Impact of Single-Gene and Co-Occurring Mutations on TMB with Potential Implications for Immunotherapy.

Background: Tumor mutational burden (TMB) has emerged as a potent biomarker for cancer immunotherapy. In this study, we systematically analyzed the impact on TMB of both single-gene and co-occurring double-gene mutations in Chinese NSCLC and CRC patients. Methods: FFPE tumor and matched blood samples of 291 NSCLC and 197 CRC patients were collected for NGS-based 450 cancer genes panel assay. Genomic alterations including single nucleotide variations (SNV), short and long insertions/deletions (Indel), copy number variations (CNV) and gene rearrangements in selected genes were assessed and tumor mutational burden (TMB) computed. Results: 37 and 81 genes were frequently mutated (each occurring in 15 or more patients) in our NSCLC and CRC cohorts, respectively, with 20 genes common to both. Mutations in most genes associated with higher TMB and exhibited similar trends between the two cohorts, examples including LRP1B, ARID1A, KMT2D, NOTCH1, FAT3, SPTA1, PRKDC etc. On the other hand, ALK- and EGFR-mutant patients possessed lower TMB in NSCLC (4.8 and 6.4, respectively) but higher in CRC. Interestingly, mutations in KRAS and TP53 saw significantly elevated TMB in NSCLC but not in CRC. In terms of double mutants, NSCLC patients with co-occurring KRAS-TP53 mutations had still higher level of TMB than either single mutant alone, consistent with reported clinical utility of immunotherapy for such patients. Our analysis produced a comprehensive list of such “synthetic rescue” pairs of genes where double mutants possessed significantly higher TMB. Finally, mutations in most gene pairs tended to be mutually dependent rather than exclusive, with consistent results between NSCLC and CRC (r = 0.41, p < e-16). A network of interactive co-mutations was assembled and functional clusters obtained. Conclusions: Gene alterations have a clear and dramatic impact on TMB, offering mechanistic clues and suggesting potential for immunotherapy. Our comprehensive analyses of single- and double-gene mutations and their impact on TMB in two very different cancer types not only revealed novel insights, but also produced many fresh cases for further investigation. Our methods are very generic and easily adaptable to other cancers. Legal entity responsible for the study: OrigiMed. Funding: OrigiMed. Disclosure: All authors have declared no conflicts of interest.