Methylation of hMLH1 in a population-based series of endometrial carcinomas.
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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.Keywords:
Microsatellite Instability
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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.
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Loss of heterozygosity and mutations in the PTEN (MMAC1) tumor suppressor gene are frequent in endometrial carcinoma. Promoter hypermethylation has recently been identified as an alternative mechanism of tumor suppressor gene inactivation in cancer, but its importance in the PTEN gene in endometrial carcinoma is unknown. The purpose of our study was to assess the frequency of promoter methylation of the PTEN gene and to determine its correlation with clinicopathologic variables in a prospective and population-based series of endometrial carcinomas with complete follow-up. Presence of PTEN promoter methylation was seen in 26 of 138 patients (19%). Methylation was significantly associated with metastatic disease (p = 0.01) and a microsatellite unstable phenotype (p = 0.006). In conclusion, we find that PTEN promoter methylation is relatively frequent in endometrial carcinoma. Its association with metastatic disease and microsatellite instability implicates its importance in the development of this tumor type. Int. J. Cancer 91:22–26, 2001. © 2001 Wiley-Liss, Inc.
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Microsatellite Instability
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Microsatellite Instability
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Colorectal carcinoma (CRC) is the second most common cause of cancer death in the United States, and the rate of CRC is nearly 1.5 times higher in African-Americans (AA) than in Caucasians. Microsatellite instability (MSI) is observed in sporadic CRC reflecting promoter hypermethylation of the DNA mismatch repair gene hMLH1, and anecdotal evidence suggests an increased incidence of MSI among AAs. Additionally, p16 can be inactivated by hypermethylation of the promoter region, abrogating its ability to regulate cell proliferation. The objective of this study is to determine the frequency of MSI and p16 gene methylation in CRC from AA patients.
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Colorectal cancers (CRC) with microsatellite instability (MSI) display unique clinicopathologic features including a mucinous pattern with frequent expression of the secreted mucins MUC2 and MUC5AC. The mechanisms responsible for this altered pattern of expression remain largely unknown. We quantified DNA methylation of mucin genes (MUC2, MUC5AC, MUC4) in colonic cancers and examined the association with clinicopathological characteristics and molecular (MSI, KRAS, BRAF, and TP53 mutations) features. A control cohort was used for validation. We detected frequent hypomethylation of MUC2 and MUC5AC in CRC. MUC2 and MUC5AC hypomethylation was associated with MUC2 and MUC5AC protein expression (p = 0.004 and p < 0.001, respectively), poor differentiation (p = 0.001 and p = 0.007, respectively) and MSI status (p < 0.01 and p < 0.001, respectively). Interestingly, MUC5AC hypomethylation was specific to MSI cancers. Moreover, it was significantly associated with BRAF mutation and CpG island methylator phenotype (p < 0.001 and p < 0.001, respectively). All these results were confirmed in the control cohort. In the multivariate analysis, MUC5AC hypomethylation was a highly predictive biomarker for MSI cancers. MUC5AC demethylation appears to be a hallmark of MSI in CRC. Determination of MUC5AC methylation status may be useful for understanding and predicting the natural history of CRC.
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Abstract Context.—Microsatellite instability (MSI) due to defective mismatch repair (MMR) genes has been reported in the majority of colorectal tumors from patients with hereditary nonpolyposis colorectal cancer syndrome and in 10% to 15% of sporadic colorectal cancers. The identification of cancers associated with MSI requires classical molecular testing as the gold standard. Objective.—The aim of this study was to evaluate the role of immunohistochemistry with antibodies directed against 4 MMR proteins as a screening tool for carcinomas with MSI. Methods.—In this study, 204 formalin-fixed, paraffin-embedded colorectal carcinomas were examined for MMR protein expression (hMLH1, hMSH2, hMSH6, and hPMS2) and analyzed for MSI (MSI-H indicates at least 2 of 6 markers affected). These results were correlated with histopathologic parameters. Results.—Immunohistochemical analysis revealed that loss of expression of at least 1 protein was present in 17% of cases. One hundred percent of carcinomas that showed high instability (MSI-H) showed loss of expression of hMLH1, hMSH2, or hMSH6. Loss of expression of 2 proteins was present in 59.4% of MSI-H cases, with only 2 combinations, namely, hMLH1/hPMS2 and hMSH2/hMSH6. Isolated loss of hMSH6 expression was present in 2 MSI-H cases. Conclusions.—These findings confirm that examination of MMR protein expression by immunohistochemistry is a simple method to diagnose colorectal cancer with MSI. Our data suggest that the study of hMSH6 may be useful, in addition to hMLH1 and hMSH2. Moreover, immunohistochemistry could represent a screening method with which to direct research on the mutations of MMR genes observed in hereditary nonpolyposis colorectal cancer syndrome.
