High frequency of microsatellite instability in young patients with head-and-neck squamous-cell carcinoma: lack of involvement of the mismatch repair genes hMLH1 AND hMSH2.

The most prevalent risk factors in the development of head-and-neck squamous-cell carcinoma (HNSCC) are excessive tobacco and alcohol consumption. In young patients with HNSCC, these risk factors are often absent. Our purpose was to investigate the risk factors, microsatellite instability (MSI) changes and status of the mismatch repair genes hMLH1 and hMSH2 in a cohort of young patients with HNSCC. Fifty-seven HNSCC tumors were examined for the presence of MSI at 16 microsatellite sites using PCR. In the young patient group (24 cases, < or = 44 years old), 100% of tumors had MSI at 1 site at least and 88% had MSI at 2 or more loci. In older patients (33 cases, > or = 45 years), MSI at 1 or more sites was found in 61% of tumors (young vs. old, p = 0.0003) and instability at 2 or more sites was found in 36% of tumors (young vs. old, p = 0.0001). The involvement of the mismatch repair genes was investigated by examining promoter methylation, exon mutation and gene expression of hMLH1 and hMSH2. All results were negative, indicating that inactivation of these 2 genes does not play a role in the development of MSI in tumors from this patient group. Furthermore, the young patient group had a significantly lower incidence of smoking (46% young, 88% old; p = 0.001) and alcohol consumption (33% young, 67% old; p = 0.0169), emphasizing the probable importance of other environmental and/or genetic factors in the development of their disease.     

BRAF mutations in colorectal carcinoma suggest two entities of microsatellite-unstable tumors

BACKGROUND: Alterations of BRAF have been implicated in the carcinogenesis of colorectal tumors with microsatellite instability (MSI). These alterations were attributed to defective DNA mismatch repair, which underlies MSI. It was the objective of this study to clarify the role of BRAF in colorectal carcinoma with MSI. METHODS: After sequencing for BRAF and KRAS in 82 colorectal tumor samples with or without MSI, mismatch repair protein expression was analyzed by immunohistochemistry, and promoter methylation of hMLH1 was analyzed with a methylation-specific polymerase chain reaction. Results were correlated with the germline status of hMLH1 or hMSH2 and clinical characteristics. RESULTS: BRAF was mutated more often in tumors with MSI than in tumors without MSI (27% vs. 5%; P = 0.016). The most prevalent BRAF alteration, V599E, occurred only in tumors with MSI. BRAF V599E was associated with more frequent hMLH1 promoter methylation (P = 0.07) and loss of hMLH1 (P = 0.02). The median age of patients with BRAF V599E was older compared with the median age of patients without this mutation (P = 0.001; 78 vs. 49 yrs). No BRAF alterations occurred in patients with germline mutations of mismatch repair genes. Five novel BRAF mutations were identified. CONCLUSIONS: Although BRAF V599E was common in colorectal carcinomas with MSI, it was not a consequence of deficient mismatch repair. The current data showed instead that the BRAF V599E mutation was associated only with a subgroup of colorectal carcinomas with MSI that were obtained from older patients without hereditary nonpolyposis colorectal carcinoma and showed epigenetic silencing of hMLH1. These results indicated that tumors with MSI caused by epigenetic MLH1 silencing have a distinct mutational background from that of tumors with genetic loss of mismatch repair, suggesting that there are two genetically distinct entities of microsatellite-instable tumors.     

Correlation of hMLH1 and HPP1 hypermethylation in gastric, but not in esophageal and cardiac adenocarcinoma

