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Published OnlineFirst October 5, 2010; DOI: 10.1158/1078-0432.CCR-10-1491
Published OnlineFirst on November 9, 2010 as 10.1158/1078-0432.CCR-10-1491
Clinical
Canceresearch
an Cancer Biology

H6 and MUTYH Deficiency Is a Frequent Event in

R

ly-Onset Colorectal Cancer

Dolores Giráldez1, Francesc Balaguer1, Luis Bujanda2, Miriam Cuatrecasas1,r Muñoz1, Virginia Alonso-Espinaco1, Mikel Larzabal2, Anna Petit1, Victoria Gonzalo1,
a Ocaña1, Leticia Moreira1, José María Enríquez-Navascués2, C. Richard Boland3, oel3, Antoni Castells1, and Sergi Castellví-Bel1
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ancer Res

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pose: Early-onset colorectal cancer (CRC) is suggestive of a hereditary predisposition. Lynchome is the most frequent CRC hereditary cause. The MUTYH gene has also been related toitary CRC. A systematic characterization of these two diseases has not been reported previouslys population.erimental Design: We studied a retrospectively collected series of 140 patients ≤50 years oldosed with nonpolyposis CRC. Demographic, clinical, and familial features were obtained. Mismatch(MMR) deficiency was determined by microsatellite instability (MSI) analysis, and immunostain-r MLH1, MSH2, MSH6, and PMS2 proteins. Germline MMR mutations were evaluated in all MMR-nt cases. Tumor samples with loss of MLH1 or MSH2 protein expression were analyzed for somaticlation. Germline MUTYH mutations were evaluated in all cases. BRAF V600E and KRAS somaticional status was also determined.ults: Fifteen tumors (11.4%) were MSI, and 20 (14.3%) showed loss of protein expression (7 for/PMS2, 2 for isolated MLH1, 3 for MSH2/MSH6, 7 for isolated MSH6, and 1 for MSH6/PMS2). Wefied 11 (7.8%) germline MMR mutations, 4 in MLH1, 1 in MSH2, and 6 in MSH6. Methylationis revealed one case with somatic MLH1 methylation. Biallelic MUTYH mutations were detectedr (2.8%) cases. KRAS and BRAF V600E mutations were present in 39 (27.9%) and 5 (3.6%) cases,tively.clusions: Loss of MSH6 expression is the predominant cause of MMR deficiency in early-onset
Con
CRC. Our findings prompt the inclusion ofMSH6 andMUTYH screening as part of the genetic counselingof these patients and their relatives. Clin Cancer Res; 16(22); 5402–13. ©2010 AACR.

represnot uMoreoof ear

orectal cancer (CRC) is the third most commonr and the second cause of cancer-related deathsst western countries (1). Although aging is therisk factor for CRC initiation and progression,

the total CRC burden occurs in indivi-ages ≤50 years (2, 3). This population

decada herconsetheirRec

presenologicwithCRCbe pocolonof vieknowentlyfor u10), irepair

s: 1Gastroenterology Department, Institut de Malaltiesbòliques, Hospital Clínic, Centro de Investigaciónde Enfermedades Hepáticas y Digestivas (CIBEREHD),onsBiomèdiquesAugustPi i Sunyer, (DIBAPS),Universityelona, Spain; 2Departments of Gastroenterology andCentro de Investigación Biomédica en Red de

páticas y Digestivas, University of Country Basque,Donostia, Spain; and 3Department of Internal Medicine,nterology, Charles A. Sammons Cancer Center andtitute, Baylor University Medical Center, Dallas, Texas

uthors: Francesc Balaguer and Sergi Castellví-Bel,epartment, Hospital Clinic, Villarroel 170, Barcelonaone: 34-93-2275418; Fax: 34-93-2279387; Email:s; [email protected].

0432.CCR-10-1491

ssociation for Cancer Research.

; 16(22) November 15, 2010

Research. on May 29, 20clincancerres.aacrjournals.org ed from

ents an important clinical problem because it issually included in the CRC screening programs.ver, epidemiologic data suggest that the incidencely-onset CRC has increased during the past threees (4, 5). Finally, early-onset CRC is suggestive ofeditary predisposition that may have importantquences not only for the index case but also forrelatives.ent studies have suggested that early-onset CRC re-ts a biologically distinct disease, with clinicopath-and molecular differences compared with patients

