Jens Plaschke

Lower Incidence of Colorectal Cancer and Later Age of Disease Onset in 27 Families With Pathogenic MSH6 Germline Mutations Compared With Families With MLH1 or MSH2 Mutations: The German Hereditary Nonpolyposis Colorectal Cancer Consortium

PURPOSE The aim of the study was the analysis of the involvement and phenotypic manifestations of MSH6 germline mutations in families suspected of hereditary nonpolyposis colorectal cancer (HNPCC). PATIENTS AND METHODS Patients were preselected among 706 families by microsatellite instability, immunohistochemistry, and/or exclusion of MLH1 or MSH2 mutations and were subjected to MSH6 mutation analysis. Clinical and molecular data of MSH6 mutation families were compared with data from families with MLH1 and MSH2 mutations. RESULTS We identified 27 families with 24 different pathogenic MSH6 germline mutations, representing 3.8% of the total of the families, and 14.7% of all families with DNA mismatch repair (MMR) gene mutations (n = 183). The median age of onset of colorectal cancer in putative mutation carriers was 10 years higher for MSH6 (54 years; 95% CI, 51 to 56) compared with MLH1 and MSH2 (44 years; 95% CI, 43 to 45; log-rank test, P = .0038). Relative to other malignant tumors, colorectal cancer was less frequent in MSH6 families compared with MLH1 and MSH2 families (Fishers exact test, P < .001). In contrast, the frequency of non-HNPCC-associated tumors was increased (Fishers exact test, P < .001). CONCLUSION Later age of disease onset and lower incidence of colorectal cancer may contribute to a lower proportion of identified MSH6 mutations in families suspected of HNPCC. However, in approximately half of these families, at least one patient developed colorectal or endometrial cancer in the fourth decade of life. Therefore, a surveillance program as stringent as that for families with MLH1 or MSH2 mutations is recommended.

DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2.

Denaturing high-performance liquid chromatography (DHPLC) is an efficient method for detection of mutations involving a single or few numbers of nucleotides, and it has been successfully used for mutation detection in disease-related genes. Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary nonpolyposis colon cancer (HNPCC), hMLH1 and hMSH2, also involve mainly point mutations. Sequence analysis is supposed to be a screening method with high sensitivity; however, it is time-consuming and expensive. We therefore decided to test sensitivity and reproducibility of DHPLC for 71 sequence variants in hMLH1 and hMSH2 initially found by sequence analysis in DNA samples of German HNPCC patients. DHPLC conditions of the PCR products were based on the melting pattern of the wild-type sequence of the corresponding PCR fragments. All but one of the 71 mutations was detected using DHPLC (sensitivity of 97%). Running time per sample averaged only 7 min, and the system is highly automated. Thus DHPLC is a rapid and sensitive method for the detection of hMLH1 and hMSH2 sequence variants.

Novel strategy for optimal sequential application of clinical criteria, immunohistochemistry and microsatellite analysis in the diagnosis of hereditary nonpolyposis colorectal cancer.

Clinical criteria, microsatellite analysis (MSA) and immunohistochemistry (IHC) are important diagnostic tools for identification of hereditary nonpolyposis colorectal cancer (HNPCC) patients who are likely to carry pathogenic germline mutations in mismatch repair genes. Based on MSA and IHC results and subsequent mutation analyses of 1,119 unrelated index patients meeting the Amsterdam II criteria or the classical Bethesda guidelines, we analyzed the value of these tools to predict MLH1 and MSH2 mutations with the aim of establishing optimal strategies for their most efficient sequential use. The overall prevalence of pathogenic germline mutations in our cohort was 20.6% (95% CI = 18.3–23.0%) and 61.8% (95% CI = 56.8–66.6%), respectively, after MSA/IHC‐based preselection. IHC was highly predictive (99.1%) and specific (99.6%) with regard to MSA. However, 14 out of 230 mutations (6%) escaped detection by IHC. Thus, IHC cannot be recommended to substitute MSA fully. Nonetheless, IHC is important to indicate the gene that is likely to be affected. To combine both methods efficiently, we propose a novel screening strategy that provides 2 alternative ways of sequential IHC and MSA application, either using IHC or MSA in the first place. A logistic regression model based on the age of the index patient at first tumor diagnosis and the number of fulfilled HNPCC criteria is used to allocate individual patients to that alternative pathway that is expected to be least expensive. A cost analysis reveals that about 25% of the costs can be saved using this strategy.

