hMLH1 and hMSH2 expression in human hepatocellular carcinoma

The role of microsatellite instability (MSI) in the pathogenesis of hepatocellular carcinoma (HCC) is incompletely defined. Although high-frequency MSI (MSI-H) is infrequently seen in HCC, some studies have suggested a role for MSI in HCC development. While MSI has been clearly defined for a subset of tumors, in particular colorectal, gastric and endometrial cancers, generally accepted criteria have not been developed for other tumors. Colorectal cancers (CRC) are classified as MSI-H if >30-40% of >5 microsatellite loci analyzed show instability. The MSI-H phenotype is associated with defective DNA mismatch repair (MMR) and is observed in the majority of tumors from patients with hereditary non-polyposis colon cancer (HNPCC) and also in 15% of sporadic CRCs. Inactivating mutations of the hMLH1 or hMSH2 genes lead to defects in MMR in HNPCC. In sporadic CRCs, MMR is usually due to hypermethylation of the hMLH-1 promoter. The role of defective MMR in hepatocellular carcinogenesis is controversial. Immunohistochemistry for hMLH1 and hMSH2 reliably indicates hMLH1 or hMSH2 loss in MSI-H CRC tumors. To investigate the role of defective MMR in HCC carcinogenesis, we performed immunohistochemistry for hMLH1 and hMSH2 on 36 HCCs. BAT26, a microsatellite marker that reliably predicts MSI-H was also examined. All 36 of the tumors stained positively for both hMLH1 and hMSH2, strongly suggesting an absence of either inactivating mutations of hMLH1 and hMSH2 or promoter hypermethylation of hMLH1. None of the tumors showed MSI at the BAT26 locus. These findings suggest that defective MMR does not contribute significantly to hepatocellular carcinogenesis.     

Immunohistochemical analysis of expression and allelotype of mismatch repair genes (hMLH1 and hMSH2) in bladder cancer

Mutation of human homologues of DNA mismatch repair (MMR) genes in tumours has been shown to be associated with the phenomenon of microsatellite instability (MSI). Several studies have reported the occurrence of MSI in bladder cancer, but evidence of involvement of MMR genes in the pathogenesis of this cancer is still unclear. We therefore utilized quantitative immunohistochemical (IHC) image analysis and PCR-based allelotype analysis to determine hMLH1 and hMSH2 genes alteration in a cohort of Egyptian bladder cancer samples. IHC analysis of 24 TCC and 12 SCC revealed marked- intra and intertumour heterogeneity in the levels of expression of the two MMR proteins. One TCC lost MLH1 expression and one lost MSH2, (1/24, 4%), and one SCC lost MSH2 (1/12, 8%). A large proportion of analysed tumours revealed a percentage positivity of less than 50% for MLH1 and MSH2 expression (44% and 69%, respectively). Complete loss of heterozygosity in three dinucleotide repeats lying within, or in close proximity to, hMLH1 and hMSH2 was rare (2/57, (4%) for MLH1; and 1/55, (2%) for MSH2), however allelic imbalance was detected in 11/57 (hMLH1) and 10/55 (hMSH2) at any of the informative microsatellite loci. These alterations in structure and expression of DNA MMR genes suggest their possible involvement in the tumorigenesis and/or progression of bladder cancer.     

BAT-26 microsatellite instability does not correlate with the loss of hMLH1 and hMSH2 protein expression in sporadic endometrial cancers

Microsatellite instability (MSI) is detected in about 20-25% of endometrial cancers (ECs). Incidence of this alteration correlates with lack of expression of certain mismatch repair genes such as hMLH1 and hMSH2. Although assessment of several markers has been proposed for identification of microsatellite unstable tumours, BAT-26, a mononucleotide microsatellite repeat, has been shown to be highly efficient when used as a single marker. The aim of the study was to evaluate instability within BAT-26 and expression of hMLH1 and hMSH2 proteins in sporadic endometrial cancer as well as to correlate these findings with histopathologic and clinical characteristics of tumours. Samples of 88 (74 endometrioid and 14 non-endometrioid) ECs were investigated for instability within BAT-26 by means of PCR and expression of hMLH1 and hMSH2 proteins using immunohistochemistry. BAT-26 MIS was discovered in 23.9% of endometrial cancers. Incidence of MSI did not correlated with grade, stage or depth of invasion. BAT-26 MSI was more frequent in non-endometrioid compared to endometrioid tumours (35.7% vs. 21.6%, respectively), but the difference was not statistically significant. Lack of hMLH1 and hMSH2 protein expression was detected in 21.6 and 15.9% of ECs, respectively, and did not correlate with clinicopathologic features of tumours. Loss of both hMLH1 and hMSH2 protein expression was similar in BAT-26 stable and unstable cancers. All cases of non-endometrioid tumours with BAT-26 MSI were positive for hMLH1. We can conclude that BAT-26 used alone may not be a reliable marker for identification of sporadic ECs with microsatellite instability induced by deficient expression of hMLH1 and hMSH2.     