Using methylation-specific real-time PCR, we determined the prevalence of aberrant methylation in the mismatch repair gene hMLH1 and in the recently described HPP1 gene among 50 esophageal, 50 cardiac and 50 gastric ADCs. Additionally, expression of hMLH1 protein was detected immunohistochemically and correlated with DNA MSI. Hypermethylation of hMLH1 was found in 14% of esophageal, 28% of cardiac and 32% of gastric ADCs, whereas HPP1 hypermethylation was found more frequently in the 3 tumor types (64% vs. 38% vs. 54%). In gastric ADC, HPP1 hypermethylation was found more frequently in tumors with concomitant hMLH1 hypermethylation (81%) than in those without hMLH1 hypermethylation (41%, p = 0.008). Complete loss of hMLH1 protein expression, which was present in 10 carcinomas (5 cardiac and 5 gastric), was invariably correlated with hMLH1 hypermethylation and MSI. In conclusion, our data indicate that MSI and loss of the mismatch repair protein hMLH1, which is mainly caused by hMLH1 gene hypermethylation, are more prevalent in stomach and cardia carcinogenesis than in that of the esophagus. Moreover, in gastric cancer, hMLH1 hypermethylation is correlated with hypermethylation of the HPP1 gene.     

Methylation of hMLH1 in a population-based series of endometrial carcinomas.

Microsatellite instability (MSI) is a characteristic feature of hereditary nonpolyposis colorectal cancer and is also observed in sporadic colorectal and endometrial cancers. Alterations in the mismatch repair genes hMLH1 and hMSH2 are important for the development of MSI. It has recently been demonstrated that hypermethylation of the hMLH1 promoter region is associated with MSI and appears to be a common mechanism for gene inactivation. For endometrial carcinoma, however, previous studies have been relatively small and have not been population based. We therefore wanted to assess the frequency and prognostic significance of hypermethylation of the hMLH1 and hMSH2 genes in conjunction with hMLH1 protein expression in a prospective and population-based series of endometrial carcinoma patients with known MSI status and complete follow-up. A total of 138 patients were studied, and methylation of hMLH1 was found in 23% of tumors with conclusive results, whereas methylation of hMSH2 was seen in only 1% of tumors. Methylation of hMLH1 was significantly correlated with MSI (P < 0.001). Loss of nuclear staining of hMLH1 protein was seen in 14% of the cases and was significantly correlated with hMLH1 methylation and MSI (P < 0.001). Normal expression of hMLH1 was seen in all of the unmethylated tumors (100%). Of the 14 MSI-positive tumors that were also methylated, all but 1 (93%) showed a loss of nuclear expression of hMLH1. None of the tumors with loss of hMLH1 expression or hMLH1 methylation were aneuploid (P for both < or = 0.05), and loss of hMLH1 expression and hMLH1 methylation was significantly correlated with lack of p53 overexpression (P for both < or = 0.05). Nuclear hMLH1 staining and hMLH1 methylation did not significantly influence survival. In conclusion, hMLH1 methylation was common and was significantly correlated with loss of hMLH1 protein expression, MSI, diploid tumors, and lack of p53 overexpression. In contrast, hMSH2 methylation was infrequent in this prospective and population-based series of endometrial carcinomas.     

Loss of hMSH2 and hMSH6 expression is frequent in sporadic endometrial carcinomas with microsatellite instability: a population-based study.

Microsatellite instability (MSI) seems to be important in the development of various human cancers including sporadic endometrial cancer. It has previously been shown that alterations in the mismatch repair gene hMLH1 seem to be important for the development of MSI in these tumors. The role of the other mismatch repair genes hMSH2 and hMSH6 has been less well studied, but investigations on patients with hereditary nonpolyposis colorectal cancer indicate that these genes also may be involved. We therefore wanted to investigate the pattern of hMSH2 and hMSH6 expression in a prospective and population-based series of endometrial carcinomas with known hMLH1 expression and MSI status. A total of 138 patients were studied, and pathological staining was seen in 19 cases (14%) for hMLH1, 26 cases (19%) for hMSH2, and 17 cases (12.3%) for hMSH6. Pathological hMLH1 expression was more frequent among tumors with high MSI (those positive for four to five of five markers), whereas pathological expression of hMSH2 and hMSH6 was more frequent among tumors with intermediate MSI (those positive for two to three of five markers). MSI was significantly correlated with pathological expression of hMLH1 (P < 0.001), hMSH2 (P = 0.04), and hMSH6 (P = 0.001). In the group with high MSI, 14 of 16 tumors (88%) showed pathological expression for at least one of the markers. The expression of hMLH1, hMSH2, or hMSH6 did not significantly influence survival. In conclusion, pathological expression of hMLH1 does not seem to account for all tumors with a MSI-positive phenotype in this population-based series of endometrial carcinomas. Our data indicate that the other mismatch repair genes hMSH2 and hMSH6 are also involved, especially in cases with intermediate MSI.     