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

Germline mutations in human mismatch repair (MMR) genes yield a predisposition for the hereditary nonpolyposis colon cancer (HNPCC) syndrome. In contrast to hMLH1 and hMSH2, little is known about the overall involvement of hMSH6 in colorectal cancer. We investigated 82 tumors from patients who fulfilled the Bethesda guidelines for HNPCC as well as 146 sporadic tumors, analyzing microsatellite instability and expression of the 4 MMR proteins hMSH6, hMSH2, hMLH1 and hPMS2. Four tumors with lost expression and 1 tumor with cytoplasmic expression of hMSH6 were identified. Sequence analysis revealed germline mutations in 4 of the 5 patients, including 1 patient with sporadic disease. The lost or reduced expression of hMSH2 and hMLH1 was always identical to its heterodimerization partners, hMSH6 and hPMS2, respectively. Furthermore, hMSH2 expression was reduced upon hMSH6 deficiency. Abnormal expression of 1 or more of the 4 proteins was always associated with a high level of microsatellite instability (MSI‐H). Conversely, all but 1 of the 44 MSI‐H tumors had abnormal expression of 1 or more of the proteins, basically excluding additional genes associated with the MSI‐H phenotype. We conclude that the involvement of somatic or epigenetic hMSH6 inactivation in colorectal cancer is rare.

Sequence analysis of the mismatch repair gene hMSH6 in the germline of patients with familial and sporadic colorectal cancer

To evaluate the involvement of hMSH6 in colorectal cancer, the complete coding sequence and flanking intron regions of the gene were analyzed by DNA sequencing in 10 patients fulfilling Bethesda Guidelines for colorectal tumors and 10 patients with sporadic colorectal carcinoma. In addition, 10 mono‐ and 10 dinucleotide repeat markers were analyzed for microsatellite instability. A protein‐truncating T insertion at codon 218 was identified in the index person of a hereditary non‐polyposis colorectal cancer (HNPCC)‐like kindred and was accompanied by a somatic T deletion in the tumor. The tumor of this patient was positive for mono‐ but negative for dinucleotide repeat instability and lacked allelic losses at loci frequently affected in colorectal carcinomas. A novel amino acid change, F340S, was found in a patient with sporadic colon and breast cancer and leukemia but was not detected in 246 chromosomes from healthy anonymous blood donors. In addition, we describe 2 silent and 15 intronic sequence variants not previously reported. Although the frequency is low, we present further evidence for hMSH6 germline mutations that predispose patients to HNPCC‐like phenotypes and suggest that mono‐ and dinucleotide repeat instability testing may be useful for distinguishing between individuals harboring an hMSH2 or hMLH1 mutation and a mutation of the hMSH6 gene. Int. J. Cancer 85:606–613, 2000.

Compound heterozygosity for two MSH6 mutations in a patient with early onset of HNPCC-associated cancers, but without hematological malignancy and brain tumor.

Heterozygous germline mutations in the human mismatch repair (MMR) genes MLH1, PMS2, MSH2 and MSH6 predispose to the hereditary non-polyposis colorectal cancer (HNPCC) syndrome. Biallelic mutations in these genes have been reported for a limited number of cases resulting in hematological malignancies, brain tumors and gastrointestinal tumors early in childhood. These tumor phenotypes are frequently associated with café-au-lait spots (CALS), one of the clinical hallmarks of neurofibromatosis type 1 (NF1). We report the first case of compound heterozygosity for two MSH6 mutations resulting in a nonconservative amino-acid change of a conserved residue and in a premature stop codon in a patient who developed rectal and endometrial cancer at ages 19 and 24 years, respectively, and presented few CALS in a single body segment. Immunohistochemistry and Western blotting revealed only residual expression of the MSH6 protein in the normal cells. The disease history resembles the HNPCC phenotype rather than a phenotype associated with biallelic MMR gene mutations. Therefore, we assume that one or both mutations abolish protein function only partially, further supported by the parents, which are both carriers of one of the mutations each, and not affected by the disease at ages 57 and 58 years. Our data suggest considering biallelic mutations in MMR genes for patients who develop HNPCC-associated tumors at an unusually young age of onset, even without hematological or brain malignancies.

Methylenetetrahydrofolate reductase polymorphisms and risk of sporadic and hereditary colorectal cancer with or without microsatellite instability

Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in the folate metabolism, which affects DNA synthesis and methylation. Low enzyme activity may reduce the capacity of DNA methylation, and possibly reduce uracil misincorporation into DNA, which can result in double strand breaks. Both processes may be critical for the oncogenic transformation of human cells. Two common amino acid-changing and enzyme activity-reducing nucleotide polymorphisms (677C --> T/Ala222Val and 1298A --> C/Glu428Ala) have been described in MTHFR. We performed estimations of the relative risk associated with these two polymorphisms in samples from 287 colorectal cancer patients, compared to 346 healthy controls. Relative risk were further determined for subpopulations of cancer patients having sporadic (n = 227) or suspected/verified hereditary disease (n = 60) and tumours exhibiting high-level microsatellite instability (n = 41) or not (n = 246). No significant differences for the relative risk of colorectal cancer were observed for the MTHFR genotypes either alone or in combination in the analysed cohorts, although the frequency of the 1298AA + AC genotypes was increased among the 60 cases with hereditary disease. Whereas our results do not support an association of high enzyme activity and increased risk of colorectal cancer in general, we can not exclude an association of patients with hereditary disease and the MTHFR 1298A --> C variant.

