Prevalence of Lynch Syndrome among Patients with Newly Diagnosed Endometrial Cancers

Prevalence of Lynch Syndrome among Patients withNewly Diagnosed Endometrial CancersCecilia Egoavil12 Cristina Alenda1 Adela Castillejo3 Artemio Paya1 Gloria Peiro1 Ana-Beatriz Saacutenchez-Heras4 Maria-Isabel Castillejo3 Estefaniacutea Rojas1 Viacutector-Manuel Barberaacute3 Sonia Ciguumlenza1 Jose-Antonio Lopez5 Oscar Pintildeero5 Maria-Jose Romaacuten5 Juan-Carlos Martiacutenez-Escoriza5 Carla Guarinos2Lucia Perez-Carbonell2 Francisco-Ignacio Aranda1 Jose-Luis Soto3

1 Pathology Alicante University Hospital Alicante Spain 2 Research Laboratory Alicante University Hospital Alicante Spain 3 Molecular GeneticsLaboratory Elche University Hospital Elche Spain 4 Genetic Counselling in Cancer Unit Elche University Hospital Elche Spain 5 Gynecology AlicanteUniversity Hospital Alicante Spain

Abstract

Background Lynch syndrome (LS) is a hereditary condition that increases the risk for endometrial and othercancers The identification of endometrial cancer (EC) patients with LS has the potential to influence life-savinginterventions We aimed to study the prevalence of LS among EC patients in our populationMethods Universal screening for LS was applied for a consecutive series EC Tumor testing using microsatelliteinstability (MSI) immunohistochemistry (IHC) for mismatch-repair (MMR) protein expression and MLH1-methylationanalysis when required was used to select LS-suspicious cases Sequencing of corresponding MMR genes wasperformedResults One hundred and seventy-three EC (average age 63 years) were screened Sixty-one patients (35) hadabnormal IHC or MSI results After MLH1 methylation analysis 27 cases were considered suspicious of LS Fromthese 22 were contacted and referred for genetic counseling Nineteen pursued genetic testing and eight werediagnosed of LS Mutations were more frequent in younger patients (lt50 yrs) Three cases had either intact IHC orMSS and reinforce the need of implement the EC screening with both techniquesConclusion The prevalence of LS among EC patients was 46 (8173) with a predictive frequency of 66 in theSpanish population Universal screening of EC for LS is recommended

Citation Egoavil C Alenda C Castillejo A Paya A Peiro G et al (2013) Prevalence of Lynch Syndrome among Patients with Newly DiagnosedEndometrial Cancers PLoS ONE 8(11) e79737 doi101371journalpone0079737

Editor Hiromu Suzuki Sapporo Medical University Japan

Received July 9 2013 Accepted September 23 2013 Published November 7 2013

Copyright copy 2013 Egoavil et al This is an open-access article distributed under the terms of the Creative Commons Attribution License which permitsunrestricted use distribution and reproduction in any medium provided the original author and source are credited

Funding This study was supported by Conselleria Sanidad Comunidad Valenciana Spain (AP17710) (httpwwwsangvaes) Biomedical ResearchFoundations of the Alicante University Hospital (PI142006 and NI022011) (httpwwwdep19sangvaes) and the Elche University Hospital Spain(FIBElx-CO1103) (httpwwwdep20sangvaes) The funders had no role in study design data collection and analysis decision to publish or preparationof the manuscript

Competing interests The authors have declared that no competing interests exist

E-mail sotomartinezjlgmailcom

Introduction

Identification of hereditary forms of neoplasias among cancerpatients is crucial for better management and prevention ofother syndrome-associated malignancies for the patients andtheir families [1] The estimated incidence and mortality countof endometrial cancer (EC) in Europe in 2012 were 58300 and24400 respectively EC accounts for approximately 4 of allcancers in women [2] The incidence is increasing andapproximately 5 of cases are thought to result from a geneticpredisposition [3]

Lynch syndrome (LS) is an autosomal dominant conditioncaused by a mutation in the mismatch repair (MMR) genes

MLH1 MSH2 MSH6 and PMS2 [4] Mutation carriers are atrisk of early onset colorectal cancer (CRC) EC and a spectrumof other tumors such as ovarian gastric small bowelpancreatic hepatobiliary brain and urothelial neoplasms [5]The cumulative lifetime risk of EC for female carriers of anMMR mutation is 50ndash60 and exceeds the risk of a CRC [6]

The identification of patients with EC and LS has thepotential to influence life-saving interventions throughpersonalized counseling and intensive cancer surveillance withearly detection screening and prevention of other LS-associated cancers Genetic testing is now an accepted part ofthe management of patients with such cancers The Mallorcagroup [7] recommended testing all cases of CRC (or individuals

PLOS ONE | wwwplosoneorg 1 November 2013 | Volume 8 | Issue 11 | e79737

with a CRC aged lt70 years) and all cases of EC (or individualswith an EC aged lt70 years) by immunohistochemistry (IHC) forMMR genes or chromosomal microsatellite instability (MSI)

The prevalence of LS among unselected cases of CRC hasbeen studied well The results indicate that 07 to 36 of allsuch cases might be caused by germline mutations in MMRgenes [8ndash12] On the contrary research on LS-related ECs isstill evolving and little is known about the genetic componentsamong patients with EC Current data on the prevalence of LSamong unselected cases of EC in North America rangebetween 18 and 45 [13ndash15] Significant differences in theprevalence of hereditary syndromes are frequently observedamong different populations [12] Here we report on theprevalence of LS in a consecutive series of patients with ECfrom the Spanish population

Materials and Methods

Ethical issuesTumor tissue and blood samples from patients with EC were

obtained from the Biobank of the Alicante University Hospital(HGUA) in Spain Written consent to be included in the Biobankwas obtained from each patient The Ethics Committee ofHGUA approved the study