hMLHl and hMSH2 somatic inactivation mechanisms in sporadic colorectal cancer patients

Much is known about the role of germline inactivation in mismatch repair (MMR) genes in hereditary non-polyposis colorectal cancer (HNPCC), but the impact of somatic MMR gene changes on sporadic colorectal cancer remains to be elucidated. In hereditary cases the hMLHl and hMSH2 genes were shown to have a great importance, and in order to examine the somatic inactivation mechanisms of the two MMR genes hMLHl and hMSH2 we screened 37 Hungarian sporadic colorectal cancer patients for allelic imbalance (AI), microsatellite instability (MSI), hMLHl promoter hypermethylation and somatic mutations. Thirteen of the examined tumours (35%) were characterized by low-level MSI and none of the cases belonged to the high MSI group. Nine (24%) and seven (19%) cases had AI at the hMLHl and hMSH2 genes, respectively. Seven tumours (19%) showed dense promoter hypermethylation of hMLHl, but only two patients had somatic mutations, one for each MMR gene. According to our study on this limited set of cases the most prominent mismatch repair inactivation mechanism in sporadic colorectal cancer patients is the hMLHl promoter hypermethylation which may have a role in the carcinogenesis of sporadic colorectal cancer.     

Microsatellite instability and hMLH1 and hMSH2 expression analysis in soft tissue sarcomas

Alterations of the size of microsatellite DNA sequences, namely microsatellite instability (MSI), have been demonstrated in some types of malignancies. We analyzed the MSI of five microsatellite markers in 40 cases of soft tissue sarcoma (STS) using high resolution fluorescent microsatellite analysis. In addition, we examined the expression of hMLH1 and hMSH2 proteins of DNA mismatch repair (MMR) genes by immunohistochemistry, and promoter methylation of the hMLH1 gene by methylation-specific PCR (MSP). MSI was recognized in 10 of 40 STS cases (25%), which consisted of 2 MSH-high (MSI-H) tumors and 8 MSI-low (MSI-L) tumors. A loss of hMLH1 expression was recognized in 7 of 40 STS cases (18%), and loss of hMSH2 expression was recognized in 3 of 40 STS cases (8%). One case showed a loss of both hMLH1 and hMSH2 expression. Promoter hypermethylation of the hMLH1 gene was detected in only 3 of 40 STS cases (8%). Of 10 cases with MSI, 5 (50%) showed a loss of hMLH1 and/or hMSH2 expression. There was a statistically significant correlation between MSI-positive tumors and the loss of hMLH1 and/or hMSH2 expression (p=0.0286). Although the frequency of MSI (25%) or a loss of hMLH1 and/or hMSH2 expression (23%) was relatively low in STS cases, a loss of hMLH1 and/or hMSH2 was recognized in 5 out of 10 MSI-positive cases (50%). These findings suggest that the inactivation of MMR gene expression might be the cause of MSI in STS cases.     

Differential expression of hMLH1 and hMSH2 is related to bladder cancer grade,stage and prognosis but not microsatellite instability

