Beyond fibroblast growth factor receptor 2 (FGFR2) fusions: Mutations and amplifications in intrahepatic cholangiocarcinoma.

4124 Background: FGFR2 alterations occur in 10-20% of iCCA. While FGFR-directed therapies are established for FGFR2 fusions, the genomic landscape and therapeutic relevance of non-fusion FGFR2 alterations remain poorly defined. Methods: Comprehensive genomic profiling was performed on 9,661 iCCA cases negative for FGFR2 rearrangements/fusions to identify FGFR2 short-variant sequence mutations and amplifications (Non- FGFR2 SV/AMP). Biomarkers included tumor mutational burden (TMB), microsatellite instability (MSI), homologous recombination deficiency deficiency signature (HRDsig), and PD-L1 expression (TPS). Genomic features were compared with FGFR2 -wild-type ( FGFR2 wt) iCCA. Clinical outcomes were assessed in a single-center cohort treated with FGFR inhibitors. Results: Non- FGFR2 SV/AMP alterations were identified in 324 cases (3.4%), consisting of SV (92.8%), AMP (6.1%), or both (1.1%). Among short variants, 66.0% localized to the extracellular domain, 23.1% to the transmembrane domain, 7.69% to the kinase domain, and 3.21% to the cytoplasmic non-kinase domain. Compared with FGFR2 wt tumors, Non- FGFR2 SV/AMP iCCA occurred more frequently in females (57.7% vs 50.0%, p = 0.02) and showed similar age, genomic ancestry, trinucleotide signatures, MSI-high status (1.6% vs 1.8%), TMB > 10 mt/Mb (2.2% vs 3.6%), PD-L1 positivity (18.8% vs 21.4%), and HRDsig positivity (4.6% vs 5.0%). Co-occurring actionable alterations ( IDH1 , ERBB2, BRAF , MTAP loss) were less frequent in Non-FGFR2 SV/AMP tumors, while BAP1 , NF2 and TSC1 alterations were enriched. Among 42 patients with non-fusion FGFR2 alterations treated at a single center, 16 (38%) received FGFR-directed therapy including pemigatinib, futibatinib, tinengotinib, lirafrugatinib and derazantinib. The objective response rate was 25% (95% CI: 7.3-52.4). Best responses included partial response (25%), stable disease (50%), and progressive disease (25%), with a median duration of response of 5.4 (95% CI: 1.9-8.9) months. Median overall survival was significantly longer in patients receiving FGFR-targeted therapy compared with those who did not (28.4 months 95% CI, 23.8-33.0 vs 14.6 months 95% CI, 6.25-23.0; p = 0.023). Recurrent FGFR2 mutations involved N549K (kinase), C382R (transmembrane), and Y375C (extracellular) domains. Conclusions: Non-fusion FGFR2 sequence mutations and amplifications define a distinct subset of iCCA with fewer alternative actionable drivers and sensitivity to FGFR-targeted therapy. These findings support consideration of FGFR inhibition beyond FGFR2 fusions and highlight the need for refined patient selection strategies.