Aberrant protein expression and frequent allelic loss of MSH3 in colorectal cancer with low-level microsatellite instability.

PURPOSE: High level of microsatellite instability (MSI-H) in colorectal cancer (CRC) is caused by the inactivation of mismatch repair (MMR) genes; however, it is unknown for tumors with low level MSI (MSI-L). The protein complex involving MSH3 preferentially recognizes insertion/deletion loops (IDLs) of two to eight bases and di- and tetranucleotide repeats are affected in the majority of MSI-L CRC. METHODS: We selected 10 and eight MSI-L CRCs from 228 and 204 patients with sporadic and hereditary disease, respectively. The tumors were analyzed for protein expression of MSH3, MSH2, MSH6, MLH1, and PMS2, and for mutations and loss of heterozygosity (LOH) in MSH3. RESULTS: Four tumors showed a markedly reduced MSH3 expression, whereas all 18 tumors had normal expression of the remaining MMR proteins. Twenty-five different sequence variants were identified. None of these results in a truncated protein, though L902W represents the first constitutional missense mutation in MSH3 predicted to be functional based on conservation among mutS homologues. All variants have also been found in normal DNA of the patients and in controls. LOH intragenic to MSH3 was evident for 12 of 16 (75%) informative tumors. CONCLUSIONS: Occurrence of sequence variants in normal DNA of the patients and in controls excludes somatic mutations and mutations specific to the CRC patient population, respectively. In contrast, the high frequency of LOH as well as the aberrant protein expression in some tumors indicates an involvement of MSH3 impairment in MSI-L CRC.

TCF-3, 4 protein expression correlates with beta-catenin expression in MSS and MSI-H colorectal cancer from HNPCC patients but not in sporadic colorectal cancers.

PURPOSE: The beta-catenin-T-cell factor-4 (TCF-4) complex is the main control switch of cell proliferation and differentiation of normal and malignant intestinal cells. The aim of our study was to analyze the protein expression of components of the Wnt pathway in microsatellite stable (MSS) and highly unstable (MSI-H) sporadic and hereditary nonpolyposis colorectal cancer (HNPCC) in human colorectal cancers. METHODS: Sixty seven colorectal tumors comprising of 15 sporadic MSS, 12 sporadic microsatellite instability colorectal tumors and 40 tumors from HNPCC patients, of which 20 were MSS and 20 MSI-H, were analyzed for the expression of APC, beta-catenin, and TCF-3, 4 proteins by immunohistochemistry. RESULTS: We found a significant difference in cytoplasmic APC expression frequency between sporadic MSS (52%) and HNPCC tumors (78%), whereas no difference was detected between MSI-H and MSS or HNPCC tumors. All tumor groups showed a similar pattern of decreased membranous staining and increased cytoplasmic and nuclear staining for beta-catenin compared to normal cells. Moreover, the TCF-3, 4 protein expression was higher (43%) in HNPCC-associated MSS tumors compared to sporadic tumors (14%; analysis of variance (ANOVA) p < 0.05). For HNPCC tumors, the subcellular beta-catenin expression (membranous, cytoplasmic, and nuclear) correlated with the nuclear TCF-3, 4 signal in MSS tumors (Spearman correlation p < 0.0007) and MSI-H tumors (Spearman correlation p < 0.0001). CONCLUSION: We have shown a previously unknown difference in TCF-3, 4 protein expression between sporadic and HNPCC MSS tumors. In addition, we found no difference in nuclear beta-catenin signal intensity, which may be caused by an alteration in Wnt pathway in MSS sporadic tumors by unknown mechanisms leading to lower TCF-3, 4 protein expression. This hypothesis has to be tested in future investigations.

Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease with high penetrance, caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2 and MLH3. Most reported pathogenic mutations are point mutations, comprising single base substitutions, small insertions and deletions. In addition, genomic rearrangements, such as large deletions and duplications not detectable by PCR and Sanger sequencing, have been identified in a significant proportion of HNPCC families, which do not carry a pathogenic MMR gene point mutation. To clarify whether genomic rearrangements in MLH1, MSH2 or MSH6 also occur in patients carrying a point mutation, we subjected normal tissue DNA of 137 colorectal cancer (CRC) patients to multiplex ligation-dependent probe amplification (MLPA) analysis. Patients fulfilled the following pre-requisites: all patients met at least one criterion of the Bethesda guidelines and their tumors exhibited high microsatellite instability (MSI-H) and/or showed loss of expression of MLH1, MSH2 or MSH6 proteins. PCR amplification and Sanger sequencing of all exons of at least one MMR gene, whose protein expression had been lost in the tumor tissue, identified 52 index patients without a point mutation (Group 1), 71 index patients with a pathogenic point mutation in MLH1 (n=38) or MSH2 (n=22) or MSH6 (n=11) (Group 2) and 14 patients with an unclassified variant in MLH1 (n=9) or MSH2 (n=3) or MSH6 (n=2) (Group 3). In 13 of 52 patients of group 1 deletions of at least one exon were identified. In addition, in group 3 one EX1_15del in MLH1 was found. No genomic rearrangement was identified in group 2 patients. Genomic rearrangements represent a significant proportion of pathogenic mutations of MMR genes in HNPCC patients. However, genomic rearrangements are rare in patients carrying point mutations in MMR genes. These findings suggest the use of genomic rearrangement tests in addition to Sanger sequencing in HNPCC patients.

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.

Absence of association between cyclin D1 (CCND1) G870A polymorphism and age of onset in hereditary nonpolyposis colorectal cancer.

CCND1 encodes cyclin D1, which plays an important role in the G1 to S phase transition of the cell cycle. A common polymorphism (c.G870A) increases alternate splicing. Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in mismatch repair (MMR) genes, mainly MSH2 and MLH1, and shows a wide range in the age of its onset (AO), suggesting the existence of other modifying genetic factors. To date, two studies have investigated the association between CCND1 G/A variation and AO in HNPCC with contradictory results in 86 and 146 MMR mutation carriers, respectively. To clarify the role of the CCND1 G/A variation in HNPCC, we performed a study in 406 individuals carrying exclusively clear cut pathogenic mutations in MSH2 or MLH1. We did not observe a significant difference in genotype frequencies of affected and unaffected mutation carriers and healthy controls. A significant association between CCND1 genotypes and AO was found neither in the global comparison (log-rank, P = 0.2981; Wilcoxon, P = 0.2567) nor in a multivariate Cox regression analysis (hazard ratios 1.111, 95%CI 0.950-1.299, P = 0.188 and 1.090, 95%CI 0.868-1.369, P = 0.459 for the additive and dominant effect, respectively). We conclude, that the CCND1 G870A sequence variation is not a genetic modifier of the phenotype of HNPCC.

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; chi(2), 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.

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 (Fisher's exact test, P < .001). In contrast, the frequency of non-HNPCC-associated tumors was increased (Fisher's 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.

Eight novel MSH6 germline mutations in patients with familial and nonfamilial colorectal cancer selected by loss of protein expression in tumor tissue.

Germline mutations in mismatch repair (MMR) genes, predominantly in MLH1 and MSH2, are responsible for hereditary nonpolyposis colorectal cancer (HNPCC), a cancer-susceptibility syndrome with high penetrance. In addition, MSH6 mutations have been reported to account for about 10% of all germline mismatch repair (MMR) gene mutations in HNPCC patients, and have been associated with a later age of onset of the disease compared to MLH1 and MSH2 mutations. Here, we report eight novel germline mutations in MSH6. The patients were selected by having developed tumors with loss of MSH6 protein expression. All tumors showed high-level microsatellite instability (MSI-H). Seven mutations resulted in premature stop codons, comprised of two nonsense mutations (c.426G>A [p.W142X], c.2105C>A [p.S702X]), two insertions (c.2611_2614dupATTA [p.I872fsX10], c.3324dupT [p.I1109fsX3]) and three deletions (c.1190_1191delAT [p.Y397fsX3], c.1632_1635delAAAA [p.E544fsX26], c.3513_3514delTA [p.1171fsX5]). In addition, an amino acid substitution of an arginine residue (c.2314C>T [p.R772W]) conserved throughout a wide variety of mutS homologs has been found in a patient not fulfilling the Bethesda criteria for HNPCC. Our results emphasize the suitability of IHC as a pre-selection tool for MSH6 mutation analysis and the high frequency of germline mutation detection in patients with MSH6-deficient tumors. In addition, our findings point towards a broad variability regarding penetrance associated with MSH6 germline mutations.

