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(American Journal of Pathology. 2000;156:1773-1779.)
© 2000 American Society for Investigative Pathology

Regular Articles

Genetic and Epigenetic Modification of MLH1 Accounts for a Major Share of Microsatellite-Unstable Colorectal Cancers

Shannon A. Kuismanen^*, Mari T. Holmberg^*, Reijo Salovaara^*, Albert de la Chapelle and Päivi Peltomäki

From the Departments of Medical Genetics^*
and Pathology,
Haartman Institute, University of Helsinki, Helsinki, Finland; and the Division of Human Cancer Genetics,
Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Microsatellite instability (MSI) is a hallmark of hereditary nonpolyposis colorectal cancer, and in these patients, results from inherited defects in DNA mismatch repair genes, mostly MSH2 and MLH1. MSI also occurs in 15% of sporadic colorectal cancers, but in these tumors, its basis is less well characterized. We investigated 46 sporadic MSI+ colorectal cancers for changes in MSH2 and MLH1 protein expression, followed by the analysis of somatic mutation, loss of heterozygosity (LOH), and promoter hypermethylation as possible underlying defects. Most cases (36/46, 78%) showed lost or reduced MLH1 expression. Among these, a majority (83%) was associated with MLH1 promoter hypermethylation, whereas the rates of LOH and somatic mutation of MLH1 were 24% and 13%, respectively. Hypermethylation and LOH were inversely correlated, suggesting that they had alternative functions in the inactivation of MLH1. MSH2 expression was lost in 7/46 (15%), and of these, 2 (29%) showed LOH and/or somatic mutation of MSH2. We conclude that most sporadic MSI+ colorectal cancers have an MLH1-associated etiology and that epigenetic modification is a major mechanism of MLH1 inactivation. Moreover, we found a significantly lower prevalence for MLH1 promoter hypermethylation in hereditary nonpolyposis colorectal cancer tumors with MLH1 germline mutations (12/26, 46%), which might explain some differences that are known to occur in the clinicopathological characteristics and tumorigenic pathways between sporadic and hereditary MSI+ colorectal cancers.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

Many previous studies have typically been restricted to only one or two possible mechanisms of DNA mismatch repair gene inactivation at a time and have used unselected series representing a mixture of hereditary and sporadic tumors. These drawbacks have made it difficult to obtain a comprehensive overview of the relative contributions of different mechanisms leading to MSI. The present investigation focused on 46 sporadic MSI+ colorectal cancers in which MSH2 and MLH1 germline mutations had been excluded by direct sequencing. A sequential approach was applied to investigate these tumors for MSH2 and MLH1 alterations including loss of protein expression, somatic mutation, LOH, and promoter hypermethylation. A cohort of MSI+ colorectal tumors from HNPCC patients was studied for comparison. We demonstrate that the MSH2 versus MLH1 genes as well as sporadic versus hereditary cases are differently involved in the inactivation by these different mechanisms, providing important insights into the basis of MSI in colorectal cancer.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Patients and Samples

Paired fresh-frozen or paraffin-derived normal colonic mucosa and colorectal tumor samples from sporadic cases were investigated, representing a collection of 509 consecutive tumors previously analyzed for MSI.¹¹ Among these were tumors that had shown high-degree MSI (MSI-H) but no germline mutations of the MSH2 or MLH1 genes by direct sequencing (n = 51). In addition, tumor and normal tissue pairs available from HNPCC patients with inherited mutations in DNA mismatch repair genes (MLH1 mutation in 26 cases and MSH2 mutation in one case^6,12,13 ) were investigated. All patients gave informed consent before sample collection, in accordance with institutional guidelines.

Immunohistochemical Analysis

Four-micrometer sections from paraffin blocks were mounted on 3-aminopropyl-triethoxy-silane (Sigma, St. Louis, MO) coated slides and dried at 37°C. The sections were deparaffinized in xylene and rehydrated through a graded alcohol series to distilled water. Antigen retrieval was performed in 0.01-mol/L citrate buffer heated in a microwave oven at high power four times for 5 minutes each. The samples were then cooled to room temperature and washed in phosphate-buffered saline. For immunohistochemical analysis, avidin-biotin-conjugated immunoperoxidase technique was applied, using a commercial Elite ABC kit (Vectastain, Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity was blocked by the incubation of the slides in hydrogen peroxide and methanol. Nonspecific antibody binding was quenched by incubation of the sections with nonimmune horse serum. Primary antibody was incubated with the sections overnight, followed by incubation in biotinylated secondary antibody and peroxidase-labeled avidin-biotin complex for 30 minutes. All antibody dilutions were in phosphate-buffered saline, pH 7.2, and incubations were carried out in humid chambers at room temperature. The following monoclonal primary antibodies were used. For MLH1, we used clone G168–728 from PharMingen (San Diego, CA), raised against full-length human MLH1 protein. For MSH2, we used clone G219–1129 from PharMingen, raised against full-length human MSH2 protein, as well as clone FE 11 from Oncogene Sciences (Uniondale, NY), raised against the COOH-terminal fragment of human MSH2 protein. Staining results were visualized by incubating the sections in 3-amino-9-ethylcarbamazole solution (Sigma) for 15 minutes at room temperature. Sections were counterstained in Mayer’s hematoxylin, rinsed in water, and mounted in aqueous media (Aquamount, BDH, Poole, UK) for microscopic evaluation and photography.

