The profile of hMLH1 methylation and microsatellite instability in colorectal and non-small cell lung cancer

Microsatellite instability (MSI) is caused mainly by dysfunction of hMLH1, where aberrant hypermethylation (HM) of its promoter region is involved. Previously, we suggested that HM in the proximal region of the hMLH1 promoter plays a critical role in progression of gastric cancer with MSI and this specific region should be analyzed for diagnostic use of hMLH1 HM. We expanded the analyses of hMLH1 HM and MSI phenotype to sporadic colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) to further evaluate the diagnostic value of hMLH1 HM. A total of 174 CRC and 94 NSCLC samples were used for hMLH1 methylation analysis by real-time methylation-specific PCR. Methylation levels were measured in three distinct regions of the promoter, designated as hMLH1-A, hMLH1-B, and hMLH1-C from distal to proximal. MSI phenotype was determined using five microsatellite markers, BAT25, BAT26, D2S123, D5S346, and D17S250. Methylation profile of the hMLH1 promoter varies between CRC and NSCLC. High methylation levels were observed in a group of CRC samples. Consequently, three patterns of methylation in the hMLH1 promoter regions were found: 1) low methylation level in all regions, 2) high methylation level in hMLH1-A with low methylation level in hMLH1-C, 3) high methylation level in all regions. In contrast, only one NSCLC showed high methylation level in hMLH1-A. Of the 134 CRCs examined, 14 (10.4%) showed MSI phenotype. No MSI phenotype was found in the initial 80 NSCLCs analyzed. Eight (57.1%) of 14 CRC with MSI showed HM in hMLH1-C, which was linked exclusively with MSI phenotype. However, the HM in hMLH1-A or -B was not sufficient for MSI. CRC with MSI phenotype was significantly more frequent in older patients and in the proximal colon, and was more evident in cases with hMLH1-C HM. The results suggested that hMLH1 HM cannot be used as an alternative diagnostic marker of MSI phenotype in sporadic CRC and NSCLC. CRC with MSI might have clinicopathologically distinct subgroups according to hMLH1-C HM status. The observed profiles of hMLH1 methylation and MSI in gastric cancer, CRC, and NSCLC were quite different from each other, facilitating the better understanding of the pathogenesis of these cancers.     

The role of hypermethylation of the hMLH1 promoter region in HNPCC versus MSI+ sporadic colorectal cancers

INTRODUCTION—Hypermethylation of the promoter region of the hMLH1 gene is associated with absent expression of MLH1 protein in sporadic colorectal cancers with microsatellite instability (MSI+), and it has been proposed that methylation may be a mechanism of inactivation in Knudson's hypothesis. The incidence of hypermethylation of the hMLH1 promoter in hereditary non-polyposis colorectal cancer (HNPCC) versus MSI+ sporadic colorectal cancer was investigated and compared.
METHODS—DNA was available from 10 HNPCC colorectal cancers (median age 58 years, range 39-67) with germline mutations in hMLH1 and 10 MSI+ sporadic colorectal cancers (mean age 79 years, range 41-85). MSI was determined by amplification of BAT26 and TGF-β RII. The methylation status of the hMLH1 promoter was studied by the polymerase chain reaction (PCR) based HpaII restriction enzyme assay technique. Evidence of allelic loss at hMLH1 was searched for in the HNPCC colorectal cancers.
RESULTS—All cases were confirmed to be MSI+. The promoter region of hMLH1 was hypermethylated in seven of 10 MSI+ sporadic cancers versus 0 of 10 HNPCC cancers (p<0.002). Evidence of loss of heterozygosity at hMLH1 was observed in eight of the 10 HNPCC colorectal cancers.
CONCLUSION—While mutations and allelic loss are responsible for the MSI+ phenotype in HNPCC cancers, the majority of MSI+ sporadic cancers are hypermethylated in the promoter region of hMLH1. These data further support our argument that tumours from HNPCC patients, which almost always acquire a raised mutation rate, mostly follow a different pathway from MSI+ sporadic tumours.


