Abstract P4-01-21: Analysis and single-cell retrieval of circulating tumor cells to monitor treatment response and assess genotype in triple-negative breast cancer
Authors: Ramirez Arturo , Campton Daniel , Mahen Elisabeth , Blau Sibel , Blau Anthony , Kaldjian Eric , Stilwell Jackie 
Published: May 2015

Abstract
Introduction: We used a high-recovery rare cell analysis and single-cell picking system to enrich, visualize, and isolate circulating tumor cells (CTCs) for genomic analysis from the blood of patients with advanced triple-negative breast cancer (TNBC) undergoing treatment with cisplatin as part of a study to intensively characterize TNBC. CTCs were evaluated regularly during treatment to monitor CTC burden and characteristics that could be associated with treatment response or disease progression, and perform single-cell mutational analysis to inform clinical decision making. Methods: Patients were enrolled in the study at the University of Washington Center for Cancer Innovation after informed consent for participation in investigation of their disease, including molecular analysis of multiple biopsies of accessible tumor. CTCs were evaluated prior to treatment and tracked longitudinally. Density-based enrichment of blood cells was performed using the AccuCyte tube, float and collector system. Collected cells were processed and applied to microscopic slides. Fluorescently labeled antibodies to cytokeratin, CD45 and EpCAM, and a nuclear dye were applied to samples using an automated slide stainer. Slides were scanned on a digital microscope and candidate CTCs identified using image analysis software. CTCs were verified by appropriate morphology and expression of epithelial and nuclear stains without CD45 expression. Other antibodies used to characterize cells included Her2, EGFR, and Ki-67. A mutation hypothesized to lead to the activation of ROS1 was identified in the cancer cells isolated from the bone marrow of one patient. CTCs were picked using our integrated semi-automated system and evaluated for the ROS1 variant using whole genome amplification followed by nested PCR and Sanger sequencing. Results: Seven patients have been enrolled to date. At least 1 CTC/mL has been found in all patients. Pre-treatment CTC levels in the patient with the ROS1 mutation were extremely high (1500/mL). One week after treatment, CTC levels spiked to more than 5000/mL. CTC counts then dropped exponentially to 9/mL after 4 months. CTC clusters and Ki-67 positive cells also decreased during therapy. Treatment with cisplatin was discontinued in this patient due to toxicity and progression, and CTC levels increased to nearly 9000/mL over 4 months. The ROS1 mutation was found in approximately 50% of individually picked CTCs before treatment with crizotinib, a ROS1 inhibitor. A second patient was found to have somatic loss of BRCA1, and was therefore treated with the PARP inhibitor, veliparib. CTC levels increased during veliparib treatment up to 13/mL. The same patient was subsequently treated with ponatinib, an FGFR inhibitor, based on the identification of two linked somatic missense mutations involving FGFR2 (S252W and Y375C). After beginning ponatinib, CTCs fell to undetectable levels. Conclusions: Analysis of CTCs may provide a non-invasive measure of cancer progression/response and the molecular evolution of tumor cells in patients with TNBC. Single-cell CTC retrieval after slide-based immunofluorescent visualization is compatible with whole genome amplification and sequencing methods.
Citation Format: Arturo Ramirez, Daniel Campton, Elisabeth Mahen, Sibel Blau, Anthony Blau, Eric Kaldjian, Jackie Stilwell. Analysis and single-cell retrieval of circulating tumor cells to monitor treatment response and assess genotype in triple-negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-21.