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

Purpose: Mutations in () predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all 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 variants. Using an cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and xenograft models of transduced isogenic clones, we assessed signaling changes across mutations.

Results: exon 2, extended , and atypical mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no codon 117/146 and only one codon 59 mutation was noted. Atypical mutations had worse overall survival than wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; = 0.014). We functionally characterized 114 variants with the FACT assay. All exon 2 and extended mutations appeared activating. Of 57 atypical 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 variants are identified. L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included variants and functionally relevant.