Abstract
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.
Similar content being viewed by others
Introduction
Most tumors arising within the context of the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, as well as about 15% of sporadic colorectal carcinomas, exhibit a type of genetic instability characterized by the accumulation of ubiquitous somatic alterations in the length of simple repeated sequences (Ionov et al, 1993). This genome-wide instability of simple repeat sequences, referred to as microsatellite instability (MSI), is seen in 14% to 39% of sporadic gastric carcinomas (SGC) (Fleisher et al, 1999; Halling et al, 1999; Kang et al, 1999; Leung et al, 1999; Oliveira et al, 1998; Santos et al, 1996; Yamamoto et al, 1999).
The MSI phenotype, as found in HNPCC, is associated with defective DNA mismatch repair (MMR) genes, such as hMLH1, hMSH2, hMSH3, and hMSH6, among others (Liu et al, 1995; Wu et al, 1997; 1999). At variance with this, mutations in MMR genes are rare in sporadic colorectal and gastric carcinomas with the MSI phenotype (Borresen et al, 1995; Liu et al, 1995; Moslein et al, 1996; Wu et al, 1997; Yamamoto et al, 1999).
An alternative mechanism to mutations for silencing gene expression is hypermethylation of the gene promoter (Costello et al, 2000; Jones et al, 1999). hMLH1 hypermethylation, with associated decreased protein expression, has been described in 44% to 100% of gastric carcinomas displaying a high level of MSI (MSI-H) (Fleisher et al, 1999; Leung et al, 1999; Suzuki et al, 1999; Toyota et al, 1999; Yamamoto et al, 1999). By contrast, this association has not been observed in microsatellite stable (MSS) tumors (Leung et al, 1999; Toyota et al, 1999; Yamamoto et al, 1999). The relationship between hMLH1 hypermethylation and MSI phenotype is less clear with regard to tumors displaying a low level of MSI (MSI-L): Leung et al (1999), Kang et al (1999), Yamamoto et al (1999) and Toyota et al (1999) reported the absence of hypermethylation of hMLH1, as well as normal protein expression, in this type of tumor. Fleisher et al (1999), however, reported the occurrence of hMLH1 hypermethylation in five out of six MSI-L tumors with associated diminished hMLH1 expression.
In a previous study, we reported that MSI SGC exhibit mutations in a series of target genes (TGFβRII, IGFIIR, and BAX) and display a particular clinicopathological profile (Oliveira et al, 1998): MSI-H tumors were found to be significantly associated with distal localization, Lauren’s intestinal and atypical histotypes, and Ming’s expansive pattern of growth. In the present study, we analyzed hMLH1 promoter hypermethylation in a series of 57 SGC, stratified into MSI-H (n = 28), MSI-L (n = 12), and MSS (n = 17). In a subset of cases, hMLH1 mutations and hMLH1 expression, as well as target gene mutations, were also evaluated. Our aims were twofold: (a) to characterize the molecular and clinicopathological features of SGC, with and without hMLH1 promoter hypermethylation, and (b) to compare molecular, namely, hMLH1 promoter methylation and target gene mutations, and clinicopathological features of MSI-L, MSI-H, and MSS tumors.
Results
MSI Status versus Clinicopathological Features
The 57 tumors were classified as MSS (n = 17), MSI-L (n = 12), and MSI-H (n = 28) using the criteria described in the “Materials and Methods” section. The comparison between MSS and MSI-H tumors showed a significant association between the MSI-H phenotype and Lauren’s intestinal and atypical histotypes (p = 0.05), Ming’s expansive pattern of growth (p = 0.04), lower pathological tumor, nodes, metastases (pTNM) stage of the tumor (p = 0.05), and the presence of mutations in target genes (p = 0.0001).
