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Cancer risk in hereditary nonpolyposis colorectal cancer due to MSH6 mutations: impact on counseling and surveillance

      Abstract

      Math Eq Hereditary nonpolyposis colorectal carcinoma (HNPCC) is caused by a mutated mismatch repair (MMR) gene. The aim of our study was to determine the cumulative risk of developing cancer in a large series of MSH6 mutation carriers. Math Eq Mutation analysis was performed in 20 families with a germline mutation in MSH6. We compared the cancer risks between MSH6 and MLH1/MSH2 mutation carriers. Microsatellite instability (MSI) analysis and immunohistochemistry (IHC) were performed in the available tumors. Math Eq A total of 146 MSH6 mutation carriers were identified. In these carriers, the cumulative risk for colorectal carcinoma was 69% for men, 30% for women, and 71% for endometrial carcinoma at 70 years of age. The risk for all HNPCC-related tumors was significantly lower in MSH6 than in MLH1 or MSH2 mutation carriers (P = 0.002). In female MSH6 mutation carriers, the risk for colorectal cancer was significantly lower (P = 0.0049) and the risk for endometrial cancer significantly higher (P = 0.02) than in MLH1 and MSH2 mutation carriers. In male carriers, the risk for colorectal cancer was lower in MSH6 mutation carriers, but the difference was not significant (P = 0.0854). MSI analysis in colorectal tumors had a sensitivity of 86% in predicting a MMR defect. IHC in all tumors had a sensitivity of 90% in predicting a mutation in MSH6. Math Eq We recommend starting colonoscopic surveillance in female MSH6 mutation carriers from age 30 years. Prophylactic hysterectomy might be considered in carriers older than 50 years. MSI and IHC analysis are sensitive tools to identify families eligible for MSH6 mutation analysis.

      Abbreviations:

      HNPCC (hereditary nonpolyposis colorectal carcinoma), IHC (immunohistochemistry), MMR (mismatch repair), MSI (microsatellite instability)
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      described 10 kindred with 9 different truncating germline MSH6 mutations. Most of these families did not fulfill the Amsterdam (I and II) criteria and were characterized by a predominance of endometrial carcinoma and a higher age at diagnosis of cancer compared with families with an MLH1 or MSH2 mutation. After this publication, more MSH6 truncating germline mutations have been reported.
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      Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations.
      ,
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      Molecular and clinical characteristics of MSH6 variants an analysis of 25 index carriers of a germline variant.
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      Atypical HNPCC owing to MSH6 germline mutations analysis of a large Dutch pedigree.
      The aims of this study were to (1) evaluate the clinical phenotype of a large series of families with an MSH6 mutation, (2) evaluate the value of MSI and IHC analysis in the identification of such families, and (3) discuss the appropriate surveillance protocol for MSH6 mutation carriers.

