Mutational analysis of MLH1 and MSH2 in 25 prospectively-acquired RER+ endometrial cancers

Mutations in the DNA mismatch repair (MMR) genes MLH1 and MSH2 have been linked to several human cancers which display the replication error (RER) phenotype. Germline mutations in these two genes have been implicated in about 90% of families with hereditary nonpolyposis colorectal cancer (HNPCC). A significant proportion of endometrial cancers, the second most common malignancy of the HNPCC syndrome, also exhibit RER. We screened 125 primary endometrial adenocarcinomas with seven microsatellite markers and identified 25 specimens with RER (20%). We used single-strand conformation variant analysis to search for mutations in MLH1 and MSH2. Direct sequencing of variants revealed only one germline mutation in MLH1 and a single somatic mutation in MSH2. However, six previously unreported sequence polymorphisms in MLH1 were identified. Four of these polymorphisms show clear population-based differences in allele frequency. In addition, a highly informative marker for MLH1 was characterized. The low frequency of mutations in MLH1 and MSH2 in this large series of cancers suggests that other MMR genes are responsible for the RER phenotype in endometrial cancers. Genes Chromosom. Cancer 18:219–227, 1997. © 1997 Wiley-Liss, Inc.     

Genetic and epigenetic modification of MLH1 accounts for a major share of microsatellite-unstable colorectal cancers

Microsatellite instability (MSI) is a hallmark of hereditary nonpolyposis colorectal cancer, and in these patients, results from inherited defects in DNA mismatch repair genes, mostly MSH2 and MLH1. MSI also occurs in 15% of sporadic colorectal cancers, but in these tumors, its basis is less well characterized. We investigated 46 sporadic MSI+ colorectal cancers for changes in MSH2 and MLH1 protein expression, followed by the analysis of somatic mutation, loss of heterozygosity (LOH), and promoter hypermethylation as possible underlying defects. Most cases (36/46, 78%) showed lost or reduced MLH1 expression. Among these, a majority (83%) was associated with MLH1 promoter hypermethylation, whereas the rates of LOH and somatic mutation of MLH1 were 24% and 13%, respectively. Hypermethylation and LOH were inversely correlated, suggesting that they had alternative functions in the inactivation of MLH1. MSH2 expression was lost in 7/46 (15%), and of these, 2 (29%) showed LOH and/or somatic mutation of MSH2. We conclude that most sporadic MSI+ colorectal cancers have an MLH1-associated etiology and that epigenetic modification is a major mechanism of MLH1 inactivation. Moreover, we found a significantly lower prevalence for MLH1 promoter hypermethylation in hereditary nonpolyposis colorectal cancer tumors with MLH1 germline mutations (12/26, 46%), which might explain some differences that are known to occur in the clinicopathological characteristics and tumorigenic pathways between sporadic and hereditary MSI+ colorectal cancers.     

Germline and somatic mutation analysis of MLH3 in MSI-positive colorectal cancer

Microsatellite instability (MSI) is characteristic of hereditary nonpolyposis colorectal cancer, and occurs in a subset (10 to 15%) of unselected colorectal cancer cases. In hereditary nonpolyposis colorectal cancer, MSI is caused by defects in five mismatch repair genes, and in sporadic cases the main cause seems to be somatic MLH1 promoter methylation. Most likely additional hereditary nonpolyposis colorectal cancer genes remain to be discovered. Genes with simple repeats in their coding region are often targets for deletions in MSI-positive tumors. Several genes (TGFbeta RII, IGFIIR, MSH3, MSH6, BAX, MBD4) with significance in tumorigenesis harbor repeats in their coding regions and are often somatically inactivated because of deletions causing frameshifts. Recently, a novel human mismatch repair gene, MLH3, was cloned and shown to be involved in mammalian mismatch repair. To evaluate the possible role of MLH3 in hereditary cancer, we performed germline single-strand conformation polymorphism-analysis for 52 patients displaying features of inherited colorectal cancer. Forty-six of these had been diagnosed with MSI-positive tumors. No germline mutations were found. Similar to MSH3 and MSH6, MLH3 harbors mononucleotide repeats, ie, (A(6))-(A(9)), in its coding region, which makes it a putative target for somatic mutations in MSI-positive tumors. To evaluate its somatic inactivation we performed a deletion search focusing on eight exonic MLH3 mononucleotide repeats in a series of 93 MSI-positive tumors. Somatic deletions were found in 8.6% of the samples, a frequency similar to one detected in neutral noncoding mononucleotide repeats. No evidence of involvement of MLH3 in MSI tumorigenesis was obtained.     

