Purpose: To discover genetic determinants of overall survival (OS) in metastatic renal cell carcinoma (mRCC) patients treated with sorafenib.
Patients and Methods: Information for 11,117 germline DNA variants from 56 genes was obtained, using both Illumina GoldenGate genotyping and imputation, from 295 patients originally enrolled in the phase III TARGET trial (155 sorafenib and 140 placebo patients). OS was this genotyping study's primary endpoint, and associations between variants and OS were tested using multivariate Cox regression. Associations were considered significant if P<0.05 and FDR q<0.1. Bioinformatic resources helped prioritize variants for functional validation. Haploreg v4 and RegulomeDB were used to help mine ENCODE data, and SIFT/PolyPhen-2 predicted functional effects of nonsynonymous variants. Dual reporter gene luciferase assays assessed variant effects on promoter activity. Isogenic cell lines were created, and direct cell proliferation assays elucidated variant effects on cell proliferation/cytotoxicity, while endothelial branch formation assays assessed variant effects on endothelial tube formation. Western blotting evaluated nonsynonymous variant effects on receptor phosphorylation, and pulse-chase experiments assessed variant effects on post-translational processing of proteins.
Results: Five intragenic variants significantly associated with OS. Among patients in the sorafenib arm, VEGFA rs1885657 (HR=17.3, 95% CI 5.7-52.7; P=1.4x10-4), ITGAV rs3816375 (HR=5.9, 95% CI 2.1-16.4; P=4.9x10-4), and WWOX rs8047917 (HR=4.1, 95% CI 1.9-8.8; P=3.3x10-4) associated with shorter OS. Among patients from both arms, FLT4 rs307826 (HR=13.8, 95% CI 3.0-62.6; P=1.2x10-4) and VEGFA rs3024987 (HR=3.0, 95% CI 1.7-5.4; P=8.3x10-5) associated with shorter OS. No significant associations were discovered in the placebo arm. Bioinformatic evidence was used to prioritize rs1885657, rs58159269 (in complete linkage disequilibrium with rs1885657), and rs307826. rs1885657 and rs58159269 increased luciferase activity in human endothelial (TIME, LPEC) and mRCC (Caki-1) cells, and rs58159269 increased cell proliferation/reduced sorafenib cytotoxicity, and reduced endothelial tube formation in TIME cells. rs307826 increased VEGFR-3 phosphorylation, and led to post-translational VEGFR-3 processing differences in HUVECs.
Conclusions: Regulatory VEGFA and FLT4 variants associated with OS in mRCC patients induce cellular alterations that affect angiogenesis and sorafenib activity. These are novel and potentially impactful clinical biomarkers for mRCC and sorafenib, and their relevance to other multikinase inhibitors used in mRCC should be tested.
Citation Format: Daniel James Crona, Andrew D. Skol, Veli-Matti Leppänen, Dylan M. Glubb, Amy S. Etheridge, Eleanor Hilliard, Carol Peña, Yuri K. Peterson, Nancy Klauber-DeMore, Kari K. Alitalo, Federico Innocenti. Clinical biomarkers of survival in renal cell carcinoma patients treated with sorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1625.