ILF2 as a predictor of cisplatin resistance and clinical outcomes in intrahepatic cholangiocarcinoma.

3122 Background: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive biliary tract cancer with increasing global incidence and limited therapeutic options, partly due to the low prevalence of actionable genomic alterations such as FGFR2 fusions (~10%) and IDH1 mutations (~13%). Using single-cell transcriptomic analyses, we identified recurrent amplification at chromosome 1q21.3 as a dominant genomic alteration in iCCA tumor cells. Given our prior evidence linking 1q21.3 amplification to DNA damage–response (DDR) processes and chemotherapy resistance in multiple cancer types, we focused on Interleukin enhancer binding Factor 2 (ILF2), a gene within 1q21.3, and systematically evaluated its multi-omic features and clinical relevance in iCCA using multiple independent clinical datasets. Methods: Single-cell RNA sequencing datasets were analyzed using Single-Cell Variational Aneuploidy (SCEVAN) to identify recurrent chromosomal alterations. Bulk RNA and DNA sequencing datasets were assessed for ILF2 copy number variation (CNV) and mRNA expression. Clinical validation was performed using independent iCCA cohorts from Keio University and SJCI (n=67) and a public dataset (GSE89748; n=49). In vitro assays validated the functional role of ILF2 in DDR and cisplatin sensitivity of iCCA cell lines (n=3). Results: SCEVAN analysis of GSE125449 (n=8) and GSE138709 (n=5) revealed that 1q21.3 was the only common highest amplified region of iCCA tumor cells (29.7% and 31.1%, respectively). TCGA-CHOL dataset (n=32) demonstrated that ILF2 CNV amplification occurred in 46.7% of cases and was strongly correlated with increased ILF2 mRNA expression (r = 0.730, p < 0.001), which was significantly higher in tumor tissue than normal liver tissue in iCCA patients (p < 0.001). In the iCCA validation cohort (n=67) and GSE89748 dataset (n=49), ILF2 mRNA level upregulation was significantly associated with shorter recurrence-free survival (log-rank test; p < 0.001 and p = 0.047, respectively), and independently predicted recurrence risk (multivariate Cox regression analysis HR = 4.63, 95% CI 1.71 – 12.6, p = 0.003 and HR = 2.73, 95% CI 1.10 – 6.80, p = 0.031, respectively). Consistently, ILF2 protein expression as assessed by multiplex immunofluorescence was significantly lower in FFPE tumor tissues from cisplatin responders than non-responders of iCCA patients (p < 0.001, n=16). Across iCCA cell lines, ILF2 interacted with DDR–related factors to suppress R-loop and γH2AX activities; both events are involved in promoting cisplatin resistance. Conclusions: ILF2 is a frequent novel functional gene strongly associated with adverse clinical outcomes and cisplatin resistance of iCCA. This supports its role as a clinically relevant and theranostic biomarker related to DDR in iCCA patients.