Keywords: hMLH1 promoter region; HNPCC; hypermethylation; colorectal cancer     

Microsatellite instability of papillary subtype of human gastric adenocarcinoma and hMLH1 promoter hypermethylation in the surrounding mucosa

Gastric cancer has striking heterogeneity in histological pattern, cellular phenotype, genotype, biomarkers, and biological behavior. We focused on the specific morphological papillary phenotype of gastric adenocarcinoma and attempted to identify its distinct molecular characteristics. In our comparative study, early stage papillary (papillary-dominant) gastric cancer showed a significantly higher and more widespread high-frequency microsatellite instability (MSI-H) than other morphological types. Analysis of mutations in a panel of five putative microsatellite instability (MSI)-associated genes in the MSI-H cases revealed that papillary or papillary-dominant cancer displays a unique profile of mutations compared to profiles previously reported in gastric cancer. Immunohistochemical staining and methylation analysis revealed that silencing of hMLH1 by methylation in its promoter region was responsible for the failure of mismatch repair in papillary-type gastric cancer, whereas aberrant promoter methylation of hMLH1 was not found in any cases without the unique mutator phenotype. Promoter hypermethylation of the hMLH1 genes was found to a lesser degree in the adjacent non-tumor mucosa in four of the 10 cases with tumor having the mutator phenotype. Microsatellite instability itself could not be detected in the adjacent non-tumor mucosa. Inactivation of hMLH1 expression by promoter hypermethylation may be an early event in carcinogenesis of this type of gastric cancer, preceding the development of the clear MSI phenotype of papillary carcinoma.     

Characterization of mutator pathway in younger-age-onset colorectal adenocarcinomas

The high-frequency microsatellite instability (MSI-H) phenotype, frequently identified in hereditary nonpolyposis colorectal cancer (HNPCC), also accounts for approximately 15% of sporadic colorectal cancers. Microsatellite instability (MSI) occurs from the mutational inactivation of the DNA mismatch repair genes, i.e. hMSH2 and hMLH1 in HNPCC, as well as from epigenetic inactivation of hMLH1 in sporadic colorectal tumors. The mutator pathway including microsatellite instability, hMLH1 promoter methylation, and hMSH2 and hMLH1 mutation patterns were identified in 21 sporadic colorectal adenocarcinoma patients younger than 30 yr excluding HNPCC. More than half of tumors showed MSI, with five MSI-H and six MSI-L (low-frequency microsatellite instability). Three of six MSI-H tumors showed the hMLH1 promoter methylation and did not express the hMLH1 protein. On the other hand, all MSI-L and all MSS (microsatellite stable) tumors expressed both hMSH2 and hMLH1 proteins. Two novel mutations, i.e. a missense mutation in hMLH1 and a splice-site alteration in hMSH2, were identified in two patients respectively. Although mutator pathway was implicated in younger-age-onset colorectal carcinogenesis, many tumors appeared to evolve from different genetic events other than hMSH2 and hMLH1 mutations frequently identified in HNPCC.     

Correlation of methylation of the hMLH1 promoter with lack of expression of hMLH1 in sporadic gastric carcinomas with replication error.

Recent studies have demonstrated that the majority of sporadic colorectal carcinomas with replication error (RER) do not harbor mutations of the hMLH1 and hMSH2 genes that account for about 70% of hereditary nonpolyposis colon cancer. Despite the absence of mutations of the hMLH1 gene, the majority of RER-positive sporadic colorectal carcinomas lack hMLH1 protein expression, which have been reported to be related to hypermethylation of the promoter region of hMLH1 gene. High frequency of microsatellite instability (MSI) has been observed in about 15% of sporadic gastric carcinomas. The relationship of tumor MSI, methylation of promoter regions of hMLH1 or hMSH2, and expression of corresponding gene products has not been studied in gastric carcinomas as thoroughly as in colorectal carcinomas. We explored the relationship between methylation of hMLH1 or hMSH2 promoter regions and its protein expression in both RER-positive and RER-negative gastric carcinomas. Of 93 cases, 20 cases comprised the RER+ group (MSI-H tumors) and the remainder comprised the RER- group (7 cases, MSI-L; 66 cases, MSS). By immunohistochemistry absence of hMLH1 protein expression was limited entirely to the RER+ group (20 of 20, 100%). All 93 cases showed hMSH2 protein expression. Nineteen (95%) of 20 RER+ tumors harbored hypermethylation of the hMLH1 promoter region whereas only four cases (5.5%) of the 73 RER- tumors did. Hypermethylation of the hMSH2 promoter region was not observed in either the RER+ group or the RER- group. These results suggest that hypermethylation of the hMLH1 promoter region may be the principal mechanism of gene inactivation in sporadic gastric carcinomas with a high frequency of MSI.     

