Retained mismatch repair protein expression occurs in approximately 6% of microsatellite instability-high cancers and is associated with missense mutations in mismatch repair genes

Abstract

Immunohistochemistry for mismatch repair protein expression is widely used as a surrogate for microsatellite instability status-an important signature for immunotherapy and germline testing. There are no systematic analyses examining the sensitivity of immunohistochemistry for microsatellite instability-high status. Mismatch repair immunohistochemistry and microsatellite instability testing were performed routinely as clinically validated assays. We classified germline/somatic mutation types as truncating (nonsense, frameshift, and in/del) versus missense and predicted pathogenicity of the latter. Discordant cases were compared with concordant groups: microsatellite instability-high/mismatch repair-deficient for mutation comparison and microsatellite stable/mismatch repair-proficient for immunohistochemical comparison. 32 of 443 (7%) microsatellite instability-high cases had immunohistochemistry. Four additional microsatellite instability-high research cases had discordant immunohistochemistry. Of 36 microsatellite instability-high cases with discordant immunohistochemistry, 30 were mismatch repair-proficient, while six (five MLH1 and one MSH2) retained expression of the defective mismatch repair protein and lost its partner. In microsatellite instability-high tumors with discordant immunohistochemistry, we observed an enrichment in deleterious missense mutations over truncating mutations, with 69% (25/36) of cases having pathogenic germline or somatic missense mutations, as opposed to only 19% (7/36) in a matched microsatellite instability-high group with concordant immunohistochemistry (p = 0.0007). In microsatellite instability-high cases with discordant immunohistochemistry and MLH1 or PMS2 abnormalities, less cells showed expression (p = 0.015 and p = 0.00095, respectively) compared with microsatellite stable/mismatch repair-proficient cases. Tumor mutation burden, MSIsensor score, and truncating mismatch repair gene mutations were similar between microsatellite instability-high cases with concordant versus discordant immunohistochemical expression. Approximately 6% of microsatellite instability-high cases have retained mismatch repair protein expression and would be missed by immunohistochemistry-based testing, hindering patient access to immunotherapy. Another 1% of microsatellite instability-high cases show isolated loss of the defective gene's dimerization partner, which may lead to germline testing of the wrong gene. These cases are enriched for pathogenic mismatch repair missense mutations.

Figure 1.

A) A microsatellite instability-high colorectal carcinoma with a nuclear dotlike or ‘nucleolar’ MLH1 expression pattern. No germline or somatic mismatch repair gene mutations were present, yet MLH1 promoter hypermethylation was present (MLH1 immunohistochemistry, Ventana, 400x original magnification). B) A microsatellite instability-high colorectal carcinoma with retain MSH6 expression in approximately 75% of tumor cells. Germline mutation analysis was negative for mismatch repair gene mutations, yet 2 somatic mutations in MSH6 were present: p. F1088Pfs*3 (c. 3260_3261dupCC) at 20.6% variant allele fraction (VAF), which is truncating, and p.T1219I (c.3656C>T) at 36.4% variant allele frequency, which is a missense mutation that is predicted to be pathogenic (MSH6 immunohistochemistry, Cell Marque, 40x original magnification).

Figure 2.

Box plot of percentage of tumor cells expressing mismatch repair proteins by immunohistochemistry. The percentage of tumor cells expressing A) MLH1, B) MSH2, C) MSH6, and D) PMS2 was in general lower for microsatellite instability-high cases with retained mismatch repair protein expression (right, green) in comparison to microsatellite stable cases (left, red).