Germline MSH2 hypermethylation
Tumors with MSI and loss of MSH2 protein expression are generally indicative of an underlying MSH2 germline mutation (inferred MSH2 mutation). Unlike the case with MLH1, MSI with MSH2 loss is rarely associated with somatic hypermethylation of the promoter. Nevertheless, in at least 30% to 40% of these cases of inferred MSH2 mutation, no germline mutation can be detected with state of the art technology. One Chinese family with tumors showing MSH2 loss was found to have allele-specific hypermethylation that appeared to have been an inherited phenomenon. Another study of a family with MSH2-deficient MSI-high tumors employed the commonly used diagnostic MLPA analysis of MSH6 and also showed reduced expression of MSH6. In doing so, a decrease in signal was observed for exon 9 of the EPCAM (TACSTD1) gene, which is near MSH2. Use of additional MLPA probes located between exon 3 of EPCAM and exon 1 of MSH2 demonstrated that the deletion spanned most 3' exons of EPCAM, but spared the MSH2 promoter. The mutation in EPCAM was found to induce the observed methylation of the MSH2 promoter by transcription across a CpG island within the promoter region. The presence of EPCAM mutations showing similar methylation-mediated MSH2 loss was found at about the same time in families from Hungary.. On the strength of these observations, EPCAM testing has already been introduced clinically for patients with loss of MSH2 protein expression in their CRCs who lack detectable MSH2 germline mutation.
A complementary and perhaps even alternative approach to MSI is to test the tumor by IHC for protein expression using monoclonal antibodies of the MSH2, MLH1, MSH6, and PMS2 proteins. Loss of expression of these proteins appears to correlate with the presence of MSI and may suggest which specific MMR gene is altered in a particular patient.[264,265,266,267]
Tumor testing for suspected LS
Either MSI or IHC can be used as a tumor-based screen for LS.[203,268,269] The choice of whether to use MSI, IHC, or both depends on local availability and expertise.
It appears that clinical practice has shifted from reliance on MSI in the early days of tumor testing to increasing, and in many cases exclusive, reliance on IHC currently. Using both of these tests increases the sensitivity of the initial screen and improves quality assurance; therefore, many laboratories assess both MSI and IHC initially. However, because these tests are so commonly regarded as simple alternatives, cost-effectiveness considerations seem to support IHC and account for its preferential use. Part of this rationale is that the information provided by IHC may direct testing toward a specific MMR gene (the one with loss of protein expression) as opposed to comprehensive testing that would be necessitated by the use of MSI alone.[203,204,269,270,271,272] Arguments for a sequential approach to increase efficiency have been made. A German consortium has proposed an algorithm suggesting a sequential approach; this is likely to depend on the different costs of MSI and IHC and the prior probability of a mutation. Data from a large U.S. study support IHC analysis as the primary screening method, emphasizing its ease of performance in routine pathology laboratories.