Abstract 3616: Validation and fitness testing of a quantitative immunoassay for HIF1α in biopsy specimens
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…
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The HIF-1α immunoassay used was validated.
“Methods for specimen collection to preserve HIF-1α in 18-gauge tumor needle biopsies and an assay to quantitatively measure HIF-1α levels in human tissue biopsy were validated (Supplementary Data section, online only) [15].”
From Pilot trial of EZN-2968, an antisense oligonucleotide inhibitor of hypoxia-inducible factor-1 alpha (HIF-1α), in patients with refractory solid tumors (2014) · cites this paper as [15]
Quantifying HIF-1α protein in human tumor biopsies remained challenging despite a validated assay.
“Even though our group has developed a validated assay to measure HIF-1α protein levels that provided reliable and reproducible results [15], quantifying HIF-1α protein in human tumor biopsy samples remained challenging.”
From Pilot trial of EZN-2968, an antisense oligonucleotide inhibitor of hypoxia-inducible factor-1 alpha (HIF-1α), in patients with refractory solid tumors (2014) · cites this paper as [15]
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Jeong, W., Rapisarda, A., Park, S. R., Kinders, R. J., Chen, A., Melillo, G., Turkbey, B., Steinberg, S. M., Choyke, P., Doroshow, J. H., et al. (2014) · Cancer Chemotherapy and Pharmacology · doi:10.1007/s00280-013-2362-z · Cited by 159 · see citing works
Self-citation
- Authors
- Park, S. R., Kinders, R. J., Khin, S., Hollingshead, M., Parchment, R. E., Tomaszewski, J. E., & Doroshow, J. H.
- Affiliations
- National Cancer Institute (US)
- Science Applications International Corporation (United States) (US)
- ORCID
- Venue
- Cancer Research
- Published
- 2012-04
- Volume
- 72
- Issue
- 8_Supplement
- Pages
- 3616-3616
- ISSN
- 0008-5472 (ISSN-L)
- 1538-7445
- OpenAlex
- W2088536642
- Cited by
- 1
- References
- 0
- Citations by year
- 2013: 1
- Type
- conference abstract
- Language
- en
- Open access
- closed
- Locations
- Cancer Research (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Health Sciences › Medicine › Hepatology › Hepatocellular Carcinoma Treatment and Prognosis
- Keywords
- HIF-1α, hypoxia, quantitative immunoassay, sample stabilization, 2-hydroxyglutarate, prolyl hydroxylase inhibition, tissue homogenization, pharmacodynamic biomarkers, topotecan, xenograft models, prostate cancer
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