      Patients and methods

       Patients

      A total of 20 families with a truncating germline mutation in the MSH6 gene were included in the study. These families originated from 2 sources. The first is a group of 214 families, tested negatively for pathogenic mutations in MLH1 or MSH2, collected for scientific purposes through The Netherlands Foundation for the Detection of Hereditary Tumours and departments of clinical genetics in The Netherlands and Norway. Most of the families collected by The Netherlands Foundation for the Detection of Hereditary Tumours were selected using the Amsterdam criteria. The families collected by departments of clinical genetics in The Netherlands and Norway were selected on the basis of familial clustering of colorectal carcinoma. The group consists of 71 Amsterdam I-positive and 143 Amsterdam I-negative families. Nine different truncating MSH6 germline mutations were identified in 10 families, as reported previously by our group.
      • Wijnen J.
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      • Vasen H.
      • van der Klift H.
      • Moller P.
      • Stormorken A.
      • Meijers-Heijboer H.
      • Lindhout D.
      • Menko F.
      • Vossen S.
      • Moslein G.
      • Tops C.
      • Brocker-Vriends A.
      • Wu Y.
      • Hofstra R.
      • Sijmons R.
      • Cornelisse C.
      • Morreau H.
      • Fodde R.
      Familial endometrial cancer in female carriers of MSH6 germline mutations.
      ,
      • Wagner A.
      • Hendriks Y.
      • Meijers-Heijboer E.J.
      • de Leeuw W.J.
      • Morreau H.
      • Hofstra R.
      • Tops C.
      • Bik E.
      • Brocker-Vriends A.H.
      • van Der Meer C.
      • Lindhout D.
      • Vasen H.F.
      • Breuning M.H.
      • Cornelisse C.J.
      • van Krimpen C.
      • Niermeijer M.F.
      • Zwinderman A.H.
      • Wijnen J.
      • Fodde R.
      Atypical HNPCC owing to MSH6 germline mutations analysis of a large Dutch pedigree.
      Eight of the 10 families agreed to participate in this study.
      The second group is composed of 12 families recruited through the departments of clinical genetics of the University Medical Centers of Leiden, Rotterdam, and Amsterdam (VU University Medical Center), The Netherlands, and at the Institute of Medical Genetics of the University Medical Center in Rome, Italy. Most of the referred families do not fulfill the Amsterdam II criteria. The families were referred for genetic analysis because of a positive family history of (colorectal) cancer. Only families with a protein truncating germline mutation in the MSH6 gene were included in this study.
      In the total group of 20 families, 17 different truncating mutations were identified (Table 1). Only 6 families fulfill the Amsterdam II criteria (Table 1).
      Table 1MSH6 Mutations and Family Characteristics
      FamilyMutationAmsterdam II criteriaConfirmed tumors with age of diagnosis in proven carriers and individuals with unknown mutation status
      1
      Previously published.26
      Previously published.34
      1784delT, L594fsX, exon 4Py77 + Py79, Py76, C84, Py59, C55 + E55, C49 + B49, E57, E60, O50, E53, E50, C50 + O51, C32, C74
      2
      Previously published.26
      467C→G, S156X, exon 3+C67, C45, C47
      3
      Previously published.26
      742C→T, R248X, exon 4+C61, C58 + C59, C59, C26
      4
      Previously published.26
      2191C→T, Q731X, exon 4C48, C49, C51
      52731C→T, R911X, exon 4C56 + E56 + C57 + C70 + Py69 + Lu70, C59
      63103C→T, R1035X, exon 4O49 + E49, Ut58
      7
      Previously published.26
      467C→G, S156X exon 3+C62 + Py73, C44, O78
      81267delT, C426fsX, exon 4E62 + C65, C63, C56, C62, E54 + C87, C85
      9
      Previously published.26
      Previously published.26,48,49
      4001G→A, R1334Q, splice donor defectC69, E57, C45 + E53 + C66, C64, E50
      101784delT, L594fsX, exon 4E58, E60, E53
      11
      Previously published.50
      2984delA, 996fsX, exon 4C48 + C67, C54, C54
      12
      Previously published.50
      1960–1961insGTGA, fsX, exon 4+C37 + St56, C61, C51
      13
      Previously published.26
      3261delC, P1087fsX, exon 5B78, C54 + E56, E51 + St73, E57, E49
      14
      Previously published.26
      IVS7-2A→C, 3647-2A→C, splice acceptor defect+E58, E50, E56, C50, E54
      153182delT, 1061fsX exon 5E50, C48
      163987–3988insGTCA, S1329fsX, exon 9E43, E50
      171444C→T, R482X, exon 4+E53 + C78 + Bl80 + Py82, C49, E49
      181614–1615delTCinsG, Y538X, exon 4E65 + C81, E55, O45
      19651–652insT, K218X, exon 4E57, C52
      20651–652insT, K218X, exon 4C61, C41
      NOTE. Boldface indicates proven carriers.
      Py, transitional cell carcinoma of the renal pyelum; C, colorectal carcinoma; E, endometrial carcinoma; B, breast carcinoma; O, ovarian carcinoma; Lu, lung cancer; St, stomach cancer; Bl, bladder cancer.
      a Previously published.
      • Wijnen J.
      • de Leeuw W.
      • Vasen H.
      • van der Klift H.
      • Moller P.
      • Stormorken A.
      • Meijers-Heijboer H.
      • Lindhout D.
      • Menko F.
      • Vossen S.
      • Moslein G.
      • Tops C.
      • Brocker-Vriends A.
      • Wu Y.
      • Hofstra R.
      • Sijmons R.
      • Cornelisse C.
      • Morreau H.
      • Fodde R.
      Familial endometrial cancer in female carriers of MSH6 germline mutations.
      b Previously published.
      • Wagner A.
      • Hendriks Y.
      • Meijers-Heijboer E.J.
      • de Leeuw W.J.
      • Morreau H.
      • Hofstra R.
      • Tops C.
      • Bik E.
      • Brocker-Vriends A.H.
      • van Der Meer C.
      • Lindhout D.
      • Vasen H.F.
      • Breuning M.H.
      • Cornelisse C.J.
      • van Krimpen C.
      • Niermeijer M.F.
      • Zwinderman A.H.
      • Wijnen J.
      • Fodde R.
      Atypical HNPCC owing to MSH6 germline mutations analysis of a large Dutch pedigree.
      c Previously published.
      • Wijnen J.
      • de Leeuw W.
      • Vasen H.
      • van der Klift H.
      • Moller P.
      • Stormorken A.
      • Meijers-Heijboer H.
      • Lindhout D.
      • Menko F.
      • Vossen S.
      • Moslein G.
      • Tops C.
      • Brocker-Vriends A.
      • Wu Y.
      • Hofstra R.
      • Sijmons R.
      • Cornelisse C.
      • Morreau H.
      • Fodde R.
      Familial endometrial cancer in female carriers of MSH6 germline mutations.
      ,
      • Menko F.H.
      • Verheijen R.H.
      • Everhardt E.
      • Louwe L.A.
      • Wijnen J.T.
      • Band S.C.
      • Felt-Bersma R.J.
      • Vasen H.F.
      • Khan P.M.
      Endometrial cancer in four sisters report of a kindred with presumed cancer family syndrome.
      ,
      • Gille J.J.
      • Hogervorst F.B.
      • Pals G.
      • Wijnen J.T.
      • van Schooten R.J.
      • Dommering C.J.
      • Meijer G.A.
      • Craanen M.E.
      • Nederlof P.M.
      • de Jong D.
      • McElgunn C.J.
      • Schouten J.P.
      • Menko F.H.
      Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach.
      d Previously published.
      • Lucci-Cordisco E.
      • Rovella V.
      • Carrara S.
      • Percesepe A.
      • Pedroni M.
      • Bellacosa A.
      • Caluseriu O.
      • Forasarig M.
      • Anti M.
      • Neri G.
      • De Leon M.P.
      • Viel A.
      • Genuardi M.
      Mutations of the “minor” mismatch repair gene MSH6 in typical and atypical hereditary nonpolyposis colorectal cancer.
      We collected clinical information, including the age at diagnosis of cancer, site of the tumor, and pathology reports for as many affected individuals as possible. In addition, we collected the results of colonoscopic and gynecologic screening of the high-risk unaffected relatives. Genetic counseling and testing were offered to all relevant relatives. MSI and IHC analyses were performed on all available tumors.