Table 1 summarizes the molecular and clinicopathological data and compares the MSI-L with MSS and MSI-H tumors. MSI-L tumors did not significantly differ from MSS tumors (Table1). By contrast, MSI-L tumors differ significantly from MSI-H tumors with regard to those same parameters that were found to be different in the comparison of MSS and MSI-H tumors (Table 1).
hMLH1 Methylation Analysis
The hMLH1 promoter region of 21 of the 28 MSI-H cases (75%) could not be digested by HpaII, indicating that all four HpaII restriction sites were methylated. Six of the 12 (50%) MSI-L cases show the same methylation status. None of 17 MSS cases had methylation of the hMLH1 promoter region (Fig. 1). The DNA isolated from all these cases were sensitive to the digestion by MspI enzyme. The DNA from the normal mucosas of the stomach were all totally digested by HpaII. The association between the MSI phenotype and methylation status of the promoter region was statistically significant (p = 0.0001). Results are summarized in Table 2.
hMLH1 Methylation Status versus Clinicopathological Features
Data on the relationship between the clinicopathological features of the 57 SGC and the methylation status of hMLH1 are summarized in Table 2. A significant association was found between the methylation status and Ming’s classification (p = 0.01): the majority of expanding tumors showed hypermethylation (62%), whereas only a minority of infiltrative tumors presented this phenotype (27%).
hMLH1 Mutations
Mutations in all exons of hMLH1 were screened for 21 cases (17 methylation+ and 4 methylation−). No germline or somatic mutations of the hMLH1 gene were found in one MSS case, and no mutations were found in the 2 MSI-L and in the 18 MSI-H cases. Although we did not find any causative mutation in this gene for any of the cases analyzed, we did find two variants in the PCR amplicons of exons 8 (10/21, 48%) and 15 (9/21, 43%). Both these variants are common polymorphisms and were previously described by Liu et al (1995).
hMLH1 Gene Expression
hMLH1 mRNA expression was detected in all three MSS cases. In the 2 MSI-L cases and in 6 of the 10 MSI-H cases, where material was available for RNA isolation, there was no expression of hMLH1. In four MSI-H cases there was residual expression of hMLH1 when compared with GAPDH (Table 3 and Fig. 2).
Mutations in Target Genes (TGFβRII, IGFIIR, BAX, and TCF4)
Mutations in target genes were screened in 50 of the 57 cases. The presence of mutations in target genes (TGFβRII, IGFIIR, BAX, and TCF4) was significantly associated with hMLH1 promoter methylation (p = 0.0001) because 20 out of 23 (87%) cases positive for mutations in target genes showed hypermethylation of this MMR gene (Table 2). Six cases had no mutations in any of the target genes but had hMLH1 promoter methylation; five of these six tumors were MSI-L and the remaining case was a MSI-H carcinoma.
The association between mutations of target genes and the methylation status of hMLH1 promoter region was statistically significant for TGFβRII (p = 0.0001), BAX (p = 0.003), and IGFIIR (p = 0.03) and not significant for TCF4 (p = 0.34) (Fig. 3).
Methylation Status of MSI-L Carcinomas versus Molecular and Clinicopathological Features
The two MSI-L/Met+ cases analyzed by reverse transcription-polymerase chain reaction (RT-PCR) showed absence of expression of hMLH1 (Table 3). No mutations in target genes were observed in MSI-L cases (5 Met+ and 3 Met−) regardless of the presence or absence of hMLH1. The comparison between MSI-L/Met+ and MSI-L/Met− cases, regarding the clinicopathological features, did not yield any significant differences (data not shown).
Discussion
Hypermethylation of the hMLH1 promoter occurs with a very high frequency in SGC exhibiting MSI. We detected an aberrant hMLH1 promoter methylation in 75% of MSI-H gastric carcinoma cases leading to the absence or diminished expression of hMLH1. This percentage fits with those previously reported for SGC (Fleisher et al, 1999; Kang et al, 1999; Leung et al, 1999; Toyota et al, 1999; Wu et al, 2000; Yamamoto et al, 1999), sporadic colorectal carcinomas (Cunningham et al, 1998; Ghimenti et al, 1999; Kang et al, 1999), and endometrial carcinomas displaying MSI (Esteller et al, 1998; Simpkins et al, 1999). The RT-PCR analysis of hMLH1 expression showed that there is a significant association between hMLH1 hypermethylation and loss of hMLH1 expression as previously reported by Deng et al (1999). Using a polymorphism described in the promoter region of hMLH1, we were able to determine that in three MSI-H cases the hypermethylation occurs as a biallelic event as previously found by Veigl et al (1998) (data not shown). The high degree of instability associated with microsatellite markers in MSI tumors prevented us from accurately assessing the loss of heterozygosity (LOH) status of the hMLH1 gene in the tumors. Our results, together with data from literature, support the hypothesis that hMLH1 promoter hypermethylation is the most prevalent mechanism of MMR deficiency in SGC.
