377 FGFR protein phosphorylation as a biomarker for FGFR-targeted therapy in cholangiocarcinoma

Objectives/Goals: Cholangiocarcinoma is an aggressive cancer with a poor prognosis. Fibroblast Growth Factor Receptor 2 fusions are therapeutic targets in ~20% of cholangiocarcinoma, which restricts their benefit to only a subset of patients. We investigate reverse phase protein array, as a novel assay for biomarker identification in cholangiocarcinoma. Methods/Study Population: Tumor samples were obtained via our registry (OSU-13053, NCT02090530). RPPA analysis was performed on tumor samples from 24 cholangiocarcinoma patients. Of these, 12 had FGFR2 fusions, 4 had FGFR2 SNVs, and 8 were FGFR wildtype. Tumor content was enriched using laser capture microdissection prior to analysis by reverse phase protein array. RPPA signal was measured by antibody binding to the phosphorylated tyrosine residue at location Y653.654 of FGFR2. Additional analysis was done for the following proteins: CD3, RSK, SGK1, STAT3, VEGFR2, 4EBP1, AKT, EGFR, eIF4G, ERK, FSR2, HLA, mTOR, p38 MAPK, p70S6K, p90RSK, PD-L1, PDGFR, RET, SHC, STAT, and ZAP70. Results/Anticipated Results: There was not a significant difference between FGFR2-phosphorylation in tumors with FGFR2 genomic alterations and tumors with wildtype FGFR. On the other hand, CD3 protein abundance was significantly lower in FGFR-altered tumors compared to wildtype, potentially indicating a cold tumor immune microenvironment in these patients. We plan to perform RPPA analysis on 61 more cases (17 FGFR-altered and 44 FGFR-wildtype) to generate a more robust sample size for subsequent analysis. Furthermore, we have access to 580 additional archival cholangiocarcinoma tumors for RPPA analysis (IRB approved protocol OSU-15030). We plan to sequence these tumors to determine FGFR status. We predict approximately 116 (20%) of these cases to be FGFR-altered. Discussion/Significance of Impact: Our results suggest that FGFR wildtype tumor may have increased FGFR pathway activation and could respond to FGFR inhibitors. Additionally, the cold tumor microenvironment in FGFR-altered tumors provide rationale for combination of FGFR inhibitors and immunotherapy. These results will be confirmed by RPPA analysis on additional cases.