Immunohistochemistry

The examination of CRC tissue for loss of expression of MMR proteins by IHC complements MSI analysis in screening for defective MMR in HNPCC. IHC is a relatively fast, easily interpretable, and inexpensive method that is performed on slides from paraffin-embedded tumor tissue. Tumor cells that demonstrate an absence of nuclear staining in the presence of positive staining in surrounding normal cells (lymphocytes and normal colonic epithelium) are interpreted as having an absence of protein expression (Figure 19-2b). Loss of MMR protein expression is highly correlated with an MSI-H phenotype, but not with MSI-L/MSS.58

In addition to defining the presence of defective MMR, an important feature of IHC analysis lies in the finding that loss of expression of a particular MMR protein is predictive of the gene most likely mutated. IHC analysis of tumor from moderate- and high-risk individuals is routinely available for hMSH2, hMLHl, hMSH6, and hPMS2. Interestingly, loss of expression of hMSH2 is accompanied by loss of hMSH6, and similarly, loss of expression of hMLH1 is accompanied by loss of hPMS2. However, loss of hMSH6 and hPMS2 can occur alone. The mechanistic reasons for the concomitant loss of these proteins are not entirely clear. However, since these combinations of proteins also make up the MutS and MutL MMR complexes, it is possible that

Figure 19-2. Immunohistochemical analysis with anti-hMLHI antibody. (a) Tumor and surrounding normal tissue showing nuclear staining for hMSH6 expression. (b) Normal tissue with positive staining, but tumor tissue shows an absence of staining, indicating a loss of hMSH6 protein expression.

Figure 19-2. Immunohistochemical analysis with anti-hMLHI antibody. (a) Tumor and surrounding normal tissue showing nuclear staining for hMSH6 expression. (b) Normal tissue with positive staining, but tumor tissue shows an absence of staining, indicating a loss of hMSH6 protein expression.

the loss of one partner of a complex has effects on the expression, protein folding, or proper cellular localization of the other partner. In practice, the loss of expression of hMSH2, loss of hMSH6 alone, or loss of hPMS2 alone is strong evidence for the presence of a germline mutation in that respective gene. Thus, efforts to determine the precise germline mutation responsible for HDMMR in an individual can be focused on a single gene. In addition, absence of MMR protein expression may indicate that an identified MMR gene mutation has functional significance.

Like MSI analysis, however, IHC analysis cannot distinguish between somatic and germline alterations. Sporadic tumors with hMLHl promoter hypermethylation also demonstrate a loss of hMLHl (and hPMS2) protein expression; hence, loss of expression of hMLHl does not establish the presence of a heritable germline gene mutation. In addition, cases have been described in which tumors with an MSI-H phenotype did not reveal a concomitant loss of protein expression.59 Thus, MSI analysis (which indicates a phenotypic defect in MMR) in conjunction with IHC (which indicates which of the MMR genes is inactivated to produce that phenotype) is likely the most effective method for screening tumors in order to identify individuals at high risk for HNPCC, and in whom mutation screening should be considered.

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