Microsatellite instability caused by hMLH1 promoter methylation increases with tumor progression in right-sided sporadic colorectal cancer

OBJECTIVE: A subset of sporadic colorectal cancers (SCRCs) exhibits microsatellite instability (MSI). Most MSI in SCRCs is caused by hMLH1 inactivation due to promoter methylation. However, the role of MSI in the progression of SCRCs remains unclear. METHODS: Thirty-two intramucosal cancers and 63 cancers with submucosal invasion were assigned to group 1 (early-stage cancer), and 30 Dukes' B and 26 Dukes' C cancers to group 2 (advanced-stage cancer). hMLH1 promoter methylation status was determined by methylation-specific PCR. MSI was determined using five markers. hMLH1 expression was determined immunohistochemically. RESULTS: MSI was found in 1 of 95 (1.1%) tumors in group 1, compared with 4 of 56 (7.1%) tumors in group 2. In right-sided tumors, the overall frequency of hMLH1-methylation-positive tumors in group 1 was not significantly different from that in group 2 (17 of 43, 39.5%, vs. 9 of 23, 39.1%). In right-sided tumors with hMLH1 promoter methylation, the frequency of MSI-positive tumors in group 1 was significantly lower than that in group 2 (1 of 17, 5.9%, vs. 4 of 9, 44.4%, p=0.0081). CONCLUSION: The frequency of MSI caused by hMLH1 promoter methylation increases with tumor progression in right-sided SCRCs.     

Microsatellite instability and hMLH1/hMSH2 expression in young endometrial carcinoma patients: associations with family history and histopathology

Endometrial cancer is the second most common malignancy in patients with hereditary nonpolyposis colorectal cancer (HNPCC). The age at diagnosis of HNPCC-associated endometrial cancer is approximately 15 years younger than for sporadic endometrial cancer. Our current study was undertaken to determine the frequency of microsatellite instability (MSI) and absence of hMLH1 or hMSH2 protein expression in young patients with endometrial carcinoma and to correlate these findings with histopathologic and clinical features. Endometrial carcinoma from 62 women (23-52 years, median age 46) were assessed for MSI. Twenty-one of the 62 (34%) tumors demonstrated MSI. Of the 21 tumors demonstrating MSI, 12 showed an absence of hMLH1 expression, 4 showed an absence of hMSH2 expression, and 5 demonstrated normal expression of both proteins. All 41 tumors without MSI demonstrated normal hMLH1 and hMSH2 expression. Two patients with MSI tumors fulfilled the Amsterdam criteria for HNPCC, while 2 had histories suggestive of HNPCC. None of the patients with tumors without MSI had a personal or family cancer history suggestive of HNPCC. The MSI phenotype was associated (p < 0.05) with high FIGO stage and grade, cribriform growth pattern, mucinous differentiation and necrosis. Our findings suggest that the frequency of HNPCC in young endometrial cancer patients is relatively low when compared with the frequency of HNPCC in young colorectal cancer patients. Defects of the MMR proteins hMSH2 or hMLH1 account for MSI in most but not all endometrial cancers from young patients.     

Analysis of Inactivation of hMLH1 by Promoter Hypermethylation and Microsatellite Instability in Gastric Carcinogenesis