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Abstract Background: Increased risk for endometrial cancer, the most common gynecological malignancy in the United States, is related to obesity, nulliparity, and postmenopausal hormone use. Several molecular pathways and mechanisms are prominently involved in endometrial carcinogenesis including mismatch repair deficiency (MMR), abnormalities in the PTEN pathway and DNA methylation. We compared DNA methylation profiles in endometrial cancers to identify markers related to MMR deficiencies and contrasted results to normal endometrial tissue to identify potential biomarkers for early detection. Methods: We conducted methylation profiling on 148 paraffin embedded endometrial cancer tissues collected in the population-based Polish Endometrial Cancer Study and 23 normal tissues included within a study of benign endometrium at Magee Women's Hospital. 1.0 mm tissue cores removed from target-rich areas of tissue blocks were used for DNA extraction and bisulfite treatment. The Illumina Golden Gate platform was used to assess CpG methylation at 1505 sites. Microsatellite instability (MSI) testing using three markers (BAT25, BAT26, and CAT25) was performed to identify MMR deficient cases. We conducted unsupervised hierarchical clustering and class comparison analyses to evaluate DNA methylation by MMR status among cancers and class comparison and pathway analyses to identify differences in methylation markers between normal and tumor tissue. Results: Unsupervised hierarchical clustering of methylation profiles in cancers showed two major clusters with significantly different proportions of MSI cases (61.4% versus 10.6%, p<0.0001). Class comparison by MSI status identified 52 differentially methylated genes with a p-value <0.001, including MLH1. Further, we observed associations between age and BMI and methylation patterns in endometrial cancers. Unsupervised hierarchical clustering showed almost perfect separation of endometrial cancers from normal endometrial tissues, reflecting differential methylation of 332 genes at a p-value = 0.001. Higher methylation levels were found in cancers compared to normal tissues, with the most significant events in the PTEN pathway. Conclusions: Analyses of methylation profiles in endometrial cancer show differences by age, BMI and MMR status. Many genes show differences in DNA methylation in endometrial cancers as compared to benign endometrial tissues. These data suggest that methylation analyses may be important for understanding etiological heterogeneity in endometrial cancer and for identifying possible markers for early detection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5012. doi:1538-7445.AM2012-5012
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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. 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. Endometrial carcinoma is the most common gynecological malignancy and is the fourth most common cancer of women in the United States.1Parker SL Tong T Bolden S Wingo PA Cancer statistics.CA Cancer J Clin. 1997; 47: 5-27Crossref PubMed Scopus (2333) Google Scholar Uterine endometrioid carcinoma (UEC) is the most common histological type. UEC often precedes or coexists with endometrial hyperplasias, which have been proposed as possible precursor lesions.2Sherman A Brown S The precursors of endometrial carcinoma.Am J Obstet Gynecol. 1979; 135: 947-954Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 3Kurman R Kaminski P Norris H The behavior of endometrial hyperplasia: a long term study of "untreated" hyperplasia in 170 patients.Cancer. 1985; 56: 403-412Crossref PubMed Scopus (1109) Google Scholar Atypical endometrial hyperplasia (AEH) is most associated with progression to carcinoma, and women with AEH without concurrent invasive carcinoma have a 30% risk of developing UEC.3Kurman R Kaminski P Norris H The behavior of endometrial hyperplasia: a long term study of "untreated" hyperplasia in 170 patients.Cancer. 