Challenges and Pitfalls in HNPCC Screening by Microsatellite Analysis and Immunohistochemistry

Abstract

Hereditary non-polyposis colorectal cancer (HNPCC) accounts for approximately 2 to 4% of the total colorectal cancer burden. For economic reasons a diagnostic "stepladder" is recommended. After evaluation of the family history , diagnostic microsatellite instability (MSI) analysis has found its place as a valuable screening tool for HNPCC. Immunohistochemical analysis can help to pinpoint the affected gene. The detection of a mutation in one of the responsible mismatch repair gene confirmed the diagnosis HNPCC. Here we demonstrate our experience of some important pitfalls that will be discussed in this study. In MSI testing, one potential source for false-negative results is intralesional heterogeneity. We demonstrate examples of a flat adenoma and a carcinoma, which required laser microdissection to correctly determine the microsatellite status. In these lesions manual microdissection, the most frequently applied method, was not sufficient. However, the number of cells obtained by using laser microdisssection can fall below a necessary minimum, which can also cause false-negative results of MSI analysis, as shown here in a mucinous carcinoma. In addition, evaluation of immunohistochemically stained tissue slides requires experience to avoid false-positive or falsenegative interpretation. A case with two synchronous colorectal cancers revealed loss of MSH2 expression in one carcinoma, whereas the second carcinoma stained positively leading to a false-negative interpretation.

Differences in Microsatellite Instability Profiles between Endometrioid and Colorectal Cancers

“…In addition, IHC allows for the specific identification of the defective MMR protein(s). However, IHC can be intact in MSI-H tumors with germline mutations when the specific mutation(s) does not disrupt antibody-reactive epitope, 19 and IHC might not be the ideal method for detecting EMC in treated patients, as chemoradiation can reduce MMR protein expression in otherwise MSS tumors. 20 There are several possible mechanisms underlying the difference in MSI profiles seen in MSI-H EMCs and CRCs that, in part, may be attributable to differences in the biology of the two different types of epithelium.…”

EGAPP supplementary evidence review: DNA testing strategies aimed at reducing morbidity and mortality from Lynch syndrome

“…There is room for improvement, however, as it is likely that none of the studies under review used the most sophisticated MSI test now possible for identifying CRC patients with Lynch syndrome. Methodological studies have shown the importance of laser microdissection, 21,25 the proportion of tumor tissue tested, and the number of cells tested. 25,26 Other studies have provided ways to improve testing of poor quality samples.…”

A systematic review and economic evaluation of diagnostic strategies for Lynch syndrome

“…4 Therefore, microsatellite analysis is commonly used as the first diagnostic screening test for LS. 18 Microsatellite instability testing involves amplification of a standardised panel of DNA markers (Bethesda/National Institutes of Health markers), although laboratories may use 10 or more markers and, more recently, a commercially available kit based on five mononucleotide markers has become popular as mononucleotide microsatellites may be the most sensitive markers for use in detecting MSI. 4 The process involves microdissection of tumour tissue, followed by extraction of DNA which is then amplified and run on a DNA fragment length analyser.…”