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 false-negative 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. In some cases, false-positive results can be obtained, if a perinuclear-staining pattern is interpreted as positive. In summary, there are several potential pitfalls in the molecular screening for HNPCC. Therefore the importance of correct interpretation of clinical data, immunohistochemistry, and microsatellite analysis in combination, performed by a pathologist with experience in molecular genetics is essential. In addition, laser microdissection of tumor areas that have been chosen by a pathologist is highly recommended in cases that cannot be resolved with manual microdissection.

Figure 1

A–D: Results of MSI analysis in patient 1 diagnosed with a flat adenoma in the cecum. A: An overview of the lesion shows slightly thickened mucosa within the marked area (box) and a minute focus of invasion (arrow). H&E, magnification, ×4. B: Immunohistochemistry showed distinct loss of MLH1 expression in the neoplastic crypts (arrows). Anti-MLH1, magnification, ×10. C: MSI-analysis after crude manual microdissection displayed MSS status. D: MSI-analysis of the mononucleotide markers BAT 25 (upper two lanes) and BAT 26 (lower two lanes) after laser microdissection disclosed the MSI-H nature particularly in the invasive part (normal tissue: lanes 1 and 3; corresponding tumor tissue: lanes 2 and 4).

Figure 2

A–D: MSI analysis in a mucinous carcinoma. A: Histological tumor section after laser microdissection of selected tumor cells. H&E, magnification, ×40. B: Crude manual dissection of the mucinous carcinoma revealed MSS status (shown: BAT25, BAT26, D5S346; normal tissue: lanes 1, 3, and 5; corresponding tumor tissue: lanes 2, 4, and 6). C and D: After laser microdissection MSI analysis revealed a loss of the second peak at the dinucleotide marker (APC, D5S346) in the tumor sample when compared to normal tissues (normal tissue, lane 1; corresponding tumor tissue, lane 2). This result, suggesting LOH, was observed only when less than 50 cells were used for the MSI-analysis, whereas laser microdissection of a larger tumor cell number revealed the correct MSI-H result (C, lane 3). D: Corresponding results for BAT 26 with the PCR artifact resulting from too few analyzed cells in lane 2 and the correct MSI result shown in lane 3.

Figure 3

A-G: Immunohistochemical staining and MSI-analyses of two synchronous colon carcinomas. A–F: The immunohistochemical analysis showed loss of MSH2 protein expression in one adenocarcinoma (B) but clearly detectable MSH2 protein expression in the second, more solid carcinoma (E) (A and D: anti-MLH1, B and E: anti-MSH2, C and F: anti-MSH6 immunostaining, magnification, ×20.). G: Results of MSI-analysis in both tumors (lanes 1 and 4, normal tissue; lanes 2 and 5, second solid tumor; lanes 3 and 6, adenocarcinoma). Less distinct shifts in the MSH2 expressing carcinoma.

Figure 4

A–D: Histological and immunohistological findings in patient 4. A: Carcinoma with dense intratumoral lymphocytic infiltrate suggestive of an MSI-H tumor. H&E, magnification, ×10. B: Strong intranuclear staining of MSH2 in the tumor cells. Anti-MSH2, mgnification, ×40. C: MLH1 immunostaining shows only a perinuclear rim-like reaction, but no distinct intranuclear staining. Anti-MLH1, magnification, ×40. D: Complete loss of PMS2 staining in the tumor cell nuclei (arrows).