In 7 of the 28 MSI-H cases, no hMLH1 hypermethylation was observed, suggesting an alternative mechanism for hMLH1 inactivation in these cases. Somatic mutations in one of the MMR genes have been detected in up to 26% of sporadic MSI cancers at various sites, namely colon and endometrium (Borresen et al, 1995; Bubb et al, 1996; Katabuchi et al, 1995; Kowalski et al, 1997; Liu et al, 1995; Moslein et al, 1996; Thibodeau et al, 1996; Wu et al, 1997). Data on MMR gene mutations is scarce in SGC. Yamamoto et al (1999) showed the presence of hMSH2 and hMLH1 somatic mutations in, respectively, 6 and 1 out of 24 cases of MSI-H SGC. In our series we did not find hMLH1 mutations in any of the tumors, including the four MSI-H cases without hypermethylation that we were able to screen. Therefore, the mechanism of hMLH1 inactivation in hypermethylation/mutation-negative gastric carcinomas remains to be elucidated.
We observed a significant association between hMLH1 promoter methylation and mutations in target genes. We think that this finding may be ascribed, partly at least, to the close relationship between MSI-H and methylation status.
We found a significant association between hMLH1 hypermethylation and Ming’s classification: hMLH1 hypermethylation was detected in 62% of the expanding tumors and in 27% of the infiltrative tumors. If this finding is confirmed in a larger series, one might have to consider the possibility that target genes other than those we looked at are linked to the expanding growth pattern of the tumors. It is tempting to advance that the “new” target genes may be involved in cell-cell or cell-matrix adhesion. No other significant associations were found between hMLH1 hypermethylation and clinicopathological features. The discrepancy between the clinicopathological features of the MSI-H cases and those of cases with hMLH1 hypermethylation may be explained by two facts: the group of tumors with hMLH1 hypermethylation includes some MSI-L tumors and not all MSI-H tumors present hMLH1 hypermethylation.
In MSI-L gastric carcinomas, we found hMLH1 hypermethylation in 50% of the tumors. These results are in agreement with those reported by Fleisher et al (1999) in gastric carcinoma and by Esteller et al (1999) in endometrial carcinoma. There are no significant differences between MSS and MSI-L tumors as regards their clinicopathological profile. Toyota et al (1999) suggested that tumor development in cases presenting the MSI-L phenotype may be accompanied by random de novo hypermethylation. This hypermethylation, however, confers no selective advantage to the tumor cells. Curiously, if this hypothesis is true, MSS tumors should also have presented a proportion of cases with aberrant methylation of the hMLH1 promoter, but we did not observe this in our series. Furthermore, in two MSI-L cases in which hMLH1 expression was evaluated, we observed that hMLH1 promoter hypermethylation led to a loss of hMLH1 expression. The aforementioned facts suggest that hMLH1 hypermethylation does not occur merely by chance in MSI-L gastric carcinomas.
The possibility that MSI-L/Met+ cases represent an intermediate step in the MMR pathway has been advanced by Esteller et al (1999) regarding the occurrence of early endometrial carcinomas. This possibility does not fit with the results obtained in the present series: only two of the six MSI-L/Met+ cases were tumors restricted to the submucosa (early carcinomas), whereas the remaining four cases were advanced tumors. Because it is difficult to conceive that these MSI-L/Met+ advanced gastric carcinomas might be seen as cases not yet expressing a complete mutator phenotype, we would rather suggest that these tumors may have followed (an) unknown MMR-independent molecular pathway(s) of progression.
