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. Preclinical studies were performed with cultured breast cancer cells spiked into human blood and analyzed on the CellSearch(®) system. Clinical data are limited to a subset of patients with ER-positive MBC from a previously reported pilot trial whose disease was progressing on fulvestrant (N = 7) or aromatase inhibitors (AIs) (N = 10). CTCs were enumerated and phenotyped for ER and B-cell lymphoma (BCL2) using the CellSearch(®) CXC kit. In preclinical modeling, tamoxifen and AIs resulted in stabilized ER expression, whereas fulvestrant eliminated it. Five of seven patients progressing on fulvestrant had ≥5CTC/7.5 ml WB. Two of these five, treated with 500 mg/month fulvestrant, had no detectable CTC-expression of ER and BCL2 (an ER regulated gene). Three patients had heterogeneous CTC-ER and BCL2 expression indicating incomplete degradation of the ER target by fulvestrant. Two of these patients received 250 mg/month whereas the third patient received 500 mg/month fulvestrant. Her cancer harbored a mutation (Y537S) in the estrogen receptor alpha gene (ESR1). All seven ER positive patients progressing on AIs had heterogeneous CTC-ER expression. These results suggest heterogeneous mechanisms of resistance to fulvestrant, including insufficient dosage, ESR1 mutation, or conversion to dependence on non-ER pathways. CTC enumeration, phenotyping, and genotyping might identify patients who would benefit from fulvestrant dose escalation versus switching to alternative therapies.

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.

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

BACKGROUND: 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. METHODS: Eighty-three evaluable patients initiating a new therapy for metastatic breast cancer were enrolled. Whole blood was collected at baseline, at one of three short term time windows (24, 48, or 72 h) after initiating treatment, and at first follow-up (3-5 weeks). CTC were isolated, enumerated, and expression of M30 and Bcl2 was determined using the CellSearch(®) System. RESULTS: At baseline, window, and 3-5 weeks post-treatment, 41/80 (51%), 40/80 (50%) and 21/75 (28%) patients had ≥5 CTC, respectively. At baseline, the proportion of CTC-apoptosis (M30) was inversely correlated with CTC number, and modestly inversely correlated with CTC-Bcl-2. As expected, higher CTC levels at baseline or first follow-up were associated with worse prognosis. Surprisingly, in patients with elevated CTC, higher levels of CTC-apoptosis were associated with worse prognosis, while higher CTC-Bcl-2 levels correlated with better outcomes. CONCLUSIONS: CTC apoptosis and expression of Bcl-2 can be analytically determined in patients with metastatic breast cancer and may have biological and clinical implications. Characterization of CTC for these and other markers could further increase the utility of CTC monitoring patients in clinical investigations of new anti-neoplastic agents.

Potential applications for circulating tumor cells expressing the insulin-like growth factor-I receptor.

PURPOSE: To detect insulin-like growth factor-IR (IGF-IR) on circulating tumor cells (CTC) as a biomarker in the clinical development of a monoclonal human antibody, CP-751,871, targeting IGF-IR. EXPERIMENTAL DESIGN: An automated sample preparation and analysis system for enumerating CTCs (CellTracks) was adapted for detecting IGF-IR-positive CTCs with a diagnostic antibody targeting a different IGF-IR epitope to CP-751,871. This assay was used in three phase I trials of CP-751,871 as a single agent or with chemotherapy and was validated using cell lines and blood samples from healthy volunteers and patients with metastatic carcinoma. RESULTS: There was no interference between the analytic and therapeutic antibodies. Eighty patients were enrolled on phase I studies of CP-751,871, with 47 (59%) patients having CTCs detected during the study. Before treatment, 26 patients (33%) had CTCs, with 23 having detectable IGF-IR-positive CTCs. CP-751,871 alone, and CP-751,871 with cytotoxic chemotherapy, decreased CTCs and IGF-IR-positive CTCs; these increased toward the end of the 21-day cycle in some patients, falling again with retreatment. CTCs were commonest in advanced hormone refractory prostate cancer (11 of 20). Detectable IGF-IR expression on CTCs before treatment with CP-751,871 and docetaxel was associated with a higher frequency of prostate-specific antigen decline by >50% (6 of 10 versus 2 of 8 patients). A relationship was observed between sustained decreases in CTC counts and prostate-specific antigen declines by >50%. CONCLUSIONS: IGF-IR expression is detectable by immunofluorescence on CTCs. These data support the further evaluation of CTCs in pharmacodynamic studies and patient selection, particularly in advanced prostate cancer.

Expression of epithelial cell adhesion molecule in carcinoma cells present in blood and primary and metastatic tumors.

The epithelial cell adhesion molecule (EpCAM) is involved in homophilic cell-cell adhesion in normal epithelia and is frequently overexpressed in primary and metastatic adenocarcinomas. It has been postulated that during detachment and dissemination of tumor cells, EpCAM may be down-regulated. Circulating tumor cells (CTC) may demonstrate this phenomenon as they have successfully escaped their local microenvironment and entered the circulation. EpCAM expression of CTC was compared to tumor cells in paraffin-embedded tissue arrays containing various benign diseases and carcinomas. EpCAM expression on CTC was determined by flow cytometry (FCM) and by immunohistochemistry (IHC) in paraffin-embedded tissue. To permit comparison of FCM results to those derived by IHC, EpCAM was quantified on cancer cell lines by FCM and then paraffin-embedded cell-blocks of these lines were used as staining guides for IHC analysis of tissue arrays. By IHC, 97% (384/397) of solid tissues analyzed had detectable EpCAM, with 72% of tissues showing antigen expression levels of > or =400,000 EpCAM molecules per cell. FCM analysis of CTC from 100 metastatic carcinoma patients with > or =2 CTC/90 microl blood showed EpCAM expression ranging from 9,900 to 246,000 (mean 49,700) antigens per cell. EpCAM expression was approximately 10-fold lower on CTC as compared to primary and metastatic tissues, suggesting that EpCAM expression is transient and dependent upon the local micro-environment. This supports the hypothesis that this adhesion molecule is down-regulated on carcinoma cells in the circulation.