       Mutation analysis

      Mutation analysis of the MSH6 gene was performed by denaturating gradient gel electrophoresis
      • Fodde R.
      • van der Luijt R.
      • Wijnen J.
      • Tops C.
      • van der Klift H.
      • van Leeuwen-Cornelisse I.
      • Griffioen G.
      • Vasen H.
      • Khan P.M.
      Eight novel inactivating germ line mutations at the APC gene identified by denaturing gradient gel electrophoresis.
      followed by sequence analysis if a variant was identified. A mutation was considered pathogenic when the nucleotide change is predicting truncation of the protein (e.g., nonsense and frameshift mutations) or when it is changing a consensus splice donor or acceptor site, confirmed by testing the mutation in splice site prediction software (Neural Network Splice Site Prediction [http://www.fruitfly.org/seq_tools/splice.html] or CBS NetGene 2 [http://www.cbs.dtu.dk/services/NetGene2]).

       Statistical analysis

      Penetrance for age was calculated using the Kaplan-Meier survival analysis method with the SPSS statistical package. Only proven carriers and only cases of cancer that were confirmed by medical records and/or pathology reports were included in the analysis. If more than one tumor developed in the same organ, only the first one diagnosed was included in the analysis. For the analysis of the cumulative risk of all HNPCC-related tumors together, only the first diagnosis was included in the analysis. For the analyses of the cumulative risk of colorectal and endometrial carcinomas, all first diagnoses in the respective organs were included. The observation time was from birth until date of diagnosis of cancer, death, or the end of the study in June 2002. No individuals were lost to follow-up.
      A Kaplan-Meier analysis was also performed in 30 families with an MLH1 mutation and 37 families with an MSH2 mutation, previously described by Vasen et al. in 2001,
      • Vasen H.F.
      • Stormorken A.
      • Menko F.H.
      • Nagengast F.M.
      • Kleibeuker J.H.
      • Griffioen G.
      • Taal B.G.
      • Moller P.
      • Wijnen J.T.
      MSH2 mutation carriers are at higher risk of cancer than MLH1 mutation carriers a study of hereditary nonpolyposis colorectal cancer families.
      in which the same detailed data were available. To evaluate whether the cancer risk differed between the 3 groups of mutation carriers, we used the Wald test criterion of the Cox proportional hazards regression model. P < 0.05 was considered statistically significant.

       MSI analysis

      MSI analysis was performed on paired tumor DNA and DNA from normal tissue using the Bethesda panel of microsatellite markers D2S123, D5S346, D17S250, BAT25, and BAT26
      • Boland C.R.
      • Thibodeau S.N.
      • Hamilton S.R.
      • Sidransky D.
      • Eshleman J.R.
      • Burt R.W.
      • Meltzer S.J.
      • Rodriguez-Bigas M.A.
      • Fodde R.
      • Ranzani G.N.
      • Srivastava S.
      A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition development of international criteria for the determination of microsatellite instability in colorectal cancer.
      with the additional BAT40 marker.
      • Hendriks Y.
      • Franken P.
      • Dierssen J.W.
      • de Leeuw W.
      • Wijnen J.
      • Dreef E.
      • Tops C.
      • Breuning M.
      • Brocker-Vriends A.
      • Vasen H.
      • Fodde R.
      • Morreau H.
      Conventional and tissue microarray immunohistochemical expression analysis of mismatch repair in hereditary colorectal tumors.
      Tumors were regarded as MSI high if at least 30% of the markers showed instability, MSI low if <30% showed instability, or microsatellite stable if none of the markers showed instability.

       IHC

      IHC staining was performed on 4-μm sections of formalin-fixed, paraffin-embedded tissues. Slides were stained with antibodies against MLH1 (clone 14; Calbiochem, Cambridge, MA), MSH2 (clone GB12; Calbiochem), and MSH6 (clone 44; Transduction Laboratories/Becton Dickinson, Lexington, KY) in a Dako Techmate 500+ automated tissue stainer using standard protocols
      • Hendriks Y.
      • Franken P.
      • Dierssen J.W.
      • de Leeuw W.
      • Wijnen J.
      • Dreef E.
      • Tops C.
      • Breuning M.
      • Brocker-Vriends A.
      • Vasen H.
      • Fodde R.
      • Morreau H.
      Conventional and tissue microarray immunohistochemical expression analysis of mismatch repair in hereditary colorectal tumors.
      and procedures as indicated by the manufacturer. Staining patterns of MMR proteins were evaluated using normal epithelial, stromal, or inflammatory cells or the centers of lymphoid follicles as internal controls. Stained slides were scored as either positive (showing nuclear staining in at least some tumor cells) or negative.

      Results

       Mutation analysis

      Mutation analysis was performed in 240 individuals (95 men and 145 women). Of the individuals tested, 55 were affected, 150 were first-degree relatives, and 35 were second-degree relatives. A mutation was identified in 119 individuals. Twenty-seven individuals were obligate carriers (13 affected and 14 not affected), based on the results of mutation analyses in their family members, and were not tested. Therefore, a total of 146 carriers were identified.
      Of the 55 affected individuals who have been tested, 4 were proven not to be carriers of the MSH6 mutation segregating in their respective families and are thus considered phenocopies. Two of these individuals developed colorectal carcinoma at 46 and 75 years of age, respectively, one woman was diagnosed with endometrial carcinoma at 45 years of age, and another women developed colorectal carcinoma at 71 years of age (and breast cancer at 50 years of age).