older onset of disease (6–8). Indeed, early-onsetis more likely to present at advanced stages, toorly differentiated, and to be located in the distal, especially in the rectum. From a molecular pointw, it represents a heterogeneous disease, includingn hereditary syndromes, familial cases, and appar-sporadic CRC. Lynch syndrome, which accountsp to 2% to 3% of the total burden of CRC (9,
s caused by germline mutations in DNA mismatch(MMR) genes (MLH1, MSH2, MSH6, and PMS2)
21. © 2010 American Association for Cancer

http://clincancerres.aacrjournals.org/

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Translational Relevance

Early-onset nonpolyposis colorectal cancer (CRC) issuggestive of a hereditary predisposition. Germlinemutations in the mismatch repair (MMR; MLH1,MSH2, MSH6, and PMS2) and the base excision repair(MUTYH) genes have been associated with early-onsetCRC. A systematic evaluation of these two diseases hasnot been reported previously in this population. Wedescribe herein that mismatch MMR deficiency is pres-ent in ∼15% of early-onset nonpolyposis CRC casesand is characterized by a high frequency of MSH6germline mutations. In addition, biallelic MUTYH-associated CRC accounts for 3% to 4% of early-onsetMMR-proficient CRCs. The results of this study promptthe inclusion of MSH6 and MUTYH screening as partof the genetic counseling of early-onset CRC.

MSH6 and MUTYH in Early-Onset Colorectal Cancer

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thought to account for up to 20% of early-onset(11–13). These cases usually show microsatelliteility (MSI), the hallmark of MMR deficiency, andf expression of the corresponding mutated proteinmunohistochemistry. Current guidelines (i.e., re-Bethesda criteria) recommend doing a moleculareening by either MSI and/or immunohistochemis-CRC patients <50 years old, regardless of familyy of CRC (10, 14).lelic mutations in the MUTYH gene, a member ofse excision repair system, represent another hered-cause of early-onset CRC (15). Although biallelicvation of this gene usually predispose to an at-ted form of colonic polyposis (MAP or MUTYH-ated polyposis; ref. 16), population-based studiesshown that up to 30% of biallelic mutations develop CRC in the absence of a polyposis phe-e (17, 18). The absence of a specific clinicopath-feature of nonpolyposis MUTYH-associated CRCit a diagnostic challenge. In this sense, it has re-been suggested that all early-onset CRC shouldted for MUTYH mutations (15). In addition, be-MUTYH-associated tumors are characterized byccumulation of somatic G:C→T:A transversions,is the hallmark of base excision repair system de-y, the analysis of this particular somatic mutationAS has also been proposed as screening method0).lly, the remaining 75% to 80% of early-onset CRCsroficient MMR and a yet unidentified genetic predis-on. Although some studies have suggested that theseare more often diploid than older onset CRC, it isontroversial if they represent a unique molecularof CRC (6, 7, 21). Interestingly, several studies have
ed a strong familial association in early-onset micro-te stable (MSS) CRC (6, 7).
anti-M(BD

acrjournals.org

Research. on May 29, 20clincancerres.aacrjournals.org Downloaded from

derstanding the molecular basis of early-onsetis critical to uncover yet unidentified genetic con-s and consequently tailor appropriate preventiveies for this disease. A comprehensive and system-pproach to identify Lynch syndrome in this cohortthe combination of immunohistochemistry for allMMR proteins and MSI analysis followed byic testing has not been attempted previously. Inon, the frequency of nonpolyposis MUTYH-associ-CRC in this population remains poorly explored.im of this study was to systematically assess thel, histologic, and molecular features of a large co-f unselected early-onset CRC patients and to an-the prevalence of known hereditary nonpolyposisomes.

rials and Methods

retrospectively recruited all patients ≤50 years oldosed with CRC who were surgically treated at twosh centers (Hospital Clínic of Barcelona and Hos-of Donostia) in 1995 to 2007 and from whom ar-formalin-fixed paraffin-embedded samples were

ble. Patients with personal history of colorectalosis or inflammatory bowel disease were excluded.opathologic data were obtained from each pa-medical record. Family history of cancer, includ-least first-degree and second-degree relatives, wased either from the medical record or by phonect. Positive family history was considered if ≥1egree or second-degree relative(s) had cancer. Toare the clinicopathologic features of the subjects in-in this study with a cohort of older-onset CRC

y), we used patients recruited in the EPICOLONt, a previously reported Spanish population-basedt of CRC (10). The study was approved by thetional ethics committee of each participating hos-Written informed consent was obtained at CRCosis on a systematic basis. In deceased cases inthe informed consent was missing, next-of-kin

nt was obtained.

isolationomic DNA from each patient was extracted fromalin-fixed paraffin-embedded tumoral andponding normal colonic mucosa with the use ofIAamp Tissue kit (Qiagen) according to the man-rers' instructions. Peripheral blood DNA, whenble, was extracted with the use of the QIAampblood Mini kit (Qiagen).

r MMR protein expressione block of formalin-fixed paraffin-embeddedr tissue was selected per case, and immunostain-as done with the use of standard protocols. Theing mouse monoclonal antibodies were used:
LH1, anti-MSH2, anti-MSH6, and anti-PMS2
Pharmingen). Tumor cells were considered to be

Clin Cancer Res; 16(22) November 15, 2010 5403



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Giráldez et al.