1985; 56: 403-412Crossref PubMed Scopus (1109) Google Scholar UEC and AEH share cytological atypia and a monoclonal pattern,4Jovanovic AS Boynton KA Mutter GL Uteri of women with endometrial carcinoma contain a histopathological spectrum of monoclonal putative precancers, some with microsatellite instability.Cancer Res. 1996; 56: 1917-1921PubMed Google Scholar, 5Esteller M Garcia A Martinez-Palones JM Xercavins J Reventos J Detection of clonality and genetic alterations in endometrial pipelle biopsy and its surgical specimen counterpart.Lab Invest. 1997; 76: 109-116PubMed Google Scholar but UEC are distinguished by the presence of invasion.3Kurman R Kaminski P Norris H The behavior of endometrial hyperplasia: a long term study of "untreated" hyperplasia in 170 patients.Cancer. 1985; 56: 403-412Crossref PubMed Scopus (1109) Google Scholar A spectrum of hyperplasia without atypia, including simple and complex hyperplasia, also exists. These lesions show a low rate of progression to UEC3Kurman R Kaminski P Norris H The behavior of endometrial hyperplasia: a long term study of "untreated" hyperplasia in 170 patients.Cancer. 1985; 56: 403-412Crossref PubMed Scopus (1109) Google Scholar and a putative polyclonal pattern.4Jovanovic AS Boynton KA Mutter GL Uteri of women with endometrial carcinoma contain a histopathological spectrum of monoclonal putative precancers, some with microsatellite instability.Cancer Res. 1996; 56: 1917-1921PubMed Google Scholar, 5Esteller M Garcia A Martinez-Palones JM Xercavins J Reventos J Detection of clonality and genetic alterations in endometrial pipelle biopsy and its surgical specimen counterpart.Lab Invest. 1997; 76: 109-116PubMed Google Scholar Very little is known about the genetic alterations underlying the biology of the precursor lesions of UEC. Only mutations in the oncogene K-ras and the tumor suppressor gene PTEN have been described in AEH.6Enomoto T Inoue M Perantoni AO Buzard GS Miki H Tanizawa O Rice JM K-ras activation in premalignant and malignant epithelial lesions of the human uterus.Cancer Res. 1991; 51: 5308-5314PubMed Google Scholar, 7Levine R Cargile CB Blazes MS van Rees B Kurman RJ Ellenson LH PTEN mutations and microsatellite instability in complex atypical hyperplasia, a precursor lesion to uterine endometrioid carcinoma.Cancer Res. 1998; 58: 3254-3258PubMed Google Scholar PTEN mutations may even precede the appearance of an evident atypia in the endometrial hyperplasia.8Maxwell GL Risinger JI Gumbs C Shaw H Bentley RC Barret JC Berchuck A Futreal PA Mutations of the PTEN tumor suppressor gene in endometrial hyperplasias.Cancer Res. 1998; 58: 2500-2503PubMed Google Scholar However, a subset of AEH displays the microsatellite instability (MSI. phenotype.5Esteller M Garcia A Martinez-Palones JM Xercavins J Reventos J Detection of clonality and genetic alterations in endometrial pipelle biopsy and its surgical specimen counterpart.Lab Invest. 1997; 76: 109-116PubMed Google Scholar, 7Levine R Cargile CB Blazes MS van Rees B Kurman RJ Ellenson LH PTEN mutations and microsatellite instability in complex atypical hyperplasia, a precursor lesion to uterine endometrioid carcinoma.Cancer Res. 1998; 58: 3254-3258PubMed Google Scholar, 9Duggan B Felix J Muderspach L Tourgeman D Zheng J Shibata D Microsatellite instability in sporadic endometrial carcinoma.J Natl Cancer Inst. 1994; 86: 1216-1221Crossref PubMed Scopus (189) Google Scholar, 10Mutter GL Boynton KA Faquin WC Ruiz RE Jovanovic AS Allelotype mapping of unstable microsatellites establishes direct lineage continuity between endometrial precancers and cancer.Cancer Res. 1996; 56: 4483-4486PubMed Google Scholar MSI was first detected in tumors from patients with hereditary non-polyposis colorectal carcinoma (HNPCC). In this setting, the cause of MSI has been attributed to germline defects in DNA mismatch repair (MMR) genes, mainly involving hMLH1 and hMSH2.11Aaltonen L Peltomaki P Leach F Sistonen P Pylkkanen L Mecklin J Jarvinen H Powell S Jen J Hamilton S Petersen G Kinzler K Vogelstein B de la Chapelle A Clues to the pathogenesis of familial colorectal cancer.Science. 