Identification of six novel MSH2 and MLH1 germline mutations in HNPCC.

Germline mutations in mismatch repair genes are responsible for hereditary nonpolyposis colorectal cancer (HNPCC), the most common hereditary cancer-susceptibility syndrome. We report six novel germline mutations, three in MSH2 and three in MLH1. All but one mutation have been found in families fulfilling the criteria of the Bethesda guidelines; two of them additionally fulfilled the Amsterdam criteria. We identified two nonsense mutations in MSH2 (c.1764T>G [p.Y588X], c.2579C>A [p.S860X]), one duplication of four nucleotides causing premature stop codon (MLH1: c.821_824dupAAGC [p.A275fsX307]), one splice site mutation resulting in skipping of exon 8 from the MLH1 transcript (c.677+3A>G), one duplication of 18 nucleotides leading to duplication of six amino acids in the mismatch-binding domain of MSH2 (c.4_21dup [p.A2_E7dup) and one missense mutation in the PMS2 interaction domain of MLH1 (c.1756G>C [p.A586P]). The three latter mutations were not found in 73, 90 and 94 healthy control individuals, respectively. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IH) revealed complete loss of expression of the affected protein in the tumor cells from the patients with the nonsense, splice-site and missense mutation. The tumor from the patient with the c.821_824dupAAGC mutation showed a reduced, rather than lost, expression of the MLH1-protein.

Seven novel MLH1 and MSH2 germline mutations in hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent hereditary form of colorectal cancer and is caused by germline mutations in mismatch repair (MMR) genes. The majority of mutations occur in MLH1 and MSH2. We report hereby seven novel germline mutations in these two genes (five in MLH1 and two in MSH2). All mutations have been found in families fulfilling criteria of the Bethesda guidelines and four of which also fulfilled the Amsterdam criteria. We identified three insertions or deletions of 1 bp leading to premature stop codons (MLH1: c.341delC, c.1413-1414insA; MSH2: c.1119delG) and three nonsense mutations (MLH1: c.67G>T [E23X], c.436C>T [Q146X]; MSH2: c.1857T>G [Y619X]). The corresponding tumors showed a high level of microsatellite instability (MSI-H) and a complete loss of expression of the affected protein. In addition, a missense mutation in MLH1 was identified (c.1984A>C [T662P]). The respective tumor also showed a high level of microsatellite instability but a reduced, rather then lost, expression of the MLH1-protein. This missense mutation was not found in 107 healthy control individuals and in 54 HNPCC patients.

Ten novel MSH2 and MLH1 germline mutations in families with HNPCC.

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common hereditary cancer-susceptibility syndromes. Germline mutations in mismatch repair genes are associated with the clinical phenotype of HNPCC. We report ten novel germline mutations, three in MSH2 and seven in MLH1. All but one mutation have been found in families fulfilling criteria of the Bethesda guidelines; four of them additionally fulfilled the Amsterdam criteria I or II. Eight mutations were considered pathogenic and predictive diagnostics in healthy family members at risk shall be undertaken; these include five frameshift mutations leading to premature stop codons, in MSH2: c.1672delT (p.S558Xfs) and c.2466_2467delTG (p.C822X) and in MLH1: c.1023delG (p.R341Xfs), c.1127_1128dupAT (p.K377Xfs) and c.1310delC (p.P437Xfs); three mutations leading to splice aberrations, in MSH2: c.1661G>C (r.1511_1661del) and in MLH1: c.677+3A>C (r.589_677del) and c.1990-2A>G predicted to result in a splice site defect. The remaining two mutations are unclassified variants with assumed pathogenicity: one missense mutation in the highly conserved ATPase domain of MLH1 (c.122A>G [p.D41G]) and one in-frame insertion of twelve nucleotides in MLH1 (c.2155_2156insATGTGTTCCACA [p.I719delinsNVFHI]). These two mutations were not found in 102 alleles of healthy control individuals. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IHC) revealed complete loss of expression of the affected protein in the tumor cells from all but three patients. The tumors from the patients with the mutations c.1127_1128dupAT and c.1990-2A>G showed a reduction of expression of the MLH1-protein, rather than complete loss. In the tumor from the patient with the missense mutation c.122A>G [p.D41G] a normal expression of the proteins coded by MLH1 and MSH2 was noticed.