In biological terms we think that the distinction between MSI-L/Met+ gastric cancers and MSS tumors should be made because the aforementioned subset of MSI-L tumors share the same genetic alterations of MSI-H tumors: hypermethylation of the promoter region and absence of expression of hMLH1. For practical purposes, no benefit appears to be achieved by making a distinction between MSS and MSI-L tumors because they share the same clinicopathological profile. Our results show 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.
Materials and Methods
Patients, Tissue Samples, and DNA Extraction
In this study we analyzed 57 tumors selected from a series of 152 gastric carcinomas consecutively resected at Hospital of S. João (Porto, Portugal) from 1988 to 1997, previously analyzed for MSI phenotype and, partly, for mutations in target genes (Oliveira et al, 1998). We analyzed all cases with MSI-H or MSI-L phenotype (see below) after excluding 16 cases because of the lack of good high-quality DNA and 10 cases because of insufficient material. We also analyzed a control group of 17 cases with MSS phenotype selected at random from the cases of MSS with good technical conditions. Hematoxylin and eosin-stained sections were used to classify the tumors according to Lauren’s classification. The pathological staging was achieved using the unified 1987 tumor, node, metastasis (TNM) system for gastric carcinoma. Orcein-stained sections were used for the detection of vascular invasion.
MSI Assays
The 57 gastric carcinomas were previously studied for MSI using a panel of at least five dinucleotide repeat sequences, as described by Santos et al (1996), and using a primer set localized on intron 5 of the hMSH2 gene, which amplifies an adenine quasi monomorphic mononucleotide repeat, BAT26, as described by Zhou et al (1998) and Oliveira et al (1998).
Cases were classified as having an MSI-H phenotype whenever they presented a high frequency of MSI (≥ 40%) and BAT26+. Cases were considered as MSI-L whenever they showed instability in less than 40% of the markers used. MSS cases showed no instability at any of the markers used.
Promoter Methylation Assays
The promoter methylation is analyzed as initially described by Kane et al (1997). In short, DNA is digested by HpaII or MspI, methylation sensitive and insensitive enzymes, respectively, after which PCR is performed amplifying the target region. Absence or presence of PCR product indicates nonmethylated or methylated promoter region, respectively. Tumor DNA samples were digested over 48 hours at 37° C in 100 μl total volume reactions, containing 250 ng of genomic DNA of each sample, no enzyme, 100 units of HpaII or 150 units of MspI. Five normal mucosas (distal to the tumor) of the stomach were analyzed as controls. To analyze the cleavage of the hMLH1 promoter region, 1 μl of each digest was amplified by PCR in 30 μl reactions containing 3 μl of 10× PCR reaction buffer (Pharmacia Biotech, Piscataway, New Jersey), 0.5 units of Taq polymerase enzyme (Pharmacia Biotech), 0.25 μm of each of the four deoxynucleotide triphosphates, and 0.4 μm of each primer (5′-CGCTCGTA-GTATTCGTGC-3′ and 5′- TCAGTGCCTCGTGCTC-AC-3′), designed to amplify nucleotides −670 to −65 of hMLH1 (Genbank Accession No. U83845). Thirty-five cycles of PCR were performed using the following conditions: 94° C, 30 seconds; 55° C, 30 seconds; 72° C, 120 seconds. The resulting amplification products of two independent experiments were analyzed by 2% agarose gel electrophoresis using standard conditions.