1993; 260: 812-816Crossref PubMed Scopus (2559) Google Scholar, 12Thibodeau SN Bren G Schaid D Microsatellite instability in cancer of the proximal colon.Science. 1993; 260: 816-819Crossref PubMed Scopus (2773) Google Scholar, 13Ionov Y Peinado MA Malkhosyan S Shibata D Perucho M Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.Nature. 1993; 363: 558-561Crossref PubMed Scopus (2384) Google Scholar, 14Liu B Parsons RE Hamilton SR Petersen GM Lynch HT Watson P Markowitz S Willson JKV Green J de la Chapelle A Kinzler KW Vogelstein B hMSH2 mutations in hereditary nonpolyposis colorectal cancer kindred's.Cancer Res. 1994; 54: 4590-4594PubMed Google Scholar, 15Kolodner RD Hall NR Lipford J Kane MF Morrison PT Finan PJ Burn J Chapman P Earabino C Merchant E Bishop T Structure of the human hMLH1 locus and analysis of a large hereditary nonpolyposis colorectal cancer kindred for MLH1 mutations.Cancer Res. 1995; 55: 242-248PubMed Google Scholar, 16Fishel R Lescoe MK Rao MRS Copeland NG Jenkins NA Garber J Kane M Kolodner R The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer.Cell. 1993; 75: 1027-1038Abstract Full Text PDF PubMed Scopus (2578) Google Scholar, 17Leach FS Nicolaides NC Papadopoulos N Liu B Jen J Parsons R Peltomaki P Sistnen P Aaltonen LA Nystrom-Lahi M Guan X-Y Zhang J Meltzer PS Yu J-W Kao F-T Chen DJ Cerosaletti KM Fournier REK Todd S Lewis T Leach RJ Naylor SL Weissenbach J Mecklin J-P Jarvinen H Petersen GM Hamilton SR Green I Jass J Watson P Lynch HT Tent JM de la Chapelle A Kinzler KW Vogelstein B Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer.Cell. 1993; 75: 1215-1225Abstract Full Text PDF PubMed Scopus (2087) Google Scholar, 18Papadopoulos N Nicolaides NC Wei Y-F Ruben SM Carter KC Rosen CA Haseltine WA Fleischmann RD Fraser CM Adams MD Venter JC Hamilton SR Petersen GM Watson P Lynch HT Peltomaki P Mecklin J-P de la Chapelle A Kinzler KW Vogelstein B Mutation of a mutL homolog in hereditary colon cancer.Science. 1994; 263: 1625-1628Crossref PubMed Scopus (1761) Google Scholar, 19Bronner CE Baker SM Morrison PT Warren G Smith LG Lescoe MK Kane M Earabino C Lipford J Lindblom A Tannergard P Bollag RJ Godwin AR Ward DC Nordenskjld MA Kolodner R Liskay RM Mutations in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary nonpolyposis colon cancer.Nature. 1994; 368: 258-261Crossref PubMed Scopus (1913) Google Scholar Other MMR genes identified include hPMS1, hPMS2, hMSH3, and hMSH6.20Nicolaides NC Papadopoulos N Liu B Wei Y-F Carter KC Ruben SM Rosen CA Hasentine WA Fleischmann RD Fraser CM Adams MD Venter JC Dunlop MG Hamilton SR Petersen GM de la Chapelle A Vogelstein B Kinzler KW Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.Nature. 1994; 371: 75-80Crossref PubMed Scopus (1429) Google Scholar, 21Nicolaides NC Papadopoulos N Liu B Parsons R Lengauer C Palombo F D'Arrigo A Markowitz S Willson JKV Kinzler KW Jiricny J Vogelstein B Mutations of GTBP in genetically unstable cells.Science. 1995; 268: 1915-1917Crossref PubMed Scopus (473) Google Scholar Endometrial carcinoma (EC) is the most common noncolorectal carcinoma occurring in women with HNPCC.22Watson P Lynch HT Extracolonic cancer in hereditary nonpolyposis colorectal cancer.Cancer. 1993; 7: 677-685Crossref Scopus (670) Google Scholar MSI has also been observed in approximately 20. of sporadic EC.9Duggan B Felix J Muderspach L Tourgeman D Zheng J Shibata D Microsatellite instability in sporadic endometrial carcinoma.J Natl Cancer Inst. 1994; 86: 1216-1221Crossref PubMed Scopus (189) Google Scholar, 23Risinger JI Berchuck A Kohler MF Watson P Lynch HT Boyd J Genetic instability of microsatellites in endometrial carcinoma.Cancer Res. 1993; 53: 1-4PubMed Google Scholar, 24Burks RT, Kessis TD, Cho KR, Hedrick L: Microsatellite instability in endometrial carcinoma. Oncogene 9:1163–1166Google Scholar, 25Kobayashi K Sagae S Kudo R Koi S Saito H Nakamura Y Microsatellite instability in endometrial carcinomas.Genes Chromosomes Cancer. 