Same-Cell Co-Occurrence of RAS Hotspot and BRAF V600E Mutations in Treatment-Naive Colorectal Cancer.

Abstract

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 <i>RAS</i> hotspot/<i>BRAF</i> 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. 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. Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: <i>NRAS</i> G13D/<i>KRAS</i> G12C, 95%; <i>KRAS</i> G12D/<i>NRAS</i> G12V, 48%; <i>BRAF</i> V600E/<i>KRAS</i> G12D, 44%; and <i>KRAS</i> G12D/<i>NRAS</i> 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%; <i>P</i> = .013). Microsatellite instability-high status was enriched in CRC-DD compared with CRC-SD (23% <i>v</i> 11.4%, <i>P</i> = .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. 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.