Improved NGS-based detection of microsatellite instability using tumor-only data

Abstract

Microsatellite instability (MSI) is a molecular signature of mismatch repair deficiency (dMMR), a predictive marker of immune checkpoint inhibitor therapy response. Despite its recognized pan-cancer value, most methods only support detection of this signature in colorectal cancer. In addition to the tissue-specific differences that impact the sensitivity of MSI detection in other tissues, the performance of most methods is also affected by patient ethnicity, tumor content, and other sample-specific properties. These limitations are particularly important when only tumor samples are available and restrict the performance and adoption of MSI testing. Here we introduce MSIdetect, a novel solution for NGS-based MSI detection. MSIdetect models the impact of indel burden and tumor content on read coverage at a set of homopolymer regions that we found are minimally impacted by sample-specific factors. We validated MSIdetect in 139 Formalin-Fixed Paraffin-Embedded (FFPE) clinical samples from colorectal and endometrial cancer as well as other more challenging tumor types, such as glioma or sebaceous adenoma or carcinoma. Based on analysis of these samples, MSIdetect displays 100% specificity and 96.3% sensitivity. Limit of detection analysis supports that MSIdetect is sensitive even in samples with relatively low tumor content and limited microsatellite instability. Finally, the results obtained using MSIdetect in tumor-only data correlate well (R=0.988) with what is obtained using tumor-normal matched pairs, demonstrating that the solution addresses the challenges posed by MSI detection from tumor-only data. The accuracy of MSI detection by MSIdetect in different cancer types coupled with the flexibility afforded by NGS-based testing will support the adoption of MSI testing in the clinical setting and increase the number of patients identified that are likely to benefit from immune checkpoint inhibitor therapy.

Keywords: MSI; Microsatellite instability; Mismatch Repair deficiency; microsatellite; next-generating sequencing; pan-cancer; tumor-only sequencing.

Figure 1

Factors limiting MSI detection in NGS workflow (A) Factors impacting detection of MSI in tumor-only NGS workflows (B) Schematic representation of the impact of increased indel burden (bottom panel) and tumor content (top panel) on the homopolymer length distribution measured by NGS at an illustrative homopolymer (MSI-H, red). Line color darkness correlates with decreased tumor content (top panel) or indel burden (bottom panel). Reference homopolymer length distribution for microsatellite stable is depicted in blue. Distribution of (C) Fraction of usable reads per total number of reads mapping to the homopolymer and (D) homopolymer score for homopolymers of the same length. MSI score obtained with MSIdetect using WES homopolymers for microsatellite stable (MSS) samples derived from (E) individuals of different ethnic origin and for samples from (F) different tumor types.

Figure 2

Properties of the MSIdetect restricted homopolymer set (A) Receiver Operating curves and corresponding Area Under the Curve (AUC) values (in the inset) for endometrial (blue), colorectal (red) and stomach (black) cancers for MSI classification by MSIdetect using WES homopolymers given the MSI status reported by TCGA. (B) Homopolymer length distribution in WES and in the restricted set. (C) Distribution of average variant population frequency observed in gnomAD for homopolymers in WES and in the restricted set with lengths ranging from 11-25 bp.

Figure 3

MSI detection in colorectal and endometrial FFPE clinical samples (A) MSIscore obtained for colorectal or endometrial cancer samples. Samples were grouped based on their respective MSI-PCR and IHC result. Each point corresponds to one sample colored by tissue of origin (refer to legend in figure) (B) MSIscore for a dilution series containing between 1 and 90% (x-axis) of DNA extracted from one MSI-H tumor DNA diluted in MSS tumor DNA in duplicates. Each point corresponds to one sample. Samples are colored according to results of MSI-PCR test (refer to legend in figure). (C) MSIscore obtained for replicate 1 and 2 for dilution series of MSI-H DNA in MSS DNA.

Figure 4

MSI detection in FFPE clinical samples (A) MSIscore obtained for dMMR or pMMR FFPE samples. Each point corresponds to one sample colored by tissue of origin (refer to legend in figure). Horizontal lines top to bottom indicates MSI-HC and MSI-LC threshold respectively (B) MSIscore obtained for glioma, sebaceoma and cholangiocarcinoma FFPE samples classified by IHC as dMMR or pMMR. Each point corresponds to one sample colored by MSI-PCR status. Horizontal lines top to bottom indicates MSI-H and MSI-LC threshold respectively. (C) MSIscore obtained for dMMR FFPE samples grouped by pairs of protein lost (x-axis) Each point corresponds to one sample colored by tissue of origin (refer to legend in figure). Horizontal lines indicate the median score for the group. (D) Histogram of the percentage of genes with detected loss of MSH2/MSH6 or MHL1/PSM2 grouped by cancer type (E) MSIscore obtained using either a global reference (y-axis) or a reference build using a matched-normal samples. Each point corresponds to one sample colored by MSI-PCR result.