Development of circulating tumor cell-endocrine therapy index in patients with hormone receptor-positive breast cancer

Abstract

Background: Endocrine therapy (ET) fails to induce a response in one half of patients with hormone receptor (HR)-positive metastatic breast cancer (MBC), and almost all will eventually become refractory to ET. Circulating tumor cells (CTC) are associated with worse prognosis in patients with MBC, but enumeration alone is insufficient to predict the absolute odds of benefit from any therapy, including ET. We developed a multiparameter CTC-Endocrine Therapy Index (CTC-ETI), which we hypothesize may predict resistance to ET in patients with HR-positive MBC.

Methods: The CTC-ETI combines enumeration and CTC expression of four markers: estrogen receptor (ER), B-cell lymphoma 2 (BCL-2), Human Epidermal Growth Factor Receptor 2 (HER2), and Ki67. The CellSearch System and reagents were used to capture CTC and measure protein expression by immunofluorescent staining on CTC.

Results: The feasibility of determining CTC-ETI was initially established in vitro and then in a prospective single-institution pilot study in patients with MBC. CTC-ETI was successfully determined in 44 of 50 (88%) patients. Eighteen (41%), 9 (20%), and 17 (39%) patients had low, intermediate, and high CTC-ETI scores, respectively. Interobserver concordance of CTC-ETI determination was from 94% to 95% (Kappa statistic, 0.90-0.91). Inter- and cell-to-cell intrapatient heterogeneity of expression of each of the CTC markers was observed. CTC biomarker expression was discordant from both primary and metastatic tissues.

Conclusions: CTC expression of ER, BCL-2, HER2, and Ki67 can be reproducibly measured with high analytical validity using the CellSearch System. The clinical implications of CTC-ETI, and of the heterogeneity of CTC biomarker expression, are being evaluated in an ongoing prospective trial.

Conflict of interest statement

Conflict of interest: D.F.H. received support from Janssen Diagnostics, LLC, the commercial vendor of CellSearch®, to support clinical and laboratory research. M.C.M., M.C.C., and D.A.C. are paid as employees of Janssen Diagnostics, LLC. D.F.H. is the inventor named on a patent application filed by the University of Michigan regarding CTC-ETI. The remaining authors have no conflict of interest to disclose.

©2014 American Association for Cancer Research.

Figures

Fig. 1

REMARK diagram for patient enrollment and distribution.

Fig. 2

CTC enumeration and biomarker staining for patients with HR positive MBC for the four biomarkers. A: ER; B: BCL-2; C: HER2; D: Ki67. In each, the top panel demonstrates CTC/7.5mL WB on a logarithmic scale for each patient, in descending order based on CTC number. The lower panel provides the percentage of CTC that stained 0 ( ▀ yellow), 1+ ( ▀ fuchsia), 2+ ( ▀ blue), or 3+ ( ▀ green). See Fig. S2 for standard control staining of cultured breast cancer cell lines for definitions of each category. For each patient, CTC levels varied slightly between among each aliquot marker. The screen failure patient is not included. Data from aliquots that were technical or analytical failures are not included, but the data from the remaining aliquots for the other biomarkers for that patient are included for analyses. The CTC enumeration on Y-axis is a logarithmic scale. Thus, patients with a single CTC/7.5 ml WB appear as 0.

Fig. 3

REMARK diagrams of tissue procurement and staining A: Primary cancers; B: Metastatic cancers.

Fig. 4

Correlation of CTC and tissue biomarker expression. A, B: ER; C, D: BCL-2; E,F: HER2; G, H: Ki67. A, C. E, G: CTC-biomarker compared to primary cancer tissue biomarker. B, D, F, H: CTC-biomarker compared to metastatic cancer tissue biomarker. For each biomarker, ● = 1–4 CTC/7.5 ml WB; ▲ =5–10 CTC/7.5 ml WB; ▀ > 10 CTC/7.5 ml WB and relative biomarker staining score (0, 1, 2, and 3+). See Methods for details. Allred Score: see Methods; for Ki67, results are expressed as % positive cells. Dotted lines represent cutoff levels for CTC-biomarker and for Allred score or % positive Ki67.