Defects in the DNA mismatch repair proteins result in microsatellite instability and malignancy in hereditary non-polyposis colorectal carcinoma (HNPCC). However, the role of mismatch repair (MMR) proteins and microsatellite instability (MSI) in transitional cell carcinoma of the bladder is less clear. In our study, the expression of 2 MMR proteins and the frequency of MSI in Transitional cell carcinoma of the bladder (TCC) were investigated. One hundred eleven patients with TCC of the bladder were studied, with complete clinicopathological data (median follow up of 5 years, range 5-16 years). Immunohistochemistry was used to detect the expression levels of hMLH1 and hMSH2. Microsatellite analysis for 14 loci (10 loci from the Bethesda consensus panel and the repeats in the TGFbetaR2, BAX, hMSH3 and hMSH6 genes) was performed on 84 tumors. Reduced expression of either MMR protein was seen in 26 of 111 tumors (23%). Reduced expression was seen more commonly in muscle invasive (p<0.03) and high grade TCC (p<0.03) than in superficial, low grade tumors. By 5 years, reduced expression of either MMR protein was associated with fewer recurrences of superficial tumors (p=0.015) and fewer relapses in all tumors (p=0.03), compared to tumors with normal expression. Nine tumors had reduced expression of both MMR proteins, analysis which suggests a synergistic reduction in expression (p=0.001). MMR expression was related to patient age, younger patients being more likely to have reduced MMR expression than older patients (p<0.01). MSI was seen at multiple loci in 1 tumor (1%) and at a single locus in 6 tumors (7%). MSI was not associated with MMR expression. Our findings indicate that reduced expression of the MMR proteins may have an important contribution in the development of a subset of TCCs and suggest a potential role for MMR expression as prognostic indicators.     

Mutations of hMLH1 and hMSH2 in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with microsatellite instability and abnormalities of mismatch repair protein expression.

PURPOSE: The relationship between germ-line mutations of hMSH2 and hMLH1, microsatellite instability (MSI), and loss of DNA mismatch repair (MMR) gene expression were studied to formulate an effective selection protocol for patients with suspected hereditary nonpolyposis colorectal cancer who should be offered genetic testing. PATIENTS AND METHODS: Patients eligible for germ-line analysis of hMLH1 and hMSH2 were selected. Tumor specimens were obtained to assess MSI and loss of MMR gene expression. RESULTS: Among 37 patients who participated in the study, two hMSH2 and two hMLH1 missense mutations (11%) were detected, none of which was found in a panel of 60 healthy volunteers. High MSI was found in five tumors (19%) and low MSI in 10 tumors (39%); 12 tumors (46%) were microsatellite stable. Four tumors demonstrated loss of hMLH1, and three tumors demonstrated loss of hMSH2 protein expression. CONCLUSION: No relationship was found between MMR gene mutations and MSI; low or no MSI was found in the four patients with germ-line mutations, and none of the five patients with high MSI demonstrated abnormalities of MMR genes. On the contrary, loss of hMLH1 or hMSH2 expression was found in the tumors from three of the four patients demonstrating germ-line mutations. These data suggest that germ-line mutations of the MMR gene can occur in people with MSI-negative tumors. Sensitive clinical criteria and the study of MMR gene expression may be useful to identify this subset of patients.     

Oncogenic pathway of sporadic colorectal cancer with novel germline missense mutations in the hMSH2 gene

The deficiency of the DNA mismatch repair (MMR) system is involved in tumorigenesis of either familial or sporadic colorectal cancers showing microsatellite instability (MSI). To investigate the involvement of the mutated hMSH2 gene in carcinogenesis, we searched for alteration of the gene in 15 MSI tumors of Japanese patients with sporadic colorectal cancer by a polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and DNA sequencing analyses. We found 20 alterations including 7 novel mutations, 6 germline and one somatic. To assume an oncogenic pathway of tumor of two patients carrying germline missense mutations, G40S located in an evolutionarily conserved amino-terminal motif and Y619C in a domain interacting with either hMSH3 or hMSH6, somatic mutations in 9 target genes of the MMR defect and in the p53 and K-ras genes and loss of heterozygosity (LOH) at the hMLH1 and p53 gene loci were then studied. In the tumor carrying G40S, other somatic hMSH2 mutations, G203R and 687delA in the (A)(7) repeat, and 5 one-bp deletions in the target genes were found, while no mutation in the p53 and K-ras genes. These results indicate that G40S may affect the hMSH2 function and the tumor may be developed by a typical MSI pathway. In another tumor with Y619C, LOH at the hMLH1 gene locus, no mutation in MMR target genes, and two-hit inactivation of the p53 gene were detected. This MSI tumor seems to be developed by another than MSI pathway. These results indicate that there are different oncogenic pathways in the MSI sporadic colorectal cancers with germline missense mutations in the hMSH2 gene. We conclude that familial colorectal cancer-suspected cases exist in a small population of sporadic colorectal cancers.     