Mutation Analysis of hMLH1
Mutational analysis of hMLH1 gene was performed by denaturing gradient gel electrophoresis (DGGE). Multiplex PCR was carried out on 500 ng of DNA in a total volume of 30 μl for 35 cycles as follows: denaturing step at 94° C for 3 minutes; 5 cycles consisting of denaturation at 94° C for 1 minute, annealing at 56° C for 1 minute, and elongation at 72° C for 1 minute; 5 cycles consisting of denaturation at 94° C for 1 minute, annealing at 53° C for 1 minute, and elongation at 72° C for 1 minute; and 25 cycles each of denaturation at 94° C for 1 minute, annealing at 50° C for 1 minute, and elongation at 72° C for 1 minute; one final elongation cycle was performed at 72° C for 5 minutes. The PCR mixture contained 1× Taq reaction buffer, 1 mm MgCl2, 0.5 unit of Taq polymerase, 0.75 mm of deoxyribonucleoside triphosphate (dNTP), and 15 pmol of each primer. The primers used for amplification of hMLH1 gene are listed in the literature (Wu et al, 1997), along with fragment sizes, melting temperatures, and PCR-annealing temperatures. To increase the amount of heteroduplex molecules, a heteroduplex step was performed after PCR amplification, ie, the samples were denatured for 10 minutes at 96° C followed by renaturation for 45 minutes at 50° C. PCR products were loaded on a polyacrylamide gel containing a denaturing gradient of urea/formamide (UF) (10% UF to 70% UF). Electrophoresis was performed overnight in TAE buffer at 59° C. Gels were stained with ethidium bromide and photographed under a UV transilluminator.
Expression Analysis of hMLH1
RT-PCR studies were performed using frozen material from 15 cases (10 MSI-H, 2 MSI-L, 3 MSS). Total RNA was extracted using standard methods. First-strand synthesis was made by random 6-mer priming using M-MLV reverse transcriptase (Boehringer Mannheim, Lewes, United Kingdom) at 37° for 60 minutes. hMLH1 mRNA expression level analysis was performed by co-amplification of the target gene (hMLH1) and of the housekeeping gene GAPDH.
Amplification of the Target Genes (TGFβRII, IGFIIR, BAX, and TCF4)
Detection of mutations in target genes was performed in 50 cases (27 MSI-H, 8 MSI-L, and 15 MSS) in which constitutional and tumor DNA was available. Of the 50 cases analyzed for TGFβRII, IGFIIR, and BAX, 34 had been analyzed previously by Oliveira et al (1998). The repeat sequence (A)9 in the putative exon 10 of TCF4 could only be analyzed in 48 cases as described by Duval et al (1999). PCR products were analyzed for mutations in 6% single mutation detection enhancement/strand conformation polymorphism analysis (MDE/SSCP) gels. The results were confirmed with radioactive PCR, and the products were run in a 10% denaturing PAA sequencing gel. The tumors were classified as harboring a mutation whenever they showed band shifts relative to the normal tissue.
Statistical Analysis
The statistical analysis of the results was performed using the χ2 test with Yates correction, Fisher’s exact test, and Student’s t test. The parameters used for this analysis are listed in Tables 1 and 2. A p value less than 0.05 was considered statistically significant.
Accession codes
References
Borresen AL, Lothe RA, Meling GI, Lystad S, Morrison P, Lipford J, Kane MF, Rognum TO, and Kolodner RD (1995). Somatic mutations in the hMSH2 gene in microsatellite unstable colorectal carcinomas. Hum Mol Genet 4: 2065–2072.
Bubb VJ, Curtis LJ, Cunningham C, Dunlop MG, Carothers AD, Morris RG, White S, Bird CC, and Wyllie AH (1996). Microsatellite instability and the role of hMSH2 in sporadic colorectal cancer. Oncogene 12: 2641–2649.
Costello JF, Fruhwald MC, Smiraglia DJ, Rush LJ, Robertson GP, Gao X, Wright FA, Feramisco JD, Peltomaki P, Lang JC, Schuller DE, Yu L, Bloomfield CD, Caligiuri MA, Yates A, Nishikawa R, Su Huang H, Petrelli NJ, Zhang X, O’Dorisio MS, Held WA, Cavenee WK, and Plass C (2000). Aberrant CpG-island methylation has non-random and tumor-type-specific patterns. Nat Genet 24: 132–138.
Cunningham JM, Christensen ER, Tester DJ, Kim CY, Roche PC, Burgart LJ, and Thibodeau SN (1998). Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res 58: 3455–3460.
