Abstract P6-04-04: Hypoxia Is Associated with Somatic Loss of BRCA1 Protein and Pathway Activity in Triple Negative Breast Cancer
Authors: Neumeister V , Li J , Lindner R , Sullivan C , Glazer P , Tuck DP , Rimm DL , Harris LN. 
Published: April 2011

Abstract
Background: Triple negative breast cancer (TNBC) has loss of BRCA1 activity either through germline mutation, epigenetic modification or negative transcriptional regulators. Yale investigators have demonstrated that tumor hypoxia leads to loss of critical DNA repair activities, including BRCA1, RAD-51 and γ-H2AX (Bindra RS, et al. Cancer Res 2005;65(24): 11597-604). This study was designed to explore the relationship of BRCA1 loss and hypoxia using Carbonic Anhydrase IX (CA-IX), a downstream target of hypoxia-induced factor 1a (HIF-1a), an accepted surrogate biomarker for tumor hypoxia, in TNBC. Methods: Two cohorts of breast tumors from Yale archival materials were studied. The first consisted of 660, unselected breast tumors, the second a cohort of 130 TNBC, both with long term clinical followup. Automated Quantitative Analysis (AQUA) was used to detect the intensity of BRCA1 and CAIX within specific subcellular compartments. CA-IX antibody M75 was provided by the J Zavada lab (1:10000), BRCA1 monoclonal antibody M110 (Ab-1) was purchased from Calbiochem (1:1000). The 130 TNBC cohort was also subjected to whole genome expression analysis. In brief, tissue core biopsies from tumor blocks were subjected to nucleic acid extraction using RecoverAll Total Nucleic Acid Isolation kit (Applied Biosystems) and 600ng total RNA were processed by the Keck Microarray Facility for the Illumina DASL platform. Statistical analysis of gene expression data was carried out in Bioconductor/R software. A set of relevant signatures was selected by enrichment analysis of modules identified by principal component analysis. Signature scores were computed as Pearson correlation between the signature vector of gene contributions and each sample's expression profile for these genes. Results: In the cohort of 660 specimens, 22 were found to have high CA-IX expression score (above cut-off value of 10). Cut-off was based on known positive cell lines and corresponding AQUA scores, as well as visual confirmation of positivity. A negative correlation (Rho=-0.6, p=0.0165) of BRCA1 nuclear protein with CA-IX level was found and this data was reproducible on a duplicate array. Of note, 14 out of 22 hypoxic breast tumors were from triple negative breast cancers (TNBC) (p=0.0034). In the triple negative cohort, CAIX staining was positive in 20 % of cases (n=15/75) and in these samples it was associated with the 2002 van ‘t Veer BRCA1 mutant signature (Rho=0.51). Positive CAIX staining was also associated with worse outcome (p=0.046) as was CAIX mRNA expression (p=0.02).
Conclusions: In human breast tumors BRCA1 nuclear expression is negatively correlated with tumor hypoxia, measured by CAIX. A subset of TNBC has higher levels of hypoxia and BRCA1 signatures. This data suggests that CAIX may be a useful biomarker for BRCA1 loss, and possibly for response to PARP inhibitor therapy. This will be evaluated in our recently completed trial of BSI-201+Irinotecan.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-04.