Kevin C Halling and Patrick C Roche 1 Introduction

Defective DNA mismatch repair (MMR) occurs in the majority of tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) and approx 15% of sporadic colorectal cancer (CRC) (1,2). In HNPCC-associated tumors, defective MMR is most often due to inactivating mutations of the DNA MMR genes hMLHl and hMSH2 (3,4). Defective MMR in sporadic CRC, on the other hand, is generally due to hypermethylation of the hMLHl promoter (5-7). As might be expected, inactivating mutations of hMSH2 and hMLHl lead to a loss of hMSH2 or hMLHl expression respectively and hypermethylation of the hMLHl promoter to a loss of hMLHl expression (5-8). One of the hallmarks of defective DNA MMR is a type of genetic instability known as microsatellite instability (MSI). Tumors with defective DNA MMR generally exhibit MSI at the majority of the loci examined (MSI-H phenotype) (8,9).

Immunohistochemical analysis for hMLHl and hMSH2 is useful for: 1) assessing colorectal tumors for defective MMR; 2) screening CRC patients for HNPCC; and 3) identifying the defective MMR gene in tumors with a MSI-H phenotype. The last mentioned utility is helpful since it identifies the gene that needs to be sequenced to find the causative mutation (or promoter hypermethylation). Immunohistochemical analysis for hMLHl and hMSH2 may eventually also be used to guide CRC patient treatment since: 1) tumors with defective MMR appear to have a better prognosis than tumors that do not, and 2) tumors with defective MMR appear to be resistant to chemotherapeutic agents such as cisplatin and N-Methyl-N'-nitro-N-nitrosoguanidine (2,10-16).

In this chapter we describe the methods that we use to perform immunohis-tochemical analysis for hMLHl and hMSH2 expression.

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

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