Maria C. Muñiz

Monitoring apoptosis and Bcl-2 on circulating tumor cells in patients with metastatic breast cancer☆

Enumeration of circulating tumor cells (CTC) from whole blood permits monitoring of patients with breast carcinoma. Analysis of apoptosis & Bcl‐2 expression in CTC might add additional prognostic and predictive information. We estimated the degree of these markers in CTC from patients being treated for metastatic breast cancer.

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

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

Tumor microvasculature targeting with dendrimer-entrapped gold nanoparticles

Monodisperse dendrimer-entrapped gold nanoparticles (diameter = 3.0 nm) were prepared using G5 poly(amidoamine) (PAMAM) dendrimer functionalized with fluorescein isothiocyanate (FI) and Arg-Gly-Asp (RGD) peptide as template; in vitro targeting efficacy to integrin receptor expressing cells was confirmed by flow cytometry, confocal microscopy, and ICP-MS.

Heterogeneous estrogen receptor expression in circulating tumor cells suggests diverse mechanisms of fulvestrant resistance

Fulvestrant is a dose dependent selective estrogen receptor (ER) down‐regulator (SERD) used in ER‐positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance.

Abstract 4154: Multi-parameter molecular characterization of circulating tumor cells (CTC): Development of a CTC-endocrine therapy index (CTC-ETI)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction. CTC can be reproducibly and reliably enumerated using a commercially-available, automated immunomagnetic system (CellSearch®; Veridex LLC). High levels of CTC predict rapid progression in patients with metastatic breast cancer (MBC). Only ∼ 50% of patients with estrogen receptor (ER) positive MBC benefit from endocrine therapy (ET). Patients with endocrine-refractory MBC are better palliated with chemotherapy. We have developed a multi-parameter assay using CellSearch® that may identify patients with ER positive MBC who are unlikely to benefit from ET and may be better served with chemotherapy. Methods. CellSearch® has four fluorescence channels. Three of these are used to distinguish CTC from WBC (DAPI, anti-cytokeratin, anti-CD45). The 4th “empty” channel was used to measure ER, Bcl-2, HER2, and Ki67 expression with antigen-specific fluorescent-labeled antibodies. These four markers were chosen because of their associations with sensitivity (ER, Bcl-2) or resistance (HER2, Ki-67) to ET. Cultured human breast cancer cells (MCF-7: ER +, BCL-2 +, HER2 -, Ki67 +; MDA-MB-231: ER -, Bcl-2 -, HER2 -, Ki67 +; BT-474: ER +, Bcl-2 +, HER2 +, Ki67 +) were spiked into 7.5 ml human whole blood from normal donors and separated and characterized using the CXC CellSearch® kit. Results. Each cell line stained appropriately for the respective markers, with appropriate negative controls, although staining was heterogeneous, even within a single cell line. Using these preliminary data, we have developed a CTC-ETI in which scores are assigned to the individual categories consisting of cell counts coupled with the relative percent and degree of cell positivity of each marker. We predict that lower scores (low or no CTC, or CTC with high % and intensity of ER and Bcl-2; low HER2 and Ki-67) will be associated with favorable response to ET. Conclusions. CellSearch® can be used to measure the expression of 4 clinically useful and proven breast cancer biomarkers. A clinical trial is underway at U. Michigan to permit calculation of CTC-ETI in patients with MBC, which will lead to a prospective trial to determine if high CTC-ETI predicts resistance and rapid progression on ET. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale ™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4154. doi:10.1158/1538-7445.AM2011-4154

P4-07-16: Development of Circulating Tumor Cell-Endocrine Therapy Index in Metastatic Breast Cancer Patients.