       Statistical analysis

      The 146 proven carriers of a pathogenic MSH6 mutation (59 men and 87 women) were included in the Kaplan-Meier analysis. Sixty-four affected carriers were identified (22 men and 42 women).
      Table 2 shows the mean risks of cancer (percentages) for all HNPCC-related tumors, for colorectal carcinoma in men and women separately, and for endometrial carcinoma as well as the 95% confidence intervals for the ages of 30, 50, and 70 years for MLH1, MSH2, and MSH6 carriers. The respective cumulative risk curves are shown in Figure 1, Figure 2, Figure 3, Figure 4. For all HNPCC-related tumors, the cumulative risks in MSH6 carriers, men and women together, differed statistically significantly from the risk of MLH1 and MSH2 (P = 0.002) (Figure 1). This is because of the higher mean age at onset. However, the cumulative risks at 70 years of age were similar for the 3 genes.
      Table 2Mean Percentage Cancer Risks at Age 30, 50, and 70 Years for Carriers of a Mutation in MLH1, MSH2, or MSH6
      GeneAge (yr) (95% confidence intervals)
      305070
      All HNPCC-related tumours
      MLH14.2 (1.6–6.8)34 (27–41)76 (62–85)
      MSH21.1 (0–2.3)50 (42–57)80 (70–86)
      MSH60.7 (0–2.1)22 (13–29)73 (60–82)
      Colorectal carcinoma in men
      MLH14.1 (0.1–7.9)31 (19–41)65 (39–80)
      MSH22.0 (0–4.4)39 (28–48)63 (49–73)
      MSH61.7 (0–5.0)17 (4.4–28)69 (42–83)
      Colorectal carcinoma in women
      MLH14.3 (0.9–7.7)26 (17–34)53 (33–66)
      MSH2030 (18–40)68 (43–82)
      MSH6010 (2.4–17)30 (12–44)
      Endometrial carcinoma
      MLH107.2 (1.4–13)27 (14–38)
      MSH2023 (12–32)40 (21–54)
      MSH6013 (5.3–22)71 (50–83)
      Figure thumbnail gr1
      Figure 1All HNPCC-related tumors; cumulative risks for MLH1, MSH2, and MSH6 mutation carriers.
      Figure thumbnail gr2
      Figure 2Colorectal carcinoma in men; cumulative risks for MLH1, MSH2, and MSH6 mutation carriers.
      Figure thumbnail gr3
      Figure 3Colorectal carcinoma in women; cumulative risks for MLH1, MSH2, and MSH6 mutation carriers.
      Figure thumbnail gr4
      Figure 4Endometrial carcinoma; cumulative risks for MLH1, MSH2, and MSH6 mutation carriers.
      In Figure 2, the age-related cumulative risk for colorectal carcinoma is shown for men only for MLH1, MSH2, and MSH6. The risks were lower in MSH6 mutation carriers, but the difference was not significantly different (P = 0.0854). The mean age at diagnosis for colorectal carcinoma in male MSH6 mutation carriers was 55 years (n = 21; range, 26–84 years) versus 43 and 44 years in MLH1 and MSH2 mutation carriers, respectively.
      In Figure 3, the age-related cumulative risk for colorectal carcinoma is shown for women only for MLH1, MSH2, and MSH6. The age-related cumulative risk was significantly lower in MSH6 mutation carriers (P = 0.0049). The mean age at diagnosis for colorectal carcinoma in female MSH6 mutation carriers was 57 years (n = 15; range, 41–81 years) versus 43 and 44 years in MLH1 and MSH2 mutation carriers, respectively.
      Of the colorectal tumors in which the exact localization in the colorectum was known, 13 (39%) were located distally and 20 (61%) were located proximally (proximal to the flexura lienalis).
      In Figure 4, the age-related cumulative risk for endometrial carcinoma is shown for MLH1, MSH2, and MSH6. The cumulative risk was significantly higher in MSH6 mutation carriers (P = 0.02) compared with the risk in MLH1 and MSH2 mutation carriers. The mean age at diagnosis of endometrial carcinoma is 54 years (n = 29; range, 43–65 years) versus 48 and 49 years in MLH1 and MSH2 mutation carriers, respectively.
      For ovarian carcinoma and transitional cell carcinoma of the upper urinary tract, cumulative risks were not calculated because the numbers were too low. The mean age at diagnosis for ovarian carcinoma was 49 years (n = 4; range, 45–51 years), and the mean age at diagnosis for transitional cell carcinoma was 72.5 years (n = 5; range, 59–82 years).
      One family (family 1
      • Wagner A.
      • Hendriks Y.
      • Meijers-Heijboer E.J.
      • de Leeuw W.J.
      • Morreau H.
      • Hofstra R.
      • Tops C.
      • Bik E.
      • Brocker-Vriends A.H.
      • van Der Meer C.
      • Lindhout D.
      • Vasen H.F.
      • Breuning M.H.
      • Cornelisse C.J.
      • van Krimpen C.
      • Niermeijer M.F.
      • Zwinderman A.H.
      • Wijnen J.
      • Fodde R.
      Atypical HNPCC owing to MSH6 germline mutations analysis of a large Dutch pedigree.
      ) was substantially more extended than the other families. To exclude the possibility that this large family biased the results, we compared the cumulative risks for the various tumors between this family and the total group. There were no substantial differences. In addition, we examined whether the degree of participation in the families influenced the results. No considerable differences in cumulative risk were found between the families with a higher and lower degree of participation. To avoid bias toward affected individuals, we performed the Kaplan-Meier analyses both with and without index patients. Because these results did not differ, we decided to include the index patients.

