Abstract LB-447: Functional validation of a genetic locus in the VEGFR-1 tyrosine kinase (TK) domain as a predictive marker for bevacizumab

Abstract

Abstract Background There are no validated biomarkers to guide patient selection for treatment with the anti-VEGF antibody bevacizumab. We recently identified a locus, consisting of 4 SNPs in the VEGFR-1 TK domain, that correlated with progression-free (PFS) and overall survival (OS) of bevacizumab-treated pancreatic cancer patients in the AViTA phase III trial. The causal SNP underlying this association and its effect on VEGFR-1 expression or function are not yet known. Methods We conducted a fine-mapping study using data from the 1000 Genomes Project to identify the causal variant in this locus. The candidate SNP was functionally validated by studying effects on VEGFR-1 RNA (RT-PCR) or protein (ELISA) expression. Transcription and translation were measured using transient transfection and rabbit reticulocyte assays. Human umbilical vein endothelial cells (HUVECs) were isolated from healthy newborns. Association of causal SNPs was replicated in bevacizumab-treated renal cancer patients from the AVOREN phase III trial via Sequenom genotyping. Results Fine-mapping identified 48 common SNPs linked to one of the 4 SNPs in the VEGFR-1 locus. In silico functional prediction revealed that only one SNP (rs7993418) was located in exon 28. No other SNPs were located in exons or functionally important regions. Remarkably, rs7993418 is a synonymous SNP introducing a shift in codon usage (TAT to TAC codon) at an evolutionary conserved tyrosine (Tyr1213Tyr). In vitro transcription/translation of VEGFR-1 cDNAs carrying either the wildtype TAT or mutant TAC codon revealed that both constructs transcribe equal amounts of mRNA, whereas a 27% increased VEGFR-1 protein expression was found for TAC versus TAT-carrying constructs (P<0.001). Overexpression of both cDNAs in HEK293T cells demonstrated a similar increase in VEGFR-1 protein, but not mRNA expression for the TAC construct (P<0.001). Since transmembrane and soluble VEGFR-1 levels strongly correlate, we studied the effect of rs7993418 on sVEGFR-1 expression in human plasma. We found 18% and 19% increased median sVEGFR-1 levels in homozygous carriers of the TAC codon in two independent cohorts (P=0.006 and P=0.014). Primary HUVECs homozygous for the TAC codon also showed increased tmVEGFR-1 and sVEGFR-1 protein expression (P=0.049 and P=0.044), leading to increased VEGFR-1 downstream ERK1/2 signaling upon stimulation with the VEGFR-1 specific ligand PlGF (P<0.05). Finally, association of rs7993418 with bevacizumab treatment outcome was also replicated in AVOREN (per-allele HR=1.8; P=0.033). Conclusions The synonymous rs7993418 SNP (Tyr1213Tyr) is a functional SNP that potentially underlies the association of the VEGFR-1 locus with bevacizumab treatment outcome. In particular, rs7993418 causes a shift in codon usage, leading to increased VEGFR-1 expression and enhanced downstream signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-447. doi:1538-7445.AM2012-LB-447