OBJECTIVE To study the microsatellite instability (MSI) and methylation state of the hMLH1 gene promoter and their mechanisms underlying the development of gastric cancer. METHODS Forty-one gastric cancer samples were obtained from patients undergoing surgery and 46 chronic atrophic gastritis tissues with dysplasia or intestinal metaplasia (IM) were obtained from patients undergoing gastro-endoscopy. Fourteen normal gastric mucosal samples were used as controls. Genomic DNA was extracted from the samples and 5 microsatellite markers were used to measure MSI. Methylation-specific PCR (MSP) was used to screen the methylation state of the samples. DNA sequencing and immunohistochemistry were performed to verify the results. RESULTS MSI was identified in 22 out of the 41 (53.7%) gastric cancers, of which 8 cases showed high-level MSI (2 or more loci altered) and 14 showed low-level MSI (1 locus altered). MSI was also detected in 12 out of 46 (26.1%) pre-cancerous lesions of the stomach, whereas it was not seen in the normal tissue. Moreover, hMLH1 hypermethylation was detected in 17 out of the 41 (41.5%) gastric cancers, 9 out of the 46 (19.6%) pre-cancerous lesions and 0 out of the 14 normal tissue. Significant differences in frequency of MSI and hMLH1 promoter methylation were observed among gastric cancers, precancerous lesions and normal gastric tissue. Gastric samples with MSI had a tendency to be hypermethylated in the hMLH1 promoter. DNA sequencing and immunohistochemistry results also confirmed that hMLH1 promoter methylation could lead to loss of the hMLH1 protein and gene silence which sequentely resulted in gene mismatch and MSI. CONCLUSION Accumulation of MSI and hMLH1 promoter methylation may be important early molecular events during gastric carcinogenesis and may contribute to the acquisition of a transformed cell phenotype and the development of gastric cancer.     

RAB32 hypermethylation and microsatellite instability in gastric and endometrial adenocarcinomas

The recently described gene, RAB32, is a ras proto-oncogene family member that encodes an A-kinase-anchoring protein. RAB32 has been found to be frequently hypermethylated in microsatellite instability-high (MSI-H) colon cancers. We sought to determine the prevalence of RAB32 hypermethylation in gastric and endometrial adenocarcinomas, the 2 other major tumor types in which MSI-H is common. Moreover, we delineated the association of RAB32 hypermethylation with microsatellite instability (MSI) and hMLH1 hypermethylation. MSI status and hypermethylation of the RAB32 and hMLH1 genes were studied in paired primary normal and tumor tissues from 48 patients with gastric cancer. An additional 80 endometrial cancer patients were studied for RAB32 methylation and MSI status. Thirteen (27%) of 48 gastric cancers demonstrated evidence of RAB32 hypermethylation. MSI status was determined in 46 of the tumors, with 7 (100%) of 7 MSI-H tumors, 1 (33%) of 3 MSI-low (MSI-L) tumors and 4 (11%) of 36 microsatellite-stable (MSS) tumors found to harbor RAB32 hypermethylation. RAB32 methylation was significantly associated with intestinal type histology and concomitant hMLH1 hypermethylation in gastric cancer. In contrast, RAB32 methylation occurred in only 1 of 80 endometrial cancers, including 20 MSI-H, 8 MSI-L and 52 MSS tumors. Hypermethylation of hMLH1 was noted in 16 (20%) of 80 endometrial tumors. We conclude that although RAB32 methylation is rare in endometrial cancers, it is strongly associated with hMLH1 hypermethylation and MSI in gastric adenocarcinomas. Given its similar involvement in colon cancer, RAB32 inactivation may represent a component of the oncogenic pathway of microsatellite-unstable gastrointestinal adenocarcinomas.     

Microsatellite instability and protein expression of the DNA mismatch repair gene, hMLH1, of lung cancer in chromate-exposed workers

Our previous studies of lung cancer in chromate-exposed workers (chromate lung cancer) have revealed that the frequency of replication error (RER) in chromate lung cancer is very high. We examined whether the RER phenotype of chromate lung cancer is due to an abnormality of DNA mismatch repair protein. We investigated the expression of a DNA mismatch repair gene, hMLH1, and hMSH2 proteins using immunohistochemistry and microsatellite instability (MSI) in 35 chromate lung cancers and 26 nonchromate lung cancers. Lung cancer without MSI or with MSI at one locus was defined as "RER(-)," lung cancer with MSI at two loci was defined as "RER(+)," and lung cancer with MSI at three or more loci was defined as "RER(++)." The repression rate of hMLH1 and hMSH2 proteins in chromate lung cancer was significantly more than that of nonchromate lung cancer (hMLH1: 56% vs. 20%, P = 0.006, hMSH2: 74% vs. 23%, P < 0.0001). In chromate lung cancer, the repression rate for hMLH1 was 43% in RER(-), 40% in RER(+), and 90% in the RER(++) group. The repression rate of hMLH1 protein in the RER(++) group was significantly higher than that in the RER(-) and RER(+) groups (P = 0.039). The inactivation of hMLH1 expression strongly correlated with the microsatellite high instability phenotype in chromate lung cancer. The genetic instability of chromate lung cancer is due to the repression of hMLH1 protein.     