       MSI analysis

      As shown in Table 3, 49 tumors, all from mutation carriers, have been tested for MSI. Eighteen of 21 (86%) of the colorectal tumors showed an MSI-high phenotype. Two of the 3 tumors with an MSI-low phenotype would have been considered microsatellite stable if the BAT40 marker had not been tested. The third MSI-low tumor showed instability of a dinucleotide marker. If MSI-low tumors are also considered, the sensitivity for MSI analysis in colorectal tumors is 100%. Of the 16 endometrial tumors tested, 11 were MSI high (69%), 4 MSI low (25%), and one microsatellite stable (6%). Two of the MSI-high tumors and 1 of the MSI-low tumors would have been considered MSI low and microsatellite stable, respectively, if the BAT40 marker had not been tested. All MSI-low endometrial tumors showed instability of one of the mononucleotide markers. Five of the 7 (71%) transitional cell carcinomas tested showed an MSI-high phenotype. The other 2 were MSI low (29%). Two ovarian tumors were MSI high. The gastric carcinoma was microsatellite stable. The breast tumor, diagnosed in a proven carrier, showed an MSI-high phenotype. One adenocarcinoma of the cervix was MSI low. MSI in all HNPCC-related tumors together has a sensitivity of 71% and 90%, respectively, if MSI-high and both MSI-high and MSI-low tumors are considered.
      Table 3Results of MSI Analyses in Tumors of MSH6 Mutation Carriers
      TumorMSI highMSI lowMicrosatellite stableTotal
      Colorectal carcinoma (%)18 (86)3 (14)021
      Endometrial carcinoma (%)11 (69)4 (25)1 (6)16
      Transitional cell carcinoma (%)5 (71)2 (29)0 (0)7
      Ovarian carcinoma2002
      Breast carcinoma1001
      Stomach carcinoma0011
      Adenocarcinoma of the cervix0101
      Total359549

       IHC

      As shown in Table 4, 40 tumors, all from mutation carriers, have been tested for MMR protein expression by IHC: 18 colorectal tumors, 15 endometrial tumors, 4 transitional cell tumors, 1 ovarian tumor, 1 breast tumor, 1 gastric tumor, and 1 adenocarcinoma of the cervix. Thirty-six of the 40 tumors (90%) showed the expected pattern of absent staining for the MSH6 protein and retained staining for both the MLH1 and MSH2 proteins.
      Table 4Results of IHC in Tumors of MSH6 Mutation Carriers
      IHC patternNo. of tumors (%)
      MLH1+, MSH2+, MSH6−36 (90)
      MLH1+, MSH2−, MSH6−2 (5)
      MLH1−, MSH2+, MSH6−1 (2.5)
      MLH1+, MSH2+, MSH6+1 (2.5)
      Total40
      +, positive staining for protein; −, negative staining for protein.
      One of the MSI-low colorectal tumors previously mentioned showed absent MSH6 staining in IHC, indicating an MMR (MSH6) mutation. Another MSI-low tumor (diagnosed at age 78 years) showed positive staining for the MLH1, MSH2, and MSH6 proteins. In the same patient, bilateral transitional cell carcinoma showed an MSI-high phenotype and absent staining for the MSH6 protein. The colon tumor in this patient is likely to have been a sporadic tumor that did not develop because of defective MMR. All endometrial and transitional cell carcinomas showed negative staining for MSH6. Two colorectal tumors from different individuals showed absent staining not only for MSH6 but also for MSH2. One of these individuals also developed an endometrial carcinoma that showed negative staining for MSH6 in combination with positive staining for MLH1 and MSH2. Another colorectal tumor showed absent staining for both MLH1 and MSH6. In 98% (39 of 40) of the tested tumors, staining for the MSH6 protein was negative. In 90% (36 of 40) of the tumors, IHC specifically indicated a mutation in the MSH6 gene by an IHC pattern with positive staining for MLH1 and MSH2 and negative staining for MSH6.