SubjectsOne hundred seventy-three consecutive patients with newly

diagnosed ECs were included in this study They werediagnosed and treated at the HGUA from 2004ndash2009 For eachcase all available hematoxylinndasheosin slides were reviewed andclassified using the 2003 World Health Organization criteria[16] Additional histopathology features recorded were thepresence of lymphovascular invasion (LVI) tumor-infiltratinglymphocytes (TILs) myometrial invasion and grade and stageaccording to the International Federation of Obstetrics andGynaecology (FIGO) The adenocarcinomas were classifiedinto endometrioid (type I) and special (type II) according toPeiroacute et al 2013 [17] A familial history of cancer was evaluatedaccording to the revised Bethesda guidelines (rBG) andAmsterdam II criteria (AmII) [18]

Immunohistochemistry of MMR proteinsIHC analysis of the expression of the MLH1 MSH2 MSH6

and PMS2 proteins was performed using a tissue microarray(TMA) Tissue cylinders of 1 mm diameter were punched outfrom selected areas and incorporated into a recipient paraffinblock using a TMA instrument (MTA-1Beecher InstrumentWisconsin USA) Four-millimeter-thick sections were preparedfrom the TMA samples The slides were placed on aAutostainer Link48 (Dako Denmark) and incubated for 30 minat room temperature with primary antibodies to MLH1 (cloneG168ndash15 130 BD Biosciences Pharmingen Franklin LakesNJ USA) MSH2 (clone 44 dilution 1100 BD TransductionLaboratories San Diego CA USA) MSH6 (clone FE11dilution 130 Calbiochem Merck Millipore Billerica MA USA)and PMS2 (clone A16ndash4 dilution 1100 BD BiosciencesPharmingen) Antibodies were detected using the EnVision

technique (Dako-Biotech) Tumor cells were judged asnegative for protein expression only if they lacked IHC stainingin a sample in which normal endometrial cells and stromal cellswere stained concurrently Results were considered unreliablein those cases where no immunostaining of normal tissue couldbe demonstrated The processed IHC slides were blindedevaluated by two pathologists (CE and CA) [19]

DNA extractionGenomic DNA was isolated from paraffin wax-embedded

tumor tissues Two 1 mm tissue cylinders were punched outfrom the tumor areas selected previously DNA from peripheralblood leukocytes or from paraffin wax-embedded nontumorousendometrium tissue was also extracted from those caseswhere LS was suspected A DNeasy Blood amp Tissue kit andQiaCube (Qiagen Valencia CA USA) automatic system wasused to isolate DNA according to the manufacturerrsquos protocol

Microsatellite analysis and MMR statusMSI status was analyzed using multiplexed polymerase

chain reaction (PCR) patterns at the monomorphic repetitivemarkers BAT26 BAT25 NR21 NR24 and NR27 [20]Amplicon detection and analysis were performed using an ABIPrism 3130 Genetic Analyzer and Genotyper software (LifeTechnologies Carlsbad CA USA) respectively A diagnosis ofMSI was considered positive when two or more markersshowed an altered pattern Tumors with MSI andor loss ofexpression of any of the MMR proteins were considered asMMR-deficient Tumors with absence of MSI and preservedMMR protein expression were considered as MMR-positivetumors

MLH1 promoter hypermethylation analysisCases with loss of MLH1 expression were tested for MLH1

methylation in the tumor DNA Any cases with such methylationwere then analyzed for MLH1 methylation in DNA from bloodcells to identify suspected constitutional MLH1 epimutations(Figure 1) We used the methylation-specific multiplex ligation-dependent probe-amplification technique (MS-MLPA KitME011 MRC-Holland Amsterdam The Netherlands) to studythe methylation status of MLH1 according to themanufacturerrsquos protocols The target regions for MLH1 genesilencing by hypermethylation are located in the C and Dregions of the MLH1 promoter (from nucleotide positions -248to -178 and -109 to +15) [21] which were tested using theprobes MLH1-3 and -4 respectively The mean results forthese two probes were calculated to obtain the methylationratio The threshold for methylated versus unmethylated statuswas set at 15 based on a previous study of MLH1 genesilencing [22] MS-MPLA fragments were analyzed using anABI Prism 3130 Genetic Analyzer and Genotyper software(Life Technologies)

Germline mutationsPatients in whom we suspected LS and were candidates for

genetic testing were referred to the Genetic Counseling divisionin our unit Suspicion of LS was based on MMR status (IHC

Lynch Syndrome in Endometrial Cancer Patients


and MSI results) and the MLH1 methylated status according toour decision tree algorithm (Figure 1) MLH1 gene testing wasperformed in cases where tumors showed loss of proteinexpression and unmethylated MLH1 MSH2 mutationalanalysis was performed in those cases with MSH2-negativestaining tumors MSH6 germline analysis was done in patientswith a lack of MSH6 protein expression but with normalexpression of MSH2 Tumors with a combined lack of MSH2and MSH6 proteins with an undetected mutation in MSH2 werealso tested for MSH6 genetic alterations Cases tested forMSH2 and MSH6 with no mutation detected were alsoanalyzed for large rearrangements at the EPCAM locusGenetic testing for PMS2 was performed only in those patientswith tumors showing loss of expression of PMS2 and normalexpression of MLH1

Germline mutation studies were performed on genomic DNAisolated from peripheral blood leucocytes or from nontumorousendometrial tissue Detection of point mutations was conductedusing PCR and direct sequencing of the whole codingsequence and intronndashexon boundaries for each gene [12]

Large rearrangements (deletions andor insertions) for MMRgenes were screened by MLPA according to the manufacturerprotocols (Salsa MLPA kits P003 P072 and P008 MRC-Holland) Confirmation testing was also performed using MLPAwith a different combination of probes (Salsa MLPA kit P248MRC-Holland) Analysis of deletions at the EPCAM locus wasalso done using MLPA (Salsa MLPA kit P072-B1 MRC-Holland) The interpretation of genetic analysis results wasbased on the American College of Medical Genetics (ACMG)Recommendations for Standards for Interpretation ofSequence Variations [23] the InSiGHT database [24] and thereferences therein were reviewed for classifying geneticvariants

Data management and statistical analysisAnalysis was carried out using R software version 2152

(The R Project for Statistical Computing Available httpwwwr-projectorg Accessed 2013 Oct 21) and the Epicalcepidemiological analysis package [25] Relevant measures ofcentral tendency (means medians and interquartile ranges for