Mononucleotide microsatellite instability and germline MSH6 mutation analysis in early onset colorectal cancer.

Germline mutations in the MSH2 and MLH1 mismatch repair genes account for most cases of hereditary non-polyposis colon cancer syndrome (HNPCC). In addition, germline MSH2 and MLH1 mutations have been detected in patients with non-HNPCC early onset colorectal cancer. Germline MSH6 mutations appear to be rare in classical HNPCC families, but their frequency in young colorectal cancer cases has not been studied previously. In a population based study of early onset colorectal cancer (<50 years) investigated for tumour microsatellite instability (MSI), we identified a subgroup of tumours with MSI for mono- but not dinucleotide repeat markers (m-MSI+ group). In contrast to tumours with classical MSI for dinucleotide markers (d-MSI+), the m-MSI+ group cancers were mainly left sided (6/7). As MSH6 mutations in yeast and human cell lines are associated with weak (and preferential mononucleotide) MSI, the complete MSH6 gene coding region was sequenced in blood DNA from the five m-MSI+ cases available for analysis. A germline nonsense mutation was identified in an isolated case of early onset colorectal cancer (age 43 years). These results support previous findings that germline MSH6 mutations may not be associated with classical MSI and suggest a role for germline MSH6 mutations in isolated early onset colorectal cancer.     

Microsatellite instability in early onset and familial colorectal cancer.

Hereditary non-polyposis colorectal cancer syndrome (HNPCC) is often considered to be the most common form of inherited colorectal cancer, although its precise incidence is unknown. The clinical diagnosis of HNPCC relies on a combination of family history and young age of onset of colorectal cancer, but as many familial aggregations of colorectal cancer do not fulfil the strict diagnostic criteria, HNPCC might be underdiagnosed. The majority of HNPCC families have germline mutations in mismatch repair (MMR) genes, such as MSH2 or MLH1, so that HNPCC cancers characteristically exhibit DNA replication errors (RERs) at microsatellite loci. Although an RER positive phenotype in tumours can also result from somatic mutations in an MMR gene, the prevalence of RER + tumours should provide a maximum estimate of the incidence of germline MMR gene mutations in patients with early onset and familial colorectal cancer. We investigated colorectal cancers for RERs from (1) a population based study of 33 patients with colorectal cancer aged 45 years or less, (2) 65 kindreds with familial colorectal cancer which only partially fulfilled the criteria for the diagnosis of HNPCC, and (3) 18 cancers from 12 HNPCC kindreds. Seven of 33 patients (21%) with colorectal cancer aged 45 years or less had an RER + cancer, with only two of these having a clear family history of HNPCC. A greater proportion of RER + tumours (5/7) occurred proximal to the splenic flexure than RER - tumours (4/26; chi2 = 6.14, p < 0.025). RERs were detected in all 18 cancers from HNPCC patients but in only six of 65 non-HNPCC familial colorectal cancer kindreds (9%; chi2 = 52.2, p < 0.0005). These findings suggest that most cancers in patients diagnosed at 45 years of age or less and familial aggregations of colorectal cancer which do not fulfil HNPCC diagnostic criteria do not have germline mutations in MSH2 and MLH1. Hence population screening for germline mutations in these genes is unlikely to be an efficient strategy for identifying people at high risk of developing colorectal cancer.     

