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Hypoxia-induced protein CAIX is associated with somatic loss of BRCA1 protein and pathway activity in triple negative breast cancer

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Abstract

The purpose of this study is to explore the relationship between tumor hypoxia assessed by CA IX protein expression and loss of BRCA1 function in triple negative breast cancer (TNBC). Protein expression of CA IX and BRCA1 was evaluated by AQUA™ technology on two breast cancer cohorts: an unselected cohort of 637 breast cancer patients and a TNBC cohort of 120 patients. Transcriptional profiling was performed on FFPE samples from the TNBC cohort to evaluate a gene expression signature associated with BRCA1 mutation (van’t Veer et al., Nature 415(6871):530–536, 2002). CA IX is expressed in 7 % of the unselected breast cancer cohort and in 25 % of the TNBCs and is significantly associated with the triple negative phenotype. CA IX protein expression and BRCA1 protein expression are inversely correlated in both cohorts. Patients expressing high levels of CA IX show significantly worse overall survival (p = 0.02). Importantly, high CA IX protein expression occurs in patients who show the BRCA1 mutant signature and low levels of BRCA1 protein. These data suggest that elevated CA IX protein in TNBC is associated with a BRCA1 mutant signature and loss of BRCA1 function. CA IX may be a useful biomarker to identify triple negative patients with defective homologous recombination, who might benefit from PARP inhibitor therapy.

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Abbreviations

FFPE:

Formalin-fixed paraffin-embedded

HIF:

Hypoxia-induced factor

PARP:

Poly(ADP-ribose) polymerase

TNBC:

Triple negative breast cancer

TMA:

Tissue microarray

AQUA:

Automated quantitative analysis

CA IX:

Carbonic anhydrase IX

HR:

Homologous recombination

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Acknowledgments

Part of this study has been funded by the Susan G. Komen Grant (Komen KG090562).

Conflict of interest

D.L. Rimm is a stockholder in and consultant to HistoRx Inc., the exclusive licensee to the Yale owned AQUA technology. All the other authors declare that they do not have any competing interests.

Ethical standards

This project was performed in compliance with the relevant ethical standards in the US.

Author information

Author notes

  1. Robert Lindner

    Present address: Heidelberg University, INF 364, 69120, Heidelberg, Germany

  2. Jia Li

    Present address: VA Connecticut Healthcare System, 950 Campbell Avenue, West Haven, CT, 06516, USA

  3. Lyndsay Harris

    Present address: Case Western Reserve University, 11100 Euclid Avenue, Weam 145, Cleveland, OH, 44106, USA

Authors and Affiliations

  1. School of Medicine, Yale University, 310 Cedar Street, New Haven, CT, 06520, USA

    Veronique M. Neumeister, Catherine A. Sullivan, Robert Lindner, Kimberley Lezon-Geyda, Jia Li, Maritza Martel, Peter M. Glazer, David P. Tuck, David L. Rimm & Lyndsay Harris

  2. Institute of Molecular Genetics, Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic

    Jan Zavada

  3. Department of Pathology, School of Medicine, Yale University, BML Rm112, 310 Cedar Street, New Haven, CT, 06517, USA

    Veronique M. Neumeister

Authors
  1. Veronique M. Neumeister
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  2. Catherine A. Sullivan
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Corresponding author

Correspondence to Veronique M. Neumeister.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10549_2012_2232_MOESM1_ESM.ppt

Supplemental Fig. 1: Antibody validation of CA IX and staining examples for BRCA1 protein. The CA IX antibody was validated with Western Blot, showing expression of CA IX at the right molecular weights in A 431 and MDA MB 468 cell lines. A faint band is also present in MDA MB 453 cells. Immunofluorescent staining and AQUA analysis of CA IX protein on a TMA containing the same cell lines confirms positivity of these cell lines as assessed via IF (A). AQUA analysis of index TMAs shows excellent reproducibility of staining runs with an r2 of 0.98 (B). Representative staining images of CA IX on cell lines (C) and breast cancer samples (D) are shown as well. The BRCA1 antibody has been validated previously and representative staining samples are shown in E. (PPT 330 kb)

10549_2012_2232_MOESM2_ESM.ppt

Supplemental Fig. 2: Analysis of BRCA1 protein expression on the unselected breast cancer cohort and the TNBC cohort. BRCA1 protein expression was analyzed in a similar manner to CA IX expression. The distribution and reproducibility/heterogeneity is shown in A and C. In both cohorts BRCA1 does not show any prognostic value as assessed with survival analysis. To be able to perform Kaplan–Meier analysis both cohorts were binarized for low and high BRCA1 expression using the median BRCA1 score (B and D). However within the TNBC cohort BRCA1 protein expression is negatively correlated with nodal status, reaching statistical significance with a p value of 0.018 (E). (PPT 359 kb)

10549_2012_2232_MOESM3_ESM.pptx

Supplemental Fig. 3: Correlation of CA IX and BRCA1 protein expression and the BRCA1 mutant signature. The 3 panels of this figure show the same correlations as Fig. 4, illustrated as 2D plots. Panel A illustrates the relationship between BRCA1 AQUA scores and the BRCA1 signature, B between CA IX AQUA scores and BRCA1 AQUA scores and panel C shows the relationship between CA IX AQUA scores and the BRCA1 signature score. (PPTX 87 kb)

Supplementary material 4 (PPTX 56 kb)

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Neumeister, V.M., Sullivan, C.A., Lindner, R. et al. Hypoxia-induced protein CAIX is associated with somatic loss of BRCA1 protein and pathway activity in triple negative breast cancer. Breast Cancer Res Treat 136, 67–75 (2012). https://doi.org/10.1007/s10549-012-2232-0

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  • DOI: https://doi.org/10.1007/s10549-012-2232-0

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