24,39-43]. A meta-analysis indicated that patients with advanced CRC tumorsexpressing high levels of TYMS had a poorer OS compared to tumors expressing lowlevels[44]. On the contrary, a subsequent prospective, blinded analysis of TYMSexpression in the adjuvant treatment of CRC concluded that TYMS expression did notshow a significant prognostic value[45]. None of the studies included in theirmultivariate analysis the mBRAF status nor the different TYMS polymorphisms.

5’UTR polymorphismsIn this study TYMS polymorphisms emerged as prognostic factors for survivaloutcomes in patients treated with surgery and adjuvant chemotherapy. More



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Table 3 Frequency of TYMS 5’UTR, 3’UTR genotypes

Genotype Total n (%)

TYMS 5’UTR 130 (100)

2R 13 (10.0)

2R/3R 78 (60.0)

2R/3RG 34 (26.1)

2R/3RG 20 (15.4)

2R/3RGLOH 8 (6.2)

2RLOH/3RG 6 (4.6)

2R/3RC 44 (33.8)

2R/3RC 24 (18.5)

2R/3RCLOH 13 (10.0)

2RLOH/3RC 7 (5.4)

3R 39 (30.0)

3RG 10 (7.7)

3RG/3RC 20 (15.4)

3RC 9 (6.9)

TYMS 3’UTR 130 (100)

ins/ins 28 (21.5)

ins/LOH 27 (20.8)

ins/del 52 (40.0)

del/LOH 7 (5.4)

del/del 16 (12.3)

TYMS: Thymidylate synthase gene; UTR: Untranslated region; SNP: Single nucleotide polymorphism; LOH:Loss of heterozygosity.

specifically, the group B (2RG/3RG, 2RG/3RCLOH, 2RG/3RGLOH, 2RGLOH/3RC)was shown to have the lowest risk of recurrence and a trend for lower risk of deathwhen compared to the other two groups A (2RG/2RG, 2RG/3RC, 3RC/3RC) and C(3RG/3RG, 3RG/3RC, 2RGLOH/3RG). Similarly, a previous study showed that5’UTR polymorphisms associated with survival. In particular, they reported that ‘lowrisk’ polymorphisms (2RG/2RG, 2RG/3RC, 3RC/3RC) were associated withimproved DFS regardless chemotherapy treatment[40]. On the contrary, a previousstudy indicated that TYMS 5’UTR polymorphisms do not predict clinical outcome ofCRC patients treated with 5-FU based chemotherapy[39]. Nevertheless, neither of thesetwo studies took into consideration a combined analysis of 3’UTR polymorphisms,LOH or mBRAF status. In addition, the categorization of the TYMS 5’UTRpolymorphisms into only two groups (high expression group: 2RG/3RG, 3RC/3RG,3RG/3RG and low expression group: 2RG/2RG, 2RG/3RC, 3RC/3RC), albeit itfacilitates statistical processing it also entails the risk of classification error. Indeed, inthis way both studies placed the 2RG/3RG with the high expression 3RG/3RG,although 2RG/3RG is a member of the group of heterozygous 5’UTR polymorphismsgroup that are generally considered to have an intermediate expression profile[27,46].Our study identified heterozygotes such as 2RG/3RG, 2RG/LOH and 3RC/LOH, asindependent good prognostic factors for recurrence and death in CRC patients treatedwith surgery and adjuvant chemotherapy.

3’UTR polymorphismsIn our study, 3’UTR polymorphism ins/LOH was found to independently increasethe risk for both relapse and death. Comparably, two other studies outlined thenegative effect of the ins allele in the therapeutic outcome of CRC patients treatedwith adjuvant chemotherapy and neoadjuvant setting in rectal cancer patients[41,47]. Onthe contrary, another study found that ins/ins with 2R/3R and any 3’UTRpolymorphism with 3R/3R predict longer DFS and OS in CRC patients treated withadjuvant 5FU-based chemotherapy[22]. However, in the later study the SNP G>C andLOH status were not taken into consideration.

