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

Abstract

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.

Figure 1.

(A) Prevalence of RAS mutations in colorectal cancer across seven tissue and one cfDNA cohort, (B) variant allele frequency of RAS mutation by class and (C) relative variant allele frequency of RAS variants by class. *Includes patients with high depth sequencing and known variant allele frequencies from MDA CMS 46, MDA T200 and Project Genie cohorts

Figure 2.

Impact of (A) RAS/BRAF mutations and (B) RAS mutation class on overall survival among patients with metastatic colorectal cancer.

Figure 3.

Functional characterization of MAPK signaling for (A) 114 RAS variants assessed using the Novellus FACT assay with (B) representative fluorescent microscopy images from the FACT assay. Values in (A) represent mean +/− 95% confidence interval.

Figure 4.

Functional annotation of RAS mutation activation status using the Ba/F3 transformation assay.

Figure 5.

Impact of cetuximab treatment on tumor volume in mouse xenograft models derived from SW48 isogenic clones carrying select RAS mutations. (A) Fold change in size of xenograft at the end of 21 days of cetuximab treatment relative to control and (B) tumor size during 21-day treatment cycle with cetuximab or control (mean +/− standard error) for each tested variant.