Cancer Biomarkers

According to recently published guidelines, microsatellite instability (MSI) testing of colorectal cancers may be clinically indicated on a significant proportion of all colorectal tumors. To date, nothing has been published regarding the reproducibility of MSI testing between laboratories. We present MSI quality control activities experience of a six center multinational consortium, as laboratories developed competency with MSI testing and interpretation. The aim of this paper is to share lessons learned and to describe the final concordance rates in scoring MSI markers within this consortium.

Missense changes constitute approximately 1/3 and 1/5 of all rare sequence constitutional variations identified in the MLH1 and MSH2 genes by mutation screening. They represent a challenge for the clinician and for the genetic counselor, who often cannot use them for the management of Lynch syndrome families. Several parameters can be evaluated to gain insight into the significance of such unclassified variants (UVs). These include analysis of microsatellite instability (MSI), immunohistochemistry of mismatch repair (MMR) proteins, segregation data, frequency of the variants in control samples, presence of other pathogenic mutations, and functional and mRNA analyses. While none of these variables can be used alone to predict the significance of UVs in a single case, combined evaluation can lead to clinically useful conclusions. This review reports available information on a sample of MLH1 and MSH2 missense UVs, for which MSI and immunohistochemical data could be retrieved from the literature. Currently, since MSI analysis is routinely performed as a diagnostic test for Lynch syndrome, tumor MSI status represents the most important factor for determining the pathogenicity of UVs in MMR genes.

Inactivation of DNA mismatch repair (MMR) is the hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) and sporadic colorectal cancers with microsatellite instability (MSI+). MMR loss results in a markedly elevated mutation rate, and many MS mutations are found in MSI+ cancers. In theory, it is possible to estimate the interval between MMR loss and cancer removal by counting numbers of cancer MS mutations – the more MS mutations, the longer the intervals since MMR loss. Using this somatic molecular clock approach, MMR loss is estimated to precede transformation (clonal expansion) and likely occurs in normal appearing colon. Surprising, ages at MMR loss are more consistent with MMR loss as a relatively late event during progression to MSI+ cancer.

Colorectal carcinoma is one of the most common cancers that occurs in the human population, resulting in a mortality rate of more than 50%. Mismatch repair (MMR) defects are mostly manifested as high levels of microsatellite instability (MSI-H); this occurs in ∼19% of all colorectal cancers including a smaller, but high-risk subgroup that is represented by hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome). The aim of molecular diagnosis is to distinguish these familial cases from of the majority of sporadic unstable cancers. Here, we review the widespread MSI-H phenotype in various tumours, assessing the genetic, epigenetic and morphological differences to distinguish between sporadic and familial unstable colorectal cancers. The most important morphological feature is the presence of tumour-infiltrating lymphocytes (TILs), the presence of which is typical in MSI cancers. The main discriminators for HNPCC patients are family history, age at onset up to 60 years and MMR defects caused by germline mutations. This is in contrast to the discriminators for sporadic colorectal cancers, which include onset at any age, BRAF mutation presence and epigenetically MMR inactivation by extensive CpG-island methylation.

This article presents a modified strategy for molecular diagnosis of HNPCC by selective inclusion of recently recognized characteristics of tumours. The clear identification of affected families can improve the strategy of early detection, therapy and prevention of colorectal cancers.

Chemotherapy for colorectal cancer is currently offered to patients based on the stage of their cancer, and there is evidence to show an overall survival benefit with 5-fluorouracil-based (5-FU) therapy for patients with lymph node metastasis who receive it. The pathogenesis of colorectal cancer involves genomic instability, with about 15% of tumors demonstrating a form of genomic instability called high-frequency microsatellite instability (MSI-H) and due to loss of DNA mismatch repair function, and the remainder of colorectal tumors lacking MSI-H with retained DNA mismatch repair function and called microsatellite stable (MSS), with a large proportion of these tumors demonstrating another form of genomic instability called chromosomal instability. There is now evidence to show that the form of genomic instability that is present in a patient's colorectal cancer may predict a survival benefit from 5-FU. In particular, patients whose colorectal tumors have MSI-H do not gain a survival benefit with 5-FU as compared to patients with MSS tumors. In vitro evidence supports these findings, as MSI-H colon cancer cell lines are more resistant to 5-FU compared to MSS cell lines. More specifically, components of the DNA mismatch repair system have been shown to recognize and bind to 5-FU that becomes incorporated into DNA and which could be a trigger to induce cell death. The binding and subsequent cell death events would be absent in colorectal tumors with MSI-H, which have lost intact DNA mismatch repair function. These findings suggest that: (a) tumor cytotoxicity of 5-FU is mediated by DNA mechanisms in addition to well-known RNA mechanisms, and (b) patients whose tumors demonstrate MSI-H may not benefit from 5-FU therapy. Future studies should include a better understanding of the cellular mechanisms of the DNA recognition of 5-FU, multi-centered prospective trials investigating the survival benefit of 5-FU based on genomic instability, and the investigation of alternative chemotherapeutic regimens for patients with MSI-H tumors to improve survival.

Microsatellite instability (MSI) is seen in many cancers and is the result of either a germline or somatic defect in the DNA mismatch repair system. Microsatellite instability is common in endometrial cancers occurring in about 25% of cases with endometrioid histology. Tumor infiltrating lymphocytes (TIL) are more prominent in colorectal cancer cases with MSI. The presence of increased TIL is associated with increased survival in these colorectal cancers, and is suggested as one possible mechanism to explain the increased survival rates in colorectal cancer patients with MSI positive cancers. Some degree of evidence indicates that increased TIL is also predictive of increased survival in endometrial cancer. The relative levels and states of activation of TIL in endometrial cancers with and without MSI has not been explored. Our previous data indicates that global gene expression patterns from MSI and non-MSI endometrial cancers are distinct, however TIL markers were not over-represented on statistically relevant gene lists that distinguish these groups. We further examined these pre-existing microarray data by directly querying transcripts present in the T-cell gene ontology (GO) group. No significant differences were observed between MSI and microsatellite stable (MSS) groups. Finally we directly examined a set of T-cell marker transcripts previously utilized to define increased activated and cytotoxic TIL in MSI positive colorectal cancers. Whereas colorectal cancers with MSI have been previously demonstrated to contain higher ratios of CD8/CD3 message levels we observed no difference in endometrial cancers. In addition, levels of CD3 indicated no increases in TIL in MSI positive cases and 2 markers of activation, granzyme B and IL-2R were not different in MSI positive and negative cancers. These data indicate that significant differences in TIL derived transcripts do not occur between endometrioid endometrial cancers with and without microsatellite instability.

Microsatellites are highly abundant short repetitive sequences found in the genomes across different species. They have gained increasing interest in recent years because length alterations in several coding as well as non-coding microsatellites are associated with a variety of different disorders. Particularly, microsatellite mutations play an important role in tumorigenesis of DNA mismatch repair deficient tumors that account for up to a 15% of colorectal, endometrial, and various other cancers. The systematic analysis of the distribution and function of affected microsatellite sequences has facilitated to unravel important steps in the selection processes that drive tumorigenesis. Here, we review the role of microsatellite mutations in the development of cancers with DNA mismatch repair deficiency, outlining biostatistical approaches for the identification of MSI target genes with relevance to MSI associated carcinogenesis. Knowledge about the biological impact of microsatellite mutations in these genes will potentially help to develop modified clinical concepts for diagnosis, prevention, and treatment of microsatellite unstable human cancers.