      Discussion

      We studied 20 families with a truncating germline MSH6 mutation to determine the age-related cumulative risk of developing cancer and to develop a tailor-made surveillance protocol. We found that the cumulative risk of all HNPCC-related tumors in MSH6 mutation carriers was significantly lower than the risk in carriers of a truncating MLH1 or MSH2 mutation. In women, the cumulative risk of colorectal cancer was significantly lower (P = 0.0049) when compared with carriers of a mutation in MLH1 or MSH2, whereas the risk of endometrial cancer was more than twice as high (P = 0.02). For both colorectal carcinoma (54 years) and endometrial carcinoma (55 years), the mean age at diagnosis was higher in female MSH6 mutation carriers compared with carriers of a mutation in MLH1 or MSH2. In men, the risk of colorectal carcinoma was also lower than in MLH1 and MSH2 mutation carriers, but the difference was not statistically significant (P = 0.084). The mean age at diagnosis (58.5 years) was more than 10 years higher in MSH6 compared with MLH1 and MSH2 mutation carriers.
      Previous studies from The Netherlands and Finland on cancer risks in carriers of an MLH1 or MSH2 mutation were possibly biased toward overestimation of the risk because most of the families were selected by using the Amsterdam criteria or on the basis of familial clustering of colorectal cancer.
      • Vasen H.F.
      • Stormorken A.
      • Menko F.H.
      • Nagengast F.M.
      • Kleibeuker J.H.
      • Griffioen G.
      • Taal B.G.
      • Moller P.
      • Wijnen J.T.
      MSH2 mutation carriers are at higher risk of cancer than MLH1 mutation carriers a study of hereditary nonpolyposis colorectal cancer families.
      ,
      • Wu Y.
      • Berends M.J.
      • Mensink R.G.
      • Kempinga C.
      • Sijmons R.H.
      • van der Zee A.G.
      • Hollema H.
      • Kleibeuker J.H.
      • Buys C.H.
      • Hofstra R.M.
      Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations.
      ,
      • Aarnio M.
      • Mecklin J.P.
      • Aaltonen L.A.
      • Nystrom-Lahti M.
      • Jarvinen H.J.
      Life-time risk of different cancers in hereditary non-polyposis colorectal cancer (HNPCC) syndrome.
      However, the only population-based study (from Scotland) reported similar risks for colorectal carcinoma in men,
      • Dunlop M.G.
      • Farrington S.M.
      • Carothers A.D.
      • Wyllie A.H.
      • Sharp L.
      • Burn J.
      • Liu B.
      • Kinzler K.W.
      • Vogelstein B.
      Cancer risk associated with germline DNA mismatch repair gene mutations.
      although the risk for developing colorectal carcinoma in women was lower compared with the findings in the Dutch and Finnish studies. Carayol et al.
      • Carayol J.
      • Khlat M.
      • Maccario J.
      • Bonaiti-Pellie C.
      Hereditary non-polyposis colorectal cancer current risks of colorectal cancer largely overestimated.
      discussed the fact that the current risks are probably overestimated in HNPCC because of the statistical method used and proposed a novel statistical approach. We have chosen the Kaplan-Meier analysis because all previous studies eligible for comparison with our data used the Kaplan-Meier analysis as well.
      • Vasen H.F.
      • Stormorken A.
      • Menko F.H.
      • Nagengast F.M.
      • Kleibeuker J.H.
      • Griffioen G.
      • Taal B.G.
      • Moller P.
      • Wijnen J.T.
      MSH2 mutation carriers are at higher risk of cancer than MLH1 mutation carriers a study of hereditary nonpolyposis colorectal cancer families.
      ,
      • Wu Y.
      • Berends M.J.
      • Mensink R.G.
      • Kempinga C.
      • Sijmons R.H.
      • van der Zee A.G.
      • Hollema H.
      • Kleibeuker J.H.
      • Buys C.H.
      • Hofstra R.M.
      Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations.
      ,
      • Berends M.J.
      • Wu Y.
      • Sijmons R.H.
      • Mensink R.G.
      • van der Sluis T.
      • Hordijk-Hos J.M.
      • de Vries E.G.
      • Hollema H.
      • Karrenbeld A.
      • Buys C.H.
      • van der Zee A.G.
      • Hofstra R.M.
      • Kleibeuker J.H.
      Molecular and clinical characteristics of MSH6 variants an analysis of 25 index carriers of a germline variant.
      The general finding of a higher age at diagnosis in MSH6 mutation carriers when compared with carriers of a mutation in MLH1 or MSH2 could be explained from the functional level of the MMR proteins. MLH1 and MSH2 are involved in MMR of both single-base mismatches and insertion-deletion loops, and repair is impaired in the absence of MLH1 or MSH2. Likewise, the MSH6 protein is involved in the repair of both single-base mismatches and insertion-deletion loops. However, in the absence of MSH6, MSH3 can partially replace its repair function and such redundancy might represent a protecting factor against accumulation of DNA damage.
      • Fishel R.
      Signaling mismatch repair in cancer.
      ,
      • Jiricny J.
      Mediating mismatch repair.
      ,
      • Lipkin S.M.
      • Wang V.
      • Jacoby R.
      • Banerjee-Basu S.
      • Baxevanis A.D.
      • Lynch H.T.
      • Elliott R.M.
      • Collins F.S.
      MLH3 a DNA mismatch repair gene associated with mammalian microsatellite instability.
      A striking finding in this study is the difference in cumulative lifetime risk of colorectal carcinoma between men and women. The same trend is described in MSH2 mutation carriers.
      • Vasen H.F.
      • Stormorken A.
      • Menko F.H.
      • Nagengast F.M.
      • Kleibeuker J.H.
      • Griffioen G.
      • Taal B.G.
      • Moller P.
      • Wijnen J.T.
      MSH2 mutation carriers are at higher risk of cancer than MLH1 mutation carriers a study of hereditary nonpolyposis colorectal cancer families.
      ,
      • Berends M.J.
      • Wu Y.
      • Sijmons R.H.
      • Mensink R.G.
      • van der Sluis T.
      • Hordijk-Hos J.M.
      • de Vries E.G.
      • Hollema H.
      • Karrenbeld A.
      • Buys C.H.
      • van der Zee A.G.
      • Hofstra R.M.
      • Kleibeuker J.H.
      Molecular and clinical characteristics of MSH6 variants an analysis of 25 index carriers of a germline variant.
      This cannot be explained by early death caused by endometrial carcinoma, before a colorectal carcinoma can develop, because endometrial carcinoma is not often the cause of death in these families.
