Genome-wide copy neutral LOH is infrequent in familial and sporadic microsatellite unstable carcinomas.

Mismatch repair deficiency in tumors can result from germ line mutations in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2), or from sporadic promoter hypermethylation of MLH1. The role of unclassified variants (UVs) in MMR genes is subject to debate. To establish the extend of chromosomal instability and copy neutral loss of heterozygosity (cnLOH), we analyzed 41 archival microsatellite unstable carcinomas, mainly colon cancer, from 23 patients with pathogenic MMR mutations, from eight patients with UVs in one of the MMR genes and 10 cases with MLH1 promoter hypermethylation. We assessed genome wide copy number abnormalities and cnLOH using SNP arrays. SNP arrays overcome the problems of detecting LOH due to instability of polymorphic microsatellite markers. All carcinomas showed relatively few chromosomal aberrations. Also cnLOH was infrequent and in Lynch syndrome carcinomas usually confined to the locus harbouring pathogenic mutations in MLH1, MSH2 or PMS2 In the carcinomas from the MMR-UV carriers such cnLOH was less common and in the carcinomas with MLH1 promoter hypermethylation no cnLOH at MLH1 occurred. MSI-H carcinomas of most MMR-UV carriers present on average with more aberrations compared to the carcinomas from pathogenic MMR mutation carriers, suggesting that another possible pathogenic MMR mutation had not been missed. The approach we describe here shows to be an excellent way to study genome-wide cnLOH in archival mismatch repair deficient tumors.

Long term follow-up of HNPCC gene mutation carriers: compliance with screening and satisfaction with counseling and screening procedures.

Hereditary non polyposis colorectal cancer (HNPCC) is a hereditary predisposition to colorectal and endometrial cancer, caused by mutations of the mismatch repair (MMR) genes MSH2, MLH1 and MSH6. Regular colonoscopy reduces the incidence of colorectal cancer in mutation carriers dramatically. The aim of this study was to evaluate the use of colonoscopy by proven HNPCC mutation carriers. We also evaluated the satisfaction with the counseling and screening procedures at the long term. A questionnaire survey was performed among 94 proven MMR gene mutation carriers. Data were analyzed using univariate and multivariate analysis. The average time of follow-up was 3,5 years (range 0.5-8.5 years). The response rate was 74%. The proportion of unaffected mutation carriers under colonoscopic screening increased from 31 to 88% upon genetic testing, and for gynecological screening from 17 to 69%. However, more than half of the responders experienced colonoscopy as unpleasant or painful. About 97% felt well informed during counseling, and 88% felt sufficiently supported. Ten percent of the responders reported a high cancer worry that was significantly (P = 0.007) associated with a high perceived cancer risk. Six responders (9%) regretted being tested. Remarkably, of 4 of these 6 a close relative died recently of cancer. Problems with obtaining a disability or life insurance or mortgage were experienced by 4 out 10 healthy carriers opting for these services. In conclusion, genetic testing for HNPCC considerably improves compliance for screening, which will result in a reduction of HNPCC-related cancer morbidity and mortality in mutation carriers. Most HNPCC gene mutation carriers cope well with their cancer susceptibility on the long term.

Introduction to molecular and clinical genetics of colorectal cancer syndromes.

The understanding of molecular genetics in the field of gastroenterology has rapidly grown over the last two decades. In recent years many genes involved in the disorders of the gastrointestinal (GI) tract such as colorectal cancer (CRC) and inflammatory bowel disease have been identified. The elucidation of the molecular genetics of these diseases made it possible to study the high-penetrance susceptibility genes for disease-causing mutations with direct implications for relatives of affected individuals. The most immediate application of these advances is the opportunity of pre-symptomatic diagnosis in relatives of affected individuals by molecular genetic testing. In this article, the most commonly employed mutation detection procedures; the outcome and use of these tests in clinical practice are discussed. We focus on the three most common hereditary colorectal cancer syndromes (CCS): Lynch syndrome, familial adenomatous polyposis and MUTYH-associated polyposis.

Diagnostic approach and management of Lynch syndrome (hereditary nonpolyposis colorectal carcinoma): a guide for clinicians.