1995; 14: 128-132Crossref PubMed Scopus (97) Google Scholar, 26Caduff RF Johnston CM Svodoba-Newman SM Poy EL Merajver SD Frank TS Clinical and pathological significance of microsatellite instability in sporadic endometrial carcinoma.Am J Pathol. 1996; 148: 1671-1678PubMed Google Scholar, 27Katabuchi H van Rees B Lambers AR Ronnett BM Blazes MS Leach FS Cho KR Hedrick L Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas.Cancer Res. 1995; 55: 5556-5560PubMed Google Scholar, 28Lim PC Tester D Cliby W Ziesmer SC Roche PC Hartmann L Thibodeau SN Podratz KC Jenkins RB Absence of mutations in DNA mismatch repair genes in sporadic endometrial tumors with microsatellite instability.Clin Cancer Res. 1996; 2: 1907-1911PubMed Google Scholar, 29Kobayashi K Matsushima M Koi S Saito H Sagae S Kudo R Nakamura Y Mutational analysis of mismatch repair genes, hMLH1 and hMSH2, in sporadic endometrial carcinomas with microsatellite instability.Jpn J Cancer Res. 1996; 87: 141-145Crossref PubMed Scopus (61) Google Scholar, 30Kowalski LD Mutch DG Herzog TJ Rader JS Goodfellow PJ Mutational analysis of MLH1 and MSH2 in 25 prospectively-acquired RER+ endometrial cancers.Genes Chromosomes Cancer. 1997; 18: 219-227Crossref PubMed Scopus (107) Google Scholar, 31Catasus Ll Machin P Matias-Guiu X Prat J Microsatellite instability in endometrial carcinomas: clinicopathological correlations in a series of 42 cases.Hum Pathol. 1998; 29: 1160-1164Abstract Full Text PDF PubMed Scopus (143) Google Scholar It was expected that UECs with MSI+ would have mutations in one of the known DNA MMR genes. However, hMSH2 or hMLH1 mutations have been found in less than 10% of UECs with MI+.27Katabuchi H van Rees B Lambers AR Ronnett BM Blazes MS Leach FS Cho KR Hedrick L Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas.Cancer Res. 1995; 55: 5556-5560PubMed Google Scholar, 28Lim PC Tester D Cliby W Ziesmer SC Roche PC Hartmann L Thibodeau SN Podratz KC Jenkins RB Absence of mutations in DNA mismatch repair genes in sporadic endometrial tumors with microsatellite instability.Clin Cancer Res. 1996; 2: 1907-1911PubMed Google Scholar, 29Kobayashi K Matsushima M Koi S Saito H Sagae S Kudo R Nakamura Y Mutational analysis of mismatch repair genes, hMLH1 and hMSH2, in sporadic endometrial carcinomas with microsatellite instability.Jpn J Cancer Res. 1996; 87: 141-145Crossref PubMed Scopus (61) Google Scholar, 30Kowalski LD Mutch DG Herzog TJ Rader JS Goodfellow PJ Mutational analysis of MLH1 and MSH2 in 25 prospectively-acquired RER+ endometrial cancers.Genes Chromosomes Cancer. 1997; 18: 219-227Crossref PubMed Scopus (107) Google Scholar Several studies have described a different pathway for inactivation of hMLH1 in colorectal tumors with MSI associated with silencing of the gene by promoter hypermethylation.32Kane MF Loda M Gaida GM Lipman J Mishra R Goldman H Jessup JM Kolodner R Methylation of the hMLH1 promoter correlates with lack of the expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines.Cancer Res. 1997; 57: 808-811PubMed Google Scholar, 33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar, 34Cunningham JM Christensen ER Tester DJ Kim CY Roche PC Burgart LJ Thibodeau SN Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability.Cancer Res. 1998; 58: 3455-3460PubMed Google Scholar, 35Veigl ML Kasturi L Olechnowicz J Ma AH Lutterbaugh JD Periyasamy S Li GM Drummond J Modrich PL Sedwick WD Markowitz SD Biallelic inactivation of hMLH1 by epigenetic silencing, a novel mechanism causing human MSI cancers.Proc Natl Acad Sci USA. 1998; 95: 8698-8702Crossref PubMed Scopus (550) Google Scholar These studies demonstrate that hMLH1 promoter hypermethylation is associated with the loss of hMLH1 expression and the MSI phenotype in colorectal cancer cell lines and sporadic colorectal carcinomas. Recent data also strongly suggest that the epigenetic silencing of hMLH1 is associated with MSI in gastric cancer.36Fleisher AS Esteller M Wang S Tamura G Suzuki H Yin J Zou TT Abraham JM Kong D Smolinski KN Shi YQ Rhyu