Microsatellite instability and alteration of the expression of hMLH1 and hMSH2 in ovarian clear cell carcinoma

Microsatellite instability (MSI) is commonly seen in tumors associated with the hereditary nonpolyposis colorectal cancer syndrome and is caused by defects in the DNA mismatch repair genes. MSI has also been observed in various sporadic cancers, including colorectal, gastric, and endometrial. The role and incidence of MSI in ovarian clear cell carcinoma remain unknown. This study was conducted to evaluate the frequency of MSI in ovarian clear cell carcinomas and to evaluate the sensitivity and specificity of immunohistochemistry in predicting mismatch-repair gene deficiency. A total of 42 ovarian clear cell carcinomas were analyzed for MSI using a panel of 5 microsatellite markers (BAT25, BAT26, D5S346, D2S123, and D17S250). Alterations in the expression of hMLH1 and hMSH2 proteins in these tumors were examined. Of the 42 ovarian clear cell tumors analyzed, 6 demonstrated a high level of MSI (MSI-H), 3 demonstrated a low level of MSI (MSI-L), and the remaining 33 exhibited microsatellite stability (MSS). No correlation was found between MSI level and patient age or tumor stage or size (P >0.05). Loss of expression of either hMLH1 or hMSH2 was observed in 4 of the 6 (67.7%) MSI-H tumors, whereas 34 of the 36 (94.4%) MSI-L or MSS tumors expressed both the hMLH1 and hMSH2 gene products. Our results indicate that MSI-H is involved in the development of a subset of ovarian clear cell carcinomas. A strong correlation exists between alterations in the expression of hMLH1 and hMSH2 and the presence of MSI-H in these tumors. However, immunohistochemical testing alone may miss a small fraction of cases with MSI-H.     

Concerted promoter hypermethylation of hMLH1, p16INK4A,and E-cadherin in gastric carcinomas with microsatellite instability

In most sporadic gastric carcinomas, microsatellite instability (MSI) originates from inactivation of the hMLH1 gene by promoter hypermethylation. However, the methylation patterns of other genes and their consequences in high MSI (MSI-H) gastric carcinomas are not well characterized. To address the aberrant promoter methylation profiles of MSI-H gastric carcinomas, promoter methylation of six genes (hMLH1, p16(INK4A), E-cadherin, Rb, RASSF1A, and VHL) and CpG island methylator phenotype (CIMP) were explored in 36 MSI-H gastric carcinomas and the results were compared with those of 43 microsatellite-stable (MSS) gastric carcinomas. Frequent promoter hypermethylation was found in hMLH1, p16(INK4A), and E-cadherin and the frequency was significantly higher in MSI-H gastric carcinomas. Promoter hypermethylation of hMLH1, E-cadherin, and p16(INK4A) was found in 89%, 78%, and 33% of MSI-H gastric carcinomas and in 16%, 32%, and 11% of MSS carcinomas, respectively (p = 0.01). Selective absent or decreased expression of the gene product related to the hypermethylated promoter was found for hMLH1 and p16(INK4A) in MSI-H carcinoma, whereas the expression of E-cadherin was generally decreased both in the MSI-H and in the MSS carcinomas. MSI-H gastric carcinomas were also related to the high CIMP (CIMP-H, three or more of the five loci examined showing methylation). Twenty-two (61%) MSI-H gastric carcinomas were CIMP-H, compared with only seven (16%) MSS carcinomas (p = 0.001). These findings indicate that hMLH1 is one of the frequent methylation targets in CIMP-H gastric carcinomas and that inactivation of hMLH1 through promoter hypermethylation results in tumours following the MSI pathway.     

Alterations of DNA mismatch repair proteins and microsatellite instability levels in gastric cancer cell lines

