Clinical and Functional Characterization of Atypical <i>KRAS</i>/<i>NRAS</i> Mutations in Metastatic Colorectal Cancer.

Abstract

Mutations in <i>KRAS/NRAS</i> (<i>RAS</i>) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all <i>RAS</i> mutations have similar impact, and atypical mutations beyond those in standard guidelines exist. We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical <i>RAS</i> variants. Using an <i>in vitro</i> cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and <i>in vivo</i> xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. <i>KRAS</i> exon 2, extended <i>RAS</i>, and atypical <i>RAS</i> mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, <i>KRAS</i> L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no <i>NRAS</i> codon 117/146 and only one <i>NRAS</i> codon 59 mutation was noted. Atypical <i>RAS</i> mutations had worse overall survival than <i>RAS/BRAF</i> wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; <i>P</i> = 0.014). We functionally characterized 114 variants with the FACT assay. All <i>KRAS</i> exon 2 and extended <i>RAS</i> mutations appeared activating. Of 57 atypical <i>RAS</i> 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. We provide best available evidence to guide treatment when atypical <i>RAS</i> variants are identified. <i>KRAS</i> L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included <i>RAS</i> variants and functionally relevant.