Figure 1 Decisionndashsupport flow diagram for patients with Lynch syndrome genetic diagnosis strategy doi 101371journalpone0079737g001



skewed data) were used to explore the data The chi-squaredtest was used to compare qualitative variables Studentrsquos t testwas used to compare normally distributed continuousvariables Significance was set at p lt 005 and results arepresented as the odds ratio (OR) and 95 confidence interval(CI)

Results

A total of 173 unselected patients with ECs was includedThe mean age at diagnosis was 633 years (range 29ndash90) Allpatients underwent biopsy or hysterectomy and paraffin wax-embedded tissues were available for IHC and moleculartesting The clinical and histopathology characteristics of thetumors are shown in Table 1 Most tumors were ofendometrioid histology (792) and FIGO grade 1 (549) Nomyometrial invasion was found in 103 of patients (15146)About 637 of cases had myometrial invasion le50 (96146)and 26 (38146) had myometrial invasion gt50 TILs werepresent in 294 of tumors (47160) and 179 of tumors hadLVI (24134) Twenty-six patients (171) had synchronousendometrial and ovarian cancers

Familial histories of cancer histopathology and molecularcharacteristics regarding LS are shown in Table 2 Eightpatients (46) had a history of colorectal lesions and 10(58) had a history of breast cancer and other neoplasias Afamilial history of cancer was available in 87 cases 42 (483)fulfilled the rBG criteria and four (46) fulfilled the AmIIcriteria Family history of cancer from the remaining 86 caseswas unavailable because unconfirmed family history were lostto follow-up or deceased

We found an altered MMR picture (loss of MMR proteinexpression andor MSI) in 61 patients (353) Loss of MLH1expression was found in 44 patients (254) From these 34(773) showed MLH1 hypermethylation in the tumorThereafter MLH1 hypermethylation analysis was done in nineof these cases where DNA from blood cells was available butgave negative results in all of them Thus all tumors with MLH1hypermethylation were considered sporadic ECs

Loss of MSH2MSH6 was detected in five patients (29)while loss of only MSH6 or only PMS2 protein expression wasobserved in eight (46) One case with both losses of MSH6and PMS2 was found (Table S1 in File S1) All these casestogether with another 10 cases with loss of MLH1 expressionand absence of MLH1 methylation were considered suspiciousof LS and suitable for genetic testing

A significant association was found between the IHC andMSI results (p lt 00001) showing concordance in 902 ofcases (156173) Discordances between IHC and MSI werefound in 17 cases (98) Loss of expression of MMR proteinsand MSS was found in 14 cases Normal expression of MMRproteins and MSI were found in three cases

We did not have evidence for a sporadic origin of thesetumors so they were also considered as suspected LS Finallya total of 27 (156) cases were included in the geneticanalysis for germline mutation screening

Comparative analysis among the suspected cases of LS andcases with MMR proficient ECs showed that the suspected

hereditary condition was more frequently found in womenyounger than 50 years (OR 284 95 CI 104ndash777) Nosignificant differences were found in any other clinical orpathological variables (Table 3) Similar results were obtainedwhen we compared patients showing abnormal MMR withthose with retained MMR function (Table S2 in File S1) withthe exception of TIL and LIV which was strongly associatedwith abnormal MMR tumors (OR 588 95 CI 280ndash1231 andOR 303 95 CI 122ndash756 respectively)

Germline mutation analyses were performed for 19 patientsof the suspected LS group (1927 704) Genetic test resultsfor the remaining eight patients were unavailable because theyrejected the testing (327 111) were lost to follow-up (327111) or deceased (227 74)

We found eight patients with pathogenic mutations (819421) representing 46 of the whole series one in MLH1three in MSH2 three in MSH6 and one in PMS2 genes (Table4) The mean age of these patients was 49 years significantly

Table 1 Clinical and pathologic characteristics

Variable N [ ]Number of patients 173 Mean age (SD) and range in years 6327(+- 1237) 29-90 N

Stage at initial diagnosis 158 I 119 753II 6 38III 31 196IV 2 13

Histology 173 Endometroid 137 792Poorly Diferenciated 8 46Papilary Serous 13 75Clear Cell 11 64Muumllerian Mixet Tumor 4 23

Grade (FIGO) 173 1 95 5492 29 1683 49 283

Myometrial invasion 146 None 15 103le50 93 637gt50 38 260

Tumor infiltrating lymphocytes 160 No 113 706Yes 47 294

Lymphovascular invasion 134 No 110 821Yes 24 179

Low Uterin Segment 173 No 157 908Yes 16 92

Synchronous ovarian cancer 152 No 126 829Yes 26 171

doi 101371journalpone0079737t001



lower than in the non-LS EC group (Table S3 in File S1)Twenty-five percent of them did not fulfill the rBth criteria andanother 25 showed negative MSI (with isolated loss ofprotein expression in MSH6 and PMS2) (Table S4 in File S1)Two patients had synchronous ovarian cancers and one had asynchronous colon cancer (Table 5) The presence ofsynchronous ovarian cancer and tumor infiltrating lymphocyteswas associated to confirmed LS cases when compared to non-LS EC (Table S3 in File S1)

Three of the mutations (375) were large deletions (two inMSH2 and one in PMS2) another three (375) werenonsense mutations (all in the MSH6 gene) and two (25)were frameshift mutations (one insertion in MLH1 and onedeletion in MSH2) Another two patients showed geneticmissense variants of unknown clinical significance both inMSH6 (c116GA pGly39Glu and c1109TC pLeu370Ser)Prediction using Polyphen-2 (httpgeneticsbwhharvardedupph2) classified both variants as probably pathogenic Inaddition both tumors showed loss of expression of MSH6 withconserved expression of MSH2 and absence of MSI

In the association study comparing mutated with nonmutatedcases only age was found to be significant Mutations weremore frequently found in patients with EC diagnosed before 50years of age (OR 1667 95 CI 101ndash58803 p = 0048) Nosignificant associations were found regarding rBG criteria IHCMSI status histopathology or the presence of synchronoustumors

Table 2 Features related to Lynch syndrome

Variable N Lynch syndrome criteria 87 Reviewed Bethesda criteria 38 4368Amsterdam II criteria 4 460No Fullfill 45 5172