KRAS and BRAFThe present study showed that the rate of mBRAF identified in our population (3.8%)



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Table 4 Associations between patient characteristics and TYMS polymorphisms

Patient characteristics Polymorphisms RR (95%CI) P value

Birth after 1942 3RG/3RG 5.128 (1.131-23.26) 0.025

3RC/3RC and 3RC/LOH 0.296 (0.088-0.988) 0.035

Male 3RG/3RG and 3RG/LOH 4.519 (1.072-19.06) 0.030

Grade III-IV 3RG/3RC 2.646 (1.167-6.024) 0.022

Stage III 3RG/3RG and 3RG/3RC and 3RG/LOH 2.198 (1.126-4.292) 0.020

3RG/3RC 4.149 (1.280-13.51) 0.008

Without any 3RG allele 0.733 (0.546-0.984) 0.050

3RC/3RC and 3RC/LOH 0.333 (1.229-0.904) 0.030

3RC/LOH 0.122 (0.015-0.986) 0.045

KRAS mutation 3RG/3RC 3.135 (1.344-7.299) 0.010

3RC/3RC and 3RC/LOH 0.241 (0.057-1.015) 0.030

TYMS: Thymidylate synthase gene; RR: Relative risk; CI: Confidence interval; LOH: Loss of heterozygosity.

was lower than expected, as previously reported rates in the adjuvant setting rangedfrom 7.9% to 17%[17,36,48]. Albeit mBRAF was not associated with the risk for relapse,mBRAF independently increased the risk of death. In agreement with our study, threeprevious studies linked mBRAF to poor survival in relation with MSI status[17,48,49]. Afourth study reported that mBRAF was an adverse prognostic factor for both DFS andOS, independently of MSI status[50]. Contrary to these studies, another study indicatedthat BRAF mutations did not confer a worse prognosis[36]. Differently to our study,none of the above studies took into consideration TYMS polymorphisms.

In this study mutated KRAS did not emerge as a predictive factor for survival in theunivariate analysis. Similar to ours, two previous studies indicated that mKRAS wasnot associated with survival in stage II/III CRC patients[48,51]. On the contrary, a morerecent study reported that the risk of recurrence was higher for mKRAS compared towild type KRAS tumors[52]. More recently, another study reported that mKRAS hadprognostic impact on DFS and OS independently of microsatellite instability status[50].None of the above studies took into consideration TYMS polymorphisms.

Other findings of the analysisWe found that patients born from 1943 onwards had more frequently thepolymorphism 3RG/3RG and high-grade malignancy tumors (RR 1.730, 95%CI: 1.088-2.747; P = 0.030). Two previous studies have also linked age to TYMS polymorphismsand protein expression in CRC[53,54]. As more data gather, the differences in thefrequency of polymorphisms among generations are of great interest. Thesedifferences could derive from epigenetic modifications induced by environmentalchanges during the course of human life[55]. Another important open question iswhether in younger generations TYMS polymorphisms associate with higher risk ofdeveloping aggressive cancer due to changes in the genetic substrate.

We report for the first time that mKRAS had a strong correlation with thepolymorphism 3RG/3RC and with polymorphisms that contain only 3RC allele(3RC/3RC, 3RC/LOH). Contrary to our findings, a previous study reported nosignificant relationship between any of the TYMS polymorphisms with tumorcharacteristics[56]. However, in the understudy grouping of TYMS polymorphisms,LOH was not considered.

LimitationsAlthough the size of this study’s patient cohort is one of the largest reported, still itmakes it difficult to analyze the large sum of polymorphisms resulting from thecombination of 3’UTR and 5’UTR polymorphisms, SNP G>C and LOH. Anotherlimitation is that subsequent chemotherapy lines following disease relapse were notincluded in the survival analysis. An important limitation is that classification ofTYMS polymorphisms into groups was based on our statistical analysis andpreviously published data but requires further validation in prospective trials.

Another important limitation is that the levels of TYMS protein expression andactivity were not examined. Although immunohistochemical analysis of TYMSprotein expression is considered important, several studies have shown that TYMSprotein expression is affected by several factors, like p53 mutation and other geneswhich proved to affect the final level of TYMS expression, like astrocyte elevated



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Table 5 Univariate Cox regression analysis for clinicopathological features and genotypes