      The current surveillance protocol used in carriers of a mutation in one of the MMR genes is colonoscopy every 1–2 years starting at the age of 20–25 years and a yearly gynecologic examination, transvaginal ultrasound examination, and blood test for assessment of CA125 levels starting at the age of 30–35 years. If transitional cell carcinoma of the upper urinary tract or stomach cancer occurs in at least 2 individuals in a family, urine cytology yearly or gastroscopy every 1–2 years, respectively, from the age of 30–35 years is recommended.
      • Vasen H.F.
      Clinical diagnosis and management of hereditary colorectal cancer syndromes.
      In the present study, we found a mean age at diagnosis of colorectal carcinoma more than 10 years higher than found in MLH1 and MSH2; the youngest age at diagnosis of colorectal cancer was 26 years in male MSH6 carriers and 41 years in female MSH6 carriers. We recommend the same colonoscopic surveillance protocol in male carriers of an MSH6 mutation as recommended in MLH1 and MSH2 mutation carriers because the cumulative risks did not differ significantly from the risk in MLH1 and MSH2 carriers. However, although this might further complicate the already-complex surveillance protocol, we recommend that female carriers of an MSH6 mutation start colonoscopy at the age of 30 years because the cumulative risk of colorectal carcinoma was significantly lower compared with carriers of a mutation in MLH1 and MSH2 and because the youngest age at diagnosis was 41 years.
      Similar to observations in MLH1 and MSH2 mutation carriers, the majority (66%) of the colon carcinomas in the families we examined were located in the proximal colon. A previous study reported that 30% of the colon carcinomas associated with MSH6 mutations were located proximally.
      • Berends M.J.
      • Wu Y.
      • Sijmons R.H.
      • Mensink R.G.
      • van der Sluis T.
      • Hordijk-Hos J.M.
      • de Vries E.G.
      • Hollema H.
      • Karrenbeld A.
      • Buys C.H.
      • van der Zee A.G.
      • Hofstra R.M.
      • Kleibeuker J.H.
      Molecular and clinical characteristics of MSH6 variants an analysis of 25 index carriers of a germline variant.
      The reason for the difference between these studies is unclear.
      We found that the cumulative risk of endometrial carcinoma increased sharply after the age of 50 years. It is still questionable whether surveillance of the endometrium will lead to the early detection of cancer and improvement of the prognosis.
      • Dove-Edwin I.
      • Boks D.
      • Goff S.
      • Kenter G.G.
      • Carpenter R.
      • Vasen H.F.
      • Thomas H.J.
      The outcome of endometrial carcinoma surveillance by ultrasound scan in women at risk of hereditary nonpolyposis colorectal carcinoma and familial colorectal carcinoma.
      Therefore, based on the substantial risk of developing this type of cancer and the overall mortality from endometrial carcinoma of approximately 14%,
      • Hickerson J.W.
      Endometrial carcinoma treatment and outcomes in the regional hospital setting.
      we advocate a liberal approach toward prophylactic hysterectomy for women with a truncating MSH6 mutation who are older than 50 years of age. For surveillance of transitional cell carcinoma, we propose starting from the age of 50 years in families in which this tumor has occurred. However, the value of urine testing for the early detection of cancer is still unknown.
      • Sijmons R.H.
      • Kiemeney L.A.
      • Witjes J.A.
      • Vasen H.F.
      Urinary tract cancer and hereditary nonpolyposis colorectal cancer risks and screening options.
      Because DNA analysis is expensive and time consuming, prescreening methods can be of great relevance to increasing the efficiency of genetic testing for the identification of the disease causing mutation. Two prescreening methods currently applied to identify families eligible for mutation analysis of the MMR genes are MSI analysis and IHC. MSI analysis in colorectal tumors caused by an MSH6 mutation has been reported to show either predominance of an MSI-high phenotype
      • Wijnen J.
      • de Leeuw W.
      • Vasen H.
      • van der Klift H.
      • Moller P.
      • Stormorken A.
      • Meijers-Heijboer H.
      • Lindhout D.
      • Menko F.
      • Vossen S.
      • Moslein G.
      • Tops C.
      • Brocker-Vriends A.
      • Wu Y.
      • Hofstra R.
      • Sijmons R.
      • Cornelisse C.
      • Morreau H.
      • Fodde R.
      Familial endometrial cancer in female carriers of MSH6 germline mutations.
      ,
      • Wagner A.
      • Hendriks Y.
      • Meijers-Heijboer E.J.
      • de Leeuw W.J.
      • Morreau H.
      • Hofstra R.
      • Tops C.
      • Bik E.
      • Brocker-Vriends A.H.
      • van Der Meer C.
      • Lindhout D.
      • Vasen H.F.
      • Breuning M.H.
      • Cornelisse C.J.
      • van Krimpen C.
      • Niermeijer M.F.
      • Zwinderman A.H.
      • Wijnen J.
      • Fodde R.
      Atypical HNPCC owing to MSH6 germline mutations analysis of a large Dutch pedigree.
      or predominance of an MSI-low phenotype.
      • Wu Y.
      • Berends M.J.
      • Mensink R.G.
      • Kempinga C.
      • Sijmons R.H.
      • van der Zee A.G.
      • Hollema H.
      • Kleibeuker J.H.
      • Buys C.H.
      • Hofstra R.M.
      Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations.
      ,
      • Berends M.J.
      • Wu Y.
      • Sijmons R.H.
      • Mensink R.G.
      • van der Sluis T.
      • Hordijk-Hos J.M.
      • de Vries E.G.
      • Hollema H.
      • Karrenbeld A.
      • Buys C.H.
      • van der Zee A.G.
      • Hofstra R.M.
      • Kleibeuker J.H.
      Molecular and clinical characteristics of MSH6 variants an analysis of 25 index carriers of a germline variant.
      We found an MSI-high phenotype in 86% of the MSH6-related colorectal carcinomas with a pattern equivalent to that found in MLH1- and MSH2-related tumors, including instability of both mononucleotide and dinucleotide markers. In the classification of MSI, we included the Bethesda panel of markers
      • Boland C.R.
      • Thibodeau S.N.
      • Hamilton S.R.
      • Sidransky D.
      • Eshleman J.R.
      • Burt R.W.
      • Meltzer S.J.
      • Rodriguez-Bigas M.A.
      • Fodde R.
      • Ranzani G.N.
      • Srivastava S.
      A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition development of international criteria for the determination of microsatellite instability in colorectal cancer.
      as well as the BAT 40 marker because it increases the sensitivity of MSI analysis, as shown in this study and a previous study performed by our group.
      • Hendriks Y.
      • Franken P.
      • Dierssen J.W.