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

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

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

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

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

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

Microsatellite instability and hMLH1/hMSH2 expression in Barrett esophagus-associated adenocarcinoma

BACKGROUND: Microsatellite instability (MSI) has been documented in malignancies associated with hereditary nonpolyposis colon carcinoma and in sporadic malignancies of the colon, stomach, and endometrium. In these malignancies, MSI is associated with defects in the DNA mismatch repair enzymes hMSH2 and hMLH1. Defects in these enzymes result in a phenotype characterized by instability of multiple microsatellite repeat sequences throughout the genome. This study sought to determine the prevalence of MSI in 80 primary Barrett esophagus-associated adenocarcinomas (BEAd) and to examine the relation of MSI with the clinical and pathologic features of the tumors. METHODS: Eighty BEAd were evaluated for the presence of MSI by using the microsatellite markers BAT25, BAT26, D10S219, D10S541, and D10S551. These tumors also were evaluated for immunohistochemical expression of hMSH2 and hMLH1. RESULTS: High levels of MSI were not found in any of the tumors examined. Furthermore, immunohistochemical expression of hMSH2 and hMLH1 was retained in all cases evaluated. Evidence of low level MSI was found in 16% of tumors. In none of these tumors, however, was MSI present in more than two of five loci. The presence of MSI did not correlate with patient age, tumor stage, degree of differentiation, or with patient survival. CONCLUSIONS: High level MSI and loss of hMLH1/hMSH2 expression is uncommon in BEAd. A subset of BEAd demonstrate low level MSI. The presence of low level MSI was not associated with the clinicopathologic features of the tumors examined.     

Allele loss occurs frequently at hMLH1, but rarely at hMSH2, in sporadic colorectal cancers with microsatellite instability.

Mutations at the hMSH2 and hMLH1 mismatch repair loci have been implicated in the pathogenesis of colorectal cancer. Tumours with two allelic mutations at a mismatch repair locus develop replication errors (RERs). In the hereditary non-polyposis colorectal cancer (HNPCC) syndrome, one mutation is inherited and the other acquired somatically: in RER+ sporadic colorectal cancers, both mutations are somatic. RER+ tumours tend to have a low frequency of allele loss, presumably because they acquire most mutations through RERs. However, before a second mismatch repair mutation has occurred somatically, there is no reason to suppose that allele loss occurs less frequently in tumours that are to become RER+. Indeed, this second mutation might itself occur by allele loss. We have searched for allele loss at the hMSH2 and hMLH1 loci in RER+ and RER- sporadic colorectal cancers. Loss occurred at the hMLH1 locus in 7/17 (41%) RER+ tumours, compared with 6/40 (15%) RER- cancers (chi2=3.82, P approximately 0.05). At hMSH2, 2/22 RER+ sporadic cancers (9%) had lost an allele, compared with 2/40 (5%) RER- cancers (chi2=0.03, P>0.5). Taken together with previous studies which focused on colorectal cancers from HNPCC families, the data suggest that allele loss at hMLH1, but not at hMSH2, contributes to defective mismatch repair in inherited and sporadic colorectal cancer.     

Genetic and epigenetic modification of mismatch repair genes hMSH2 and hMLH1 in sporadic breast cancer with microsatellite instability

Breast cancer is the most common cancer in women, but its pathogenesis is still unclear. Microsatellite instability (MSI) has been identified in breast cancer cells, suggesting an association with mismatch repair defects. To test this hypothesis, we investigated MSI, protein expression of hMSH2 and hMLH1, as well as genetic and epigenetic modifications of these two genes in 32 sporadic breast tumors. MSI was identified in 15 cases. Immunohistochemistry analysis revealed that all MSI cases but one had lower than normal expression of hMSH2 (nine cases), hMLH1 (12 cases), or both (seven cases). In tumors with MSI, both genetic and epigenetic modifications of these mismatch repair genes were also identified. Eight cases harbored mutations or polymorphisms in hMSH2 and hMLH1, and 10 exhibited hypermethylation in the promoter region of hMLH1. These results suggest that both genetic and epigenetic alterations of hMSH2 and especially of hMLH1 contribute to genomic instability and tumorigenesis in sporadic breast cancer.     

