Genomic characterization and clinical associations of rare co-mutations in <i>RAS</i> and <i>RAF</i> genes in colorectal cancer.

Abstract

e15581 Background: In colorectal cancer (CRC), mutations in RAS family genes and BRAF are typically mutually exclusive. However, with the widespread application of next-generation sequencing (NGS), case reports of such co-mutations are accumulating, warranting a systematic investigation of their genomic features, cooperative mechanisms, and clinical significance. Methods: A retrospective analysis was performed on a CRC patient cohort who underwent DNA-based NGS using a 733-gene panel (full-length coverage) between 2023 and 2025. Samples were primarily formalin-fixed, paraffin-embedded (FFPE) tissues, mostly from primary sites. Results: Among 1379 CRC patients, 834 harbored RAS and/or RAF mutations. Seventeen patients (1.23%, 17/1379) exhibited concurrent RAS and RAF mutations. The co-mutation patterns were: KRAS+BRAF in 13 patients (76.47%), NRAS+BRAF in 3 (17.65%), and KRAS+ARAF in 1 (5.88%). The cohort included 11 males (64.71%) with a median age of 61 years (range: 49-88), and 15 cases (88.24%) originated in the colon. KRAS variant sites included G12C/D/V (n = 6), A146T/V (n = 4), G13C/A59S/Q61H/K147E (each n = 1). NRAS variants included G12C/D (n = 2) and Q61L (n = 1). These RAS mutations clustered in critical functional domains (exons 2-4), disrupting the P-loop, Switch II region, and G-domain, thereby abrogating guanosine triphosphatase (GTPase) activity and causing constitutive activation. BRAF mutations comprised: Class I V600E (n = 5); Class II G469A/R (n = 2) and K601N (n = 3); Class III D594E/N (n = 3) and G466E (n = 1); and unclassified variants F247L and F468S (each n = 1). These mutations predominantly localized to the kinase domain (exons 11/15), affecting key functional regions including the P-loop, A-loop. The co-occurrence of RAS and BRAF may lead to additive MAPK pathway activation, with cooperative mechanisms varying by BRAF mutation class: (1) RAS + Class III BRAF (low kinase activity): RAS activation provides sustained upstream signaling, potentially maintaining moderate but persistent pathway output. (2) RAS + Class II BRAF (moderate kinase activity, RAS-independent): Co-mutation may amplify signaling via positive feedback or enhanced dimerization. (3) RAS + Class I BRAF V600E (high kinase activity): Both are strong activators; this rare combination might lead to excessive signaling, possibly subject to negative selection during tumor evolution. Conclusions: Co-mutations in RAS and RAF represent a rare but noteworthy genomic event in CRC. They likely confer resistance to single-agent targeted therapies, such as EGFR monoclonal antibodies and BRAF inhibitors. Future therapeutic strategies should consider multi-level MAPK pathway inhibition (combining MEK inhibitors or RAF dimer inhibitors). The precise biological functions and clinical prognosis associated with these co-mutations require further validation with treatment response and survival data.