Methylation Analysis

A polymerase chain reaction-based assay was applied that relies on the inability of the HpaII restriction enzyme to cut CCGG sequences with the internal cytosine methylated. Four HpaII sites contained at the MLH1 promoter region were studied using the primers and conditions described in Kuismanen et al.¹⁰

LOH Analysis

Three microsatellite markers from the MLH1 region were used that showed the highest rates of deletions in a previous study.² Marker D3S1611 is located in intron 12 of MLH1 (our unpublished data), whereas D3S1029 and D3S1283 flank the gene on either side at 5-cM distance. For MSH2, D2S391 was used, flanked by D2S2259 and D2S123, together encompassing a region of 9 cM around MSH2 (ftp://ftp.genethon.fr). LOH at 1–3 loci was recorded as LOH, whereas constitutional homozygosity and/or MSI at all three loci were interpreted as an uninformative result. In some cases, additional LOH data were obtained by denaturant gradient gel electrophoresis, taking advantage of intragenic point mutations and sequence polymorphisms.

Mutation Analysis

All cases included in the present study had been screened for germline mutations of MSH2 and MLH1.^6,11-13 Thirty-one sporadic colorectal cancer samples and two HNPCC cases were screened for somatic mutations in MSH2 and MLH1 by two-dimensional DNA electrophoresis, as described in Wu et al.⁶

Statistical Analysis

Fisher’s exact test (two-tailed) was used to test statistical significance for the observed differences between groups.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Basis of MSI in Sporadic Colorectal Tumors

Fifty-one MSI+ colorectal tumors that showed no germline mutation in the two major HNPCC-associated genes, MSH2 and MLH1, were subjected to immunohistochemical analysis of the MSH2 and MLH1 proteins, and the results were fully interpretable in 46 cases (Table 1 and Figure 1 ). All but 9 (80%) showed the involvement of either MLH1 or MSH2 or both. The predominant involvement of MLH1 was suggested by the fact that most cases (36/46, 78%) were associated with lost or reduced MLH1 expression, and only 7 (15%) showed decreased expression of MSH2. Our findings are compatible with those of Thibodeau et al,¹⁴ who found reduced expression of MLH1 in 91% of unselected colorectal carcinomas with the MSI-H phenotype. By contrast, Dietmaier et al¹⁵ reported a roughly equal involvement of MSH2 and MLH1 by immunohistochemical analysis of MSI-H tumors (8 and 6 tumors, respectively, showing reduced expression). This discrepancy could be due to the fact that the series studied by Dietmaier et al¹⁵ was a combination of sporadic and hereditary cases, whereas our tumors were truly sporadic based on the absence of MSH2 and MLH1 germline mutations.

View larger version (15K): [in this window] [in a new window]   Figure 1. Graphic presentation of the concurrence of the MLH1 and MSH2 changes in a series of 46 MSI+ sporadic colorectal cancers. The number of cases showing each type of alteration is given within the bars.

In the MSH2-associated group, a somatic defect (loss or mutation of MSH2) was detected in 2/7 tumors (29%). In one case (tumor no. 56), biallelic inactivation of MSH2 was suggested as there was a somatic mutation in one allele and loss of the other allele.⁶ The possibility of promoter hypermethylation underlying MSH2 inactivation was not addressed in the present investigation, as two earlier studies^8,9 had demonstrated that, unlike MLH1, the MSH2 promoter is not prone to hypermethylation in MSI+ tumors. Although most MSI+ tumors were associated with a reduction of either MLH1 or MSH2 protein alone, the expression of both was decreased in six cases, and in half of these (tumors no. 10, 11, and 56), somatic hits affecting both MSH2 and MLH1 were detected.