Germline and somatic mutation analyses in the DNA mismatch repair gene MLH3: Evidence for somatic mutation in colorectal cancers

DNA mismatch repair is of considerable scientific and medical importance because of its essential role in maintaining genomic integrity, and its association with hereditary non-polyposis colon cancer (HNPCC). Germline mutations in five mismatch repair genes (MLH1, MSH2, PMS1, PMS2, and MSH6) have been associated with HNPCC susceptibility. Our laboratory recently identified MLH3, a novel DNA mismatch repair gene. We screened the MLH3 coding sequence in 60 probands with increased genetic risk factors for colorectal cancer susceptibility and no mutations in the other candidate genes. No definite MLH3 germline mutations were found. We subsequently screened 36 colon tumors, and discovered an appreciable frequency of somatic MLH3 coding mutations in MSI-H tumors (25%). In four of six tumors, evidence of biallelic inactivation was noted. Furthermore, MLH3 nonsense mutations were identified in two of 12 microsatellite stable (MSS) tumors with 14q24 loss of heterozygosity. While our analyses do not exclude the existence of germline MLH3 mutations in patients with increased genetic risk factors for colorectal cancer susceptibility, they suggest such mutations are uncommon in this patient population. The finding of an appreciable frequency of somatic MLH3 mutations is consistent with a possible role for this gene in the progression of colorectal cancer tumorigenesis. Hum Mutat 17:389-396, 2001. Published 2001 Wiley-Liss, Inc.     

Microsatellite instability as a predictor of a mutation in a DNA mismatch repair gene in familial colorectal cancer

Germline alterations in human DNA mismatch repair genes are associated with hereditary nonpolyposis colorectal cancer (HNPCC). Mutation analysis of the genes reveals carriers with a high risk of colorectal cancer, who will benefit from surveillance. We wanted to find the best predictive parameter of a germline mutation in those genes among patients with familial colorectal cancer. Affected members from a total of 83 unrelated colorectal cancer families previously analyzed for mutations in MSH2 and MLH1 were used to evaluate different parameters' ability to predict a germline mutation. We studied various clinical criteria such as family structure, age of onset, and prevalence of endometrial cancer, as well as microsatellite instability in the tumors from the families. In total, 124 tumors from 59 of the families were tested for microsatellite instability (MSI) using PCR-based mono- and dinucleotide markers to establish whether the families could be scored as MSI-positive or -negative. The finding of MSI-positive tumors in a family was the best predictor of a germline mutation, and was found in 73% of the MSI-positive, but in less than 3% of the MSI-negative families (P < 0.0001). In contrast, MSI in unselected colorectal cancer is not as useful, since most of these MSI-positive tumors are sporadic. The finding of microsatellite instability in colorectal tumors seems efficient enough even to select those with germline mutations among families fulfilling HNPCC Amsterdam criteria, once used in identification of the DNA mismatch repair genes. Genes Chromosomes Cancer 27:17-25, 2000.     

Identification of a breast tumor with microsatellite instability in a potential carrier of the hereditary non-polyposis colon cancer trait

Allelic expansion at microsatellite loci in colorectal tumor DNA indicates a genomic instability caused by defects in DNA mismatch repair. This is observed in a high proportion of tumors from individuals affected by hereditary non-polyposis colorectal carcinoma, but to a lesser extent in sporadic colorectal tumors. In this study we screened 46 colorectal tumors for replication errors (RER). Tumors from six patients were found to be RER positive, two of which had a marked family history of colon cancer. In both cases the RER+ phenotype was detected in colon tumors from other family members, suggesting a germline mutation in mismatch repair genes. Additionally, RER+ phenotype, distinct from that of the colon and sporadic breast tumors, was found in malignant breast tissue from the mother of one proband.     

