Mutational analysis of microsatellite-stable gastrointestinal cancer with high tumour mutational burden: a retrospective cohort study

Abstract

Background: Genomic signatures contributing to high tumour mutational burden (TMB-H) independent from mismatch-repair deficiency (dMMR) or microsatellite instability-high (MSI-H) status are not well studied. We aimed to characterise molecular features of microsatellite stable (MSS) TMB-H gastrointestinal tumours.

Methods: Molecular alterations of 48 606 gastrointestinal tumours from Caris Life Sciences (CARIS) identified with next-generation sequencing were compared among MSS-TMB-H, dMMR/MSI-H, and MSS-TMB-low (L) tumours, using χ² or Fisher's exact tests. Antitumour immune response within the tumour environment was predicted by analysing the infiltration of immune cells and immune signatures using The Cancer Genome Atlas database. The Kaplan-Meier method and the log-rank test were used to evaluate the impact of gene alterations on the efficacy of immune checkpoint inhibitors in MSS gastrointestinal cancers from the CARIS database, a Memorial Sloan Kettering Cancer Center cohort, and a Peking University Cancer Hospital cohort.

Findings: MSS-TMB-H was observed in 1600 (3·29%) of 48 606 tumours, dMMR/MSI-H in 2272 (4·67%), and MSS-TMB-L in 44 734 (92·03%). Gene mutations in SMAD2, MTOR, NFE2L2, RB1, KEAP1, TERT, and RASA1 might impair antitumour immune response despite TMB-H, while mutations in 16 other genes (CDC73, CTNNA1, ERBB4, EZH2, JAK2, MAP2K1, MAP2K4, PIK3R1, POLE, PPP2R1A, PPP2R2A, PTPN11, RAF1, RUNX1, STAG2, and XPO1) were related to TMB-H with enhanced antitumour immune response independent of dMMR/MSI-H, constructing a predictive model (modified TMB [mTMB]) for immune checkpoint inhibitor efficacy. Patients with any mutation in the mTMB gene signature, in comparison with patients with mTMB wildtype tumours, showed a superior survival benefit from immune checkpoint inhibitors in MSS gastrointestinal cancers in the CARIS cohort (n=95, median overall survival 18·77 months [95% CI 17·30-20·23] vs 7·03 months [5·73-8·34]; hazard ratio 0·55 [95% CI 0·31-0·99], p=0·044). In addition, copy number amplification in chromosome 11q13 (eg, CCND1, FGF genes) was more prevalent in MSS-TMB-H tumours than in the dMMR/MSI-H or MSS-TMB-L subgroups.

Interpretation: Not all mutations related to TMB-H can enhance antitumour immune response. More composite biomarkers should be investigated (eg, mTMB signature) to tailor treatment with immune checkpoint inhibitors. Our data also provide novel insights for the combination of immune checkpoint inhibitors and drugs targeting cyclin D1 or FGFs.

Funding: US National Cancer Institute, Gloria Borges WunderGlo Foundation, Dhont Family Foundation, Gene Gregg Pancreas Research Fund, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Victoria and Philip Wilson Research Fund, Fong Research Project, Ming Hsieh Research Fund, Shanghai Sailing Program, China National Postdoctoral Program for Innovative Talents, China Postdoctoral Science Foundation, National Natural Science Foundation of China.

Figure 1.. The relationship between dMMR/MSI-H and TMB-H status in GI cancers.

A. An overview of the distribution of MSI and TMB status in GI cancers. Of 48606 total patients, 1600 patients are MSS&TMB-H, 2272 patients are dMMR/MSI-H, 44734 patients are MSS&TMB-L. TMB-H cutoff: 10 mut/Mb. B. Distribution of MSI and TMB status amongst various tumor histologies (n=12) in GI cancers. Abbreviations: MSS, microsatellite stability; MSI-H, microsatellite instability-High; dMMR, mismatch-repair deficiency; TMB-H/L, tumor mutation burden-high/low; GI cancer, gastrointestinal cancer.

Figure 2.. Distinct genomic mutations among dMMR/MSI-H, MSS&TMB-H, MSS&TMB-L subgroups in GI cancers.

A. The landscape of gene mutations related to TMB-H, independent of dMMR/MSI-H in GI cancers (All q<0.0001, Foldchange>1.5, ranked by q value). B. The landscape of genes with similar mutation rates between dMMR/MSI-H and MSS&TMB-H subgroups, but significantly lower in the MSS&TMB-L subgroup (All q<0.0001, Foldchange>3, ranked by q value). C. The landscape of genes with significantly lower mutation rates in the MSS&TMB-H subgroup than in the dMMR/MSI-H subgroup, but significantly higher mutation rates than in MSS&TMB-L subgroup (All q<0.0001, ranked by Q value). Abbreviations: MSS, microsatellite stability; MSI-H, microsatellite instability-High; dMMR, mismatch-repair deficiency; TMB-H/L, tumor mutation burden-high/low; GI cancer, gastrointestinal cancer.

Figure 3.. Distinct features of copy number amplifications among dMMR/MSI-H, MSS&TMB-H and MSS&TMB-L in GI cancers (Ranked by q value, q<0.05).

Abbreviations: MSS, Microsatellite stability; MSI-H, Microsatellite instability-high; dMMR, mismatch-repair deficiency; TMB-H/L, tumor mutation burden-high/low; GI cancer, gastrointestinal cancer.

Figure 4.. Differences of gene fusions, HER2 status and PD-L1 expression among dMMR/MSI-H, MSS&TMB-H and MSS&TMB-L subgroups in GI cancers.

A. Comparison of actionable gene fusions among MSS&TMB-H, dMMR/MSI-H and MSS&TMB-L subgroups (All P<0.0001). B. Comparison of HER2 positivity evaluated by CISH, IHC and copy number analysis among MSS&TMB-H, dMMR/MSI-H and MSS&TMB-L subgroups (All P<0.0001). C. Comparison of PD-L1 expression among MSS&TMB-H, dMMR/MSI-H and MSS&TMB-L subgroups (All P<0.0001). For PD-L1 expression, Dako Link 48 platform for Dako 22C3 pharmDx kits and Ventana Benchmark Ultra platform for SP142 assay kit were used for gastroesophageal cancer and other GI cancers respectively. Abbreviations: MSS, microsatellite stability; MSI-H, microsatellite instability-High; dMMR, deficient mismatch repair; TMB-H/L, tumor mutation burden-high/low; IHC, immunohistochemical staining, IHC; CISH, Chromogenic In Situ Hybridization; Copy number amplification, CNA; GI cancer, gastrointestinal cancer.

Figure 5.. The impact of gene mutations in mTMB signature on tumor immune environment and ICI efficacy in GI cancers.

A. The difference of mutation counts between GI tumors with and without gene mutations in mTMB signature. B. The difference of neoantigens between GI tumors with and without gene mutations in mTMB signature. C. The difference of BCR repertoires between GI tumors with and without gene mutations in mTMB signature. D. The association of mTMB signature with immune signature in GI cancers. E. The impact of gene mutations in mTMB signature on the infiltration of immune cells in GI cancers using CIBERSORT. F. The impact of gene mutations in mTMB signature on the overall survival in patients with MSS GI cancers who received ICIs in the CARIS database. Abbreviations: MSS, microsatellite stability; MSI-H, microsatellite instability-High; TMB-H/L, tumor mutation burden-high/low; TCGA, The Cancer Genome Atlas; GI cancer, gastrointestinal cancer; mTMB, modified TMB; ICI, immune checkpoint inhibitor.