Personal History of Colorectal Lesions 8 Colon cancer 6 347

Polyps 2 116

Personal History Others Tumors 15 Breast cancer 10 578

Lung cancer 2 116

Pleura 1 058

Thyrod cancer (Medular) 1 058

Urothelial Cancer 1 058

IHC protein expression Loss of MLH1MSH2MSH6PMS2 173 No 115 6647Yes 58 3353

MSI Status 173 MSS 126 7283MSI 47 2717

Mismatch Repair status 173 Proficient 112 6474Deficient 61 3526

Calculated from whole series (n=173)doi 101371journalpone0079737t002

Discussion

A small proportion of ECs might be the result of a geneticrisk condition [3] LS is the main syndrome involved in suchcases [56] although the existence of a familial site-specific ECgenetic entity separate from LS has been sugested [26] Thecurrent data about the prevalence of LS among patients with

Table 3 Comparative analysis of patients with suspectedLynch syndrome and MMR proficient endometrial cancers

Suspected LS MMR proficient pNumber of patients 27 146 Mean age (SD) 5770 (1245) 6431 (1212) 001 N N OR (IC 95) p

Age lt50 yrs 7 2593 16 1096 284 (104-777) 004ge50 yrs 20 7407 130 8904

Histology Endometroid (type I) 22 8148 123 8425 082 (028-239) 072Special (type II) 5 1852 23 1575

Grade (FIGO) High 9 3333 40 2740 132 (05-319) 053Low 18 6667 106 7260

Myometrialinvasion

(n=146) gt50 6 2500 32 2623 093 (034-257) 090le50 18 7500 90 7377

Tumor infiltratinglymphocytes

(n=160) Yes 9 3333 38 2857 125 (052-303) 062No 18 6667 95 7143

Lymphovascular invasion

(n=134) Yes 7 3043 17 1532 242 (087-676) 009No 16 6957 94 8468

Low UterinSegment

Yes 4 1481 12 822 194 (057-654) 028No 23 8519 134 9178

Synchronous ovariancancer

(n=152) Yes 5 1852 22 1760 106 (036-311) 091No 22 8148 103 8240

Reviewed Bethesda criteria

(n=86) Fullfill 14 6087 28 4444 194 (073-514) 018No Fullfill 9 3913 35 5556

Amsterdam IIcriteria





EC is limited and restricted to populations from the UnitedStates [13ndash15] Differences in the prevalence of geneticdiseases are frequently observed between differentpopulations especially for syndromes where the penetrance isincomplete and other genetic and environmental factors mightact as penetrance modifiers

We aimed to determine the prevalence of LS among patientswith ECs in our Spanish population to establish properscreening strategies for identifying individuals with geneticpredispositions to other tumors consequently to identify familymembers at risk and establish personalized follow-uprecommendations

We used a prevalence study using establishedrecommendations and consensus algorithms for screening andmutation analysis [7] To maximize our mutation detectionsensitivity no limit on age at diagnosis was considered IHCand MSI analysis were performed for all tumors MLH1methylation was tested in tumors and also in blood whenrequired and mutational analysis was done for the fourcommon MMR and EPCAM genes

Our IHC and MSI screening detected 353 (61173) oftumors with deficient MMR which is higher than those 228(124543) and 253 (62245) found by Hampel et al2006 andMoline et al 2013 respectively We found normal IHCexpression of MMR proteins together with MSI in 361 cases(49) Similar results were obtained by Hampel et al 2006(63 696) [13] Moreover we found that about 23 (1461)

Table 4 IHC patterns of suspected Lynch syndrome

IHC pattern Suspected LynchGermline mutation analyzedcases

Loss of MLH1 no Methylated 10 16

Loss of MSH2MSH6 5 34

Loss of MSH6 7 25

Loss of PMS2 1 11

Loss of MSH6PMS2 1 01

MSI + normal IHC 3 12

TOTAL 27 819


of tumors had loss of expression of an MMR protein and MSIThe expected percentage of loss of expression and MSI in theHampel (2006) study [13] would be about 15 Interestinglywe found that two unrelated patients with these apparentdiscordances had the same mutation at MSH6 (c1367GApTrp456) suggesting that in some circumstances the natureof the second hit could be determinant for the tumorrsquos IHC andMSI characteristics (Table 5) Our data shows that in patientswith EC up to 279 of cases with MMR deficiency might haveeither normal IHC results or MSI and reinforces the need forimplementing screening with both techniques

Comparative analysis of our MMR deficient versus positivecases did show a significant association with LVI and TILs asdescribed elsewhere [2728] However no association wasfound between age at diagnosis and the rBG or AmII criteria(Table S2 in File S1) These results could be explained by thefrequent occurrence of somatic MMR deficiency in ECs [13]MLH1 methylation and BRAF mutation analyses allow one to

identify sporadic forms of CRC among MMR-deficient tumorsWe found no case mutated in BRAF (data not shown) Thusthis gene does not have a major role in EC and should not beincluded in the screening algorithm [15] In contrast MLH1promoter methylation was present in the majority of tumorsalong with the loss of expression of MLH1 (773 3444) Thisproportion was significantly lower than the 94 (7984) foundby Hampel et al 2006 [13] Differences among the two cohortsand different methodologies for the methylation analysis (MS-MLPA vs MS-PCR) might have contributed to this disparity

Constitutional epimutations to the MLH1 gene in cases of ECare very rare [29] None of the nine cases analyzed with tumormethylation had MLH1 methylation in blood DNA Thus atumor with MLH1 methylation is unlikely to be LS-associated Incontrast a tumor that shows loss of MLH1 and PMS2 by IHCwith no evidence of methylation is likely to be associated withLS [1]

After the initial screening we selected 27 patients (1561)suspected of LS The mean age in this group was 577 yearsand patients younger than 50 were more frequent (Table 3) Noother pathological or clinical characteristic was found to beassociated with suspected LS cases Currently there havebeen attempts to identify pathological factors in LS-associatedEC Some authors have identified the tumor localizationreporting a significantly higher prevalence of lower uterine