Clin C5404


ive for protein expression only if they lackedng in a sample in which healthy colonocytestromal cells were stained. If no immunostainingalthy tissue could be shown, the results wereered undetermined.

r MSI analysisI status was assessed with the use of five mononucle-markers (22): BAT25, BAT26, NR21, NR24, andO27 (MSI Analysis System version 1.2, Promega)ing to the manufacturers' instructions. Tumors withility at ≥3 markers were classified as high (MSI) andshowing instability at ≤2 markers as MSS. Research-oring immunostaining were blinded to the MSI, and vice versa.

line MMR mutational analysisents found to have tumors with MSI and/or lackR protein expression underwent germline genetic

g for MMR genes. Gene screening selection wason the immunostaining results (MLH1 for MLH1/

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loss, MSH2 for MSH2/MSH6 loss, and MSH6 fored MSH6 loss). First, genomic rearrangementsanalyzed by multiple ligation probe amplification-Holland, the Netherlands) according to the manu-ers' instructions. Multiple ligation probe amplifica-esults were confirmed by the same technique withse of a confirmation kit and by an independentd. Afterwards, we analyzed the coding sequencingxon-intron boundaries of MLH1, MSH2, and MSH6gle-strand conformational polymorphism (SSCP)irect sequencing of abnormal band shifts withe of standard protocols (ABI 3100 Genetic Analyzer,ed Biosystems). Primer sequences are availablequest.yPhen software (http://genetics.bwh.harvard.edu/ndex.html) was used to test the potential patho-role of missense variants. This prediction is ano tool that predicts the possible effect of an aminoubstitution on the structure and function of a hu-
protein with the use of physical and comparativeerations (23).
1. Clinicopathologic features of the early-on
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ean (SD) 44
5.6) 6 (5.9) (5.6) 0.2 (%) ales 66 (47.1) 8 (40) 58 (48.3) 0.489 es 7 52.9) 2 (60) (51.7) location*, n (%) imal 3 25) 4 (70) (17.5) 0.0 5 ( 1 21 001al 59 (42.1) 4 (20) 55 (45.8) tum 4 32.9) 2 (10) (36.7) umor stage†, n (%)
0.7)
0 (0) (0.8) 0.9 1 8.6) 1 (5) (9.3) 3 23.6) 5 (25) (23.7) 3 ( 2862 (44.3) 9 (45) 53 (44.9) 3 21.4) 5 (25) (21.2)
of differentiation‡, n (%)
l/moderate 11 91.8) 2 (80) (93.5) 0.1 r 1 8.2) 3 (20) (6.5) production, n (%) 2 18.6) 7 (35) (15.8) 0.0 ronous CRC§ 2.9) 1 (5) (2.5) 0.4 history of CRC∥ 3 26.3) 8 (42.1) (23.7) 0.1 history of endometrial cancer∥ 5.8) 2 (10.5) (5.1) 0.3 ily 8 ( 6 07
ily history of Lynch spectrum extracolonic tumors∥ 27 (19.7) 5 (26.3) 22 (18.6) 0.533illment of Amsterdam II criteria∥ 8 (5.8) 4 (21.1) 4 (3.4) 0.013

h respect to the splenic flexure.sed on 138 patients.sed on 122 patients.sed on 138 patients.

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and MSH2 methylation analysisumor samples with loss of MLH1 or MSH2 proteinsion, DNA methylation status of the CpG island ofgenes was established by PCR analysis of bisulfite-ied genomic DNA (EZ DNA Methylation-Gold kit,Research) with the use of pyrosequencing (PSQA Pyrosequencing System, QIAGEN). Cases with

tic methylation in any of these genes were alsofor methylation in the germline (either normal

ic mucosa or blood). Primer sequences are avail-n request.

line MUTYH gene mutation analysispatients were screened for four MUTYH mutationslent in the Spanish population (Y176C, G393D,elC, 1220_1221insGG; Genbank access NM_012222)ele-specific TaqMan probes and resolved on a 7300imePCRSystem(AppliedBiosystems). Inheterozygotesy of these mutations, the coding region and exon-boundaries of the MUTYH gene were screened byand direct sequencing of abnormal band shifts,viously described (17). Primer details are availableuest.

