Abstract P1-01-04: Comprehensive genomic characterization of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) sheds light on the biology of blood-borne metastasis
Authors: Magbanua MJ , Hauranieh L , Roy R , Wolf D , Benz S , Vaske C , Pendyala P , Sosa E , Scott J , Lee JS , Ordonez A , Ho B , Solanki T , VantVeer L , Rugo H , Park J 
Published: March 2017

Abstract
Background: CTCs offer a relatively non-invasive source of metastatic tissue for molecular analysis. To elucidate the underlying biology of blood-borne metastasis, we profiled CTCs from MBC patients (pts).
Methods: CTCs were isolated by IE/FACS (immunomagnetic enrichment/fluorescence-activated cell sorting). Expression of 64 cancer-related genes in CTCs was analyzed via microfluidic-based multiplex QPCR. Genome-wide copy number (CN) analysis by array comparative genomic hybridization (ACGH) was performed on CTCs isolated from the same tumor-enriched blood samples. The Illumina platform was utilized for next generation sequencing and data was analyzed using NantOmics analysis pipeline and Nexus 8.0 software. Mutations were confirmed by Sanger sequencing or by digital droplet PCR.
Results: Expression profiles of CTCs from 105 MBC pts clustered away from those of blood, indicating high-purity isolation of CTCs by IE/FACS. In addition to EPCAM, tumor-related genes, e.g., CCND1, MUC1, and TTF3 were upregulated in CTCs. Approximately 70% of the CTC samples were considered ER-positive, of which 47% were ER+HER2+, and 22% ER+HER2-. Among the ER+HER2- samples, about two-thirds (68%) had low proliferative (MKI67) status. HER2-positive and triple-negative CTCs accounted for 27% and 30% of the samples, respectively. Furthermore, 30% of the samples were assigned to luminal A, 6% to luminal B, 13% to Her2-enriched, 33% to basal-like, and 12% to normal-like subtypes. Expression profiling of CTCs in 74 serial blood samples from 28 pts showed fluctuations in expression at the gene-level, while subtype calls were mostly consistent across time points.
CTCs from 49 of the 105 pts analyzed by ACGH revealed numerous genomic aberrations such as 1q/8q gains and 8p/16q losses, consistent with breast cancer origin. CN profiles grouped into three major clusters: CTCs exhibiting low genomic instability, 8q gain, and 1q gain/11q loss. ERBB2 and CCND1 were upregulated in CTCs showing increased genomic alterations.
Changes in ER (n=102) and HER2 (n=130) status between CTCs and matched primary tumors (PT) were observed in 27% and 23% of the pts, respectively, indicating that biomarker status may change during disease progression.
Comparative analysis of CN data from low-pass whole genome sequencing (WGS) of CTCs vs. matched PTs (n=7 pairs) demonstrated clonal-relatedness as well as some genetic divergence. WGS (38x) and whole exome sequencing (140x) analysis of CTCs from an index pt diagnosed with invasive lobular carcinoma detected numerous genomic aberrations, including a copy loss and a frameshift mutation in E-cadherin (CDH1). Interestingly, analysis of CN and mutation data revealed that CTCs were more closely related to the lymph node metastases than to the PT. Single-cell sequencing of CTCs revealed uniformity in genome-wide CN alterations, while cell-to-cell heterogeneity was observed only when single-cell expression profiles were analyzed.
Conclusions: Comprehensive molecular characterization provided novel insights into the biology of breast CTCs. Further CTC profiling may open avenues for discovery and development of novel biomarkers for personalized medicine and strategies to prevent metastasis.
Citation Format: Magbanua MJ, Hauranieh L, Roy R, Wolf D, Benz S, Vaske C, Pendyala P, Sosa E, Scott J, Lee JS, Ordonez A, Ho B, Solanki T, VantVeer L, Rugo H, Park J. Comprehensive genomic characterization of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) sheds light on the biology of blood-borne metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-04.