Table 5 Characteristics of patients with germline mutation

Case rBG Age MSI status IHC loss Gene Nucleotide nomenclature Protein nomenclature Synchronous tumorlesionsEnd131 Yes 41 MSI MLH1PMS2 MLH1 c2154_2157insAACA pHis718Glnfs5 Colonic PolypEnd111 Yes 45 MSI MSH2MSH6 MSH2 c1-_645+del Deletion of exon 1-3 p Colon cancerEnd091 Yes 40 MSI MSH2MSH6 MSH2 c1226_1227delAG pGln409Argfs7 Ovarian cancerEnd003 Yes 60 MSI MSH2MSH6 MSH2 c1387-_1661del Deletion of exon 9-10 p NoneEnd014 No 61 MSI MSH6 MSH6 c2731CT pArg911 NoneEnd088 Yes 45 MSS MSH6 MSH6 c1367GA pTrp456 Ovarian cancerEnd137 No 56 MSI No loss MSH6 c1367GA pTrp456 NoneEnd034 Yes 44 MSS PMS2 PMS2 c538-_705+del Deletion of exon 6 p None

Not described at InSiGHT databasedoi 101371journalpone0079737t005



segment tumors in patients with LS [3031] Westin et alconcluded that screening for LS should be considered in caseswith ECs originating in the lower uterine segment [30] Thislocation can be a source of diagnostic consternation because itcan host both endometrial and endocervical carcinomasresulting in tumor misclassification [32]

Mutations were found in eight patients (421) with a meanage of 49 years MSH6 and MSH2 were mutated in six cases(Table 5) Two cases with mutations MSH6 (ages 56 and 61years) did not fulfill the rBG criteria and no other synchronoustumor was present at diagnosis Thus about 25 of patientswith EC associated with LS might appear to have sporadictumors and go undiagnosed when the rBG criteria are used totest a suspicion of LS LS-related ECs commonly result frommutations in MSH6 and occur at later ages than do mutationsin MLH1 and MSH2 [3334] For patients with LS-related ECsthe risk of developing a second cancer following the initial ECdiagnosis is estimated at 25 in 10 years and 50 at 15 years[3536] The 20-year cumulative risk of cancer after endometrialcancer has been recently reported with 48 risk for CRC 11for cancer of the kidney renal pelvis or ureter 9 for urinarybladder cancer and 11 for breast cancer [37] Amongpatients with LS 50 of the ECs present before a diagnosis ofCRC if the diagnoses are not synchronous Therefore EC canserve as a lsquosentinelrsquo cancer for patients and potentially for theirfamily members [38]

In our study mutations were found more frequently inpatients with ECs diagnosed before 50 years of age and noassociation was found regarding the rBG criteria IHC or MSIstatus histopathology and the presence of synchronoustumors However it is possible that the limited sample sizecould be hiding any other association

There are several possibilities to explain those cases withundetected germline mutations First presence of mutations atuntested regulatory regions of the analyzed genes second anMMR deficiency caused by somatic biallelic inactivation thirdgenetic mosaicism fourth germline mutations in other genesdirectly or indirectly involved with MMR function such asSETD2 [39] POLE and POLD1 [40] and finally other unknowngenetic or epigenetic mechanisms (Figure 1)

Many institutions and policy groups are considering whetherto implement screening for patients with LS among patientswith ECs Different screening strategies for women with EChave been shown to be cost effective [4142] IHC triage of allcases of EC can identify most mutation carriers but atconsiderable cost The inclusion of at least one first-degreerelative with an LS-associated cancer at any age might provecost effective [42] Nevertheless it is important to stress that

multiple local factors might interfere in the efficiency of anyprocess New approaches for making the universal screeningof patients with EC by IHC more cost effective are in progressRecent data suggest that a two-antibody panel testing forPMS2 and MSH6 is as effective as the four-antibody panel fordetecting MMR abnormalities [4344] We observed that toincrease sensibility in LS diagnosis an analysis of MSIcombined with IHC should be considered because about 5(361) of patients with suspected LS and one-eighth ofconfirmed LS cases in our study had MSI with intact IHC

The prevalence of LS we found among patients with EC was46 (8173) with a predictive frequency of 66 for theSpanish population This prediction was made extrapolating thefound frequency of mutated cases and considering the possibleabsence of lost cases Is important to note that the presentwork is a prevalence study conducted in a single hospital fromSpain therefore extrapolation of data to the whole Spanishpopulation may be biased

Previous studies from North American populations showedprevalence rates ranging from 18 to 45 [13ndash15] In ourpopulation we found that the prevalence of LS among patientswith EC is sixfold higher than the prevalence of LS amongpatients with CRCs (46 vs 07) [12]

In conclusion we found a high prevalence of LS amongpatients with ECs (46ndash66) Unlike CRC only the patientrsquosage at diagnosis was found to be associated with LSConsistent with these results we consider that universalscreening of all patients with ECs by IHC MSI and MLH1methylation analysis should be recommended

Supporting Information

File S1 Supporting informationTable S1 Result of the pre-genetic test screening Table S2Factors associated with mismatch repair profile Table S3Comparative analysis of patients with Lynch syndrome andnon-LS endometrial cancers Table S4 Lynch syndromerelated variables and Bethesda guidelines(DOC)

Author Contributions

Conceived and designed the experiments CA JLS AP CEPerformed the experiments CE AC ER MIC VMB CG LPC GPAnalyzed the data CE CA JLS AP FIA Contributed reagentsmaterialsanalysis tools ASH JCME MJR JAL SC CE GP CAOP Wrote the manuscript CE JLS CA

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3 Gruber SB Thompson WD (1996) A population-based study ofendometrial cancer and familial risk in younger women Cancer and

Steroid Hormone Study Group Cancer Epidemiol Biomarkers Prev 5411-417

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5 Lynch HT de la Chapelle A (2003) Hereditary colorectal cancer N EnglJ Med 348 919ndash932 doi101056NEJMra012242 PubMed 12621137

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8 Aaltonen LA Salovaara R Kristo P Canzian F Hemminki A et al(1998) Incidence of hereditary nonpolyposis colorectal cancer and thefeasibility of molecular screening for the disease N Engl J Med 3381481-1487 doi101056NEJM199805213382101 PubMed 9593786