tic BRAF V600E and KRAS mutation analysisBRAF V600E mutational analysis was done by

specific TaqMan probes as previously described.r details are available on request. The mutationalpot of KRAS (codons 12 and 13) was analyzedrect sequencing of both strands with the use ofard protocols (ABI 3100 Genetic Analyzer, Ap-Biosystems). Amplification was done with thef a coamplification-at-lower-denaturation-tempera-CR method (24). Primer details are available onst.

tical analysisa were analyzed with the use of the SPSS 13 statis-oftware. Quantitative variables were analyzed withse of Student's t-test. Qualitative variables werezed with the use of either the chi-square test or's test. A two-sided P-value of <0.05 was regardednificant.

lts

t characteristicsrecruited 140 patients ≤50 years diagnosed withClinicopathologic features are shown in Table 1.age at diagnosis was 44.1 years (SD, 5.6 y); 105) tumors were located distal to the splenic flexure,6 (32.9%) were in the rectum. The majority of cases%) were diagnosed at advanced stages (III-IV); 10) showed poorly differentiated tumors, and 26%) had mucinous features. Family history of CRCer first-degree or second-degree relatives was present
(26.3%) patients, and 8 (5.8%) cases fulfilled therdam II criteria.
quentMSH6

acrjournals.org


tic MMR deficiencyR deficiency was evaluated by both immunohisto-istry and tumor MSI analysis in all cases. MMR defi-was defined as loss of protein expression in any ofMR proteins and/or having a MSI tumor. Fifteen tu-(10.7%) were MSI, and 20 (14.3%) showed loss ofin expression (7 for MLH1/PMS2, 2 for isolated, 3 for MSH2/MSH6, 7 for isolated MSH6, and 1SH6/PMS2). Clinicopathologic features of patientsMR deficiency are summarized in Table 2, and aary of the molecular results of this study is shownle 3. All MSI cases had loss of protein expression;

ver, two cases with loss of MLH1 and two cases withf MSH6 showed MSS tumors. In one case with isolat-s of MSH6, MSI could not be determined. According-MR deficiency in our cohort was present in 20) cases.

shown in Table 1, compared with MMR-proficientrs, cases with MMR deficiency were more likely tooximal (70% versus 17.5%; P = 0.0001), andd a trend toward having more mucinous featuresversus 15.8%; P = 0.059) and being more frequentlyy differentiated (20% versus 6.5%; P = 0.106). Theency of MMR deficiency according to patient ages follows: 21 to 30 years, 25% (1 of 4); 31 to 4016.7% (4 of 24); and 41 to 50 years, 13.4% (152). On family history, patients with MMR-deficientrs fulfilled more frequently the Amsterdam II criteria

versus 3.4%; P = 0.013) and showed a trend to-more frequently having a family history of CRC% versus 23.7%; P = 0.1). Compared with MLH1H2 deficiency, MSH6-deficient tumors were moreently located in the distal colon (50% versus; P = 0.16) and displayed less family history of14.3% versus 58.3%; P = 0.14), although the differ-did not reach statistical significance.

line MMR mutationsidentified 11 (7.8%) cases with sequence variants inMLH1 (4 cases), MSH2 (1 case), or MSH6 (6 cases).cteristics of these patients are shown in Table 2. For, three cases carried a nonsense mutation (p.R226X,7X, p.Q700X), and a missense variant was present inase (p.S692F). Based on an in silico approach, thise was predicted as “benign” but with a borderlinegenic prediction. For MSH2, we identified a com-deletion of exon 7, whereas for MSH6, four of sixponded to missense variants (p.S1279P, p.I1115T,2A, p.P656L) and two to nonsense mutations32X and p.Q344X; Fig. 1). All four MSH6 missensets were predicted to be “possibly damaging” withe of an in silico approach. Overall, the mutation de-n rate was 55% (11 of 20), and MMR-deficient casesr without identified germline mutation did not dif-any clinicopathologic or familial feature (data not). However, family history of CRC was more fre-
ly seen in MLH1/MSH2 mutation carriers than inones (100% versus 0%; P = 0.02).



