Purpose: Mitogen-activated protein kinase pathway-activating mutations occur in the majority of colorectal cancer (CRC) cases and show mutual exclusivity. We identified 47 epidermal growth factor receptor/BRAF inhibitor-naive CRC patients with dual RAS hotspot/ BRAF V600E mutations (CRC-DD) from a cohort of 4,561 CRC patients with clinical next-generation sequencing results. We aimed to define the molecular phenotypes of the CRC-DD and to test if the dual RAS hotspot/BRAF V600E mutations coexist within the same cell.
Materials and Methods: We developed a single-cell genotyping method with a mutation detection rate of 96.3% and a genotype prediction accuracy of 92.1%. Mutations in the CRC-DD cohort were analyzed for clonality, allelic imbalance, copy number, and overall survival.
Results: Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: NRAS G13D/ KRAS G12C, 95%; KRAS G12D/ NRAS G12V, 48%; BRAF V600E/ KRAS G12D, 44%; and KRAS G12D/ NRAS G13V, 14%, respectively. Allelic imbalance favoring the oncogenic allele was less frequent in CRC-DD (24 of 76, 31.5%, somatic mutations) compared with a curated cohort of CRC with a single-driver mutation (CRC-SD; 119 of 232 mutations, 51.3%; P = .013). Microsatellite instability-high status was enriched in CRC-DD compared with CRC-SD (23% v 11.4%, P = .028). Of the seven CRC-DD cases with multiregional sequencing, five retained both driver mutations throughout all sequenced tumor sites. Both CRC-DD cases with discordant multiregional sequencing were microsatellite instability-high.
Conclusion: Our findings indicate that dual-driver mutations occur in a rare subset of CRC, often within the same tumor cells and across multiple tumor sites. Their presence and a lower rate of allelic imbalance may be related to dose-dependent signaling within the mitogen-activated protein kinase pathway.
Competing Interests: Ahmet ZehirStock and Other Ownership Interests: Arcus Biosciences, Mirati TherapeuticsHonoraria: Illumina Luciano G. MartelottoPatents, Royalties, Other Intellectual Property: I'm and advisor for OmniScope, a startup tech company that develop test for detecting immune systems responses Rona YaegerConsulting or Advisory Role: Array BioPharma, Natera, Mirati TherapeuticsResearch Funding: Array BioPharma (Inst), Boehringer Ingelheim (Inst), Pfizer (Inst), Mirati Therapeutics (Inst) Britta WeigeltStock and Other Ownership Interests: Repare TherapeuticsConsulting or Advisory Role: Genentech/Roche, Invicro, Ventana Medical Systems, Volition RX, PAIGE, Goldman Sachs, Repare Therapeutics, Lilly, Repare Therapeutics Jorge S. Reis-FilhoLeadership: Grupo OncoclinicasStock and Other Ownership Interests: Repare Therapeutics, PAIGE.AIConsulting or Advisory Role: Genentech/Roche, Invicro, Ventana Medical Systems, Volition RX, Paige.AI, Goldman Sachs, Novartis, Repare Therapeutics, Lilly, Personalis Jaclyn F. HechtmanEmployment: NeoGenomics LaboratoriesStock and Other Ownership Interests: NeoGenomics LaboratoriesHonoraria: WebMD, Illumina, BayerConsulting or Advisory Role: Cor2Ed, Axiom Healthcare Strategies, BayerResearch Funding: Bayer, Lilly, Boehringer IngelheimNo other potential conflicts of interest were reported.