Mean age of tumor onset in hereditary nonpolyposis colorectal cancer (HNPCC) families correlates with the presence of mutations in DNA mismatch repair genes

Fourteen Italian families affected with hereditary nonpolyposis colorectal cancer (HNPCC) were screened for germline mutations at three DNA mismatch repair (MMR) genes, MSH2, MLH1, and GTBP, by using a combination of different methods that included an in vitro synthesized protein assay, single-strand conformation polymorphism analysis, and direct sequencing. DNA alterations were observed in six instances, including a single base deletion in MSH2 exon 14, an A-to-G transition in the splice donor site of MLH1 exon 6, and two missense mutations in MLH1 exons 5 and 9. A previously reported common mutation affecting the splice donor site of MSH2 exon 5 was identified in two families. No mutations were detected in the GTBP gene. In total, eight of 16 Italian HNPCC families (50%), including two previously reported kindreds, were found to carry a mutation in MMR genes. We compared the mean age of colorectal cancer onset in the index cases (three patients for each family) between the two groups of kindreds, those with identified mutation vs. those without, and found that the first had a significantly lower value (43.0 vs. 53.7 years, P = 0.014). This finding suggests that HNPCC families with a more advanced age of tumor onset are less likely to be associated with known MMR genes. Genes Chromsom. Cancer 19:135–142, 1997. © 1997 Wiley-Liss Inc.     

Adrenocortical adenocarcinoma in an MSH2 carrier: Coincidence or causal relation?

A woman is described who developed an ovarian adenocarcinoma, 3 metachronous colorectal adenocarcinomas, and a primary adrenocortical adenocarcinoma. Genetic investigation of the mismatch repair genes MLH1 and MSH2 showed a germline mutation in MSH2. Colorectal and ovarian carcinoma belong to the tumor spectrum of hereditary nonpolyposis colorectal cancer (HNPCC). Adrenocortical adenocarcinoma, however, has never been described as 1 of the HNPCC-associated tumors. To investigate whether the adrenocortical adenocarcinoma in this patient was caused by the MSH2 germline mutation, determination of microsatellite instability (MSI) and immunohistochemical analysis were performed on 1 of the colorectal tumors and the adrenocortical adenocarcinoma. MSI and general loss of MSH2 protein expression could be seen in the colorectal tumor but not in the adrenocortical adenocarcinoma. Therefore, it is highly unlikely that the adrenocortical adenocarcinoma found in this patient was due to her genetic predisposition for HNPCC. H P 31:1522-1527.     

PMS2 involvement in patients suspected of Lynch syndrome

It is well‐established that germline mutations in the mismatch repair genes MLH1, MSH2, and MSH6 cause Lynch syndrome. However, mutations in these three genes do not account for all Lynch syndrome (suspected) families. Recently, it was shown that germline mutations in another mismatch repair gene, PMS2, play a far more important role in Lynch syndrome than initially thought. To explore this further, we determined the prevalence of pathogenic germline PMS2 mutations in a series of Lynch syndrome‐suspected patients. Ninety‐seven patients who had early‐onset microsatellite instable colorectal or endometrial cancer, or multiple Lynch syndrome‐associated tumors and/or were from an Amsterdam Criteria II‐positive family were selected for this study. These patients carried no pathogenic germline mutation in MLH1, MSH2, or MSH6. When available, tumors were investigated for immunohistochemical staining (IHC) for PMS2. PMS2 was screened in all patients by exon‐by‐exon sequencing. We identified four patients with a pathogenic PMS2 mutation (4%) among the 97 patients we selected. IHC of PMS2 was informative in one of the mutation carriers, and in this case, the tumor showed loss of PMS2 expression. In conclusion, our study confirms the finding of previous studies that PMS2 is more frequently involved in Lynch syndrome than originally expected. © 2009 Wiley‐Liss, Inc.     

