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

JCO Precis Oncol. 2022 Mar:6:e2100365. doi: 10.1200/PO.21.00365.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Colorectal Neoplasms* / genetics
  • Humans
  • Microsatellite Instability
  • Mitogen-Activated Protein Kinases / genetics
  • Mutation / genetics
  • Proto-Oncogene Proteins B-raf* / genetics
  • Proto-Oncogene Proteins p21(ras) / genetics


  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Mitogen-Activated Protein Kinases
  • Proto-Oncogene Proteins p21(ras)