Alterations in DNA mismatch repair (MMR) proteins result in microsatellite instability (MSI), increased mutation accumulation at target genes and cancer development. About one-third of gastric cancers display high-level microsatellite instability (MSI-High) and low-level microsatellite instability (MSI-Low) is frequently detected. To determine whether variations in the levels of MMR proteins or mutations in the main DNA MMR genes are associated with MSI-Low and MSI-High in gastric cancer cell lines, the MSI status (MSI-High, MSI-Low or MS-Stable (MSS)) of 14 gastric cancer lines was determined using multiple clone analysis with a panel of five microsatellite markers. Protein levels of hMLH1, hMSH2, hMSH6, hPMS2 and hPMS1 were determined by Western blot. Sequence analysis of hMLH1 and hMSH2 was performed and the methylation status of the hMLH1 promoter was examined. The cell lines SNU1 and SNU638 showed MSI-High, decreased to essentially absent hMLH1 and hPMS2 and reduced hPMS1 and hMSH6 protein levels. The hMLH1 promoter region was hypermethylated in SNU638 cells. The MKN28, MKN87, KATOIII and SNU601 cell lines showed MSI-Low. The MMR protein levels of cells with MSI-Low status was similar to the levels detected in MSS cells. A marked decrease in the expression levels of MutL MMR proteins (hMLH1, hPMS2 and hPMS1) is associated with high levels of MSI mutations in gastric cancer cells. Gastric cancer cell lines with MSI-Low status do not show significant changes in the levels of the main DNA MMR proteins or mutations in the DNA mismatch repair genes hMSH2 and hMLH1. These well-characterized gastric cancer cell lines are a valuable resource to further our understanding of DNA MMR deficiency in cancer development, progression and prognosis.     

hMLH1 Promoter Hypermethylation Is an Early Event in Human Endometrial Tumorigenesis

It has recently been suggested that silencing of the hMLH1 gene by promoter hypermethylation is the mechanism underlying the presence of the microsatellite instability (MSI) phenotype in sporadic colon and endometrial carcinomas. To determine whether hMLH1 promoter hypermethylation is a relatively early event in endometrial tumorigenesis we evaluated endometrial hyperplasia (EH) characterized as simple, complex, and atypical (the direct precursor of endometrial carcinoma) for hMLH1 aberrant methylation. In addition, we studied the hMLH1, hMSH2, hMSH3, and hMSH6 promoter methylation and MSI status of those endometrial carcinomas with synchronous hyperplasias and those without them. We found that 11 of 12 (91%) cases of endometrial carcinoma (EC) displaying MSI had hMLH1 promoter hypermethylation, whereas aberrant methylation of any of the other mismatch repair genes was not observed. All 15 cases of EC without MSI were unmethylated at hMLH1. Abnormal methylation of hMLH1 was also present in 8 of 116 (7%) cases of EH and was restricted primarily to the atypical endometrial hyperplasia (AEH) type with coexisting endometrial carcinoma. In this set, half of EH methylated at hMLH1 displayed MSI, whereas none of the unmethylated EH had MSI. Our data suggest that hypermethylation of hMLH1 can be an early event in the pathogenesis of EC, preceding the development of an apparent MSI phenotype in a subset of cases.     

MLH1 promoter methylation and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers

Defective DNA mismatch repair in human tumors leads to genome-wide instability of microsatellite repeats and a molecular phenotype referred to as microsatellite instability (MSI). MSI has been reported in a variety of cancers and is a consistent feature of tumors from patients with hereditary non-polyposis colorectal cancer. Approximately 20% of cancers of the uterine endometrium, the fifth most common cancer of women world-wide, exhibit MSI. Although the frequency of MSI is higher in endometrial cancers than in any other common malignancy, the genetic basis of MSI in these tumors has remained elusive. We investigated the role that methylation of the MLH1 DNA mismatch repair gene plays in the genesis of MSI in a large series of sporadic endometrial cancers. The MLH1 promoter was methylated in 41 of 53 (77%) MSI-positive cancers investigated. In MSI-negative tumors on the other hand, there was evidence for limited methylation in only one of 11 tumors studied. Immunohistochemical investigation of a subset of the tumors revealed that methylation of the MLH1 promoter in MSI-positive tumors was associated with loss of MLH1 expression. Immunohistochemistry proved that two MSI-positive tumors lacking MLH1 methylation failed to express the MSH2 mismatch repair gene. Both of these cancers came from women who had family and medical histories suggestive of inherited cancer susceptibility. These observations suggest that epigenetic changes in the MLH1 locus account for MSI in most cases of sporadic endometrial cancers and provide additional evidence that the MSH2 gene may contribute substantially to inherited forms of endometrial cancer.     