MLH1Am

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GermBia

(2.8%of bibialleanytwo

Table linico ic an r fea f pat with

Case Age/S Locat Stage MSI mmun hemis

MLH1 PMS2 MSH2 MSH6

69D 32M Rec IV MSS84D 44F Ascen III MSI

111D 42M Caec III MSI

98 41F Rec III MSI

16 50F Caec II MSS ND

6 31F Trans III MSI

25 30M Ascen II MSI89 45F Caec I MSI

85D 46F Ascen III MSI

17 41M Ascen IV MSI

35D 45M Tranv IV MSI

86 48M Caec III MSI

75 48M Desce IV MSI101 37M Trans III MSS112 46M Sigm ND MSS117 46F Sigm ND ND ND154D 40M Ascen IV MSI

40D 43F Trans III MSI7491

(Continued on the following page)

Giráldez et al.

Clin C5406


and MSH2 methylation analysisong 12 patients showing loss of either MLH1SH2 expression, we only found somatic MLH1lation in one case (Table 2). This patient corre-ed to a 30-year-old male with a stage II tumord in the ascending colon and without family his-f any cancer. In this case, neither a pathogenic
germline mutation nor a germline MLH1 methyl-was evident.
in thpatien



line MUTYH mutationsllelic MUTYH mutations were found in 4 of 140) patients (Table 3). Clinicopathologic featuresallelic carriers are shown in Table 4. Overall,lic MUTYH mutation carriers did not showspecific clinicopathologic feature, althoughtumors were located in the rectum and one

2. C
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olon 12A wt 1% — MLHEndom

ovaolon wt wt 2% — MLH1

ectum 13D wt 1% — MLH1 p.R226Xolonolon36,

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ndom(gra

13D wt 1% —

olono wt wt 31% —ndom wt wt 1% —sophvariu 13D wt 1% —toma

Colo olon wt wt — 2% SH2olon

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wt wt — 2% No

variuColo o wt wt — 3%Skin

o wt wt — — MSH6nkno wt wt — — MSH6Skin G12V wt — — MSHo wt wt — — MSHeuke ND wt — — MSHrosta

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showed a polyposis phenotype. On family histo-o of them showed a recessive pattern of inheri-of either CRC or colorectal polyposis. We could
ine the KRAS mutational status in two of thets, and one of them showed a G:C→T:A trans-
(2.85erozyg

acrjournals.org


n in codon 12, the hallmark of base excision re-eficiency.noallelic MUTYH mutations were identified in four

2. Clinicopa
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39, 40)
n (38) C (father, 55)
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ch (grandmother, 50)(brother, 50) No R
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ma hypophisist, 50)
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etrium G ndmother, 52)
No

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(uncle, 60) No N No E etrium (mother, 52)
NoNo

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agus (brother, 62) No O m (mother, 72) G No
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ch (grandmother, 70) n (41) C (mother, 67)
C
(grandmother, 50)m (cousin, 45)tatic tumor
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nknown origin er, 60)
On (48) N

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No
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p.S1279P No U wn p.I1115T No tumor (father, 93) 6 p.P202A No N No L mia (father, 62)
6 p.R732X6 p.P656L

P
te (grandfather, 70) No S ch (aunt, 60) p.Q344X toma wt ND — — MSH6No Colon (mother, 91) wt wt — — NoNo CLL (father, 80) wt wt — — No
reviations: ND, not determined; wt, wild-type; CLL, chronic lymphocytic leukemia.lid cells indicate loss of protein expression.ected relative and age at diagnosis are indicated between parentheses. Only the affected side of the family is described.matic methylation analysis done by pyrosequencing.

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casesMMRassociwas p0.014140 (malesfoundGly13Gly12(two,and G

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Giráldez et al.

Clin C5408


nse mutation in MSH6. This patient was a 40-year-male with a stage IV ascending colon tumor withnd isolated loss of MSH6. The family history wasarkable for CRC or any other Lynch syndrome–ated tumors.

tic BRAF and KRAS mutationsF V600E mutation was detected in 5 of 140 (3.6%)(Table 3). We found no BRAF mutation in any-deficient tumor, and the only clinical featureated with the presence of the BRAF V600E mutationroximal tumor location (80% versus 22.7%; P =). Somatic KRAS mutations were detected in 39 of27.9%) cases (Table 3) and were associated with fe-(64.1% versus 40.9%; P = 0.016). The frequency ofmutations was as follows: Gly12Asp (nine, 23.0%),Asp (eight, 20.5%), Gly12Cys (seven, 18.0%),Val (six, 15.4%), Gly12Ala (four, 10.2%), Gly12Ser
5.1%), Gly13Ser (one, 2.6%), Gly13Tryp (one, 2.6%),ly13Val (one, 2.6%).
In tpathohortoveralin thimolechigh fMUTYand artationsioncharafrequeand frologicLyn

genesfrequeAccurfor thcause(26).da cricy tesof bei(10).at earficienreasostudieCRCto 50MSI salyzedMLH1analyCRC,in thepopulquenc

le 3. Summary of molecular results

(%

s o /140LH 7LH 2SH 3SHSHnd 3teSI 15SS 116nd 9mli /140LH 4SH 1SH 6mli /14039 217 139 139 317 1at /140od 28od 11

rmline testing done only in MMR-deficient patients.