中国人家族性结直肠癌错配修复基因大片段变异分析

目的分析中国人家庭性结直肠癌错配修复基因大片段变异的特点。方法 采用多重连接探针扩增技术，分析32例具有家庭史结直肠癌、20例散发性结直肠癌患者错配修复基因MSH2的16个外显子、MLH1的19个外显子及7个其它基因外显子的拷贝数。研究工作包括：（1）双盲法分析阴性和阳性对照样本，完成方法学可靠性检验；（2）分析结直肠癌患者外周血细胞DNA，筛查MSH2和MLH1基因大片段变异。结果 多重连接探针扩增技术分析系统稳定检出阳性对照样本的DNA大片段缺失；在3/32（9.4%）具有家庭聚集性结直肠癌患者中检出遗传性MSH2基因DNA大片段缺失。而在20例散发性结直肠癌患者未检出这类突变。结论 中国人家族性结直肠癌患者中错配修复基因的大片段变异是频发事件，对此类患者的遗传检测应包含错配修复基因大片段变异的筛查。     

Microsatellite instability in the peripheral blood leukocytes of HNPCC patients

Most hereditary nonpolyposis colorectal cancer (HNPCC) patients inherit a defective allele of a mismatch repair (MMR) gene, usually MLH1 or MSH2, resulting in high levels of microsatellite instability (MSI‐H) in the tumors. Presence of MSI in the normal tissues of mutation carriers has been controversial. Here we directly compare MSI in the peripheral blood leukocyte (PBL) DNA of seven HNPCC patients carrying different types of pathogenic MMR mutations in MLH1 and MSH2 genes with the PBL DNA of normal age‐matched controls and of patients with sporadic colorectal cancer (SCRC). Small pool PCR (SP‐PCR) was used studying three microsatellite loci for at least 100 alleles each in most samples. The average frequencies of mutant microsatellite fragments in each HNPCC patient (0.04–0.24) were significantly higher (p<0.01) relative to their age‐matched normal controls with mutant frequencies (MF) from 0.00 to 0.06, or SCRC patients (MF from 0.01–0.03). The data support the conclusions that higher MF in the PBL DNA of HNPCC patients is real and reproducible, may vary in extent according to the type of germline MMR mutation and the age of the individual, and provide a possible genetic explanation for anticipation in HNPCC families. Hum Mutat 31:317–324, 2010. © 2010 Wiley‐Liss, Inc.     

MLH1 and MSH2 protein immunohistochemistry is useful for detection of hereditary non-polyposis colorectal cancer in young patients

AIMS: Hereditary non-polyposis colorectal cancer is related to germline mutations of DNA mismatch repair genes MLH1 and MSH2, which result in microsatellite instability and loss of protein expression of the corresponding mutated gene in the tumour tissue. METHODS AND RESULTS: MLH1 and MSH2 protein expression was studied by immunohistochemistry in paraffin-embedded surgical samples of 100 colorectal adenocarcinomas occurring before 50 years of age. Absence of tumour cell nuclear staining with positive internal control (normal mucosa, lymphoid follicles) was considered negative. Loss of MLH1 or MSH2 expression was found in 20 cases with microsatellite instability in 15 cases. Twelve of these patients had a family history of colorectal cancer. Compared with MLH1- and MSH2-positive cases, MLH1- or MSH2-deficient colorectal adenocarcinomas were significantly associated on multivariate analysis with a younger age (38 vs. 43 years, P;0.0224), a larger tumour size (60 +/- 6 vs. 46 +/- 2 mm, P=0.0291), an expanding margin (85% vs. 51%, P=0.0159), a higher number of tumour-infiltrating lymphocytes assessed by CD3 immunostaining (202 +/- 48 vs. 33 +/- 4 CD3+ lymphocytes/10 high-power fields, P=0.0039), and a grade 2 Crohn's like lymphoid reaction (70% vs. 9%, P=0.0037). The two groups were not different for tumour site, differentiation, pTNM stage, vascular and perineural invasion, peripheral adenomatous residue, and 5-year survival rates. CONCLUSIONS: MLH1- or MSH2-deficient colorectal carcinomas of young patients exhibit pathological and molecular features similar to hereditary non-polyposis colorectal cancer. This suggests that MLH1 and MSH2 immunohistochemistry is valuable for detecting hereditary non-polyposis colorectal cancer in young patients.     

