The biochemical basis of microsatellite instability and abnormal immunohistochemistry and clinical behavior in Lynch syndrome: from bench to bedside.

Lynch syndrome is an inherited disease caused by a germline mutation in one of four DNA mismatch repair (MMR) genes. The clinical manifestations can be somewhat variable depending upon which gene is involved, and where the mutation occurs. Moreover, the approach to the diagnosis of Lynch syndrome is becoming more complex as more is learned about the disease, and one needs to understand how the DNA MMR proteins function, and what makes them malfunction, to have an optimal appreciation of how to interpret diagnostic studies such as microsatellite instability and immunohistochemistry of the DNA MMR proteins. Finally, an understanding of the role of the DNA MMR system in regulation of the cell cycle and the response to DNA damage helps illuminate the differences in natural history and response to chemotherapeutic agents seen in Lynch syndrome.

Characterization of sporadic colon cancer by patterns of genomic instability.

Colorectal cancer (CRC) can progress through two pathways of genomic instability: chromosomal (CIN) and microsatellite instability (MSI). We hypothesized that these two pathways are not always independent and that some tumors therefore show a significant degree of overlap between these two mechanisms. We classified 209 high-risk stage II and stage III sporadic CRCs based on their MSI status, using a National Cancer Institute-recommended panel of microsatellite markers, and also identified MSI-associated mutations of CRC target genes such as TGFbetaRII. Evidence for CIN was gathered by identifying loss of heterozygosity (LOH) events on chromosomal arms 1p, 2p, 3p, 5q, 17p, and 18q, which are regions harboring mismatch-repair and tumor-suppressor genes that are significant in CRC development. Results of all molecular markers tested were correlated with clinicopathological variables of the cohort, including treatment outcome. Of the 209 cases, 65% cancers were microsatellite stable, 21% were MSI-low, and 14% were MSI-high (MSI-H). Overall, 51% of the tumors had at least one LOH event, with most frequent chromosomal losses observed on 18q (72.5%), followed by 5q (22%), 17p (21%), and 3p (14%). Interestingly, we observed a significant degree of overlap between MSI and CIN pathways. Of 107 cancers with LOH events, 7 (6.5%) were also MSI-H, and of 30 cancers that were MSI-H, 7 (23.3%) also had one or more LOH events. We also found that 37.8% of microsatellite-stable cancers had no LOH events identified, thus comprising a subgroup of tumors that were not representative of either of these two pathways of genomic instability. Our data suggest that molecular mechanisms of genomic instability are not necessarily independent and may not be fully defined by either the MSI or CIN pathways.

Frequent loss of hMLH1 by promoter hypermethylation leads to microsatellite instability in adenomatous polyps of patients with a single first-degree member affected by colon cancer.

The first-degree relatives of patients affected by colorectal cancer, who do not belong to familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer families, have a doubled risk of developing tumors of the large intestine. We have previously demonstrated that subjects with a single first-degree relative (SFDR) with colon cancer have a doubled risk for developing colorectal adenomas, and in these cases, polyps recur more frequently. The mechanism underlying this predisposition has not been clarified. In this study, we evaluated the frequency of microsatellite instability (MSI) using the five markers suggested by the National Cancer Institute workshop, target gene mutations, hMLH1 and hMSH2 expression, and hMLH1 promoter hypermethylation in the adenomas of patients with and without a SFDR affected by colon cancer. Seventy polyps were obtained from 70 patients: 27 with a single FDR with colon cancer and 43 without such a history. Of the 70 polyps, 12 were MSI-H (17.1%), 20 were MSI-L (28.6%), and 30 were microsatellite stable (42.9%). Of the 27 patients with positive family history, 8 polyps (29.6%) were MSI-H compared with those with negative history in which 4 polyps (9.3%) were MSI-H (P < 0.02). Of the 12 MSI-H polyps, all of the polyps obtained from patients with positive family history had loss of hMLH1 immunostaining versus one with negative family history (P < 0.02). Of the MSI-H polyps, 2 had a somatic frameshift mutation of the MBD4 gene, 1 of MSH6, 1 of BAX, and 2 of transforming growth factor betaRII. Furthermore, 6 of 8 polyps from patients with positive family history with MSI-H and loss of MLH1 had hypermethylation of the MLH1promoter versus none of the MSI-H with negative family history (P < 0.02). All 6 polyps of the 27 from SFDR positive subjects, with hMLH1 promoter hypermethylation loss of hMLH1 and MSI, were located in the right colon (P < 0.02). Hypermethylation of the promoter of hMLH1, consequent loss of hMLH1 expression, and MSI are at the basis of approximately 25% of adenomatous polyps developed in subjects with a SFDR affected by colorectal cancer.