Deng G, Chen A, Hong J, Chae HS, and Kim YS (1999). Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res 59: 2029–2033.
Duval A, Gayet J, Zhou XP, Iacopetta B, Thomas G, and Hamelin R (1999). Frequent frameshift mutations of the TCF-4 gene in colorectal cancers with microsatellite instability. Cancer Res 59: 4213–4215.
Esteller M, Levine R, Baylin SB, Ellenson LH, and Herman JG (1998). MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas. Oncogene 17: 2413–2417.
Esteller M, Catasus L, Matias-Guiu X, Mutter GL, Prat J, Baylin SB, and Herman JG (1999). hMLH1 promoter hypermethylation is an early event in human endometrial tumorigenesis. Am J Pathol 155: 1767–1772.
Fleisher 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, and Meltzer SJ (1999). Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability. Cancer Res 59: 1090–1095.
Ghimenti C, Tannergard P, Wahlberg S, Liu T, Giulianotti PG, Mosca F, Fornaciari G, Bevilacqua G, Lindblom A, and Caligo MA (1999). Microsatellite instability and mismatch repair gene inactivation in sporadic pancreatic and colon tumors. Br J Cancer 80: 11–16.
Halling KC, Harper J, Moskaluk CA, Thibodeau SN, Petroni GR, Yustein AS, Tosi P, Minacci C, Roviello F, Piva P, Hamilton SR, Jackson CE, and Powell SM (1999). Origin of microsatellite instability in gastric cancer. Am J Pathol 155: 205–211.
Ionov Y, Peinado MA, Malkhosyan S, Shibata D, and Perucho M (1993). Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363: 558–561.
Jones PA (1999). The DNA methylation paradox. Trends Genet 15: 34–37.
Kang GH, Shim YH, and Ro JY (1999). Correlation of methylation of the hMLH1 promoter with lack of expression of hMLH1 in sporadic gastric carcinomas with replication error. Lab Invest 79: 903–909.
Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, Jessup JM, and Kolodner R (1997). Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res 57: 808–811.
Katabuchi H, van Rees B, Lambers AR, Ronnett BM, Blazes MS, Leach FS, Cho KR, and Hedrick L (1995). Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas. Cancer Res 55: 5556–5560.
Kowalski LD, Mutch DG, Herzog TJ, Rader JS, and Goodfellow PJ (1997). Mutational analysis of hMLH1 and hMSH2 in 25 prospectively acquired RER+ endometrial cancers. Genes Chrom Cancer 18: 219–227.
Leung SY, Yuen ST, Chung LP, Chu KM, Chan AS, and Ho JC (1999). hMLH1 promoter methylation and lack of hMLH1 expression in sporadic gastric carcinomas with high-frequency microsatellite instability. Cancer Res 59: 159–164.
Liu B, Nicolaides NC, Markowitz S, Willson JK, Parsons RE, Jen J, Papadopoulos N, Peltomaki P, de la Chapelle A, Hamilton SR, Kinzler KW, and Vogelstein B (1995). Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability. Nat Genet 9: 48–55.
Moslein G, Tester DJ, Lindor NM, Honchel R, Cunningham JM, French AJ, Halling KC, Schwab M, Goretzki P, and Thibodeau SN (1996). Microsatellite instability and mutation analysis of hMSH2 and hMLH1 in patients with sporadic familial and hereditary colorectal cancer. Hum Mol Genet 5: 1245–1252.
Oliveira C, Seruca R, Seixas M, and Sobrinho-Simões M (1998). The clinicopathological features of gastric carcinomas with microsatellite instability may be mediated by mutations of different “target genes.” A study of the TGFβRII, IGFIIR, and BAX genes. Am J Pathol 153: 1211–1219.
Santos NR, Seruca R, Constância M, Seixas M, and Sobrinho-Simões M (1996). Microsatellite instability at multiple loci in gastric carcinoma: Clinicopathological implications and prognosis. Gastroenterology 110: 38–44.
