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. 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). 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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