Microsatellite Instability (MSI) Detection in DNA from FFPE Tissues

Microsatellite sequences are genome-wide dispersed stretches of short tandem nucleotide repeats. Because of their repetitive nature, microsatellites are prone to undergo shortening or extension during DNA replication because of polymerase slippage or misalignment of template strands (microsatellite instability, MSI). As spontaneous mutation rate increases dramatically in the presence of a defective mismatch repair (MMR) system, MSI represents an ideal phenotypic indicator of an MMR defect. MSI occurs in approximately 15% of colorectal cancers, including those arising in the Hereditary Non-Polyposis CRC familiar syndrome (HNPCC or Lynch Syndrome). MSI tumours feature a series of molecular and clinicopathological signatures that are distinct from non-MSI ones. MSI testing therefore enables identification of patients having a unique prognosis and a different response to particular drug therapies. This chapter provides three methodological approaches for assessing MSI in FFPE samples: a basic method involving amplification of the NCI-validated microsatellite marker sequences, with PAGE run and silver stain detection of PCR products; a multiplex PCR amplification of five mononucleotide markers alternative to the NCI panel, coupled with DHPLC (Denaturating High-Performance Liquid Chromatography) analysis of PCR products; and a multiplex PCR amplification of the five mononucleotide markers coupled with capillary electrophoresis.