MG Powell SM James SP Wilson KT Herman JG Meltzer SJ Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability.Cancer Res. 1999; 59: 1090-1095PubMed Google Scholar, 37Leung SY Yuen ST Chung LP Chu KM Chan AS Ho JC hMLH1 promoter methylation and lack of hMLH1 expression in sporadic gastric carcinomas with high-frequency microsatellite instability.Cancer Res. 1999; 59: 159-164PubMed Google Scholar In addition, the demethylation of the hMLH1 gene in cell lines, including one endometrial carcinoma cell line, using the agent 5-azacytidine results in reactivation of hMLH1 expression and restoration of the MMR activity.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar We have also recently described the strong association between hMLH1 promoter hypermethylation and the presence of a MSI phenotype in sporadic cases of UEC.38Esteller M Levine R Baylin SB Hedrick Ellenson L Herman JG MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas.Oncogene. 1998; 17: 2413-2417Crossref PubMed Scopus (427) Google Scholar This finding has also been corroborated lately in two independent studies.39Gurin CC Federici MG Kang L Boyd J Causes and consequences of microsatellite instability in endometrial carcinoma.Cancer Res. 1999; 59: 462-466PubMed Google Scholar, 40Simpkins SB Bocker T Swisher EM Mutch DG Gersell DJ Kovatich AJ Palazzo JP Fishel R Goodfellow PJ MLH1 promoter methylation, and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers.Hum Mol Genet. 1999; 8: 661-666Crossref PubMed Scopus (265) Google Scholar Methylation of normally unmethylated CpG islands in the promoter regions of tumor suppressor genes including p16, p15, VHL, and E-cadherin correlates with loss of transcription and suggests an alternative mechanism for tumor suppressor gene inactivation.41Baylin SB Herman JG Graff JR Vertino PM Issa JP Alterations in DNA methylation: a fundamental aspect of neoplasia.Adv Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar All of the above findings led us to investigate promoter methylation status in relation to the MSI phenotype in a large series of endometrial hyperplasias and carcinomas. Our results suggest that hMLH1 promoter hypermethylation is an early event in endometrial tumorigenesis, being present in AEH, and preceding in some cases a detectable MSI phenotype. Twenty-seven endometrial carcinomas and 116 endometrial hyperplasias were obtained from the Departments of Pathology of Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) and Brigham and Women's Hospital (Boston, MA). All tumors and hyperplasias were examined and classified according to the criteria of the World Health Organization. Concerning our series of hyperplasias, 62 cases of simple endometrial hyperplasia without atypia, 33 cases of complex endometrial hyperplasia without atypia, and 21 cases of complex hyperplasia with atypia were included in the study. No cases of simple endometrial hyperplasia with atypia were found in this set of samples. DNA was isolated from tumors and hyperplasias and matching normal DNA by standard methods from fresh frozen biopsies, paraffin-embedded material, and peripheral blood. Microdissection was performed on some sections to separate neoplastic and nonneoplastic tissues. For MSI analysis five dinucleotide microsatellite DNA sequences (D5S107, D10S197, D12S79, D12S95, and D18S58) on chromosomes 5,10, 12, and 18 were amplified by the PCR using MapPairs (Research Genetics Inc.) according to the same conditions previously described.31Catasus Ll Machin P Matias-Guiu X Prat J Microsatellite instability in endometrial carcinomas: clinicopathological correlations in a series of 42 cases.Hum Pathol. 