Introduction: Only ∼ 50% of patients (pts) with estrogen receptor (ER) positive metastatic breast cancer (MBC) benefit from endocrine therapy (ET). Currently only clinical judgment can be used to identify pts with endocrine-refractory MBC, who are better palliated with chemotherapy. Circulating Tumor Cells (CTC) are reliably enumerated using an automated immunomagnetic system (CellSearch®; Veridex LLC). High CTC levels predict rapid progression in pts with MBC. We have developed a multi-parameter assay, the CTC-Endocrine Therapy Index (CTC-ETI) using CellSearch® that may identify pts with ER positive MBC who are unlikely to benefit from ET and may be better served with chemotherapy. CTC-ETI scores are assigned based on CTC levels coupled with the relative percent and degree of marker positivity on the CTC. We report preliminary results from a pilot single institutional study. Methods: CellSearch® has 4 fluorescence channels. Three distinguish CTC from WBC (DAPI, anti-cytokeratin, anti-CD45). The 4 th “empty” channel was used to measure ER, BCL-2, HER-2, and Ki-67 expression with fluorescent-labeled antibodies. These 4 markers reflect sensitivity (ER, BCL-2) or resistance (HER-2, Ki-67) to ET. Forty ml of blood was drawn into 4 CellSave® tubes from pts with progressive MBC. Whole blood from 4 tubes was pooled and divided into 4 different 7.5 ml aliquots of blood, which were processed and characterized for CTC counts and each of the four molecular markers using the CXC CellSearch® kit. Results: 21 pts have been accrued to the feasibility study. One patient was ineligible. Five of 20 pts had low CTC counts ( Conclusions: ER, BCL-2, HER-2, and Ki-67 can be accurately determined on CTC using the 4 th channel in the CellSearch® system to calculate CTC-ETI. We predict that lower CTC-ETI scores (low or no CTC, or CTC with high CTC ER and BCL-2 and low CTC HER-2 and Ki-67) could be associated with favorable response to ET. Successful completion of the feasibility study will lead to a prospective trial to determine if high CTC-ETI at baseline predicts resistance and rapid progression on ET in women starting a new endocrine therapy for MBC. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale ™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-16.

Abstract PD6-4: Heterogeneity of expression of estrogen receptor by circulating tumor cells suggests diverse mechanisms of resistance to fulvestrant in metastatic breast cancer patients

Introduction: Fulvestrant is a selective estrogen receptor down-regulator (SERD). Recent studies have shown that the efficacy of fulvestrant is dose-related. However, at the higher dose (500 mg/month) most cancers develop resistance and progress. We previously reported expression of several markers, including estrogen receptor (ER) and BCL-2, on breast cancer circulating tumor cells (CTC) using CellSearch®. We now report pilot data showing inter-patient heterogeneity of these markers on CTC in patients with known ER positive breast cancer whose disease is progressing on fulvestrant. Methods: We conducted a pilot trial to determine the analytical validity of measuring expression of markers of endocrine sensitivity (ER, BCL-2) or resistance (HER-2, Ki-67) with fluorescent-labeled antibodies using the CellSearch® system. Patients with ER positive metastatic breast cancer (MBC) whose disease was progressing on any type of therapy were eligible after signed informed consent. This report is limited to the subjects who were progressing on fulvestrant. Whole blood (WB) was characterized for CTC counts and each of the four molecular markers using the CXC CellSearch® kit. Results : Of 50 enrolled patients, seven were progressing on fulvestrant. Two patients had no detectable CTC, while five patients had an average of ≥5 CTC/7.5 mL WB. Results are shown in a table below: View this table: These exploratory data suggest widely different mechanisms of resistance to fulvestrant in different patients with ER positive MBC. In two of the patients (29, 45) treated with 500 mg/month, both CTC-ER and CTC-BCL-2 expression were absent, suggesting no signaling through the ER pathway. We hypothesize either that fulvestrant was actively down-regulating ER, but the cancers had adopted other growth and survival pathways, or that ER negative, hormone-independent clones had evolved. In the other three cases, ER was clearly present with evidence of signaling, based on BCL-2 expression. Two of these patients (2, 17) were on the lower dose of fulvestrant, now considered to be less effective. However, the third (8) was on the higher dose and yet still had evidence of ER signaling. This observation suggests that some patients may benefit from even higher doses of SERD therapy. Conclusions: These pilot results suggest heterogeneous biological or pharmacological mechanisms of resistance to SERD therapy. These data suggest that CTC-ER and CTC-BCL-2 expression could serve as pharmacodynamic monitoring tools for dose escalation of fulvestrant or other SERDs. Further molecular analysis might provide biological bases for resistance to fulvestrant. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD6-4.

