Abstract 1388: Multi-template analysis in metastatic breast cancer blood samples
Authors: William M. Strauss, Chris Carter, Erich Klem, Jill Simmons, Keerthi Gogineni, Ruth O’Regan, Laura Austin, Paul W. Dempsey, Massimo Cristofanilli
Journal: Cancer Research (2016)

Abstract Introduction The presence of Circulating Tumor Cells (CTC) has been observed in advanced cancer patients and studies have indicated that these cells contribute to the process of metastasis. Historically CTCs with metastatic potential have been characterized as cells that are EpCAM positive, Cytokeratin positive (CK+), and CD45 negative. We report the results of a prospective study challenging these historical definitions of circulating tumor cells. Description In a prospective study, patients with metastatic breast cancer there were about to start a new systemic therapy were enrolled The samples and representative tissue were collected at baseline. The patients were predominantly female (97%) and all had stage IV disease. From these whole blood samples a LiquidBiopsy® was performed using the “MultiTemplate” format which directly compares CTC, circulating cell free (CCF), and WBC patient-matched DNA in a NGS based resequencing test. Of 22 patients, 18 biopsy samples were successfully evaluated; the balance had insufficient tissue or DNA for analysis. Summary The LiquidBiopsy compared two phenotypic definitions for CTC capture. Tumor cell populations were enriched using epithelial targeted capture and compared to a novel cocktail of antibodies. The cocktail of reagents gave 77% average recovery of engineered samples when a target receptor was present on cell lines representing the spectrum of breast cancer subtypes. This enrichment protocol was applied to serial patient samples. Epithelial based enrichment served as a control and showed an average recovered purity of 10.7% CK+ cells. By comparison, cocktail selection enriched populations with on average 8.8% CK+ cell populations but a larger range of cells than epithelial selection. Importantly, the median number of CK+ cells recovered from cocktail capture was almost twice that of epithelial capture alone. (median of 35.8 vs 22.1 for cocktail and epithelial respectively). The median CD45+ non-target cells background was 80 and 155 cells respectively. This background supports sequencing detection of mutations present at &amp;gt;1%. ccfDNA sample was purified from the same tube of blood. The average concentration of patient matched ccfDNA recovered was 7.3 ng/mL. The combined results of ctcDNA and ccfDNA template sequencing gave productive samples showed similar detection frequency (55% vs 58%), were temporally flexible, and were complementary both to each other and the “gold standard” (FFPE). Conclusions The data from this prospective clinical study reveals subsets of CTC, including an important cohort that is not detected using the standard definition for epithelial CTCs. Furthermore, we present evidence for the successful use of a “Multi-Template” approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and justifies the redefinition of CTC phenotype based upon cell surface biomarkers and mutation content. Citation Format: William M. Strauss, Chris Carter, Erich Klem, Jill Simmons, Keerthi Gogineni, Ruth O’Regan, Laura Austin, Paul W. Dempsey, Massimo Cristofanilli. Multi-template analysis in metastatic breast cancer blood samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1388.