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.

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

“…We find that concurrent BRAF V600E and RAS hotspot mutations are extremely rare and are only seen in MSI-H CRC. Four cases (9%) showed co-occurrence between a typical KRAS mutation and an atypical KRAS V14I of cases, as has also been seen by Pietrantonio et al 18 This mutation likely confers higher MAPK activity than wild-type KRAS , as recently proposed by Loree et al 19 Furthermore, we show that dual MAPK pathway driver mutations co-occur within the same tumor cell at a relatively high frequency. Our findings contradict the classic belief that MAPK pathway driver mutations such as RAS hotspot mutations and BRAF V600E occur only in mutual exclusivity of each other.…”

Identification of actionable targets for breast cancer intervention using a diversity outbred mouse model

“…We interrogated the clinical significance of candidate genes identified in human Chr 2, Chr X, Chr 6 and Chr 19 ( Table S2 ) by analyzing their association with breast cancer outcome using the Real World Outcomes data (Caris Life Sciences). 38 The analysis was conducted in a cohort of 3,533 women with primary breast cancer and a second cohort of 4,870 women with metastatic breast cancer. Whole transcriptome sequencing was performed with mRNA isolated from formalin-fixed paraffin-embedded tumor samples using the Illumina NextSeq 600 platform (Illumina, Inc., San Diego, CA) in a CAP-accredited, CLIA certificated laboratory (Caris Life Sciences).…”

“…We queried the deidentified real-world evidence (RWE) dataset from the Caris Life Sciences using CODEai, a real-word clinic-genomic data platform that integrates patient’s molecular data with cancer treatment and clinical information obtained from insurance claims data. 38 Breast cancer patients with whole transcriptome sequencing performed on mRNA isolated from a formalin-fixed paraffin-embedded tumor sample using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) were included in the analysis. Cohorts included 4,920 patients with metastatic disease and 3,568 patients with primary tumors.…”

Development and Validation of a Targeted ‘Liquid’ NGS Panel for Treatment Customization in Patients with Metastatic Colorectal Cancer

“…Interestingly, at first line disease progression (PD1), our targeted NGS panel detected an ‘atypical’ KRAS mutation, namely L19F. This mutation has been described to have a potential intermediate phenotype, associated with increased RAS pathway signaling, but limited oncogenic potential and continued response to cetuximab [ 41 ]. This ‘anti-EGFR naïve’ patient was challenged with anti-EGFR at second line, based on the baseline RAS wild-type status, and was progression-free at 4 months of treatment before being lost to follow-up.…”