opathologic features of early-onset MMR andexcision repair–proficient CRCnext evaluated the clinicopathologic features of ear-set CRC with both MMR and base excision repairiency, and compared them with a population-basedt of patients >50 years diagnosed with CRC and re-d in the EPICOLON project (Table 5). Comparedlder-onset CRC, MMR-proficient and base excision–proficient early-onset CRC was more frequently lo-in the distal colon (82.7% versus 72%; P = 0.021),frequently diagnosed at advanced stages (66.6% ver-.1%; P < 0.0001), and was associated to a stronger

es (23.7% versus 16.4%; P = 0.06).

ssion

his study we have systematically assessed the clinico-logic, molecular, and familial features of a large co-of early-onset nonpolyposis CRC showing that,l, the frequency of known hereditary CRC syndromess population is ∼10%. This cohort depicts a distinctular profile of MMR deficiency characterized by arequency of germline mutations in MSH6. BiallelicH mutations account for 3% of early-onset CRCe clinically indistinguishable from non–MUTYHmu-carriers. Early-onset CRC with MMR and base exci-repair proficiency, the predominant group, iscterized by distal location, advanced stage, lowncy of mutations in the BRAF-KRAS-MEKK pathway,equent family history of CRC, suggesting a distinct bi-al entity.ch syndrome, caused by mutations in the MMR(i.e., MLH1, MSH2, MSH6, and PMS2) is the mostnt monogenic hereditary CRC syndrome (9, 25).ate diagnosis of this syndrome is of great benefite management of both patients and relatives be-surveillance has proven to be highly effectiveThe current guidelines, based on the revised Bethes-teria (14), recommend doing tumor MMR deficien-ting to preselect those cases with higher probabilityng germline mutation carriers in one of these genesPrevious studies have revealed that CRC diagnosedly ages have a high probability of having MMR de-cy, ranging from 17% to 73% (11, 12, 27–30). Then for this wide range is the heterogeneity of thes. First, the threshold to consider “early-onset”varies between different studies, ranging from 24years. Second, the methods used to assess the

tatus are highly heterogeneous, and the proteins an-by immunohistochemistry are usually restricted toand MSH2. Finally, the majority of studies have

zed either high-risk or general populations ofand it is known that the rate of MSI is much lowerlatter (29). Our study, based on a large unselected

n/N
)
f MMR protein expression, n (%) 20
(14.3) 1/PMS2 (5) 1 (1.4) 2/MSH6 (2.1) 6 (5) 76/PMS2 1 (0.7) etermined (2.1) sting, n (%)
(10.7)
(82.9)
etermined
(6.4) ne MMR mutations*, n (%) 11 (7.8) 1 (2.8) 2 (0.7) 6 (4.3) ne MUTYH mutations, n (%) 8 (5.7) 3D/G393D (1.4) 6C/1138delC (0.7) 3D/T474fs488X (0.7) 3D/- (2.1) 6C/- (0.7) ic KRAS mutations, n (%) 39 (27.9) on 12 (20) on 13 (7.9) etermined (7.9) nd 11
atic BRAF V600E mutation, n (%) 5/140 (3.6)ndetermined 3 (2.1)

ation of CRC ≤50 years, shows that the overall fre-y of MMR deficiency in this population is ∼15%.

Clinical Cancer Research



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te of MSI was slightly lower than previous reports,tent with a lower frequency of MSI previously re-in a Spanish population (10). We have found a

ct profile of MMR deficiency characterized by a highncy of isolated loss of MSH6, accounting for 40%MMR-deficient tumors. This frequency is much

r than previously reported because germline muta-in MSH6 are usually responsible for only 10% toof all cases of Lynch syndrome (9). Interestingly,ared with tumors with loss of MLH1 or MSH2 ex-on, those with loss of MSH6 were predominantlyd in the distal colon (4 of 6) and lacked family his-f CRC (31). Of note, 25% of them were MSS andbe missed if MSI is used as a single screening ap-. In clinical practice, all these features make thesediagnostic challenge, and they probably remain un-ized. These results highlight the significance of aatic evaluation of MMR deficiency in an unselected

anucleotide repeats used as controls. Bottom, MSH6 sequence electrophero

ation of CRC to recognize unusual features of hered-yndromes. In this sense, these results also underline

