Advantage of HSP110 (T17) marker inclusion for microsatellite instability (MSI) detection in colorectal cancer patients

Abstract

Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Microsatellite instability (MSI) is a genetic pathway leading to CRC, associated with particular clinicopathological features, and recently a major biomarker of immunotherapy response. There is little information the frequency MSI among Brazilian CRC patients, and it is still debatable the ideal methodology for MSI screening in countries with limited resources. We proposed to evaluate MSI by molecular and immunohistochemistry (IHC) methods, to compare both methodologies and also to assess the inclusion of a novel microsatellite marker, HSP110 (T17). The molecular MSI evaluation was performed using a PCR-multiplex panel in a total of 1013 CRC patients. Mismatch repair (MMR) proteins (MLH1, MSH2, MSH6 and PMS2) expression were evaluated by IHC. HSP110 (T17) marker was analyzed by fragment analysis. Molecularly, 89.5% of cases were MSI-negative and 10.5% were MSI-positive. The IHC showed that 88.9% of cases exhibited MMR-proficient status, 10.2% were MMR-deficient and 0.9% was inconclusive. Genotyping of the HSP110 (T17) in 106 MSI-positive and 215 MSI-negative cases showed its alteration only among the MSI-positive cases. We observed agreement (0.956, Kappa Test) between both molecular and IHC methodologies, with only eight discordant results, and in this subset of cases the HSP110 (T17) corroborate the molecular findings. This study suggests the use of molecular assays over IHC for MSI analysis and proposes the inclusion HSP110 (T17) marker as a complementary analysis in discordant cases.

Keywords: HSP110 (T17); colorectal cancer; immunohistochemistry; microsatellite instability; molecular diagnostic.

Figure 1

(A) Percentage of MMR IHC status for 996 Brazilian CRC patients; Positive IHC status: 88.9% (886/996), Negative IHC status: 10.2% (102/996) and Inconclusive IHC status 0.9% (8/996). (B) Percentage of molecular MSI status for 1013 Brazilian CRC patients; MSS (microsatellite stability): 85.8% (870/1013), MSI-L (low-microsatellite instability): 3.7% (37/1013) and MSI-H (high-microsatellite instability): 10.5% (106/1013).

Figure 2

(A) Fragment analysis of peaks for molecular MSI analysis in one MSS CRC sample with five markers (NR27, NR21, NR24, BAT25 and BAT26) within of the quasi-monomorphic variation range (QMVR). (B) Fragment analysis of peaks for molecular MSI analysis in one MSI-H CRC sample with five markers (NR27, NR21, NR24, BAT25 and BAT26) outside of the QMVR. Arrow indicates the allele outside of the QMVR. bp – base pair.

Figure 3

(A) The allele 131 of HSP110 (T17) marker was the most observed in 54.7% (234/428) of analyzed alleles from healthy individuals blood DNA. (B) Two alleles (131 and 126) of HSP110 (T17) marker were observed in MSI-H CRC tumor DNA showing alteration in this marker. Arrow indicates the allele outside of the QMVR. bp – base pair.