Causes of microsatellite instability in colorectal tumors: implications for hereditary non-polyposis colorectal cancer screening

Microsatellite instability (MSI) analysis was performed using a "reference panel" of microsatellite markers in 345 unselected primary colorectal cancers (CRC). Thirty-five (10%) tumors were classified as high MSI (MSI-H). We identified 6 (17%) MSI-H tumors with germline mutations in mismatch repair (MMR) genes (tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome) and 29 (83%) MSI-H tumors without germline MMR mutations (sporadic MSI-H tumors). Hypermethylation of the hMLH1 promoter was found in 26/29 (90%) sporadic MSI-H tumors but only in 1/6 (17%) HNPCC tumors (P<.001). Somatic alterations were identified in both MMR genes in HNPCC tumors but mainly in the hMSH2 gene in sporadic MSI-H tumors. LOH at MMR loci was detected in 3/6 (50%) HNPCC tumors and in 4/26 (15%) informative sporadic MSI-H tumors. These results together indicate different mode of inactivation of MMR genes in sporadic MSI-H tumors versus MSI-H tumors in HNPCC patients. We therefore propose that MSI analysis of newly diagnosed primary CRC followed by methylation analysis of hMLH1 promoter in MSI-H tumors and mutational analysis of MMR genes in MSI-H tumors lacking hMLH1 promoter methylation might be an efficient molecular genetic approach for HNPCC screening.     

Microsatellite Instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer

Immunohistochemical expression analysis of mismatch repair gene products has been suggested for the prediction of hereditary nonpolyposis colorectal cancer (HNPCC) carrier status in cancer families and the selection of microsatellite instability (MSI)-positive tumors in sporadic colorectal cancer. In this study, we aimed to evaluate hMSH2 and hMLH1 immunohistochemistry in familial and sporadic colorectal cancer. We found that immunohistochemistry allowed us to identify patients with germline mutations in hMSH2 and many cases with germline mutations in hMLH1. However, some missense and truncating mutations may be missed. In addition, hMLH1 promoter methylation, commonly occurring in familial and sporadic MSI-positive colorectal cancer, can complicate the interpretation of immunohistochemical expression analyses. Our results suggest that immunohistochemistry cannot replace testing for MSI to predict HNPCC carrier status or identify MSI-positive sporadic colorectal cancer.     

MSI-L gastric carcinomas share the hMLH1 methylation status of MSI-H carcinomas but not their clinicopathological profile

Sporadic gastric carcinomas (SGC) with microsatellite instability (MSI) exhibit mutations in target genes and display a particular clinicopathological profile. In SGC the MSI phenotype has been associated with hMLH1 promoter hypermethylation. Fifty-seven SGC, classified as high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), were analyzed for hMLH1 promoter methylation status and clinicopathological features. hMLH1 mutations and hMLH1 expression, as well as target gene mutations, were also evaluated. Our aims were to characterize the molecular and clinicopathological features of SGC, with and without hMLH1 promoter hypermethylation, and to compare the molecular and clinicopathological features of MSI-L, MSI-H, and MSS tumors in an attempt to clarify the place of MSI-L tumors in the mismatch repair (MMR) pathway. Hypermethylation of hMLH1 promoter occurred in 27 of 57 SGC (47.3%) and was significantly associated with MSI status, target gene mutations, and expansive pattern of growth of the tumors. Seventy-five percent of the MSI-H and 50% of MSI-L carcinomas showed hypermethylation (Met+) of hMLH1 in contrast to 0% in MSS carcinomas. No hMLH1 expression was observed in MSI-L/Met+ and MSI-H/Met+ cases. MSS and MSI-L tumors share the same clinicopathological profile regardless of the methylation status of the latter and are distinct from MSI-H tumors. We conclude that mutations in target genes, more than hypermethylation or absence of expression of hMLH1, are the link between MSI status and most of the clinicopathological features of SGC.     

Frequent hypermethylation of the hMLH1 gene promoter in differentiated-type tumors of the stomach with the gastric foveolar phenotype

