Development and validation of an immunohistochemistry assay to assess glucocorticoid receptor expression for clinical trials of mifepristone in breast cancer
Authors Baker G, Murphy T, Block T, Nguyen D, Lynch F
Received 1 July 2015
Accepted for publication 21 September 2015
Published 4 December 2015 Volume 2015:7 Pages 361—368
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Jaya Mallela
Peer reviewer comments 3
Editor who approved publication: Professor Kenan Onel
Gabrielle M Baker,1 Tiffany Murphy,2 Thaddeus Block,3,4 Dat Nguyen,3 Frank J Lynch2
1Department of Pathology, The University of Chicago, Chicago, IL, USA; 2QualTek Molecular Laboratories, Newtown, PA, USA; 3Corcept Therapeutics, Menlo Park, CA, USA; 4Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA
Background: Glucocorticoid receptor (GR) activity has been associated with chemotherapy resistance and poor outcomes in patients with triple negative breast cancer (TNBC). The aim of this study was to develop an immunohistochemistry (IHC) assay to assess GR expression in archival formalin-fixed, paraffin-embedded human invasive breast carcinoma samples.
Methods: An optimized GR assay protocol was developed using rabbit monoclonal antibody to GR clone D8H2. Precision and reproducibility of the GR IHC assay was determined by conducting multiple staining runs of four invasive breast carcinoma samples using replicate serial sections. Assay sensitivity was examined in 50 TNBC samples (>10 mm) obtained from a tumor bank, and 43 paired TNBC samples from a tissue microarray (TMA) (1.5 mm). GR positivity was assessed using a percent scoring approach with a ≥10% cutoff for nuclear staining of tumor cells at any intensity. Analysis of the paired TMA cores was performed by averaging the scores of the two cores for each case.
Results: Equivalent cellular patterns of GR reactivity were observed in all replicates from the multiple staining runs; coefficients of variation did not exceed 4.7% for average H-scores greater than 3.4, thus meeting the criteria for assay precision and reproducibility (coefficient of variation ≤20%). GR expression in TNBC single-tissue samples and TMA cores was characterized as mostly nuclear, with some concurrent cytoplasmic reactivity. Eighty-four percent of the 49 evaluable TNBC samples and 60% of the 42 evaluable paired TMA samples were positive for GR expression.
Conclusion: A robust and reproducible GR IHC assay was successfully developed for use in invasive breast carcinoma tissues. Differences in GR expression between larger single tissues and smaller TMA cores illustrate the heterogeneity of the disease, as well as potential intratumoral heterogeneity. This assay is currently being utilized in clinical trials of mifepristone, a GR antagonist, in patients with TNBC.
Keywords: glucocorticoid receptor, immunohistochemistry, triple negative breast cancer, mifepristone
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