Molecular profiling of colorectal cancer in a clinical diagnostic cohort: RAS/RAF alterations, microsatellite instability, and mismatch repair status.

e15644 Background: Molecular characterization of colorectal cancer (CRC) is essential for therapeutic stratification, particularly for selecting patients eligible for anti-EGFR therapy. Beyond canonical hotspot mutations in KRAS and BRAF, evaluation of microsatellite instability (MSI) and deficient mismatch repair (dMMR) provides prognostic information and guides immunotherapy. Because RAS and certain RAF alterations predict resistance to EGFR-directed antibodies, comprehensive gene assessment is routine. Rare non-hotspot RAS/RAF variants are increasingly identified and present interpretive challenges. This study describes the molecular landscape of CRC in a real-world diagnostic cohort, emphasizing standardized variant curation and implications for anti-EGFR therapy. Methods: We retrospectively analyzed 285 CRC specimens submitted for routine molecular testing. Somatic variants in KRAS, NRAS, BRAF, and PIK3CA were detected using Sanger-sequencing. MSI testing was performed by PCR-fragment analysis, and MMR protein expression (MLH1, PMS2, MSH2, MSH6) by immunohistochemistry (IHC). Variant interpretation followed ACMG and AMP/ASCO/CAP somatic guidelines. Results: KRAS mutations were identified in 37.0% of tumors, including rare KRAS p.Gln22Lys (Q22K, 2 cases) and p.Lys147Asn (K147N, 1 case). BRAF mutations occurred in 7.5% of cases, mainly p.Val600Glu (V600E), with one p.Asn581Ile (N581I). NRAS and PIK3CA mutations were 1.1% and 5.7%, respectively. Overall, 51.3% of tumors harbored one mutation in KRAS, BRAF, NRAS, or PIK3CA. MSI tumors were 37.9% and largely concordant with dMMR IHC (32.3%). Among mutation-positive cases, 13.8% were dMMR and 16.6% MSI-H. 15% of dMMR-positive cases were associated with Lynch syndrome. KRAS Q22K and BRAF N581I were classified as oncogenic gain-of-function variants, while KRAS K147N was likely oncogenic gain-of-function. Alterations occurred across diverse histopathologic subtypes. Conclusions: This cohort highlights the clinical significance of molecular testing in CRC. Canonical hotspot mutations in KRAS, NRAS, PIK3CA and BRAF, detected in 51.3% of cases, are critical for guiding anti-EGFR therapy, as these mutations predict resistance to EGFR-directed monoclonal antibodies. Additionally, 13.8% of mutation-positive tumors were dMMR and 16.6% MSI-H, identifying patients who may benefit from immune checkpoint inhibitors. Rare non-hotspot gain-of-function variants—including KRAS Q22K, BRAF N581I, and KRAS K147N—were also observed and may impact therapy selection. Overall, comprehensive molecular testing, including both hotspot mutation analysis and MSI/dMMR evaluation, is essential for informed treatment planning in CRC.