Methylation of the hMLH1 promoter but no hMLH1 mutations in sporadic gastric carcinomas with high-level microsatellite instability

Microsatellite instability (MSI) in tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in mismatch repair (MMR) genes, principally hMSH2 and hMLH1. In contrast, somatic mutations in MMR genes are relatively rare in sporadic MSI(+) colon cancers. Rather, the majority of mutation-negative, MSI(+) cases involve hypermethylation of the hMLH1 promoter and subsequent lack of expression of hMLH1. The details of the mechanisms of this epigenetic gene silencing remain to be elucidated. In some colon cancer cell lines, hMLH1 promoter methylation is accompanied by mutation of 1 of the 2 alleles, whereas in other cell lines and tumors, such combinations have not been reported. To contribute to the characterization of MSI in gastric cancer and to directly investigate whether hMLH1 promoter methylation is accompanied by gene mutation in these cancers, we have analyzed 42 gastric tumors and corresponding normal tissue for MSI, hypermethylation of the hMLH1 promoter, and mutations in hMLH1 as well as hMSH2. We found that 10 (23.8%) of 42 cases of sporadic gastric cancer were MSI(+) and that 8 had at least 2 of 12 altered microsatellite loci. All samples with at least 2 altered loci exhibited methylation of the hMLH1 promoter region, but none had detectable mutations in hMLH1 or hMSH2. Our results confirm the importance of methylation of the hMLH1 promoter region in MSI(+) gastric tumors and suggest that methylation takes place in the absence of hMLH1 mutations in these tumors.     

Distinct methylation pattern and microsatellite instability in sporadic gastric cancer.

Aberrant 5' CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor-suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLH1 in association of promoter hypermethylation. In the present study, we investigated the 5' CpG island hypermethylation of hMLH1, E-cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation-specific PCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLH1 methylation, 16 (26.2%) and 25 (40.9%) showed E-cadherin and p16 methylation respectively, and 8 (13.1%) presented high-frequency MSI (MSI-H). Of the 8 MSI-H patients, 5 presented hMLH1 methylation, whereas no low-frequency MSI (MSI-L) and microsatellite stable (MSS) cases exhibited hMLH1 methylation (5/8 vs. 0/43, p < 0.00001). Furthermore, these patients also presented E-cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLH1 methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these cancers.     

Microsatellite instability is linked to loss of hMLH1 expression in advanced gastric cancers: lack of a relationship with the histological type and phenotype

Background It has been suggested that the prevalence of microsatellite instability (MSI) is high in intramucosal differentiated gastric cancers with gastric foveolar phenotypic expression, and that these tumors are prone to lose their glandular structures and progress to undifferentiated-type lesions. To test this hypothesis, we examined the relationships among human MutL homologue 1 (hMLH1) expression (which is linked to MSI), the phenotype, and the histological type in patients with advanced and intramucosal gastric cancer.Methods We analyzed hMLH1 expression by immunohistochemistry in 70 advanced and 30 intramucosal gastric cancers with histological evaluation and assessment of the phenotype, and Cdx2 expression determined by immunohistochemistry. The MSI status was also examined in 20 cases.Results Thirteen (18.6%) advanced and 5 (16.7%) intramucosal gastric cancers were judged to be hMLH1-negative. In the advanced cases, no association was observed between the histological type and the phenotype and loss of hMLH1. In the intramucosal cases, MUC5AC expression was observed in all 5 hMLH1-negative differentiated-type cancers. However, no hMLH1-negative lesions were detected in the intramucosal undifferentiated cancers (0/14; P < 0.05 vs differentiated types). In the advanced cases, MSI-positivity (MSI +) and loss of hMLH1 expression did correlate (P < 0.0001), while no association was observed between MSI +, histological type, and phenotype.Conclusion Our data support the hypothesis that, phenotypically, some MSI-positive differentiated gastric cancers of gastric foveolar phenotypic expression may easily change, from gastric to intestinal phenotypic expression, also changing, histologically, from differentiated to undifferentiated type with progression.     