ABSTRACT diagnostic workup of familial colorectal cancer is an elaborate and time consuming process in which the family and several medical specialists closely collaborate. However, establishing a diagnosis can be very rewarding. If a mutation is detected in the family, a satisfactory explanation can be provided for an accumulation of tumors at young age, and often of untimely death. Appropriate presymptomatic testing can be offered to reduce mortality among at-risk family members, and relatives not at risk can avoid uncertainty and needlessly intensive surveillance. We show the differential diagnostic considerations when an individual with a family history of colorectal carcinoma is encountered, with emphasis on Lynch syndrome (Hereditary Nonpolyposis Colorectal Carcinoma [HNPCC]). Practical recommendations for laboratory workup of suspected Lynch syndrome, including analysis of tumor tissue by microsatellite instability analysis and immunohistochemistry, and germline DNA analysis are given. Furthermore, the clinical management after a molecular diagnosis has been made is described. The diagnostic scheme presented here allows efficient and effective analysis of colorectal carcinoma cases with (suspected) Lynch syndrome, making optimal use of currently available technology.

Heterozygous mutations in PMS2 cause hereditary nonpolyposis colorectal carcinoma (Lynch syndrome).

BACKGROUND & AIMS: The role of the mismatch repair gene PMS2 in hereditary nonpolyposis colorectal carcinoma (HNPCC) is not fully clarified. To date, only 7 different heterozygous truncating PMS2 mutations have been reported in HNPCC-suspected families. Our aim was to further assess the role of PMS2 in HNPCC. METHODS: We performed Southern blot analysis in 112 patients from MLH1-, MSH2-, and MSH6-negative HNPCC-like families. A subgroup (n = 38) of these patients was analyzed by denaturing gradient gel electrophoresis (DGGE). In a second study group consisting of 775 index patients with familial colorectal cancer, we performed immunohistochemistry using antibodies against MLH1, MSH2, MSH6, and PMS2 proteins. In 8 of 775 tumors, only loss of PMS2 expression was found. In these cases, we performed Southern blot analysis and DGGE. Segregation analysis was performed in the families with a (possibly) deleterious mutation. RESULTS: Seven novel mutations were identified: 4 genomic rearrangements and 3 truncating point mutations. Three of these 7 families fulfill the Amsterdam II criteria. The pattern of inheritance is autosomal dominant with a milder phenotype compared with families with pathogenic MLH1 or MSH2 mutations. Microsatellite instability and immunohistochemical analysis performed in HNPCC-related tumors from proven carriers showed a microsatellite instability high phenotype and loss of PMS2 protein expression in all tumors. CONCLUSIONS: We show that heterozygous truncating mutations in PMS2 do play a role in a small subset of HNPCC-like families. PMS2 mutation analysis is indicated in patients diagnosed with a colorectal tumor with absent staining for the PMS2 protein.

Molecular analysis of hereditary nonpolyposis colorectal cancer in the United States: high mutation detection rate among clinically selected families and characterization of an American founder genomic deletion of the MSH2 gene.

The identification of germline mutations in families with HNPCC is hampered by genetic heterogeneity and clinical variability. In previous studies, MSH2 and MLH1 mutations were found in approximately two-thirds of the Amsterdam-criteria-positive families and in much lower percentages of the Amsterdam-criteria-negative families. Therefore, a considerable proportion of HNPCC seems not to be accounted for by the major mismatch repair (MMR) genes. Does the latter result from a lack of sensitivity of mutation detection techniques, or do additional genes underlie the remaining cases? In this study we address these questions by thoroughly investigating a cohort of clinically selected North American families with HNPCC. We analyzed 59 clinically well-defined U.S. families with HNPCC for MSH2, MLH1, and MSH6 mutations. To maximize mutation detection, different techniques were employed, including denaturing gradient gel electrophoresis, Southern analysis, microsatellite instability, immunohistochemistry, and monoallelic expression analysis. In 45 (92%) of the 49 Amsterdam-criteria-positive families and in 7 (70%) of the 10 Amsterdam-criteria-negative families, a mutation was detected in one of the three analyzed MMR genes. Forty-nine mutations were in MSH2 or MLH1, and only three were in MSH6. A considerable proportion (27%) of the mutations were genomic rearrangements (12 in MSH2 and 2 in MLH1). Notably, a deletion encompassing exons 1-6 of MSH2 was detected in seven apparently unrelated families (12% of the total cohort) and was subsequently proven to be a founder. Screening of a second U.S. cohort with HNPCC from Ohio allowed the identification of two additional kindreds with the identical founder deletion. In the present study, we show that optimal mutation detection in HNPCC is achieved by combining accurate and expert clinical selection with an extensive mutation detection strategy. Notably, we identified a common North American deletion in MSH2, accounting for approximately 10% of our cohort. Genealogical, molecular, and haplotype studies showed that this deletion represents a North American founder mutation that could be traced back to the 19th century.

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

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.