Androgen receptor expression on circulating tumor cells in metastatic breast cancer

Abstract

Purpose: Androgen receptor (AR) is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+) breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs) can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer.

Methods: Blood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK), and biomarkers of interest (AR, estrogen receptor [ER], and HER2) on all nucleated cells. The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM) using the Epic Sciences CTC platform. Data were analyzed using established digital pathology algorithms.

Results: Of 68 patients, 51 (75%) had at least 1 CTC, and 49 of these 51 (96%) had hormone-receptor-positive (HR+)/HER2-negative primary tumors. AR was expressed in CK+ CTCs in 10 patients. Of these 10 patients, 3 also had ER expression in CK+ CTCs. Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns. AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells.

Conclusions: CTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer. The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers. Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their heterogeneity.

Conflict of interest statement

Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: NTU has a legal agreement with Epic Sciences for using their technology for analysis. Epic Sciences paid salary only for Rachel Krupa, Mahipal V. Suraneni, Ryon P. Graf, Jerry Lee, Stephanie Greene, Angel Rodriguez, Lyndsey Dugan, Jessica Louw, Yipeng Wang, Mark Landers and Ryan Dittamore at Epic Sciences. No one from The University of Texas MD Anderson Cancer Center has received direct financial support from Epic Sciences. This affiliation with Epic Sciences does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. AR expression by IHC staining.

Representative images of AR-positive (A) and AR-negative (B) tumors. Arrows: tumors; arrowhead, normal breast epithelium. Original magnifications, x100.

Fig 2. Representative images of CTC subtypes identified by the Epic Sciences CTC platform.

Representative fluorescence microscopy images of subtypes of CTCs identified by the Epic Sciences CTC platform. Blood samples from patients with metastatic breast cancer were deposited on glass slides and stained with a cocktail of DAPI and antibodies against CK, CD45, and AR. After staining, CTCs were detected using a digital pathology algorithm and classified into CTC subtypes on the basis of marker expression profile into CK+ CTCs, CK- CTCs, CTC clusters, and apoptotic CTCs. The top panel shows an AR+CK+ CTC with AR expression localized in the nucleus.

Fig 3. Prevalence of AR+ and ER+ CTCs in metastatic breast cancer samples.

(A) Dot plot depicting AR expression in CTCs identified in patients with metastatic breast cancer with respect to the threshold for AR positivity (indicated by dotted line). Each dot represents a single CTC, and the color indicates the subtype, defined as CK+, CK-, CK+ cluster, or apoptotic (Apop). AR+ CTCs were detected in 10 of 68 patients tested. The 2 patients with only apoptotic AR+ CTCs were excluded. Interpatient heterogeneity was observed in levels of AR expression and subtypes of CTCs. N-term, N-terminal region. (B) Dot plot depicting ER expression in CTCs in the 10 patients with AR+ CTCs with respect to the threshold for ER positivity (indicated by dotted line). Each dot represents a single CTC, and the color indicates the subtype, defined as CK+, CK-, CK+ cluster, or apoptotic (Apop). ER+ CTCs were identified in 3 of 10 patients tested.

Fig 4. Single-cell CNV analysis of CTCs.

Seventy eight CTCs with various biomarker (AR, ER and HER2) positive and negative and 1 white blood cell (germline control) detected in the sample from patient 11 were sequenced and analyzed for the presence of CNVs. (A) Characteristics of CTCs sequenced for CNV analysis according to AR, ER, and HER2 expression. (B) Representative examples of the 3 different CNV patterns identified in patient #11. The bottom figure is the CNV profile of the WBC. (C) Incidences of the 3 CNV patterns in CTCs according to AR, ER, and HER2 expression.