Simpkins SB, Bocker T, Swisher EM, Mutch DG, Gersell DJ, Kovatich AJ, Palazzo JP, Fishel R, and Goodfellow PJ (1999). MLH1 promoter methylation and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers. Hum Mol Genet 8: 661–666.
Suzuki H, Itoh F, Toyota M, Kikuchi T, Kakiuchi H, Hinoda Y, and Imai K (1999). Distinct methylation pattern and microsatellite instability in sporadic gastric cancer. Int J Cancer 83: 309–313.
Thibodeau SN, French AJ, Roche PC, Cunningham JM, Tester DJ, Lindor NM, Moslein G, Baker SM, Liskay RM, Burgart LJ, Honchel R, and Halling KC (1996). Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes. Cancer Res 56: 4836–4840.
Toyota M, Ahuja N, Suzuki H, Itoh F, Ohe-Toyota M, Imai K, Baylin S B, and Issa JP (1999). Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype. Cancer Res 59: 5438–5442.
Veigl ML, Kasturi L, Olechnowicz J, Ma AH, Lutterbaugh JD, Periyasamy S, Li GM, Drummond J, Modrich PL, Sedwick WD, and Markowitz SD (1998). Biallelic inactivation of hMLH1 by epigenetic gene silencing a novel mechanism causing human MSI cancers. Proc Natl Acad Sci USA 95: 8698–8702.
Wu MS, Lee CW, Shun CT, Wang HP, Lee WJ, Chang MC, Sheu JC, and Lin JT (2000). Distinct clinicopathological and genetic profiles in sporadic gastric cancer with different mutator phenotypes. Genes Chrom Cancer 27: 403–411.
Wu Y, Berends MJ, Mensink RG, Kempinga C, Sijmons RH, van Der Zee AG, Hollema H, Kleibeuker JH, Buys CH, and Hofstra RM (1999). Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations. Am J Hum Genet 65: 1291–1298.
Wu Y, Nystrom Lathi M, Osinga J, Looman MW, Peltomaki P, Aaltonen LA, de la Chapelle A, Hofstra RM, and Buys CH (1997). MSH2 and MLH1 mutations in sporadic replication error-positive colorectal carcinoma as assessed by two-dimensional DNA electrophoresis. Genes Chrom Cancer 18: 269–278.
Yamamoto H, Perez-Piteira J, Yoshida T, Terada M, Itoh F, Imai K, and Perucho M (1999). Gastric Cancers of the microsatellite mutator phenotype display characteristic genetic and clinical features. Gastroenterology 116: 1348–1357.
Zhou XP, Hoang JM, Li YJ, Seruca R, Carneiro F, Sobrinho-Simoes M, Lothe RA, Gleeson CM, Russell SE, Muzeau F, Flejou JF, Hoang-Xuan K, Lidereau R, Thomas G, and Hamelin R (1998). Determination of the replication error phenotype in human tumors without the requirement for matching normal DNA by analysis of mononucleotide repeat microsatellites. Genes Chrom Cancer 21: 101–107.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Pinto, M., Oliveira, C., Machado, J. et al. MSI-L Gastric Carcinomas Share the hMLH1 Methylation Status of MSI-H Carcinomas but Not Their Clinicopathological Profile. Lab Invest 80, 1915–1923 (2000). https://doi.org/10.1038/labinvest.3780201
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1038/labinvest.3780201
This article is cited by
-
Gastric biomarkers: a global review
World Journal of Surgical Oncology (2016)
-
Low-Frequency Microsatellite Instability in Genomic Di-Nucleotide Sequences Correlates with Lymphatic Invasion and Poor Prognosis in Gastric Cancer
Cell Biochemistry and Biophysics (2015)
-
Biomarkers for gastric cancer: prognostic, predictive or targets of therapy?
Virchows Archiv (2014)
-
Clinical aspect and molecular mechanism of DNA aneuploidy in gastric cancers
Journal of Gastroenterology (2012)
-
Alterations of DNA mismatch repair proteins and microsatellite instability levels in gastric cancer cell lines
Laboratory Investigation (2004)