American Association for Cancer Research
10780432ccr141913-sup-135179_1_supp_2674294_ncxrh1.pptx (11.17 MB)

Supplementary Figures from Development of Circulating Tumor Cell-Endocrine Therapy Index in Patients with Hormone Receptor–Positive Breast Cancer

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posted on 2023-03-31, 18:47 authored by Costanza Paoletti, Maria C. Muñiz, Dafydd G. Thomas, Kent A. Griffith, Kelley M. Kidwell, Nahomi Tokudome, Martha E. Brown, Kimberly Aung, M. Craig Miller, Dorothy L. Blossom, Anne F. Schott, N. Lynn Henry, James M. Rae, Mark C. Connelly, David A. Chianese, Daniel F. Hayes

Supplementary Figures. Figure S1. Clinical trial study design. Figure S2. Examples of biomarker staining of cultured human breast cancer cell lines after spike and capture from normal human blood using the CellSearch® system. A semi-quantitative scale was established based on relative expression of each biomarker: negative (0), weak (1+), intermediate (2+), and high (3+). Footnote: *hormone depleted condition. Figure S3. Demonstration of appropriate antibody staining of cultured human breast cancer cells with known positive and negative biomarker expression after spike and capture from normal human blood using the CellSearch® System. A: ER staining for MCF-7 and Sk-Br-3. B: BCL- 2 staining for MCF-7 and Sk-Br-3. C: HER2 staining for Sk-Br-3 and MCF-7. D: Ki-67 staining for MCF-7. Figure S4. Composite "CTC-Bio-Score" for different cell lines. A: Hormone depleted MCF-7 cells (Luminal-type). B: MDA-MB-231 (Basal-like type). C: Sk-Br-3 (HER2 positive). Figure S5. Heterogeneity of ER expression within the cultured MCF-7 human breast cancer cell line. Cells were cultured in hormone depleted media for 48 hours, harvested, and spiked into 7.5 ml of normal human blood. The blood was then processed using CellSearch® and stained for DAPI, CK, CD45, and ER. Examples of strong (2+, 3+), weak (1+), and no staining (0) are displayed. Apparent staining in the bottom panel is due to high gain analysis by the automated fluorescent microscopy, as indicated by very high background. Figure S6. Coefficient of variation of CTC-enumeration by mean CTC count. Figure S7. Heterogeneity between primary tissue and CTC for patient #4. The CTC galleries displayed were selected from over 1,000 images to illustrate CTC-ER heterogeneity.



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. Clin Cancer Res; 21(11); 2487–98. ©2014 AACR.See related commentary by Mathew et al., p. 2421