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.

A novel strumpellin mutation and potential pitfalls in the molecular diagnosis of hereditary spastic paraplegia type SPG8.

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous, neurodegenerative movement disorder. A total of eight KIAA0196/strumpellin variants have thus far been associated with SPG8, a rare dominant HSP. We present a novel strumpellin alteration in a small family with clinically pure HSP. We corroborated its causality by comparing it to rare benign variants at several levels, and, along this line, also re-considered previous genetic reports on SPG8. These analyses identified significant challenges in the interpretation of strumpellin alterations, and suggested that at least two of the few families claimed to suffer from SPG8 may have been genetically misdiagnosed.

A novel missense mutation in CACNA1A evaluated by in silico protein modeling is associated with non-episodic spinocerebellar ataxia with slow progression.

Spinocerebellar ataxia type 6 (SCA6), episodic ataxia type 2 (EA2) and familial hemiplegic migraine type 1 (FHM1) are allelic disorders of the gene CACNA1A encoding the P/Q subunit of a voltage gated calcium channel. While SCA6 is related to repeat expansions affecting the C-terminal part of the protein, EA2 and FHM phenotypes are usually associated with nonsense and missense mutations leading to impaired channel properties. In three unrelated families with dominant cerebellar ataxia, symptoms cosegregated with CACNA1A missense mutations of evolutionary highly conserved amino acids (exchanges p.E668K, p.R583Q and p.D302N). To evaluate pathogenic effects, in silico, protein modeling analyses were performed which indicate structural alterations of the novel mutation p.E668K within the homologous domain 2 affecting CACNA1A protein function. The phenotype is characterised by a very slowly progressive ataxia, while ataxic episodes or migraine are uncommon. These findings enlarge the phenotypic spectrum of CACNA1A mutations.

Microsatellite instability and loss of heterozygosity in squamous cell carcinoma of the head and neck.

BACKGROUND: Microsatellite instability (MSI) in head and neck squamous cell carcinoma (HNSCC) has been reported with a wide range of frequencies. The aim of our study was to disclose the frequency and basis of MSI in HNSCC and to correlate MSI and findings on loss of heterozygosity (LOH) with the clinical data. METHODS: We analyzed MSI and LOH in 91 tumors. All tumors presenting instability were analyzed for the expression of mismatch repair genes (MMR) proteins. RESULTS: Low-level microsatellite instability (MSI-L) was seen in 7.7% of the HNSCC. None of the MSI-L tumors had aberrant MMR protein expression. LOH rates up to 57% were identified for different regions on chromosome 3p. For the marker D10S197, we found a significant correlation between LOH and tumor stage IV. CONCLUSION: Our results indicate that MMR gene inactivation is rare among primary HNSCC. In contrast, the MSI-L phenotype plays a role in a small subset of tumors. LOH on chromosome arm 3p and 10p12 seems to be involved in tumorigenesis and progression HNSCC, respectively.

N-Acetyltransferase (NAT) 2 acetylator status and age of tumour onset in patients with sporadic and familial, microsatellite stable (MSS) colorectal cancer.