Variable HR DFS 95%CI P value HR OS 95%CI P value

Age < 65 yr 1.513 0.873-2.621 0.140 1.229 0.682-2.213 0.492

Rectal Ca 1.550 0.890-2.703 0.121 1.282 0.713-2.306 0.406

Stage III vs I%II 2.532 1.368-4.695 0.003 1.877 1.009-3.494 0.047

Grade III and IV vs I and II 1.984 1.143-3.436 0.015 2.097 1.166-3.770 0.013

KRAS mutation 1.330 0.761-2.326 0.321 1.283 0.702-2.346 0.418

BRAF V600E mutation 1.276 0.310-5.255 0.736 2.743 0.845-8.902 0.093

TYMS 5’UTR 0.397 0.766

2R 1 1

2R/3R 1.213 0.498-2.958 0.671 0.745 0.332-1.672 0.475

3R 1.690 0.678-4.213 0.260 0.846 0.355-2.020 0.707

TYMS 5’UTR 0.596 0.615

2RG/3RG 1 1

2RG/2RG 1.038 0.377-2.858 0.942 1.750 0.672-4.559 0.252

2RG/3RC 1.523 0.684-3.394 0.303 1.625 0.702-3.760 0.257

3RC/3RC 1.414 0.482-4.148 0.528 0.680 0.146-3.162 0.623

3RG/3RC 2.128 0.902-5.018 0.085 1.782 0.686-4.625 0.235

3RG/3RG 1.489 0.466-4.672 0.502 1.996 0.610-6.532 0.253

TYMS 5’UTR 0.204 0.589

2RG/3RG 1 1

2RG/2RG 1.702 0.343-8.441 0.515 3.322 0.787-14.03 0.102

2RG/3RC 2.935 0.778-11.08 0.112 3.034 0.803-11.46 0.102

2RG/3RCLOH 5.387 1.427-20.34 0.013 3.879 0.967-15.56 0.056

2RG/3RGLOH 2.138 0.431-10.60 0.352 2.026 0.408-10.06 0.388

2RGLOH/3RC 3.178 0.640-15.78 0.157 3.109 0.626-15.44 0.165

2RGLOH/3RG 7.402 1.648-33.24 0.009 6.127 1.358-27.64 0.018

3RC/3RC 4.326 1.031-18.15 0.045 1.733 0.288-10.42 0.548

3RG/3RC 4.865 1.336-17.72 0.016 3.438 0.888-13.32 0.074

3RG/3RG 3.413 0.761-15.30 0.109 3.994 0.887-17.99 0.071

TYMS 5’UTR groups 0.130 0.223

A1 1.136 0.574-2.251 0.714 1.882 0.917-3.861 0.085

B2 1 1

C3 1.908 0.980-3.713 0.057 1.309 0.637-2.692 0.464

TYMS 3’UTR 0.791 0.846

del/del 1.170 0.496-2.760 0.721 1.145 0.456-2.873 0.773

ins/del 1.244 0.664-2.329 0.495 1.219 0.622-2.387 0.564

ins/ins 1 1

TYMS 3’UTR 0.299 0.391

del/del 0.624 0.244-1.595 0.324 0.634 0.228-1.761 0.382

del/LOH 0.374 0.086-1.630 0.190 0.408 0.093-1.797 0.236

ins/del 0.634 0.329-1.224 0.175 0.743 0.372-1.482 0.399

ins/LOH 1 1

ins/ins 0.417 0.172-1.016 0.054 0.391 0.140-1.087 0.072

ins/ins vs ELSE 0.593 0.267-1.318 0.200 0.511 0.201-1.297 0.158

ins/LOH vs ELSE 1.807 1.000-3.266 0.050 1.650 0.877-3.104 0.120

ins/del vs ELSE 0.976 0.556-1.713 0.933 1.131 0.626-2.044 0.684

del/del vs ELSE 0.964 0.411-2.262 0.934 0.907 0.358-2.299 0.837

del/LOH vs ELSE 0.565 0.137-2.327 0.430 0.570 0.138-2.359 0.438

LOH 1.480 0.833-2.629 0.181 1.350 0.732-2.487 0.336

SNP G->C 1.542 0.878-2.707 0.132 1.108 0.617-1.992 0.731

1Low expression profile;2Medium expression profile;3High expression profile. DFS: Disease-free survival; OS: Overall survival; HR: Hazard ratios; CI: Confidence



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interval; Ca: Cancer; TYMS: Thymidylate synthase gene; UTR: Untranslated region; LOH: Loss ofheterozygosity; 5FU: 5-fluorouracil; SNP: Single nucleotide polymorphism.

gene-1 (AEG-1) and enolase superfamily member 1 (ENOSF1) during the course of thedisease[57-60]. It has been reported that there is discordance in TYMS mRNA expressionand TYMS protein levels between primary tumors and their metastasis[61-63].Furthermore, the binding of TYMS protein to its own mRNA, as well as the binding ofTYMS to p53 mRNA causes translational repression, in an autoregulatory translationalmanner[64-66]. Other significant prognostic and predictive markers such as NRAS,PIK3CA exon 20 and MMR/MSI were not included in this analysis[64-66].

In conclusion, the group of TYMS polymorphisms 2RG/3RG, 2RG/LOH and3RC/LOH and the absence of ins/LOH was associated with better prognosis in CRCpatients treated with adjuvant chemotherapy while mBRAF was associated withincreased risk of death. Proof of concept, prospective studies are required to validateour findings.

ACKNOWLEDGEMENTSWe dedicate this manuscript to the late Petros Karakitsos, our mentor and colleaguewho founded the lab of cellular and molecular biology where this work was carriedout. He will always be remembered with love.