MSH2ture (

acrjournals.org


ed to include MSH6 deficiency evaluation in the as-ent of early-onset CRC.ur population of early-onset CRC, the overall fre-y of Lynch syndrome, defined by the presence of aine mutation in one of the MMR genes, was 7.8%,is similar to a previous report of the frequencyH1/MSH2/MSH6 mutations in a series of CRCears old (32). Although we did not do MMR geneticg in the whole cohort, it is unlikely that we missednidentified germline mutations due to our syste-approach to MMR deficiency. On the MSH6 germ-utations, the predominant mutated gene in this

lation, two changes corresponded to pathogenicnse mutations (p.R732X and p.Q344X), and fourmissense variants (p.P202A, p.P656L, p.I1115T,.S1279P). The decreased frequency of frameshiftions and increased levels of point mutations associ-with MSH6 deficiency, compared with MLH1 or

howing the p.S1279P mutation.

MSH6 mismatch repair deficiency. Top, isolated loss of MSH6 expression by immunohistochemistry; left shows normal expression of MSH2, and rightloss of MSH6 expression. Middle, MSI testing in case 75 showing instability for all five mononucleotide repeats. Penta D and Penta D correspond

deficiency, is a well-documented fact in the litera-33). All four missense variants were predicted to be




Table 4. Clinicopathologic and molecular features of MUTYH mutation carriers

Case Age Sex Tumorlocation

Stage Differentiation Mucinousproduction

MSIstatus

MMR IHC Othertumors

Synchronousadenomas (no.)

Family history* KRASstatus

MUTYHmutation

52 46 M Rectum III Moderately No MSS Normal Colon (46) Yes (5) Colon (brother, 43) wt Y176C/1138delCLarynx (father, 33)

Breast (mother, 50)Thyroid (mother, 70)Breast (maternal aunt, 60)

95D 43 F Rectum III Well No MSS Normal No No Breast (mother, 67) ND G393D/G393D

137D 47 F Sigmoid II Well No MSS Normal No Yes (1) No ND G393D/G393D

31D 45 M Ascending II Well No MSS ND No Yes (2) Colonic polyposis(brother, ?)

G12C G393D/T474fs488X

Colonic polyposis (sister, ?)Colon (uncle, 80)Colon (cousin, 48)

38D 48 F Rectum I ND No MSS Normal Multinodulargoiter

No No G12C Y176C/-

154D 40 M Ascending IV Well No MSI Loss ofMSH6

No No Leukemia (father, 62) ND G393D/-Prostate (grandfather, 70)

31 50 M Rectum IV Poorly Yes MSS Normal No No No wt G393D/-2 33 F Rectum III Moderately No MSS Normal No No Endometrium (sister, 42) wt G393D/-

Endometrium(grandmother, 42)

Abbreviation: IHC, immunohistochemistry.*Affected relative and age at diagnosis are indicated between parentheses.

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genic with the use of an in silico approach, and nei-he nonsense mutation nor missense variants havepreviously reported in any Lynch syndrome muta-atabase. The finding of a high number of MSH6ions in an early-onset CRC cohort is in agreementrecent report that found germline MSH6 mutationsf 38 (18.4%) patients without family history ofr who were diagnosed with CRC before the age ofars. On MLH1 and MSH2 germline mutations, wedentified one carrier of a MLH1 missense variant2F). This case was a 32-year-old patient with a stageS rectal tumor with loss of MLH1/PMS2 and ave family history of CRC. This missense variant is, and it is likely pathogenic because it had a border-rediction by PolyPhen.ough we identified 20 patients with MMR deficien-ermline mutation in the MMR genes could only beed in 11 patients (mutation detection rate of 55%).requency is consistent with previous reports ineven when patients were selected from high-risk

clinics, the mutation detection rate was no more0% to 60% (10, 27–30, 34). This fact constitutesproblem in clinical practice, and in preventive strat-these cases are usually considered as mutation car-Possible causes for these cases without an identifiedular alteration could be undetected mutations byt analytical methods or somatic events that affectalleles of a MMR gene.ther relevant finding of our study is that biallelicions in the MUTYH gene account for ∼3% ofnset CRC without apparent polyposis. Base exci-repair deficiency due to biallelic inactivation ofH was initially associated with the developmentattenuated form of colonic polyposis (16). How-
several subsequent population-based studiesd that ∼30% of biallelic mutation carriers devel-
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acrjournals.org