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.     

Reduced likelihood of metastases in patients with microsatellite-unstable colorectal cancer.

PURPOSE: The outcome of patients with colorectal cancer is more favorable when the tumor exhibits high-frequency microsatellite instability (MSI). Although associated with earlier-stage tumors, MSI has been proposed as an independent predictor of survival. We tested the prognostic value of MSI in a large series of patients diagnosed with colorectal cancer in the last decade. EXPERIMENTAL DESIGN: The survival of 893 consecutive patients with colorectal cancer characterized by microsatellite status was analyzed. The 89 (10%) patients with MSI cancer were classified according to tumor mismatch repair (MMR) defect, MMR germ-line mutation, hMLH1 and p16 promoter methylation, BRAF and K-ras mutations, and frameshifts of target genes. RESULTS: The colorectal cancer-specific survival was significantly (P = 0.02) better in patients with MSI cancer than in those with stable tumor (MSS). MSI did not predict a significantly lower risk of cancer-related death if tumor stage was included in the multivariate analysis [hazard ratio, 0.72; 95% confidence interval (95% CI), 0.40-1.29; P = 0.27]. Instead, MSI was strongly associated with a decreased likelihood of lymph node (odds ratio, 0.31; 95% CI, 0.17-0.56; P < 0.001) and distant organ (odds ratio, 0.13; 95% CI, 0.05-0.33; P < 0.001) metastases at diagnosis, independently of tumor pathologic features. Molecular predictors of reduced metastatic risk, and then of more favorable prognosis, included TGFbetaRII mutation for all MSI tumors, hMSH2 deficiency for hereditary non-polyposis colorectal cancer, and absence of p16 methylation for sporadic hMLH1-deficient cancers. CONCLUSIONS: Tumor MSI is a stage-dependent predictor of survival in patients with colorectal cancer. The decreased likelihood of metastases in patients with MSI cancer is associated with specific genetic and epigenetic changes of the primary tumor.     

Genetic and clinical features of human pancreatic ductal adenocarcinomas with widespread microsatellite instability

The incidences of microsatellite instability (MSI) and underlying DNA mismatch repair (MMR) defects in pancreatic carcinogenesis have not been well established. We analyzed 100 sporadic and 3 hereditary pancreatic ductal adenocarcinomas for MSI, and high-frequency MSI (MSI-H) and low-frequency MSI (MSI-L) tumors were further analyzed for frameshift mutations of possible target genes and for promoter methylation and mutation of DNA MMR genes, including hMLH1, hMSH2, hMSH3, and hMSH6 genes. Among the 100 sporadic tumors, 13 (13%) were MSI-H, 13 (13%) were MSI-L, and 74 (74%) were microsatellite stable (MSS) tumors. All of the three hereditary tumors from hereditary nonpolyposis colorectal cancer (HNPCC) patients were MSI-H. MSI-H tumors were significantly associated with poor differentiation and the presence of wild-type K-RAS and p53 genes. Patients with MSI-H tumors had a significantly longer overall survival time than did those with MSI-L or MSS tumors (P = 0.0057). Frameshift mutations of hMSH3, hMLH3, BRCA-2, TGF-beta type II receptor, and BAX genes were detected in MSI-H tumors. Hypermethylation of the hMLH1 promoter was observed in 6 (46%) of the 13 sporadic MSI-H tumors but not in any of the 3 hereditary MSI-H tumors or 13 MSI-L tumors. All of the 3 HNPCC cases had germ-line hMLH1 mutation accompanied by loss of heterogeneity or other mutation in the tumor. Our results suggest that pancreatic carcinomas with MSI-H represent a distinctive oncogenic pathway because they exhibit peculiar clinical, pathological, and molecular characteristics. Our results also suggest the principal involvement of epigenetic or genetic inactivation of the hMLH1 gene in the pathogenesis of pancreatic carcinoma with MSI-H.     