9 Salovaara R Loukola A Kristo P Kaumlaumlriaumlinen H Ahtola H et al (2000)Population-based molecular detection of hereditary nonpolyposiscolorectal cancer J Clin Oncol 18 2193-2200 PubMed 10829038

10 Hampel H Frankel WL Martin E Arnold M Khanduja K et al (2005)Screening for the Lynch syndrome (hereditary nonpolyposis colorectalcancer) N Engl J Med 352 1851-1860 doi101056NEJMoa043146PubMed 15872200

11 Hampel H Frankel WL Martin E Arnold M Khanduja K et al (2008)Feasibility of screening for Lynch syndrome among patients withcolorectal cancer J Clin Oncol 26 5783-5788 doi101200JCO2008175950 PubMed 18809606

12 Peacuterez- Carbonell L Ruiz-Ponte C Guarinos C Alenda C Payaacute A et al(2012) Comparison between universal molecular screening for Lynchsyndrome and revised Bethesda guidelines in a large population-basedcohort of patients with colorectal cancer Gut 61 865-872 doi101136gutjnl-2011-300041 PubMed 21868491

13 Hampel H Frankel W Panescu J Lockman J Sotamaa K et al (2006)Screening for Lynch syndrome (hereditary nonpolyposis colorectalcancer) among endometrial cancer patients Cancer Res 667810-7817 doi1011580008-5472CAN-06-1114 PubMed 16885385

14 Hampel H Panescu J Lockman J Sotamaa K Fix D et al (2007)Comment on Screening for Lynch Syndrome (Hereditary NonpolyposisColorectal Cancer) among Endometrial Cancer Patients Cancer Res67 9603 doi1011580008-5472CAN-07-2308 PubMed 17909073

15 Moline J Mahdi H Yang B Biscotti C Roma AA et al (2013)Implementation of Tumor Testing for Lynch Syndrome in EndometrialCancers at a Large Academic Medical Center Gynecol Oncol 130121-126 doi101016jygyno201304022 PubMed 23612316

16 Silverberg SG Kurman RJ Nogales F (2003) Tumours of the uterinecorpus In FA TavassoliP Devilee Pathology and genetics of tumoursof the breast and female genital organs Lyon IARC Press pp217-228

17 Peiroacute G Peiroacute FM Ortiz-Martiacutenez F Planelles M Saacutenchez-Tejada L etal (2013) Association of mammalian target of rapamycin withaggressive type II endometrial carcinomas and poor outcome apotential target treatment Hum Pathol 44 218-225 doi101016jhumpath201205008 PubMed 22955108

18 Umar A Boland CR Terdiman JP Syngal S de la Chapelle A et al(2004) Revised Bethesda Guidelines for hereditary nonpolyposiscolorectal cancer (Lynch syndrome) and microsatellite instability J NatlCancer Inst 96 261-268 doi101093jncidjh034 PubMed 14970275

19 Jover R Payaacute A Alenda C Poveda MJ Peiroacute G et al (2004) Defectivemismatch-repair colorectal cancer clinicopathologic characteristics andusefulness of immunohistochemical analysis for diagnosis Am J ClinPathol 122 389-394 doi101309V9PGK2Y260VFVULR PubMed15362369

20 Suraweera N Duval A Reperant M Vaury C Furlan D et al (2002)Evaluation of tumor microsatellite instability using fivequasimonomorphic mononucleotide repeats and pentaplex PCRGastroenterology 123 1804-1811 doi101053gast200237070PubMed 12454837

21 Deng G Chen A Hong J Chae HS Kim YS (1999) Methylation of CpGin a small region of the hMLH1 promoter invariably correlates with theabsence of gene expression Cancer Res 59 2029-2033 PubMed10232580

22 Peacuterez-Carbonell L Alenda C Payaacute A Castillejo A Barberaacute VM et al(2010) Methylation analysis of MLH1 improves the selection of patientsfor genetic testing in Lynch syndrome J Mol Diagn 12 498-504 doi102353jmoldx2010090212 PubMed 20489114

23 Richards CS Bale S Bellissimo DB Das S Grody WW et al (2008)Molecular Subcommittee of the ACMG Laboratory Quality AssuranceCommittee ACMG recommendations for standards for interpretationand reporting of sequence variations Revisions2007 Genet Med 10294-300 doi101097GIM0b013e31816b5cae PubMed 18414213

24 Ou J Niessen RC Vonk J Westers H Hofstra RM et al (2008) Adatabase to support the interpretation of human mismatch repair genevariants Hum Mutat 29 1337-1341 doi101002humu20907PubMed 18951442

25 Chongsuvivatwong Virasakdi (2012) epicalc Epidemiologicalcalculator R package version 21510 Retrieved onpublished at whilstDecember year 1111 from httpCRANR-projectorgpackage= epicalc

26 Cook LS Nelson HE Stidley CA Dong Y Round PJ et al (2013)Endometrial cancer and a family history of cancer Gynecol Oncol 130334-339 doi101016jygyno201304053 PubMed 23632205

27 Shia J Ellis NA Paty PB Nash GM Qin J et al (2003) Value ofhistopathology in predicting microsatellite instability in hereditarynonpolyposis colon cancer and sporadic colon cancer Am J SurgPathol 27 1407ndash1417 doi10109700000478-200311000-00002PubMed 14576473

28 Ryan P Mulligan AM Aronson M Ferguson SE Bapat B et al (2012)Comparison of clinical schemas and morphologic features in predictingLynch syndrome in mutation-positive patients with endometrial cancerencountered in the context of familial gastrointestinal cancer registriesCancer 118 681-688 doi101002cncr26323 PubMed 21721000

29 Hitchins MP Ward RL (2009) Constitutional (germline) MLH1epimutation as an aetiological mechanism for hereditary non-polyposiscolorectal cancer J Med Genet 46 793-802

30 Westin SN Lacour RA Urbauer DL Luthra R Bodurka DC et al(2008) Carcinoma of the lower uterine segment a newly describedassociation with Lynch syndrome J Clin Oncol 26 5965-5971 doi101200JCO2008186296 PubMed 19001318