1998; 29: 1160-1164Abstract Full Text PDF PubMed Scopus (143) Google Scholar Reconfirmation of MSI in endometrial cancers and determination of MSI in hyperplasias were performed using primer BAT-26. BAT-26 is a repeat of 26 deoxyadenosines localized within intron 5 of the MSH2 gene. BAT-26 was amplified by polymerase chain reaction (PCR) and analyzed by single-strand conformation polymorphism (SSCP) analysis as described.42Hoang JM Cottu PH Thuille B Salmon RJ Thomas G Hamelin R BAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines.Cancer Res. 1997; 57: 300-303PubMed Google Scholar DNA bands were visualized by silver staining method. DNA methylation patterns in the CpG islands of hMLH1, hMSH2, hMSH6, and hMSH3 genes were determined by MSP.43Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5174) Google Scholar MSP distinguishes unmethylated from methylated alleles in a given gene based on sequence changes produced after bisulfite treatment of DNA, which converts unmethylated, but not methylated, cytosines to uracil, and subsequent PCR using primers designed for either methylated or unmethylated DNA.43Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5174) Google Scholar Primer sequences of hMLH1 and MSH2 for the MSP analysis have been previously described.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar, 38Esteller M Levine R Baylin SB Hedrick Ellenson L Herman JG MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas.Oncogene. 1998; 17: 2413-2417Crossref PubMed Scopus (427) Google Scholar Primer sequences of hMSH6 for the unmethylated reaction were 5′-TTT GGG TTT TTT TGG TGG AGT GT-3′ (sense) and 5′-CTT AAA AAA AAA ACT ATA CAA AAT ACT CTA TCA CA-3′ (antisense) and for the methylated reaction 5′-TTT TTT CGG CGG AGC GC-3′ (sense) and 5′-AAA AAA AAA CTA TAC AAA ATA CTC TAT CGC-3′ (antisense). Primer sequences of hMSH3 for the unmethylated reaction were 5′-GGT TTG TGT TTT TTG TTA GGT TTT GTT-3′ (sense) and 5′-CTA AAA ACA ACA AAA CCA CCC AAC A-3′ (antisense. and for the methylated reaction 5′-CGT TTT TCG TTA GGT TTT GTC GTC-3′ (sense) and 5′-AAC GAA ACC GCC CGA CG-3′ (antisense). SW48 DNA, which has previously been characterized as methylated at the hMLH1 locus,32Kane MF Loda M Gaida GM Lipman J Mishra R Goldman H Jessup JM Kolodner R Methylation of the hMLH1 promoter correlates with lack of the expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines.Cancer Res. 1997; 57: 808-811PubMed Google Scholar, 33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar was used as a positive control for methylated alleles of hMLH1. Because there are no previous reports for cell lines with hypermethylation of hMSH2, hMSH6, and hMSH3, placental DNA treated in vitro with Sss I methyltransferase was used as a positive control for methylated alleles of the three genes. DNA from normal lymphocytes was used as negative control for methylated genes. Ten microliters of each PCR reaction were loaded directly onto nondenaturing 6% polyacrylamide gels, stained with ethidium bromide, and visualized under UV illumination. To check for a specific cytosine methylation within the hMLH1 promoter region studied, we digested with the restriction enzyme BstU I following bisulfite modification as described.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar, 44Sadri R Hornsby PJ Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification.Nucleic Acids Res. 1996; 24: 5058-5059Crossref PubMed Scopus (72) Google Scholar The BstU I recognition site, CGCG, will remain CGCG if both C's are methylated after bisulfite treatment and amplification, but will become TGTG if they are unmethylated. The colorectal carcinoma cell lines RKO and SW480 were used as positive and negative controls for hMLH1 promoter hypermethylation.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar To determine whether the locus containing the hMLH1 gene was deleted in tumors, LOH was assessed. Five different (CA)n repeats (Research Genetics, Inc., Huntsville, AL) localized on chromosome 3 (D3S1314, D3S1286, D3S1283, D3S1298, and D3S1611) were used to screen endometrial cancers for allelic loss in hMLH1 gene. D3S1611 is actually located within an intron of hMLH1.18Papadopoulos N Nicolaides NC Wei Y-F Ruben SM Carter KC Rosen CA Haseltine WA Fleischmann RD Fraser CM Adams MD Venter JC Hamilton SR Petersen GM Watson P Lynch HT Peltomaki P Mecklin J-P de la Chapelle A Kinzler KW Vogelstein B Mutation of a mutL homolog in hereditary colon cancer.Science. 