Frequent microsatellite instability in primary esophageal carcinoma associated with extraesophageal primary carcinoma

Patients with esophageal squamous cell carcinoma (ESCC) frequently develop other primary cancers, such as gastric cancer and head and neck cancer. Details of carcinogenesis in patients with multiple primaries that include esophageal carcinoma with other primary carcinoma (ECOPC) remain uncertain. We examined microsatellite instability (MSI) status, frameshift mutation in target genes of MSI, mismatch repair protein expression and hypermethylation of the hMLH1 promoter region in ECOPC patients to better understand the underlying carcinogenic processes. High frequency MSI (MSI-H) was found in 15 (44.1%) of 34 patients with ECOPC, but in only 6 (14.3%) of 42 patients with esophageal cancer alone (p < 0.01). Frameshift mutations in TGFbetaRII, BAX, MSH3 and MSH6 genes respectively were present in 4, 1, 2 and 2 of 34 ECOPC patients. Immunohistochemical study showed that 12 (80.0%) of 15 MSI-H tumors showed loss of expression of either hMLH1 or hMSH2. In addition, 6 of 9 tumors (66.7%) that showed reduced hMLH1 expression also had hypermethylation of the hMLH1 promoter region. Our findings suggested that carcinogenesis in ECOPC was closely associated with the MSI pathway because of mismatch repair protein deficiency.     

Clinical and biologic heterogeneity of hereditary nonpolyposis colorectal cancer

MMR gene mutations and MSI are not found in all clinically diagnosed HNPCC families. We evaluated whether MMR genotyping and tumor MSI analysis could identify distinct clinical subgroups among HNPCC families. Twenty-nine clinical HNPCC families were divided into 3 groups: A, families with hMLH1 or hMSH2 gene mutations; B, MMR gene mutations not present but MSI present in at least 50% of tumors tested; C, mutational and MSI analyses negative. We evaluated tumor spectrum, age at onset, risk of cancer in the follow-up and survival for CRC in the 3 groups. Tumors of the target organs in HNPCC (colon and rectum, endometrium, ovary, small bowel, stomach, renal pelvis and ureter) were more frequent in the first 2 groups than in the latter. Colon cancer was more frequently located in the proximal colon and showed an earlier age at onset in families with MMR gene mutation or with MSI than in families with stable tumors. Comparing the occurrence of tumors in the follow-up, in the first 2 groups patients younger than 50 years had a higher RR, which was particularly marked for CRC (RR = 18.6 for group A vs. group C, RR = 16.7 for group B vs. group C). CRC patients in the first 2 groups had a better clinical prognosis. The results of molecular analysis could distinguish, within clinically defined HNPCC families, different subgroups to which specific programs of surveillance could be addressed.     

Assessment of microsatellite instability status for the prediction of metachronous recurrence after initial endoscopic submucosal dissection for early gastric cancer

The technique of endoscopic submucosal dissection (ESD) has been developed for en bloc resection of early gastric cancer (EGC); however, little is known about the risk of metachronous cancer in the remnant stomach after initial ESD. In this study, we investigated the correlation between microsatellite instability (MSI) status and the incidence of metachronous recurrence of gastric cancer. According to the genetic/molecular background determined with MSI status and expression levels of hMLH1 and p53 tumour suppressor, 110 EGCs removed with ESD were subclassified into three groups: the mutator/MSI-type (8%), suppressor/p53-type (45%) and unclassified type (47%). Interestingly, patients with the mutator/MSI-type tumour had a high incidence (67%) of metachronous recurrence of gastric cancer within a 3-year observation after initial ESD, which was significantly higher than those with the suppressor/p53-type and unclassified type tumours (P<0.01). Although we investigated mucin phenotypes, there was no correlation between mucin phenotype and the recurrence of EGC. These findings suggest that subclassification of molecular pathological pathways in EGCs is required for the assessment of patients with a high risk of recurrent gastric cancer. The information delivered from our investigation is expected to be of value for decisions about therapy and surveillance after ESD.