Personalized Medicine and Imaging Circulating Tumor Cell Analysis in Metastatic Triple-Negative Breast Cancers Mark Jesus M. Magbanua 1 , Lisa A. Carey 2 , Amy DeLuca 1 , Jimmy Hwang 1 , Janet H. Scott 1 , Mothaffar F. Rimawi 3 , Erica L. Mayer 4 , P. Kelly Marcom 5 , Minetta C. Liu 6 , Francisco J. Esteva 7 , John W. Park 1 , and Hope S. Rugo 1 for the Translational Breast Cancer Research Consortium Abstract Purpose: Recent developments in rare-cell technology have led to improved blood-based assays that allow for the reliable detec- tion, enumeration, and more recently, genomic profiling of circu- lating tumor cells (CTC). We evaluated two different approaches for enumeration of CTCs in a prospective therapeutic study of patients with metastatic triple-negative breast cancer (TNBC). Experimental Design: The CellSearch system, a commercially available and U.S. Food and Drug Administration (FDA)–cleared assay for CTC enumeration, and IE/FC, an alternative method using EPCAM-based immunomagnetic enrichment and flow cytometry that maintains cell viability, were used to enumerate CTCs in the blood of patients with metastatic TNBC. CTC num- bers were assessed at baseline and 7 to 14 days after initiation of therapy with cetuximab  carboplatin in a phase II multicenter clinical trial (TBCRC 001). Results: CTC numbers from two methods were significantly correlated at baseline (r ¼ 0.62) and at 7 to 14 days (r ¼ 0.53). Baseline CTCs showed no association with time-to-progression (TTP), whereas CTCs at 7 to 14 days were significantly corre- lated with TTP (CellSearch P ¼ 0.02; IE/FC P ¼ 0.03). CTCs at both time points were significantly associated with overall survival (OS) [CellSearch: baseline (P ¼ 0.0001) and 7 to 14 days (P < 0.0001); IE/FC: baseline (P ¼ 0.0009) and 7 to 14 days (P ¼ 0.0086)]. Conclusions: Our findings demonstrate that CTC enumeration by two different assays was highly concordant. In addition, results of both assays were significantly correlated with TTP and OS in patients with TNBC. The IE/FC method is also easily adapted to isolation of pure populations of CTCs for genomic profiling. Clin Cancer Res; 21(5); 1098–105. Ó2014 AACR. Introduction The detection of rare circulating tumor cells (CTC) in the blood of patients with cancer is extremely challenging. Several CTC enumeration studies using the FDA-cleared methodology, CellSearch (Veridex) have demonstrated the predictive and prognostic value of CTCs in metastatic breast cancer (1–9). Patients with CTCs at or above the threshold of 5 per 7.5 mL blood before treatment and those who fail to clear the cells during treatment have a significantly worse outcome than those who maintain a CTC count <5 after starting systemic therapy (2). However, a recent randomized phase III trial found that enumerating CTCs using the commercially available CellSearch assay to support treatment decisions did not lead to improved outcomes (10). Moreover, most commercial assays use meth- ods that cannot capture viable cells, limiting their utility in correlative science analyses and in the emerging use of blood- based assays to identify resistance mechanisms (11). Novel CTC detection assays have been developed that can be used for genomic and other molecular profiling (11, 12). Whether these newer methods identify the same CTC population and provide similar clinical implications as the CellSearch system has not been evaluated. The CellSearch system involves a two-step process, initial EPCAM-based immunomagnetic enrichment is followed by immunofluorescence microscopy. Here, CTCs are defined as nucleated cells, which express cytokeratin but do not express the leukocyte-specific marker, CD45. Our group has developed a similar enumeration method, referred to as IE/FC, which also involves an EPCAM-based immunomagnetic enrichment (IE) step. However, instead of microscopy, our method uses flow cytometric (FC) analysis and maintains cell viability (13). During flow cytometry, events that are positive for EPCAM and a nuclear stain but negative for CD45 are considered CTCs. In this study, we performed a head-to-head comparison of these two CTC enumeration methods in the context of a multicenter clinical trial (Translational Breast Cancer Research Consortium, TBCRC 001, clinicaltrials.gov: NCT00232505) 1 University of California San Francisco Helen Diller Family Compre- hensive Cancer Center, San Francisco, California. 2 Lineberger Com- prehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina. 3 Baylor University College of Medicine, Houston,Tex- as. 4 Dana-Farber Cancer Institute, Boston, Massachusetts. 5 Duke Uni- versity Medical Center, Durham, North Carolina. 6 Georgetown Univer- sity, Washington, District of Columbia. 7 Laura & Isaac Perlmutter Cancer Center, New York University Langone Medical Center, New York, New York. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Current Address for M.C. Liu: Mayo Clinic, Rochester, Minnesota. Corresponding Author: Hope S. Rugo, University of California San Francisco Helen Diller Family Comprehensive Cancer Center, Box 1710 1600 Divisadero St. San Francisco, CA 94115. Phone: 415-353-7618; Fax: 415-353-9571; E-mail: hrugo@medicine.ucsf.edu doi: 10.1158/1078-0432.CCR-14-1948 Ó2014 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 21(5) March 1, 2015 1098 on June 10, 2020. © 2015 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 18, 2014; DOI: 10.1158/1078-0432.CCR-14-1948 on June 10, 2020. © 2015 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 18, 2014; DOI: 10.1158/1078-0432.CCR-14-1948 on June 10, 2020. © 2015 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 18, 2014; DOI: 10.1158/1078-0432.CCR-14-1948 on June 10, 2020. © 2015 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst December 18, 2014; DOI: 10.1158/1078-0432.CCR-14-1948