Development and validation of an immunohistochemistry assay to assess glucocorticoid receptor expression for clinical trials of mifepristone in breast cancer

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Abstract

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 intra-tumoral heterogeneity. This assay is currently being utilized in clinical trials of mifepristone, a GR antagonist, in patients with TNBC.

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Most cited references 22

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American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version).

F Sweep, Joseph Hayes, … (2010)

To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers. The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance. Up to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate (false negative or false positive). Most of the issues with testing have occurred because of variation in pre-analytic variables, thresholds for positivity, and interpretation criteria. The Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences. A testing algorithm that relies on accurate, reproducible assay performance is proposed. Elements to reliably reduce assay variation are specified. It is recommended that ER and PgR assays be considered positive if there are at least 1% positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal (normal epithelial elements) and external controls. The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of randomized clinical trials.

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Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies.

K S McCarty, L S Miller, E B Cox … (1985)

Attempts at histochemical localization of estrogen receptor with anti-steroid antibody or some fluoresceinated estrogens have given unacceptable sensitivities and specificities when compared with biochemical methods or clinical response. In the present study a monoclonal antibody against estrogen receptor (H222 Sp gamma) was used on cryostat sections of freshly frozen breast tumors with a peroxidase-antiperoxidase immunoperoxidase technique. Biochemical receptor analyses were by dextran-coated charcoal analyses. Tumors from three separate cohorts of patients were studied as follows: population A, 62 primary breast cancers from 1983; population B, 72 primary lesions stored from 1976 to 1983; and population C, 23 patients with metastases, treated with hormonal therapy. Distinct staining was seen in the cell nucleus. A semiquantitative relationship was seen between histochemical score assessment of staining and biochemical assay in each cohort. The sensitivity and specificity using a threshold of 75 for the histochemical score and more than 20 femtomoles/mg of protein for dextran-coated charcoal analyses were as follows: population A, specificity, 89%, and sensitivity, 95%; population B, specificity, 94%, and sensitivity 88%; and for population C, the comparison was with objective clinical response yielding specificity, 89%, and sensitivity, 93%.

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Gene-expression molecular subtyping of triple-negative breast cancer tumours: importance of immune response

Pascal Jézéquel, Delphine Loussouarn, Catherine Guérin-Charbonnel … (2015)

Introduction Triple-negative breast cancers need to be refined in order to identify therapeutic subgroups of patients. Methods We conducted an unsupervised analysis of microarray gene-expression profiles of 107 triple-negative breast cancer patients and undertook robust functional annotation of the molecular entities found by means of numerous approaches including immunohistochemistry and gene-expression signatures. A triple-negative external cohort (n = 87) was used for validation. Results Fuzzy clustering separated triple-negative tumours into three clusters: C1 (22.4%), C2 (44.9%) and C3 (32.7%). C1 patients were older (mean = 64.6 years) than C2 (mean = 56.8 years; P = 0.03) and C3 patients (mean = 51.9 years; P = 0.0004). Histological grade and Nottingham prognostic index were higher in C2 and C3 than in C1 (P < 0.0001 for both comparisons). Significant event-free survival (P = 0.03) was found according to cluster membership: patients belonging to C3 had a better outcome than patients in C1 (P = 0.01) and C2 (P = 0.02). Event-free survival analysis results were confirmed when our cohort was pooled with the external cohort (n = 194; P = 0.01). Functional annotation showed that 22% of triple-negative patients were not basal-like (C1). C1 was enriched in luminal subtypes and positive androgen receptor (luminal androgen receptor). C2 could be considered as an almost pure basal-like cluster. C3, enriched in basal-like subtypes but to a lesser extent, included 26% of claudin-low subtypes. Dissection of immune response showed that high immune response and low M2-like macrophages were a hallmark of C3, and that these patients had a better event-free survival than C2 patients, characterized by low immune response and high M2-like macrophages: P = 0.02 for our cohort, and P = 0.03 for pooled cohorts. Conclusions We identified three subtypes of triple-negative patients: luminal androgen receptor (22%), basal-like with low immune response and high M2-like macrophages (45%), and basal-enriched with high immune response and low M2-like macrophages (33%). We noted out that macrophages and other immune effectors offer a variety of therapeutic targets in breast cancer, and particularly in triple-negative basal-like tumours. Furthermore, we showed that CK5 antibody was better suited than CK5/6 antibody to subtype triple-negative patients. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0550-y) contains supplementary material, which is available to authorized users.

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Author and article information

Journal

Journal ID (nlm-ta): Cancer Manag Res

Journal ID (iso-abbrev): Cancer Manag Res

Journal ID (publisher-id): Cancer Management and Research

Title: Cancer Management and Research

Publisher: Dove Medical Press

ISSN (Electronic): 1179-1322

Publication date Collection: 2015

Publication date (Electronic): 04 December 2015

Volume: 7

Pages: 361-368

Affiliations

[1 ]Department of Pathology, The University of Chicago, Chicago, IL, USA

[2 ]QualTek Molecular Laboratories, Newtown, PA, USA

[3 ]Corcept Therapeutics, Menlo Park, CA, USA

[4 ]Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA

Author notes

Correspondence: Gabrielle M Baker, Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, MC 6101, Room P-633, Chicago, IL 60637, USA, Tel +1 773 834 8068, Fax +1 773 702 4633, Email gabrielle.baker@ 123456uchospitals.edu

Article

Publisher ID: cmar-7-361

DOI: 10.2147/CMAR.S91546

PMC ID: 4675647

PubMed ID: 26673410

SO-VID: 8a984fae-6a45-4529-a0ef-0757b45c5e7d

License:

The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.

Development and validation of an immunohistochemistry assay to assess glucocorticoid receptor expression for clinical trials of mifepristone in breast cancer

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Most cited references 22

American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version).

Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies.

Gene-expression molecular subtyping of triple-negative breast cancer tumours: importance of immune response

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