Hypermethylation of the hMLH1 mismatch repair gene promoter has been revealed to lead to microsatellite instability (MSI). Previously, we demonstrated a high prevalence of MSI in differentiated-type gastric tumors showing distinct features of gastric foveolar epithelium (foveolar type). To clarify the significance of hMLH1 promoter hypermethylation in the development of this tumor type, we studied promoter methylation status and expression of hMLH1 in foveolar-type tumors and their surrounding non-neoplastic mucosae, as well as in tumors with other cellular phenotypes. The results were compared to MSI status. After phenotypical analyses using mucin histochemistry and immunohistochemistry, 41 differentiated-type tumors with distinct cellular phenotypes were classified into three categories: foveolar type, intestinal type (tumors with the distinct cellular phenotype of the intestine), and combined type (tumors with both foveolar and intestinal phenotypes). Methylation-specific polymerase chain reaction (MSP) was performed to determine the methylation status of hMLH1 promoter. hMLH1 protein expression was immunohistochemically examined. MSI was detected in 57% of the foveolar type, 8% of the intestinal type, and 67% of the combined-type tumors. Hypermethylation of hMLH1 promoter was found in 74% of the foveolar type, 33% of the intestinal type, and 83% of the combined-type tumors. Of 18 MSI-positive tumors, all but one were hypermethylated. Methylation status of hMLH1 promoter correlated well with protein expression in foveolar-type tumors. Moreover, hypermethylation was also detected frequently (71%) in the non-neoplastic surrounding mucosa of the hypermethylated tumors. Hypermethylation of hMLH1 promoter is an initial, vital event in the development of foveolar-type tumors of the stomach.     

A cost-effective algorithm for hereditary nonpolyposis colorectal cancer detection

Colorectal cancer with microsatellite instability (MSI) may occur sporadically or be inherited in cases of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. However, there is no consensus as to which patients must be tested and how to test MSI. In this study, MSI was tested by immunohistochemical analysis and by polymerase chain reaction in 148 cases of colorectal cancer, and methylation of the hMLH1 promoter was examined. MSI status was correlated with tumor phenotype. We found that localization, tumor infiltrating lymphocytes, and mucinous differentiation were predictive of high-frequency MSI (MSI-H) colorectal cancer and might be used to select cases for MSI analysis. Immunohistochemical analysis detected most MSI-H colorectal cancer and might constitute the first step in MSI detection. Absence of hMLH1 promoter methylation in MSI-H colorectal cancer could be predictive of hereditary colorectal cancer, and, hence, methylation analysis might constitute the second step in the identification of patients with HNPCC.     

Promoter hypermethylation and inactivation of hMLH1, a DNA mismatch repair gene, in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a multistage process during which adverse genetic alterations accumulate resulting in loss of cell cycle control, selective cell overgrowth, and ultimately formation of malignancy. Among various genetic alterations in HNSCC is increased microsatellite instability (MSI). hMLH1 is one of the major mismatch DNA repair genes, the inactivation of which caused increased MSI in a variety of human cancers including HNSCC. While somatic mutation is a major mechanism of the hMLH1 gene inactivation in hereditary form of human cancer, promoter hypermethylation appears to be primarily involved in the inactivation of the hMLH1 gene in sporadic form of human cancers. In the current study, we analyzed 78 cases of HNSCC for hMLH1 protein expression and promoter hypermethylation by IHC and methylation-specific PCR (MSP). Twenty-four of 78 cases (31%) of HNSCC contained markedly reduced levels of the hMLH1 protein. Based on the IHC results, 8 cases without and 8 with hMLH1 protein expression (total of 16) were further analyzed by MSP. Seven of 8 cases (88%) that were negative for the hMLH1 protein displayed promoter hypermethylation, whereas 7 of 7 cases (100%) strongly positive for the protein were free of promoter methylation. This study confirms our previous conclusion that promoter hypermethylation represents a major mechanism of the hMLH1 gene inactivation in HNSCC.     

Methylation of the hMLH1 promoter in multiple gastric carcinomas with microsatellite instability

Hypermethylation of the hMLH1 promoter is observed in the majority of sporadic gastric carcinomas with high frequency microsatellite instability (MSI), and it contributes to the genesis of MSI-positive gastric carcinoma. Multiple gastric carcinoma is known to have a higher frequency of MSI positivity than single gastric carcinoma. However, the molecular basis of MSI in these tumors remains obscure. We investigated the role of hMLH1 promoter hypermethylation in the genesis of multiple gastric carcinoma with MSI. We analyzed 33 tumors from 15 patients with multiple gastric carcinoma (12 double tumors and three triple tumors) for MSI, expression of hMLH1 and hMSH2, and hypermethylation of hMLH1 and hMSH2 promoters. High frequency MSI was found in seven out of 33 tumors (21%) in five out of 15 patients (33%). All of the tumors with high frequency MSI had a lack of hMLH1 expression, with the presence of hMSH2 expression, while all the tumors with no MSI or low frequency MSI were positive for both hMLH1 and hMSH2. All of the tumors with no expression of hMLH1 had hMLH1 hypermethylation, whereas hMLH1 hypermethylation was observed in two out of 26 (8%) tumors with no or low frequency MSI. None of the tumors showed hMSH2 hypermethylation. These results suggest that epigenetic changes in the hMLH1 promoter account for the genesis of multiple gastric carcinoma with high frequency MSI.     