1994; 263: 1625-1628Crossref PubMed Scopus (1761) Google Scholar PCR amplification of marker loci was carried out on paired normal-tumor DNAs in 25-μl reaction volume containing 100 ng template DNA, 10 pmol each forward and reverse primers, 0.2 mmol/L each dNTP, 1.5 units of Taq DNA polymerase (Ecotaq, Ecogen, Langhorne, PA) and 1.5 to 3 mmol/L MgCl2, dependent on primer. Thirty PCR cycles were used, with each cycle consisting of 30 seconds at 95 °C, 30 seconds at 58 °C, and 1 minute at 72 °C. The denatured samples were loaded onto a 8. denaturing polyacrylamide gels containing 8.3 Mol/L urea, and run at 1800 V for 4.5 hours at room temperature. PCR products were capillary-blotted onto Hybond-N membrane (Amersham, Buckinghamshire, UK) and DNA was fixed by alkali treatment. The membranes were analyzed using the ECL Gene Detection System (ECL Amersham) according to the specifications of the Amersham kit. Allelic loss of heterozygosity (LOH) was defined as a visible reduction of 50. or more in the band intensity of the tumor sample when compared to the corresponding normal band. We investigated 27 UECs previously characterized for the MSI phenotype10Mutter GL Boynton KA Faquin WC Ruiz RE Jovanovic AS Allelotype mapping of unstable microsatellites establishes direct lineage continuity between endometrial precancers and cancer.Cancer Res. 1996; 56: 4483-4486PubMed Google Scholar, 31Catasus Ll Machin P Matias-Guiu X Prat J Microsatellite instability in endometrial carcinomas: clinicopathological correlations in a series of 42 cases.Hum Pathol. 1998; 29: 1160-1164Abstract Full Text PDF PubMed Scopus (143) Google Scholar for promoter hypermethylation of hMLH1 using MSP.33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar, 38Esteller M Levine R Baylin SB Hedrick Ellenson L Herman JG MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas.Oncogene. 1998; 17: 2413-2417Crossref PubMed Scopus (427) Google Scholar Methylation of the promoter region studied for hMLH1 correlates with the loss of hMLH1 expression in primary sporadic colorectal cancer using MSP33Herman JG Uma A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1677) Google Scholar and another PCR-based approach.32Kane MF Loda M Gaida GM Lipman J Mishra R Goldman H Jessup JM Kolodner R Methylation of the hMLH1 promoter correlates with lack of the expression of hML
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Objective To explore the clinicopathologic features of sporadic colorectal cancers with mismatch repair deficiency and the application of the methods detecting the status of mismatch repair deficiency. Methods Detecting the methylation of the hMLH1 promotor, microsatellite instability and the expression of the hMLH1 and hMSH2 proteins among 71 sporadic colorectal cancer patients, evaluating these three approaches and summarizing the clinicopathologic characteristics of sporadic mismatch repair deficient colorectal cancers. Results The positive rate of the hypermethylation of the hMLH1 promotor, microsatellite instability and the expression of the hMLH1 and hMSH2 proteins were 9.9, 9.9 and 71 percent respectively. Sporadic colorectal cancers with the hypermethylation of the hMLH1 promotor or microsatellite instability were more likely to happen to the colon and to be poorly- differentiated adenocarcinomas. Sporadic colorectal cancers with the loss of hMLH1 or hMSH2 proteins were prone to be poorly- differentiated adenocarcinomas. There was a close relationship among the hypermethylation of the hMLH1 promotor, microsatellite instability and the silencing expression of the hMLH1 proteins. Conclusions Sporadic colorectal cancers with mismatch repair deficiency are inclined to occur to the colon and to have poor differentiation. The hypermethylation of the hMLH1 promotor, microsatellite instability and the loss of the hMLH1 or hMSH2 proteins are correlated intimately to each other.
Microsatellite Instability
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