Yann R Parc and Kevin C Hailing 1 Introduction

Microsatellites are tandem repeats of simple sequences that occur abundantly and are randomly interspersed throughout the human genome. They typically consist of 10-50 copies of 1-6 bp motifs, and are characterized by a high degree of polymorphism. Despite the variability observed among individuals, microsatellite are replicated faithfully at each cell division in normal germline and somatic cells (1).

In 1993, three research groups described a novel type of genomic instability, which has been called microsatellite instability (MSI) or replication error (RER), in a subset of sporadic colorectal cancers and in most tumors from patients with HNPCC (2-4). Microsatellite instability is a change of any length (due to either insertion or deletion of repeating units) in a microsatellite within a tumor when compared to normal tissue.

In CRC, three MSI phenotypes have been described (5,6). The MSI-H phe-notype is characterized by MSI at >30-40% of the loci examined, the MSI-L phenotype by MSI at <30-40% of the loci examined and the MSS phenotype by an absence of MSI at any of the loci examined. In sporadic CRC the MSI-H phenotype is associated with distinct clinicopathologic features (e.g., proximal tumor site, high grade, diploidy, favorable survival) (3,5,6). The MSI-L and MSS phenotypes, on the other hand, are not associated with distinct clinicopathologic features (5).

The MSI-H phenotype is the result of defective DNA mismatch repair. Inactivating mutations of one of at least five different DNA MMR genes (hMLHl, hMSH2, PMS1, PMS2, and hMSH6/GTPBP) are the cause of the MSI-H phenotype in HNPCC-associated tumors (7-11). Hypermethylation of

From: Methods in Molecular Medicine, vol. 50: Colorectal Cancer: Methods and Protocols Edited by: S. M. Powell © Humana Press Inc., Totowa, NJ

the hMLHl promoter is the etiology of the MSI-H phenotype in over 90% of sporadic CRC (12-14).

MSI has been reported in a variety of other malignancies (15). However, among sporadic tumors, the MSI-H phenotype appears to be most common in cancers of the stomach, endometrium and upper urinary tract (16). Interestingly, these are also the same types of tumors that frequently occur in HNPCC patients (17).

MSI analysis can be used to: 1) identify tumors with defective MMR (i.e., the tumors with the MSI-H phenotype), 2) as a screening test for the identification of colorectal cancer patients that may have HNPCC (18-21), and 3) to identify colorectal cancer patients that may have a favorable prognosis (i.e., the MSI-H tumors) (3,22-25). Additionally, the identification of the MSI-H phenotype in CRC may have treatment implications beyond prognostication since some studies suggest that tumors with the MSI-H phenotype are resistant to chemotherapeutic agents such as cisplatin and N-Methyl-N'-nitro-N-nitrosoguanidine (26-28).

In this chapter we describe methods that we use in our laboratory to assess tumors for MSI. Additional information on technical guidelines for the detection of MSI can be found in the report of a recent National Cancer Institute workshop on microsatellite instability (21).

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