RNA-seq Reveals Differences in Expressed Tumor Mutational Burden in Colorectal and Endometrial Cancer With and Without Defective DNA Mismatch Repair.

Tumor mutational burden (TMB) is an emerging biomarker for predicting immunotherapy response. We set up to use RNA sequencing (RNA-seq) for determining TMB in microsatellite stable (MSS) and unstable-high (MSI-H) cases from Formalin Fixed Paraffin Embedded (FFPE) tissues. Tumors and paired normal tissue from 46 MSI-H and 12 MSS cases were included. Out of the MSI-H tumors, 29 had defective DNA Mismatch Repair (dMMR) mutations and 17 had MLH1 promoter hypermethylation (HM). TMB was measured using the expressed somatic nucleotide variants (eTMB). We developed a method to accurately measure eTMB that removes FFPE derived artifacts by leveraging mutational signatures. There was a significant difference in the eTMB observed between MSI-H and MSS cases; MSI-H cases had a median of 27.3 mutations/Mb compared to 6.7 mutations/Mb for MSS cases (p=3.5x10-9). Among tumors with dMMR the tumors with MMR mutations had a significantly higher eTMB (p=0.037) than tumors with HM: a median of 28.1 mutations/Mb vs. a median of 17.5 mutations/Mb. Multivariate analysis showed that MSI status, tissue type (endometrial or colorectal) and patient ages are significantly associated with eTMB. Additionally, using Whole Exome Sequencing on a subset of these patients, we determined that DNA TMB correlates well with eTMB (Spearman correlation of 0.83). These results demonstrate that RNA-seq can be used to measure eTMB in FFPE tumor specimens.