Oxidative stress inactivates the human DNA mismatch repair system.

In the human DNA mismatch repair (MMR) system, hMSH2 forms the hMutSalpha and hMutSbeta complexes with hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2 form the hMutLalpha heterodimer. These complexes, together with other components in the MMR system, correct single-base mismatches and small insertion/deletion loops that occur during DNA replication. Microsatellite instability (MSI) occurs when the loops in DNA microsatellites are not corrected because of a malfunctioning MMR system. Low-frequency MSI (MSI-L) is seen in some chronically inflamed tissues in the absence of genetic inactivation of the MMR system. We hypothesize that oxidative stress associated with chronic inflammation might damage protein components of the MMR system, leading to its functional inactivation. In this study, we demonstrate that noncytotoxic levels of H2O2 inactivate both single-base mismatch and loop repair activities of the MMR system in a dose-dependent fashion. On the basis of in vitro complementation assays using recombinant MMR proteins, we show that this inactivation is most likely due to oxidative damage to hMutSalpha, hMutSbeta, and hMutLalpha protein complexes. We speculate that inactivation of the MMR function in response to oxidative stress may be responsible for the MSI-L seen in nonneoplastic and cancer tissues associated with chronic inflammation.

Epigenetic inactivation of RUNX3 in microsatellite unstable sporadic colon cancers.

Runt domain transcription factors are important targets of TGF-beta superfamily proteins and play a crucial role in mammalian development. Three mammalian runt-related genes, RUNX1, RUNX2 and RUNX3, have been described. RUNX3 has been shown to be a putative tumor suppressor gene localized to chromosome 1p36, a region showing frequent loss of heterozygosity events in colon, gastric, breast and ovarian cancers. Because of the important role of TGF-beta signaling in the human colon, we hypothesized that RUNX3 may serve as a key tumor suppressor in human colon cancers and colon cancer-derived cell lines. We examined RUNX3 expression and the frequency of RUNX3 promoter hypermethylation in 17 colon cancer cell lines and 91 sporadic colorectal cancers. Semiquantitative analysis of RUNX3 transcripts was performed by RT-PCR and de novo methylation of the RUNX3 promoter was studied by a methylation-specific PCR (MSP) assay. Nineteen of 91 informative tumors (21%) and 11 of 17 (65%) colon cancer cell lines exhibited hypermethylation of the RUNX3 promoter. Interestingly, RUNX3 promoter hypermethylation was more common in tumors exhibiting high frequency of microsatellite instability (MSI-H) (33% of MSI-H vs. 12% of MSI-L/MSS tumors; p = 0.012). Hypermethylation of the RUNX3 promoter correlated with loss of mRNA transcripts in all cell lines. RUNX3 promoter methylation was reversed and its expression restored in SW48 and HCT15 colon cancer cells after treatment with the demethylating agent 5-aza-2'-deoxycytidine, indicating that loss of expression is caused by epigenetic inactivation in colon carcinogenesis. This is the first demonstration of frequent de novo hypermethylation of the RUNX3 promoter in sporadic colon cancers. The significant association of RUNX3 promoter hypermethylation with MSI-H colon cancers suggests that RUNX3 is a novel target of methylation, along with the hMLH1 gene, in the evolution of MSI-H colorectal cancers.