Three independent genetic profiles based on mucin expression in early differentiated-type gastric cancers--a new concept of genetic carcinogenesis of early differentiated-type adenocarcinomas.

Recent molecular studies have shown that the genetic profiles of differentiated-type adenocarcinomas of the stomach are associated with distinct cellular mucin phenotypes (gastric- intestinal- and mixed-phenotypes). Therefore, we examined whether these cellular mucin phenotypes reflect specific molecular genetic alterations, and whether the phenotypes can be used to help categorize the intramucosal neoplasias of gastric tumors. We subclassified tumors into four cellular phenotypes using immunohistochemical mucin analysis. In all, 62 early gastric carcinomas (gastric-phenotype, 13; intestinal-phenotype, 17; mixed-phenotype, 31; unclassified-phenotype, 1) were examined using a combination of polymerase chain reaction microsatellite assays and immunohistochemical analysis in order to detect chromosomal allelic losses of multiple cancer-related chromosomal loci (1p, 3p. 4p, 5q, 8p, 9p, 13p, 17p, 18q and 22q), microsatellite instability (MSI), and overexpression of the p53 protein. In addition, we analyzed the relationship between MSI status and hMLH1 promoter hypermethylation, which is thought to be a cause of high MSI status. For gastric phenotype cancers, the frequency of 3p allelic loss was higher than that of other microsatellite markers, whereas 5q allelic loss was frequently found in intestinal phenotype cancers. The genetic profile of mixed phenotype cancers is comprised of two distinct genetic types: LOH and MSI types. In the former, 5q, 3p and 18q allelic losses are seen frequently in intramucosal carcinomas. On the other hand, 17p, 1p and 9p allelic losses are associated with the development of submucosal carcinomas. MSI was observed only in mixed phenotype cancers (six of 31 mixed phenotype cancers). Overexpression of the p53 protein is common in differentiated-type gastric cancers. In addition, the MSI status of the tumor cells was correlated with the extent of hypermethylation of the hMLH1 promoter. We suggest that the cellular mucin phenotypes of the differentiated-type adenocarcinomas result from distinct genetic alterations.     

Detection of microsatellite instability by real time PCR and hybridization probe melting point analysis

Microsatellite alterations can be found in a number of tumors. There are two types of alterations: loss of heterozygosity (LOH), which can be detected in the majority of colorectal cancers (CRC), and microsatellite instability (MSI). MSI occurs in about 15% of CRC with a mutator phenotype and are the hallmark of hereditary nonpolyposis colorectal cancers (HNPCC). Furthermore, MSI can also be detected in other tumors which are part of the HNPCC tumor spectrum (eg, gastric, ovarian, and endometrial carcinomas). Usually, a set of microsatellite markers is amplified by PCR followed by gel or capillary electrophoresis to separate PCR amplicons and by detection of the markers using autoradiography (Thibodeau et al, 1993), silver staining (Schlegel et al, 1996), or fluorescence techniques (Gyapay et al, 1996; Mansfield et al, 1994). We have established a technique to detect MSI by LightCycler PCR and melting point analysis using sequence-specific hybridization probes (HyProbes) labeled with LightCycler dyes, LCRed640 and LCRed705. Amplification of microsatellites by real-time PCR is followed by melting point analysis to display alterations in the length of repetitive sequences, thereby avoiding any electrophoretical separation of amplified DNA. Two mononucleotide markers (BAT25 and BAT26) were tested in 81 formalin-fixed and paraffin-embedded colorectal cancer samples with matched normal tissues from 21 MSI tumors and 60 tumors with microsatellite stability. Amplification and melting point determination of BAT26 and BAT25 was possible in 129/162 (80%) and 123/162 (76%) formalin-fixed and paraffin-embedded tissue samples, respectively. MSI could be detected specifically with both BAT25 and BAT26 markers only in MSI-high tumors (> or =40% MSI rate, determined with microsatellite reference panel, BAT25, BAT26, D5S346, D2S123, D17S250; Boland et al, 1998; Dietmaier et al, 1997). This new technique allows MSI detection within less than a hour and provides a basis for fast, high-throughput MSI analysis.