31 Masuda K Banno K Hirasawa A Yanokura M Tsuji K et al (2012)Relationship of lower uterine segment cancer with Lynch syndrome anovel case with an hMLH1 germline mutation Oncol Rep 28(5)1537-1543 PubMed 22940821

32 Mills AM Liou S Kong CS Longacre TA (2012) Are women withendocervical adenocarcinoma at risk for lynch syndrome Evaluation of101 cases including unusual subtypes and lower uterine segmenttumors Int J Gynecol Pathol 31 463-469 doi101097PGP0b013e31824a1dad PubMed 22833088

33 Buttin BM Powell MA Mutch DG Babb SA Huettner PC et al (2004)Penetrance and expressivity of MSH6 germline mutations in sevenkindreds not ascertained by family history Am J Hum Genet 74 1262ndash1269 doi101086421332 PubMed 15098177

34 Hendriks YM Wagner A Morreau H Menko F Stormorken A et al(2004) Cancer risk in hereditary nonpolyposis colon cancer due toMSH6 mutations impact on counseling and surveillanceGastroenterology 1 17ndash25

35 Lynch HT Harris RE Lynch PM Guirgis HA Lynch JF Bardawil WA(1977) Role of heredity in multiple primary cancer Cancer 401849-1854 doi1010021097-0142(197710)404+ PubMed 907988

36 Mecklin JP Jaumlrvinen HJ (1986) Clinical features of colorectalcarcinoma in cancer family syndrome Dis Colon Rectum 29 160-164doi101007BF02555012 PubMed 3943429

37 Win AK Lindor NM Winship I Tucker KM Buchanan DD et al (2013)Risks of colorectal cancer and other cancers after endometrial cancerfor women with Lynch syndrome J Natl Cancer Inst 105 274-279 doi101093jncidjs525 PubMed 23385444

38 Lu KH Dinh M Kohlmann W Watson P Green J et al (2005)Gynecologic cancer as a lsquosentinel cancerrsquo for women with hereditarynonpolyposis colorectal cancer syndrome Obstet Gynecol 105569-574 doi10109701AOG000015488544002ae PubMed15738026

39 Li F Mao G Tong D Huang J Gu L et al (2013) The Histone MarkH3K36me3 Regulates Human DNA Mismatch Repair through ItsInteraction with MutSα Cell 153 590-600 doi101016jcell201303025 PubMed 23622243

40 Church DN Briggs SE Palles C Domingo E Kearsey SJ et al ( 2013)DNA polymerase varepsilon and δ exonuclease domain mutations inendometrial cancer Hum Mol Genet 22 2820-2828 doi101093hmgddt131 PubMed 23528559

41 Resnick K Straughn JM Jr Backes F Hampel H Matthews KS CohnDE (2009) Lynch syndrome screening strategies among newlydiagnosed endometrial cancer patients Obstet Gynecol 114 530-536doi101097AOG0b013e3181b11ecc PubMed 19701031

42 Kwon JS Scott JL Gilks CB Daniels MS Sun CC Lu KH (2011)Testing women with endometrial cancer to detect Lynch syndrome JClin Oncol 29 2247-2252 doi101200JCO2010329979 PubMed21537049

43 Shia J Tang LH Vakiani E Guillem JG Stadler ZK et al (2009)Immunohistochemistry as first-line screening for detecting colorectalcancer patients at risk for hereditary nonpolyposis colorectal cancersyndrome a 2-antibody panel may be as predictive as a 4-antibodypanel Am J Surg Pathol 33 1639-1645 doi101097PAS0b013e3181b15aa2 PubMed 19701074

44 Mojtahed A Schrijver I Ford JM Longacre TA Pai RK (2011) A two-antibody mismatch repair protein immunohistochemistry screening



approach for colorectal carcinomas skin sebaceous tumours andgynecologic tract carcinomas Mod Pathol 24 1004-1014 doi101038modpathol201155 PubMed 21499234



with a CRC aged lt70 years) and all cases of EC (or individualswith an EC aged lt70 years) by immunohistochemistry (IHC) forMMR genes or chromosomal microsatellite instability (MSI)

The prevalence of LS among unselected cases of CRC hasbeen studied well The results indicate that 07 to 36 of allsuch cases might be caused by germline mutations in MMRgenes [8ndash12] On the contrary research on LS-related ECs isstill evolving and little is known about the genetic componentsamong patients with EC Current data on the prevalence of LSamong unselected cases of EC in North America rangebetween 18 and 45 [13ndash15] Significant differences in theprevalence of hereditary syndromes are frequently observedamong different populations [12] Here we report on theprevalence of LS in a consecutive series of patients with ECfrom the Spanish population

Materials and Methods

Ethical issuesTumor tissue and blood samples from patients with EC were

obtained from the Biobank of the Alicante University Hospital(HGUA) in Spain Written consent to be included in the Biobankwas obtained from each patient The Ethics Committee ofHGUA approved the study

SubjectsOne hundred seventy-three consecutive patients with newly

diagnosed ECs were included in this study They werediagnosed and treated at the HGUA from 2004ndash2009 For eachcase all available hematoxylinndasheosin slides were reviewed andclassified using the 2003 World Health Organization criteria[16] Additional histopathology features recorded were thepresence of lymphovascular invasion (LVI) tumor-infiltratinglymphocytes (TILs) myometrial invasion and grade and stageaccording to the International Federation of Obstetrics andGynaecology (FIGO) The adenocarcinomas were classifiedinto endometrioid (type I) and special (type II) according toPeiroacute et al 2013 [17] A familial history of cancer was evaluatedaccording to the revised Bethesda guidelines (rBG) andAmsterdam II criteria (AmII) [18]