Despite the MSI+ phenotype, the expression of MLH1 and MSH2 was unaltered in 9 cases. The expression of a mutated nonfunctional protein remains a possibility in this group, since only one tumor was available for mutation analysis (with no mutation present). Alternatively, defects in other genes, such as the DNA mismatch repair genes MSH3 and MSH6¹⁷ or DNA polymerase delta¹⁸ may account for MSI in these tumors. As a whole, our present and previous¹⁰ data combined with reports by others^7-9 suggest that in a vast majority of cases, promoter hypermethylation is associated with immunohistochemically and/or biochemically demonstrable inactivation of MLH1 as a functional consequence. However, this is not invariably the case, as illustrated by the fact that hypermethylation was present in six tumors without any apparent reduction in protein expression. It is possible that hypermethylation affected only one of the two MLH1 alleles, as proposed,⁹ or despite affecting some CpG (in this case, HpaII) sites, left some other sites intact whose methylation might be necessary for the silencing of this gene.¹⁹ Finally, recent observations^10,20 suggest that MLH1 promoter methylation should be viewed as being part of a more widespread hypermethylation tendency that characterizes MSI+ colorectal tumors, but is rare in MSI- tumors. Indeed, the frequency of MLH1 promoter hypermethylation without reduced protein expression (6/36, 17%) was comparable to the frequency of MLH1 hypermethylation in MSI- tumors (11%), as determined previously.¹⁰ Thus, although typically clones with inactive MLH1 and defective DNA mismatch repair after MLH1 promoter hypermethylation would be selected for during tumorigenesis, hypermethylation might occasionally have other targets, and the occurrence of MLH1 promoter hypermethylation in the same cells could be merely coincidental and without functional consequences.

Somatic Events in Hereditary MSI+ Colorectal Cancers

To investigate whether the presence of a germline mutation as an inherited defect in every cell had an effect on the nature of the somatic lesion, we investigated colorectal tumors from 27 HNPCC patients for LOH and promoter hypermethylation (Table 2) . All cases were included in which the predisposing mutation was known and samples were available. With one exception, all were MLH1-linked, reflecting the fact that MLH1 germline mutations predominate in the studied population, for reasons that are not fully understood.¹³ Among 26 MLH1-linked HNPCC tumors, promoter hypermethylation was significantly less prevalent than in the sporadic MSI+ tumors showing reduced expression of MLH1 (12/26, 46% vs. 30/36, 83%, P = 0.003). By contrast, the LOH rates were comparable (7/21, 33% in HNPCC vs. 7/29, 24% in sporadic MSI+ tumors). Combining hereditary and sporadic MSI+ tumors, hypermethylation and LOH were mutually exclusive (LOH was present in 5/34, 15% vs. 9/16, 56% among informative cases with versus without hypermethylation, P = 0.005), suggesting that they had similar functions in MLH1 inactivation. In particular, with just one exception, HNPCC tumors carrying the inherited mutation as the first hit did not show promoter hypermethylation at all, if LOH was present (Table 2) , suggesting that the wild-type allele rather than the mutated allele was the preferential target for hypermethylation, the putative second hit. In the single MSH2-linked case, the germline mutation was accompanied by LOH as an acquired event.

MLH1 promoter hypermethylation was a feature of proximal tumors in the sporadic MSI+ group, whereas the HNPCC group showed no statistically significant association between DNA methylation status and tumor location (Table 3) . If, as suggested,^10,20 MLH1 promoter hypermethylation represents a more generalized phenomenon that is associated with tumor formation preferentially in the proximal colon, and if such hypermethylation is significantly more common in sporadic than in hereditary MSI+ tumors (this study), this might explain the relatively higher percentage of proximal tumors in the former group (83 to 94% vs. 70%, respectively²¹ ). The different prevalence for hypermethylation combined with its possible regulatory consequences might thus be reflected in pathogenetic differences between the two groups of tumors despite a similar MSI+ phenotype. An example of a gene whose regulation by hypermethylation could make a difference is APC, an important gatekeeper of colon tumorigenesis²² ; in fact, this gene was recently found to show promoter hypermethylation specifically in proximal tumors.²³ LOH in the MLH1 region was associated with distal sporadic MSI+ tumors (Table 3) , which is in agreement with the inverse correlation between LOH and MLH1 promoter hypermethylation, as noted previously. By contrast, the LOH status showed no correlation with tumor location among HNPCC tumors. It is possible that LOH in general is important in the development of distal sporadic tumors,²⁴ no matter whether MSI+ or MSI-, whereas in HNPCC, the inherited DNA mismatch repair deficiency per se is probably a strong independent determinant of tumor location.

	Acknowledgements

 
We thank Drs. Lauri Aaltonen and Anu-Liisa Moisio for helpful discussions and Saila Saarinen for expert technical assistance.

	Footnotes

 
Address reprint requests to Dr. Päivi Peltomäki, Division of Human Cancer Genetics, Comprehensive Cancer Center, The Ohio State University, 690 Medical Research Facility, 420 W. 12th Avenue, Columbus, OH 43210. E-mail: peltomaki-1{at}medctr.osu.edu

Supported by the Nordic Cancer Union, the Sigrid Juselius Foundation, the European Commission (contract BMH4-CT-96–0772), the National Institutes of Health (grants CA67941, CA 82282, and P30 CA16058), and the Ohio Cancer Research Associates.

S. A. K. and M. T. H. contributed equally to this work.

Accepted for publication January 24, 2000.

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