Clinical and Functional Characterization of Atypical KRAS/ NRAS Mutations in Metastatic Colorectal Cancer

Clin Cancer Res. 2021 Aug 15;27(16):4587-4598. doi: 10.1158/1078-0432.CCR-21-0180. Epub 2021 Jun 11.


Purpose: Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist.

Experimental design: We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations.

Results: KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts.

Conclusions: We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

Publication types

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

MeSH terms

  • Aged
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / pathology
  • Female
  • GTP Phosphohydrolases / genetics*
  • Humans
  • Male
  • Membrane Proteins / genetics*
  • Middle Aged
  • Mutation*
  • Neoplasm Metastasis
  • Proto-Oncogene Proteins p21(ras) / genetics*


  • KRAS protein, human
  • Membrane Proteins
  • GTP Phosphohydrolases
  • NRAS protein, human
  • Proto-Oncogene Proteins p21(ras)