Immunohistochemistry of MMR proteinsIHC analysis of the expression of the MLH1 MSH2 MSH6

and PMS2 proteins was performed using a tissue microarray(TMA) Tissue cylinders of 1 mm diameter were punched outfrom selected areas and incorporated into a recipient paraffinblock using a TMA instrument (MTA-1Beecher InstrumentWisconsin USA) Four-millimeter-thick sections were preparedfrom the TMA samples The slides were placed on aAutostainer Link48 (Dako Denmark) and incubated for 30 minat room temperature with primary antibodies to MLH1 (cloneG168ndash15 130 BD Biosciences Pharmingen Franklin LakesNJ USA) MSH2 (clone 44 dilution 1100 BD TransductionLaboratories San Diego CA USA) MSH6 (clone FE11dilution 130 Calbiochem Merck Millipore Billerica MA USA)and PMS2 (clone A16ndash4 dilution 1100 BD BiosciencesPharmingen) Antibodies were detected using the EnVision

technique (Dako-Biotech) Tumor cells were judged asnegative for protein expression only if they lacked IHC stainingin a sample in which normal endometrial cells and stromal cellswere stained concurrently Results were considered unreliablein those cases where no immunostaining of normal tissue couldbe demonstrated The processed IHC slides were blindedevaluated by two pathologists (CE and CA) [19]

DNA extractionGenomic DNA was isolated from paraffin wax-embedded

tumor tissues Two 1 mm tissue cylinders were punched outfrom the tumor areas selected previously DNA from peripheralblood leukocytes or from paraffin wax-embedded nontumorousendometrium tissue was also extracted from those caseswhere LS was suspected A DNeasy Blood amp Tissue kit andQiaCube (Qiagen Valencia CA USA) automatic system wasused to isolate DNA according to the manufacturerrsquos protocol

Microsatellite analysis and MMR statusMSI status was analyzed using multiplexed polymerase

chain reaction (PCR) patterns at the monomorphic repetitivemarkers BAT26 BAT25 NR21 NR24 and NR27 [20]Amplicon detection and analysis were performed using an ABIPrism 3130 Genetic Analyzer and Genotyper software (LifeTechnologies Carlsbad CA USA) respectively A diagnosis ofMSI was considered positive when two or more markersshowed an altered pattern Tumors with MSI andor loss ofexpression of any of the MMR proteins were considered asMMR-deficient Tumors with absence of MSI and preservedMMR protein expression were considered as MMR-positivetumors

MLH1 promoter hypermethylation analysisCases with loss of MLH1 expression were tested for MLH1

methylation in the tumor DNA Any cases with such methylationwere then analyzed for MLH1 methylation in DNA from bloodcells to identify suspected constitutional MLH1 epimutations(Figure 1) We used the methylation-specific multiplex ligation-dependent probe-amplification technique (MS-MLPA KitME011 MRC-Holland Amsterdam The Netherlands) to studythe methylation status of MLH1 according to themanufacturerrsquos protocols The target regions for MLH1 genesilencing by hypermethylation are located in the C and Dregions of the MLH1 promoter (from nucleotide positions -248to -178 and -109 to +15) [21] which were tested using theprobes MLH1-3 and -4 respectively The mean results forthese two probes were calculated to obtain the methylationratio The threshold for methylated versus unmethylated statuswas set at 15 based on a previous study of MLH1 genesilencing [22] MS-MPLA fragments were analyzed using anABI Prism 3130 Genetic Analyzer and Genotyper software(Life Technologies)

Germline mutationsPatients in whom we suspected LS and were candidates for

genetic testing were referred to the Genetic Counseling divisionin our unit Suspicion of LS was based on MMR status (IHC












Results















Grade (FIGO) 173 1 95 5492 29 1683 49 283















Polyps 2 116


Lung cancer 2 116

Pleura 1 058







Discussion




Age lt50 yrs 7 2593 16 1096 284 (104-777) 004ge50 yrs 20 7407 130 8904



Myometrialinvasion

(n=146) gt50 6 2500 32 2623 093 (034-257) 090le50 18 7500 90 7377


(n=160) Yes 9 3333 38 2857 125 (052-303) 062No 18 6667 95 7143


(n=134) Yes 7 3043 17 1532 242 (087-676) 009No 16 6957 94 8468

Low UterinSegment

Yes 4 1481 12 822 194 (057-654) 028No 23 8519 134 9178


(n=152) Yes 5 1852 22 1760 106 (036-311) 091No 22 8148 103 8240
















Loss of MSH6 7 25

Loss of PMS2 1 11



TOTAL 27 819

























References






























































Results















Grade (FIGO) 173 1 95 5492 29 1683 49 283















Polyps 2 116


Lung cancer 2 116

Pleura 1 058







Discussion




Age lt50 yrs 7 2593 16 1096 284 (104-777) 004ge50 yrs 20 7407 130 8904



Myometrialinvasion

(n=146) gt50 6 2500 32 2623 093 (034-257) 090le50 18 7500 90 7377


(n=160) Yes 9 3333 38 2857 125 (052-303) 062No 18 6667 95 7143


(n=134) Yes 7 3043 17 1532 242 (087-676) 009No 16 6957 94 8468

Low UterinSegment

Yes 4 1481 12 822 194 (057-654) 028No 23 8519 134 9178


(n=152) Yes 5 1852 22 1760 106 (036-311) 091No 22 8148 103 8240
















Loss of MSH6 7 25

Loss of PMS2 1 11



TOTAL 27 819

























References



























































Polyps 2 116


Lung cancer 2 116

Pleura 1 058







Discussion




Age lt50 yrs 7 2593 16 1096 284 (104-777) 004ge50 yrs 20 7407 130 8904



Myometrialinvasion

(n=146) gt50 6 2500 32 2623 093 (034-257) 090le50 18 7500 90 7377


(n=160) Yes 9 3333 38 2857 125 (052-303) 062No 18 6667 95 7143


(n=134) Yes 7 3043 17 1532 242 (087-676) 009No 16 6957 94 8468

Low UterinSegment

Yes 4 1481 12 822 194 (057-654) 028No 23 8519 134 9178


(n=152) Yes 5 1852 22 1760 106 (036-311) 091No 22 8148 103 8240
















Loss of MSH6 7 25

Loss of PMS2 1 11



TOTAL 27 819

























References





























































Loss of MSH6 7 25

Loss of PMS2 1 11



TOTAL 27 819

























References


































































References





















































Prevalence of Lynch Syndrome among Patients with Newly Diagnosed Endometrial Cancers

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Transcript of Prevalence of Lynch Syndrome among Patients with Newly Diagnosed Endometrial Cancers