Abstract 3616: Validation and fitness testing of a quantitative immunoassay for HIF1α in biopsy specimens

Abstract

Abstract HIF1α is an important marker of hypoxia in human tumors that is altered in a number of cancers, but a reproducible method to determine changes in HIF1α protein expression in human tumor biopsy specimens has not been available. While HIF1α is being used as a pharmacodynamic marker in clinical trials in the NCI (CTEP# 8610, 8856, 8880), an important limitation in measuring the change in HIF1α induced by cancer therapeutic agents is the lability of the protein, in the presence of oxygen, upon collection of the sample. We have devised a method of specimen collection, handling, and extraction that preserves and stabilizes HIF1α levels in tumor biopsies. Degassed collection buffer yielded a significant increase of recovered HIF1α levels compared to the standard method of flash-freezing. The addition of 2-hydroxyglutarate, an inhibitor of prolyl hydroxylase domain enzyme that is involved in the degradation pathway of HIF1α under normoxic conditions, to the collection buffer showed a trend for improved yield of the HIF1α recovery. Sonication and homogenization with ceramic beads were superior to homogenization with metallic beads or tissue grinding. Employing this specimen-handling method allowed validation of a two-site immunoassay for HIF1α quantitation in solid-tissue extracts generated from tumor biopsies. Intra-assay variability was less than 10%, and inter-assay variability was less than 20%. Accuracy, assessed by spike recovery, was 99 +/− 7%. HIF1α readings declined linearly with decreasing sample load over a range of 1 to 10 µg protein per well. Fitness for purpose was demonstrated by quantifying a reduction in HIF1α protein levels following topotecan or indenoisoquinoline NSC 743400 treatment in an A375 xenograft model, followed by assessment of other xenograft models. HIF1α was also demonstrated to be upregulated under low oxygen tension culture conditions in DU145 human prostate cancer cells. Funded by NCI Contract No. HHSN261200800001E. 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 3616. doi:1538-7445.AM2012-3616