Anthony, H.; Seoighe, C.

Subclonal diversity within a tumor is highly relevant for tumor evolution and treatment. This diversity is often referred to as intratumoral heterogeneity and is known to complicate the interpretation of single-test biomarkers. Microsatellite instability (MSI) is one such biomarker, which is used to help guide immune checkpoint inhibitor treatment through the classification of samples as either having high microsatellite instability (MSI-H) or as being microsatellite stable (MSS). One area that has yet to be addressed in depth is whether MSI itself is a heterogeneous phenomenon, such that only some subclones are MSI-H. To investigate heterogeneity in MSI status, we curated and analyzed data from several single-cell RNA sequencing studies that had paired clinical MSI status and developed a computational pipeline to infer MSI-H cells and quantify heterogeneity In MSI status. We found evidence of heterogeneity in MSI status both in individuals originally classified as MSI-H and MSS. Out of 49 individuals, 15 showed evidence of divergence in MSI status between distinct clusters of cancer cells and most had distinct MSI-H and MSS subclones. These results raise questions about the current practice of treating MSI as a binary biomarker, ignoring heterogeneity between cancer subclones. Accounting for heterogeneity may lead to improved biomarker performance and, potentially, help explain reports of intrinsic treatment resistance and low overall responder rate in MSI-H cancers. Further studies are warranted to determine the frequency of heterogeneity in this biomarker at the population level, and whether the presence of both MSI-H and MSS subclones can have clinical implications.