Microsatellite Instability Testing and Prognostic Implications in Colorectal Cancer

Abstract

Given the crucial predictive implications of microsatellite instability (MSI) in colorectal cancer (CRC), MSI screening is commonly performed in those with and at risk for CRC. Here, we compared results from immunohistochemistry (IHC) and the droplet digital PCR (ddPCR) MSI assay on formalin-fixed paraffin-embedded tumor samples from 48 patients who underwent surgery for colon and rectal cancer by calculating Cohen's kappa measurement (k), revealing high agreement between the methods (k = 0.915). We performed Kaplan-Meier survival analyses and univariate and multivariate Cox regression to assess the prognostic significance of ddPCR-based MSI and to identify clinicopathological features associated with CRC outcome. Patients with MSI-high had better overall survival (OS; p = 0.038) and disease-free survival (DFS; p = 0.049) than those with microsatellite stability (MSS). When stratified by primary tumor location, right-sided CRC patients with MSI-high showed improved DFS, relative to those with MSS (p < 0.001), but left-sided CRC patients did not. In multivariate analyses, MSI-high was associated with improved OS (hazard ratio (HR) = 0.221, 95% confidence interval (CI): 0.026-0.870, p = 0.042), whereas the loss of DNA mismatch repair protein MutL homolog 1 (MLH1) expression was associated with worse OS (HR = 0.133, 95% CI: 0.001-1.152, p = 0.049). Our results suggest ddPCR is a promising tool for MSI detection. Given the opposing effects of MSI-high and MLH1 loss on OS, both ddPCR and IHC may be complementary for the prognostic assessment of CRC.

Keywords: colorectal cancer (CRC); formalin-fixed paraffin-embedded (FFPE) tissue samples; microsatellite instability (MSI); mismatch repair (MMR); primary site of tumors; prognosis.

Figure 1

Representative immunohistochemical staining of MMR proteins in tumors. Expression of MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), and PMS1 homolog 2 (PMS2) in formalin-fixed paraffin-embedded (FFPE) tissue samples identified as normal expression (top row, labeled as high) and loss of staining (bottom row, labeled as low); magnification, 20×.

Figure 2

Two-dimensional droplet fluorescence plots from the three Bio-Rad ddPCR™ MSI assays (i.e., for BAT25 and BAT26, NR21 and NR24, and Mono27) performed on four patient samples (P4, P9, P19, and P45). Wild-type molecules are indicated by orange clusters, and unstable microsatellite molecules are indicated by blue clusters. The individual targets are shown on each plot.

Figure 3

Kaplan–Meier survival analyses for CRC patients according to MSI status. (A,B) Kaplan–Meier curves of overall survival (A) and disease-free survival (B) in patients with MSI-high (MSI-H; pink line) or microsatellite-stable (MSS; blue line) tumors. (C,D) Kaplan–Meier curves of disease-free survival in patients with MSI-H (pink line) or MSS (blue line) tumors for those with left-sided (C) and right-sided (D) primary CRC.

Figure 4

(A,B) Kaplan–Meier analyses comparing overall survival of n = 348 colon cancer (A) and n = 378 rectal cancer (B) patients from the cBioPortal for Cancer Genomics (http://www.cBioPortal.org, accessed on 30 November 2023 [31,32]) with MSI-high (red line) and MSS (blue line). Differences between groups were analyzed using the log-rank test, and p-values are shown. (C,D) Bar plot showing the proportions of MSI-H and MSS samples arising from various primary tumor locations in the colon cancer (C) and rectal cancer (D) cohorts.