      • de Leeuw W.
      • Wijnen J.
      • Dreef E.
      • Tops C.
      • Breuning M.
      • Brocker-Vriends A.
      • Vasen H.
      • Fodde R.
      • Morreau H.
      Conventional and tissue microarray immunohistochemical expression analysis of mismatch repair in hereditary colorectal tumors.
      If the MSI-low tumors are included, the sensitivity of MSI analysis is 100% in colorectal tumors. In endometrial tumors obtained from MSH6 mutation carriers, MSI analyses have been reported to show predominantly MSI-low phenotypes with mainly instability of mononucleotide repeats.
      • de Leeuw W.J.
      • Dierssen J.
      • Vasen H.F.
      • Wijnen J.T.
      • Kenter G.G.
      • Meijers-Heijboer H.
      • Brocker-Vriends A.
      • Stormorken A.
      • Moller P.
      • Menko F.
      • Cornelisse C.J.
      • Morreau H.
      Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients.
      ,
      • Wijnen J.
      • de Leeuw W.
      • Vasen H.
      • van der Klift H.
      • Moller P.
      • Stormorken A.
      • Meijers-Heijboer H.
      • Lindhout D.
      • Menko F.
      • Vossen S.
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      • Brocker-Vriends A.
      • Wu Y.
      • Hofstra R.
      • Sijmons R.
      • Cornelisse C.
      • Morreau H.
      • Fodde R.
      Familial endometrial cancer in female carriers of MSH6 germline mutations.
      ,
      • Wu Y.
      • Berends M.J.
      • Mensink R.G.
      • Kempinga C.
      • Sijmons R.H.
      • van der Zee A.G.
      • Hollema H.
      • Kleibeuker J.H.
      • Buys C.H.
      • Hofstra R.M.
      Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations.
      ,
      • Berends M.J.
      • Wu Y.
      • Sijmons R.H.
      • Mensink R.G.
      • van der Sluis T.
      • Hordijk-Hos J.M.
      • de Vries E.G.
      • Hollema H.
      • Karrenbeld A.
      • Buys C.H.
      • van der Zee A.G.
      • Hofstra R.M.
      • Kleibeuker J.H.
      Molecular and clinical characteristics of MSH6 variants an analysis of 25 index carriers of a germline variant.
      Accordingly, in the present study, we found an MSI-low phenotype in a substantial proportion (25% [4 of 16]). An MSI-high phenotype predominated in the other types of carcinoma tested. MSI in all HNPCC-related tumors together has a sensitivity of 71% and 90%, respectively, if MSI-high and both MSI-high and MSI-low tumors are considered. IHC in both colorectal and endometrial tumors has been reported to show positive staining of the MLH1 and MSH2 proteins and absent staining for MSH6.
      • de Leeuw W.J.
      • Dierssen J.
      • Vasen H.F.
      • Wijnen J.T.
      • Kenter G.G.
      • Meijers-Heijboer H.
      • Brocker-Vriends A.
      • Stormorken A.
      • Moller P.
      • Menko F.
      • Cornelisse C.J.
      • Morreau H.
      Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients.
      ,
      • Berends M.J.
      • Wu Y.
      • Sijmons R.H.
      • Mensink R.G.
      • van der Sluis T.
      • Hordijk-Hos J.M.
      • de Vries E.G.
      • Hollema H.
      • Karrenbeld A.
      • Buys C.H.
      • van der Zee A.G.
      • Hofstra R.M.
      • Kleibeuker J.H.
      Molecular and clinical characteristics of MSH6 variants an analysis of 25 index carriers of a germline variant.
      ,
      • Wagner A.
      • Hendriks Y.
      • Meijers-Heijboer E.J.
      • de Leeuw W.J.
      • Morreau H.
      • Hofstra R.
      • Tops C.
      • Bik E.
      • Brocker-Vriends A.H.
      • van Der Meer C.
      • Lindhout D.
      • Vasen H.F.
      • Breuning M.H.
      • Cornelisse C.J.
      • van Krimpen C.
      • Niermeijer M.F.
      • Zwinderman A.H.
      • Wijnen J.
      • Fodde R.
      Atypical HNPCC owing to MSH6 germline mutations analysis of a large Dutch pedigree.
      ,
      • Plaschke J.
      • Kruger S.
      • Pistorius S.
      • Theissig F.
      • Saeger H.D.
      • Schackert H.K.
      Involvement of hMSH6 in the development of hereditary and sporadic colorectal cancer revealed by immunostaining is based on germline mutations, but rarely on somatic inactivation.
      We found an almost 100% sensitivity in predicting an MMR defect, including a mutation in MSH6. In 90% of the tumors, IHC specifically predicted a germline mutation in the MSH6 gene. Two colorectal tumors from different individuals showed absent staining not only for MSH6 but also for MSH2. A possible explanation is that in the colorectal tumor of one of these patients, both the C-8 tract in MSH6 and the A-8 tract of MSH3 were shown to be somatically instable in MSI analysis. As a result, both the MSH2-MSH6 and the MSH2-MSH3 heterodimer might be less frequently formed, which will add to loss of expression of MSH2.
      In our clinic, IHC is the first step in prescreening families that fulfill the Amsterdam criteria because the yield of mutation analysis is high and IHC directly indicates which gene to test. If IHC is positive for all tested proteins, MSI analysis is performed. On the other hand, MSI analysis is the first step in prescreening families that do not fulfill the Amsterdam criteria. When an MSI-high or MSI-low phenotype, especially with instability of a mononucleotide marker, is found in an HNPCC-related tumor, IHC of the MMR proteins is the second step. In case of an MSS tumor, IHC of MSH6 is performed. Our results in this study confirm that this approach has a high sensitivity for identifying families with an MSH6 mutation.
      In conclusion, the present study shows that female MSH6 mutation carriers develop colorectal carcinoma at a significantly higher age than reported for MLH1 and MSH2 mutation carriers and that the cumulative risk is significantly lower. Based on these findings, we recommend starting colonoscopic surveillance from a higher age than recommended in MLH1 and MSH2 families in female MSH6 carriers. Secondly, we found a dramatic increase in the risk of developing endometrial carcinoma after the age of 50 years in female MSH6 mutation carriers and therefore recommend a liberal approach toward hysterectomy for women above this age. Finally, we show that both MSI analysis and IHC for the MMR proteins are very sensitive prescreening methods for identifying families eligible for mutation analysis of the MSH6 gene.
      This study underscores the distinct phenotype in MSH6 families and provides guidelines for the identification, counseling, and management of these families.

      Acknowledgements

      The authors thank the 20 families that participated in this study.

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