INTRODUCTION: N-Acetyltransferase (NAT) 2 is an important enzyme involved in the metabolism of different xenobiotics, including potential carcinogens. Allelic variants of the NAT2 gene are determined by a pattern of single nucleotide polymorphisms (SNPs) resulting in slow (SA), intermediate (IA) or rapid acetylator (RA) phenotypes and causing the individual differences in the NAT2 metabolic capacity. To clarify the potential modifying role of the NAT2 acetylator status in microsatellite stable (MSS) colorectal cancer (CRC), we studied 140 patients with sporadic CRC (group 1) and 69 patients with CRC who met at least one criterion of the revised Bethesda guidelines (group 2). OBSERVATIONS: We did not observe any significant difference in the NAT2 acetylator status frequency between patients in both groups and 100 healthy controls (P=0.486). Regardless of a younger median age of tumour onset (AO) of 41 years in group 2 patients compared to 64 years in group 1 patients, no significant difference in AO was found between RA and SA status patients in both groups. The median AO in group 1 was 65 years in patients with RA and 63 years with SA status (P=0.065). The median AO in group 2 was 40 years in patients with RA and 42 years with SA status (P=0.814). Multivariate Cox regression analysis revealed that neither the NAT2 acetylator status (P=0.064 and 0.810, respectively) nor the gender (P=0.165 and 0.918, respectively) was a risk factor for the AO in both groups. These data do not support the hypothesis that the NAT2 acetylatorship acts as a modifying factor on the AO in sporadic and familial, microsatellite stable CRC.

Occult endometrial cancer and decision making for prophylactic hysterectomy in hereditary nonpolyposis colorectal cancer patients.

BACKGROUND AND OBJECTIVE: Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent form of hereditary colorectal cancer. In addition to the high lifetime risk for colorectal cancer in mutation carriers, there is also a remarkably increased risk for endometrial cancer (EC). METHODS: In this retrospective study, clinical and molecular approach to the individual decision making as to whether or not to perform a prophylactic hysterectomy in a subset of HNPCC patients is discussed. 147 female patients meeting at least one criterion of the Bethesda guidelines were included in this analysis between 1995 and 2003. After clinical and genetic counseling, patients gave informed written consent and microsatellite analysis, immunohistochemistry and sequencing of the mismatch repair genes MLH1, MSH2 and MSH6 was performed. RESULTS: 11 of the analyzed patients had a personal history of EC and had undergone previous hysterectomy at ages 26 to 62 years. Prophylactic hysterectomy with oophorectomy was considered in postmenopausal women meeting the Amsterdam criteria and/or carrying a disease causing mismatch repair gene mutation who were operated on because of diagnosed colorectal cancer in our center for hereditary cancer. This procedure was performed in 4 patients. None of them had shown any symptoms of a gynecologic malignancy. Preoperative gynecological examination showed no evidence for EC or ovarian cancer in these patients. Postoperative histological examination showed EC stage T1b N0 M0 in 2 patients. CONCLUSIONS: Since the efficiency of gynecological surveillance is uncertain, prophylactic hysterectomy could be an option for a subset of HNPCC patients and mutation carriers.

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.

Molecular mechanisms associated with chromosomal and microsatellite instability in sporadic glioblastoma multiforme.

OBJECTIVE: Two chromosomal instability (CIN) pathways are described in glioblastoma multiforme (GBM), type 1 and type 2, which can be observed in up to 70% of the cases. Microsatellite instability (MSI) plays a pathogenic role in sporadic cancers such as colon, gastric and endometrial carcinomas with deficient mismatch repair (MMR). We aimed to perform a comprehensive analysis of the relationship between CIN and MSI mechanisms in sporadic glioblastomas. METHODS: 129 GBMs were examined (109 newly diagnosed and 20 relapses) investigating MSI, immunohistochemical expression of MMR proteins as well as sequencing and promoter methylation of hMLH1. We characterized the molecular changes frequently correlated with CIN in MSI+ GBMs and compared them with 26 microsatellite-stable tumors. RESULTS: Low-level MSI was observed in 11 of 129 (8.5%) cases and was higher in relapses than in primary GBMs (25 vs. 5.5%, p = 0.027). High-level MSI was not found in any case. A deficient expression of MLH1 and PMS2 without hMLH1 inactivation was observed only in one giant cell GBM. 55% of the MSI+ GBMs showed a profile which did not correspond to one of the known CIN pathways. An inverse association was observed between MSI and mutations of both p53 and PTEN. CONCLUSIONS: Our data suggest that CIN and MSI contribute to the genomic instability in GBMs via independent pathways. Since MSI was significantly more frequent in relapses, it might play a role in the malignant progression of GBM.