Not hMSH2 but hMLH1 is frequently silenced by hypermethylation in endometrial cancer but rarely silenced in pancreatic cancer with microsatellite instability

Disruption of the DNA mismatch repair (MMR) system has been found to play an important role in sporadic human cancers of several organs such as colorectum, stomach, endometrium, and pancreas. In cancers of the former three organs, disruption of the MMR system is mainly caused by hypermethylation of the hMLH1 gene. We investigated the expression of the hMLH1 and hMSH2 proteins immunohistochemically in pancreatic and endometrial cancers with high frequency microsatellite instability (MSI-H). Loss of expression of hMLH1 was found in none of seven pancreatic cancer, whereas eight (57%) of 14 endometrial cancer showed loss of expression of hMLH1. On the other hand, one (14%) of seven pancreatic cancers and two (14%) of 14 endometrial cancers showed loss of hMSH2 expression. We further analyzed the methylation status at the promoter region of the hMLH1 and hMSH2 genes and found hypermethylation of hMLH1 at the promoter region in the great majority of endometrial cancers with loss of expression. However, no pancreatic cancer showed hypermethylation. We then further analyzed 22 pancreatic cancer cell lines and obtained similar results. These results suggested that MSI-H in pancreatic cancer is probably caused by different mechanisms from those of other sporadic cancers with MSI-H.     

Thymidylate synthase expression in colon carcinomas with microsatellite instability.

PURPOSE: Colon cancer cells with high-frequency microsatellite instability (MSI-H) display resistance to 5-fluorouracil (5-FU) that can be reversed by restoring DNA mismatch repair (MMR) proficiency. Given that thymidylate synthase (TS) is inhibited by 5-FU, we studied the relationship between MSI and TS expression, and the prognostic effect of these and other markers (i.e., p53 and 17p allelic imbalance). EXPERIMENTAL DESIGN: Dukes' stage B2 and C colon carcinomas (n = 320) from participants in 5-FU-based adjuvant therapy trials were analyzed for MSI and 17p allelic imbalance. Expression of MMR (hMLH1, hMSH2), TS, and p53 proteins were analyzed by immunohistochemistry. Correlations between markers and associations with overall survival were determined. RESULTS: Of 320 cancers studied, 60 (19%) were MSI-H. TS expression variables were similar in MSI-H and microsatellite stable/low-frequency MSI (MSS/MSI-L) cancers, and unrelated to MMR proteins. MSI-H tumors had lower stage (P = 0.0007), fewer metastatic lymph nodes (P = 0.004), and improved overall survival (P = 0.01). Loss of MMR proteins was also associated with better overall survival (P = 0.006). None of the TS variables were prognostic. Histologic grade (P = 0.0008) and nodal status (P = 0.0002) were associated with overall survival, in contrast to 17p allelic imbalance or p53. Only MSI status or loss of MMR proteins, histologic grade, and tumor stage were independent markers for overall survival. CONCLUSIONS: MSI-H tumors show earlier stage at presentation and better stage-adjusted survival rates. MSI status and TS expression were unrelated and TS was not prognostic, suggesting that TS levels cannot explain therapeutic resistance to 5-FU reported in MSI-H colon cancers.     

Combined deficiency of hMLH1, hMSH2, hMSH3 and hMSH6 is an independent prognostic factor in colorectal cancer

We examined biological and clinicopathological significance of individual and combined hMLH1, hMSH2, hMSH3 and hMSH6 expression with immunohistochemistry in 301 unselected colorectal cancers. Weak hMLH1 expression was correlated to microsatellite instability (P=0.04), negative p53 expression (P=0.005) and mucinous carcinomas (P=0.02). Weak hMSH2 expression was related to negative ras (P<0.001) and p53 expression (P=0.005), and better survival (P=0.03). hMSH2, hMSH3 and hMSH6, as well as hMLH1, hMSH2, hMSH3 and hMSH6, were combined into a 'functional' and a 'less-functional' group, respectively. Both 'less-functional' groups were/tended to be associated with microsatellite instability, negative ras and p53 expression, and better survival. In summary, hMLH1 and hMSH2 were more important when investigated individually, and the combined groups were more related to the mutator pathway, suggesting that combined deficiencies of the proteins are more efficiently involved in the mutator pathway. Our result from weak versus strong staining may suggest that the intensity of staining should be considered in future studies on mismatch repair proteins.