Loss of MSH3 protein expression is frequent in MLH1-deficient colorectal cancer and is associated with disease progression

Mononucleotide repeat sequences are particularly prone to frameshift mutations in tumors with biallelic inactivation of the mismatch repair (MMR) genes MLH1 or MSH2. In these tumors, several genes harboring mononucleotide repeats in their coding region have been proposed as targets involved in tumor progression, among which are also the MMR genes MSH3 and MSH6. We have analyzed the expression of the MSH3 and MSH6 proteins by immunohistochemistry in 31 colorectal carcinomas in which MLH1 was inactivated. Loss of MSH3 expression was identified in 15 tumors (48.5%), whereas all tumors expressed MSH6. Frameshift mutations at coding microsatellites were more frequent in MSH3 (16 of 31) than in MSH6 (3 of 31; Fisher’s exact test, P < 0.001). Frameshift mutations and allelic losses of MSH3 were more frequent in MSH3-negative tumors compared with those with normal expression (22 mutations in 30 alleles versus 8 mutations in 28 alleles; χ2, P = 0.001). Biallelic inactivation was evident or inferred for 60% of MSH3-negative tumors but none of the tumors with normal MSH3 expression. In contrast, we did not identify frameshift mutations in the (A)8 tract of MSH3 in a control group of 18 colorectal carcinomas in which the MMR deficiency was based on the inactivation of MSH2. As it has been suggested that mutations of MSH3 might play a role in tumor progression, we studied the association between MSH3 expression and disease stage assessed by lymph node and distant metastases status. Dukes stages C and D were more frequent in primary tumors with loss of MSH3 expression (9 of 13), compared with tumors with retained expression (1 of 14; Fisher’s exact test, P = 0.001), suggesting that MSH3 abrogation may be a predictor of metastatic disease or even favor tumor cell spread in MLH1-deficient colorectal cancers.

The p53 codon 72 variation is associated with the age of onset of hereditary non-polyposis colorectal cancer (HNPCC)

The polymorphic variants at codon 72 of the p53 gene were shown to be functionally distinct in vitro, whereby the arginine (arg) variant induces apoptosis more efficiently than the proline (pro) variant. From the evidence that the DNA mismatch repair system and p53 interact to maintain genomic integrity, we hypothesized that the codon 72 variation may influence the age of onset of disease in HNPCC patients. We tested 538 patients for p53 codon 72 variants, including 167 unrelated patients with pathogenic germline mutations in MSH2 or MLH1 and colorectal carcinoma as first tumour, 126 patients with sporadic microsatellite stable colorectal cancers, and 245 healthy controls. The median age of onset was 41, 36, and 32 years for MSH2 or MLH1 mutation carriers with arg/arg, arg/pro, and pro/pro genotypes, respectively. The log rank test revealed significant differences in the age of onset between arg/arg and pro/pro individuals (p = 0.0002) and in arg/pro versus arg/arg and pro/pro individuals (p = 0.0026 and p = 0.0217, respectively). A Cox regression model indicated an additive mode of inheritance. No significant differences in age of onset were observed among different genotype carriers with microsatellite stable tumours. Our results suggest that p53 codon 72 genotypes are associated with the age of onset of colorectal carcinoma in a mismatch repair deficient background in a dose dependent manner. These findings may be relevant for preventive strategies in HNPCC.

An intronic germline transition in the HNPCC gene hMSH2 is associated with sporadic colorectal cancer

The aim of this study was to determine whether an intronic germline substitution in the hereditary non-polyposis colorectal cancer (HNPCC) gene hMSH2 represents a genetic risk factor for sporadic CRC. Possible effects of this substitution were investigated by assessment of microsatellite instability and hMSH2 cDNA sequencing. Constitutional DNA from patients with sporadic CRC and healthy controls from the same region in Germany was analysed for the intronic germline T-->C transition six bases upstream of exon 13 of hMSH2. 29 of 106 patients (27%) were found to harbour the germline T-->C transition as opposed to only 13 of 125 controls (10%; P < 0.001; OR 3.2, CI 1.58-6.63). CRCs from patients with the substitution displayed neither clinical HNPCC-like features nor an increased rate of microsatellite instability. No abnormal cDNA sequence was found at the exon 12-13 border. These data suggest a 3.2-fold increased risk of sporadic CRC for individuals with the intronic hMSH2 transition. However, this substitution might not be pathogenic itself, but may be linked to a locus nearby that is.