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Table 6 Multivariate Cox regression analysis for clinicopathological features and selected genotypes

Variable HR DFS 95%CI P value HR OS 95%CI P value

Stage III vs I and II 2.432 1.279-4.625 0.007

Grade III and IV vs I and II 1.715 0.951-3.091 0.073 1.860 0.982-3.525 0.057

TYMS 5’UTR groups 0.031 0.052

A 3.122 1.193-8.169 0.020 2.715 1.093-6.739 0.031

B 1 1

C 2.919 1.258-6.772 0.013 2.540 1.098-5.876 0.029

TYMS 3’UTR groups

A (without ins/LOH) 1

B (ins/LOH) 4.124 1.744-9.753 0.001 3.335 1.474-7.548 0.004

BRAF V600E mutation 4.500 1.241-16.32 0.022

DFS: Disease-free survival; OS: Overall survival; HR: Hazard ratio; CI: Confidence interval; TYMS: Thymidylate synthase gene; UTR: Untranslated region;LOH: Loss of heterozygosity.

Figure 1

Figure 1 Kaplan-Meier curves for disease free survival and overall survival according to thymidylate synthase polymorphisms: A: Disease free survival(DFS) according to 5’ untranslated region (UTR); B: Overall survival (OS) according to 5’UTR; C: DFS according to 3’UTR; D: OS according to 3’UTR.aP <0.05 vs Group A and C; bP < 0.005. LOH: Loss of heterozygosity.



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Figure 2

Figure 2 Kaplan-Meier survival curve for overall survival according to BRAF mutation status (V600E vs WT - wild type).aP < 0.05.

ARTICLE HIGHLIGHTSResearch backgroundA large proportion of patients with colorectal cancer (CRC) do not benefit from fluoro-pyrimidine-based adjuvant chemotherapy (FBAC). Fluoropyrimidines are thymidylate synthase(TYMS) inhibitors. Single nucleotide polymorphism (SNP) and various polymorphisms havebeen discovered in the 5’ untranslated region (UTR) and in the 3’UTR of the TYMS gene andtheir association with the survival of CRC patients is under consideration but with conflictingresults. Molecular profiling could help clinicians to identify patients with CRC who may benefitfrom adjuvant chemotherapy, as shown by the associations of BRAF mutations with inferiorsurvival in CRC patients after adjuvant chemotherapy. Also, although KRAS mutations havebeen found to be associated with poor patient survival, their role in the adjuvant setting is underinvestigation

Research motivationThere is a need to study the association of the numerous combinations of TYMS polymorphisms(3’UTR, 5’UTR and SNP) with CRC patient survival in a multivariate model includingclinicopathological patients’ features and KRAS/BRAF mutations. The loss of heterozygosity(LOH) affects polymorphisms and should be included in such a study.

Research objectivesThis study aimed to investigate the association of all known TYMS gene polymorphisms, LOH,KRAS and BRAF mutations with the survival of CRC patients treated with adjuvantchemotherapy.

Research methodsFormalin-fixed paraffin-embedded tissues of 130 consecutive patients treated with FBAC wereanalysed for the detection of TYMS polymorphisms, mKRAS and mBRAF. Patients wereclassified according to 5’UTR TYMS polymorphisms and the predicted expression profile, intothree groups (high, medium and low expression), utilizing the current literature. Thiscategorization could reduce classification errors. Based on the presence or absence of the 3’UTRpolymorphism ins/LOH patients were allocated into two groups (high and low risk of relapse),utilizing the results from univariate analysis of the 3’UTR TYMS polymorphisms. Cox regressionmodels examined the associated 5-year survival outcomes

Research resultsIn this study, where BRAF, TYMS polymorphisms including SNP G>C and LOH were taken intoconsideration, both 3’UTR and 5’UTR polymorphisms emerged as independent prognosticfactors of survival outcome after adjuvant chemotherapy for CRC. More specifically, the groupof patients with tumors bearing 5’UTR polymorphisms 2RG/3RG, 2RG/LOH and 3RC/LOHwas associated with better survival. On the contrary, patients with ins/LOH polymorphism inthe 3’UTR had worse survival outcome. Also, mBRAF was found to correlate independentlywith worse prognosis.

Research conclusionsKnowledge of TYMS gene polymorphisms and BRAF status indicates prognosis and could aidclinicians to distinguish the group of patients in need for adjuvant chemotherapy.

Research perspectivesThe study of the effect on the survival of CRC patients of the numerous genotypes resulting fromthe combinations of the 3’UTR and 5’UTR polymorphisms, the SNP and LOH requires largerprospective studies. These studies could validate our findings. Also, they could facilitate the



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grouping of the TYMS polymorphisms in more than just two groups and thus reduce theclassification errors.

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