CRC in the absence of a polyposis phenotype (17,5). Accordingly, it has been suggested that MU-testing should be considered in early-onset CRCts with intact DNA MMR, regardless of familyy or number of colonic polyps (15, 17). We pre-ly showed that in a population-based cohort ofonset MMR-proficient CRC without polyposis,equency of biallelic mutation carriers was 4.6%Because our mutational screening strategy wason the MUTYH mutational profile in the Spanishation (17), and we did not do systematic whole-sequencing, the described frequencies are likely toderestimated. Considering that biallelic MUTYHion carriers are at risk for multiple cancers (36),esults highlight the fact that MUTYH should bein early-onset CRC regardless of family history

lonic polyps.ally, our study emphasizes that the genetic basismajority of early-onset CRC, if any, remains un-

n. Eighty-three percent of patients included in thisshowed no evidence of either MMR or base exci-repair deficiency. Compared with older patients,und that this subset of early-onset CRC patientsmore frequent distal tumors (especially in the rec-they are usually diagnosed at advanced stages,ore interestingly, they are associated with a high-quency of family history of CRC. These featuresonsistent with previous reports suggesting theeness of these patients. Boardman et al. analyzedequency of chromosomal instability in a group oftients ≤50 years old with MSS CRC and com-them with a series of 90 patients ≥65 yearsith MSS CRC. MSS tumors in the young groupmore often diploid (46%) than those in older
ts (26%; P = 0.006; ref. 6). Other studies havesimilar results (7), although it is still a controversial
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es, n/N (%)
(48.2) 059 (41.4) location*, n/N (%) imal (17.2) 059 (28) al (46.5) 059 (36.6) tum (36.2) 059 (35.3) umor stage, n/N (%) 1
(33.3)
024 (55.9) (66.6) 024 (44.1)
egree of differentiation, n/N (%)
(6.8) 961 (7.5) r d 7/103 72/ 1in production, n/N (%) 19/116 (16.4) 113/958 (11.8) 0.176ily history of CRC†, n/N (%) 27/114 (23.7) 174/1,059 (16.4) 0.06
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21). We found that the frequency of mutations inRAF-KRAS-MEKK pathway in this population islower than the one reported in unselected CRC8), suggesting that this pathway does not play

tral role in early-onset CRC carcinogenesis. Futures focused on the understanding of the molecularnisms involved in the pathogenesis of early-onsetproficient tumors are needed, and the higher fre-y of family history of CRC in this subgroupts the presence of yet unidentified cancer suscep-alleles.

summary, our study represents the first systematicpt to describe the frequency of known hereditarysyndromes in an unselected cohort of nonpolypo-rly-onset CRC. Our results show that MMR defi-accounts for ∼15% of this population and is

cterized by a high frequency of germline MSH6ions, which frequently shows unusual features forsyndrome. Accordingly, in addition to MLH1/immunohistochemistry, MSH6 should not be

oked in the molecular prescreening of these pa-If no MMR is found, biallelic MUTYH mutationssponsible for ∼3% to 4% of cases, and screeninge most common mutations in this gene is recom-ed. Finally, a better understanding of the molecu-sis of early-onset CRC neither due to MMR nor
excision repair deficiency is critical to be able to Rece
identification of patients with hereditary nonpolyposis colorectalcer. Jama 2005;293:1986–94.fe R, Kim H, Hsieh ET, et al. Tumor microsatellite instability and

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mprove genetic counseling of these patients andrelatives.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

thank all the investigators of the EPICOLON project for theirration and Susana Moyano for her assistance in recruiting thes included in the study.

Support

o de Investigación Sanitaria/FEDER grants 08/0024, 07/0359, and0020/0021; Ministerio de Ciencia e Innovación grant SAF 07-64873;ión Española contra el Cáncer (Fundación Científica y Junta dena); Fundación Olga Torres, Agència de Gestió d'Ajuts Universitariserca grant 2009 SGR 849; Fundación de Investigación Médica Mutuaña; Fundación Caja Madrid and Societat Catalana de Digestologiagrant (F. Balaguer); Centro de Investigación Biomédica en Red de

edades Hepáticas y Digestivas contracts (V. Alonso-Espinaco andoz); and Fondo de Investigación Sanitaria contract CP 03-0070tellví-Bel). Centro de Investigación Biomédica en Red de Enferme-epáticas y Digestivas (CIBEREHD) is funded by Instituto de Salud

III.costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedsement in accordance with 18 U.S.C. Section 1734 solely tothis fact.

ived 06/01/2010; revised 08/27/2010; accepted 09/17/2010;
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Published OnlineFirst October 5, 2010.Clin Cancer Res María Dolores Giráldez, Francesc Balaguer, Luis Bujanda, et al. Early-Onset Colorectal CancerMSH6 and MUTYH Deficiency Is a Frequent Event in

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