Abstract P1-04-01: Significance of circulating tumor cells in metastatic triple negative breast cancer: Results of an open label, randomized, phase II trial of nanoparticle albumin-bound paclitaxel with or without the anti-death receptor 5 tigatuzumab (TBCRC 019)

Background: Circulating Tumor cells (CTCs) are prognostic at baseline and first follow-up in patients with metastatic breast cancer (MBC). Using the most commonly used assay (CellSearch®), we have previously reported the ability to detect apoptotic vs. non-apoptotic CTCs in patients with MBC. However, there has been concern regarding the performance of the CellSearch® assay in patients with triple negative (TN) MBC. We hypothesized that CellSearch® is an effective assay in patients with TN MBC, and that CTC-apoptosis might further separate prognosis. Therefore, we studied CTCs in patients with TN MBC who participated in a prospective randomized phase II trial testing for activity of tigatuzumab (TIG) in combination with nanoparticle albumin-bound paclitaxel (nab-PAC) conducted by the Translational Breast Cancer Research Consortium (overall results reported by Forero A., et al, ASCO 2013). Methods: Whole blood (WB) was drawn into a CellSave tube at baseline, day 15, and day 29 and CTC counts were determined using the CXC CellSearch® kit. Apoptosis was characterized by staining with a monoclonal antibody that detects a neo-epitope on fragmented cytokeratin (M-30) and independently by visual inspection (nucleic condensation and/or fragmentation, as well as granular cytokeratin). Association between levels of CTCs and CTC-apoptosis with the overall response rate (ORR) and progression free survival (PFS) at baseline, day 15, and day 29 was assessed using logistic regression, Kaplan Meier curves, and Cox proportional hazards modeling. Results: Of the 60 patients entered into the trial, 52 were evaluable for CTCs. Of these, 19/52 (36.5%), 14/52 (26.9%), and 13/49 (26.5%) had elevated CTCs (≥5CTC/7.5 ml WB) at baseline, day 15, and day 29, respectively. Patients with elevated CTCs at each time point had worse PFS than patients with low or no CTCs. Hazard rates (HR) at baseline, day 15, and day 29 were 2.38 (95% CI: 1.27-4.45, p = 0.007), 2.87 (95% CI: 1.46-5.66, p = 0.002), and 3.40 (95% CI: 1.68-6.89, p = 0.001), respectively. The odds of overall response for those who had elevated CTCs compared to those who did not at baseline, day 15, and day 29, were 0.25 (95% CI: 0.073-0.81, p = 0.024), 0.18 (95% CI: 0.04-0.67, p = 0.014), and 0.06 (95% CI: 0.01-0.28, p = 0.001), respectively. There was no apparent prognostic effect comparing the degree of CTC-apoptosis vs. non-apoptosis. Conclusions: Similar to observations in other intrinsic subgroups, CTCs were detected in a large fraction of TN MBC patients at baseline using CellSearch® assay, and reductions in CTC levels reflected response. In these homogenously prospectively enrolled TN MBC patients, regardless of treatment, CTCs are prognostic at baseline, day 15, and day 29. It does not appear that analysis of CTC-apoptosis is prognostic. Supported by Susan G. Komen for the Cure, Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP), Triple Negative Breast Cancer Foundation, The AVON Foundation, and The Breast Cancer Research Foundation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-01.