Protein expression profiles of deoxyribonucleic acid mismatch repair genes: Association with clinicopathological characteristics of Malaysian Lynch syndrome patients

Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer, accounts for approximately 1–5% of all colorectal cancers. Germline mutations in a group of deoxyribonucleic acid (DNA) mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS1, and PMS2) are responsible for Lynch syndrome cases. This study focuses on the determination of MMR (MLH1, MSH2, MSH6, and PMS2) protein expression profile by immunohistochemical analysis and its association with clinicopathological characteristics in clinically diagnosed Malaysian Lynch syndrome patients. Fifty patients who fulfilled any of the revised Bethesda Guidelines criteria were recruited from four collaborating centers in Malaysia. Clinicopathological information of clinically diagnosed Lynch syndrome cases that underwent bowel resection was reviewed. Immunohistochemical analysis for MLH1, MSH2, MSH6, and PMS2 proteins were performed on paraffin-embedded carcinomatous tissues. Colorectal cancer protein expression analysis for MLH1, MSH2, MSH6, and PMS2 antigens showed absence of expression of any MMR proteins in 18 out of 50 clinically diagnosed Lynch syndrome patients (36.0%). There was a significant association between abnormal MMR protein expression with tumor size (p = 0.012), histological differentiation of cancers (p = 0.012), and growth pattern of tumor (p = 0.01). Abnormal expression of MMR protein in colorectal cancers in clinically diagnosed Lynch syndrome patients was associated with specific clinicopathological characteristics such as tumor size, histological differentiation of cancers, and growth pattern of tumor. Immunohistochemical analysis proved to be an advantageous pre-screening tool for Lynch syndrome in Malaysian patients and highly predictive of a germline mutation in DNA MMR genes.     

Histologic features distinguish microsatellite-high from microsatellite-low and microsatellite-stable colorectal carcinomas, but do not differentiate germline mutations from methylation of the MLH1 promoter

The detection of microsatellite-unstable (microsatellite instability [MSI]) colorectal carcinomas (CRCs) has prognostic value and can help screen for Lynch syndrome. We determined which histologic features are associated with MSI status and presence of germline mutation and/or methylation of MLH1 promoter. Patients diagnosed with CRC were offered participation in the Columbus-area hereditary nonpolyposis colorectal cancer syndrome study regardless of age or family history. Tumors were evaluated for MSI using a modified Bethesda panel of microsatellite markers. Methylation status of the MLH1 promoter was evaluated by methylation-specific polymerase chain reaction and bisulfite PCR followed by restriction digestion of tumor DNA. All patients with microsatellite-unstable tumors underwent mutation analysis of the MLH1, MSH2, and MSH6 genes by full sequencing of genomic DNA and by multiplex ligation probe assay of MLH1 and MSH2. Histologic end points were tumor type, grade, percentage of mucin, border, and lymphoid host response. Of the 482 CRCs, 87 were MSI with 69 MSI high (MSI-H), 18 MSI low (MSI-L), and 395 microsatellite stable (MSS). Of 87 MSI tumors, 12 had germline mutations and 34 had methylation of the MLH1 promoter. Younger age, but not histologic features, was significantly associated with a germline mutation. Percentage of mucin, histologic type, grade, and lymphoid host response differed significantly between MSI-H when compared with MSI-L or MSS. No difference was found between MSI-L versus MSS. Histologic features are associated with MSI-H CRC and are helpful to differentiate MSI-H from MSI-L and MSS. These features are not useful to distinguish MSI-L from MSS carcinomas, and those with a deleterious germline hereditary nonpolyposis colorectal cancer syndrome mutation from those with methylation of the MLH1 promoter region.