Beyond microsatellite testing: assessment of tumor mutational burden identifies subsets of colorectal cancer who may respond to immune checkpoint inhibition

Abstract

Background: The clinical application of PD1/PD-L1 targeting checkpoint inhibitors in colorectal cancer (CRC) has largely focused on a subset of microsatellite instable (MSI-high) patients. However, the proposed genotype that sensitizes these patients to immunotherapy is not captured by MSI status alone. Estimation of tumor mutational burden (TMB) from comprehensive genomic profiling is validated against whole exome sequencing and linked to checkpoint response in metastatic melanoma, urothelial bladder cancer and non-small cell lung carcinoma. We sought to explore the subset of microsatellite stable (MSS) CRC patients with high TMB, and identify the specific genomic signatures associated with this phenotype. Furthermore, we explore the ability to quantify TMB as a potential predictive biomarker of PD1/PD-L1 therapy in CRC.

Methods: Formalin-fixed, paraffin embedded tissue sections from 6,004 cases of CRC were sequenced with a CLIA-approved CGP assay. MSI and TMB statuses were computationally determined using validated methods. The cutoff for TMB-high was defined according to the lower bound value that satisfied the 90% probability interval based on the TMB distribution across all MSI-High patients.

Results: MSS tumors were observed in 5,702 of 6,004 (95.0%) cases and MSI-H tumors were observed in 302 (5.0%) cases. All but one (99.7%) MSI-H cases were TMB-high (range, 6.3-746.9 mut/Mb) and 5,538 of 5,702 (97.0%) MSS cases were TMB-low (range, 0.0-10.8 mut/Mb). Consequently, 164 of 5,702 (2.9%) MSS cases were confirmed as TMB-high (range, 11.7-707.2 mut/Mb), representing an increase in the target population that may respond to checkpoint inhibitor therapy by 54% (466 vs. 302, respectively). Response to PD-1 inhibitor is demonstrated in MSS/TMB-high cases.

Conclusions: Concurrent TMB assessment accurately classifies MSI tumors as TMB-high and simultaneously identifies nearly 3% or CRC as MSS/TMB-high. This subgroup may expand the population of CRC who may benefit from immune checkpoint inhibitor based therapeutic approaches.

Keywords: PD-1; colorectal cancer; immunotherapy; microsatellite instability; tumor mutational burden.

Figure 1

Distribution of TMB across 6,004 advanced colorectal cancer cases stratified by microsatellite instability status. TMB distribution, represented on a log 10 scale, of all 6,004 CRC cases. TMB-high, defined as ≥12 mutations/Mb, is represented by the dashed red line. The box plot distributions reveal the overlap of TMB-high amongst both subsets of both MSI-H and MSS cases. The fraction of MSS cases that are TMB-high is 2.9%, or 164 of 5,702 cases. The fraction of MSI-H cases that are TMB-high is 99.7%, or 301 of 302 cases. TMB, tumor mutational burden; CRC, colorectal cancer; MSI-H, microsatellite-high; MSS, microsatellite stable.

Figure 2

Microsatellite stable CRC case 1 with TMB-high (223 mutations/Mb) demonstrating response to immunotherapy. Response to anti-PD-1 monotherapy in a patient with MSS advanced colorectal cancer that was found to be TMB-high (223 mutations/Mb). Partial response after 4 cycles of pembrolizumab is highlighted by 50% reduction in hepatic metastatic disease (A vs. B) and significant improvement in pulmonary metastasis (C vs. D). CRC, colorectal cancer; TMB, tumor mutational burden; MSS, microsatellite stable.