Abstract P5-10-07: The LiquidBiopsy in metastatic breast cancer (MBC): A novel diagnostic platform for next generation sequencing (NGS) of circulating tumor cells (CTCs)
Authors: Strauss William , Winer-Jones Jessamine , Austin Laura , Dempsey Paul W , Cristofanilli Massimo 
Published: May 2015

Abstract
Background: Effective treatments for advanced breast cancer are limited leaving palliative care as the only option for patients. The development of effective treatments has been challenging in part due to the biologic heterogeneity of the disease. The challenges to improve patient outcomes are represented by the availability of diagnostic technologies allowing real-time access to the genetic drivers of disease in each individual patients and the ability to match such information with novel targeted therapies. Blood based tests have a long history of providing real-time evidence in medicine. Thus blood derived templates could fill this role in genetic diagnosis. To perform, any technology for molecular analysis of tumor cells from blood must overcome limiting amounts of template and high signal to noise. Current efforts are directed to develop blood based tests for circulating biomarkers that can describe the molecular changes in breast cancer. We investigated the ability to productively sequence tumor cells recovered from whole blood.
Methods: EpCAM expressing Circulating tumor cells (CTCs) were recovered from 49 patients with metastatic breast cancer on the LiquidBiopsy™ platform (Cynvenio Biosystems, Inc). CTC events were characterized using a nuclear stain (DAPI), Cytokeratin expression and absence of CD45. DNA template recovered from this population was case control sequenced with the Life Technologies AmpliSeq targeted resequencing panel on an Ion Torrent Platform using a CLIA validated sequencing pipeline and a sensitivity of 1%. Somatic single nucleotide variants (SNV) present in CTC but not the germline were identified. Critically, the false positive score for 31 normal donors thus evaluated was zero.
Results: Circulating target cells were detected in all 49 patients with metastatic BC (CTC Median 28, range 2 - 4098). Upon resequencing, shared germline polymorphisms were observed in CTC and germline populations. In contrast, SNV were detected in EpCAM enriched populations but not germline in 32% of samples (allele frequency 1.0%-49.6% with 2417-22068 x coverage). Enumeration of cytokeratin expressing cells was predictive of neither the presence nor the allele frequency of detectable SNV. Interestingly mutations in the gene for PIK3ca accounted for 20% of the mutations observed including variants at K111E, E542K, E545K, and H1047L. Mutations were also observed in TP53 (8%), and MET, FGFR2, SMAD4. Twenty samples had tissue biopsy sequence data available and of those, 11 contained mutations that were evaluable in the CTC. All but one mutation were absent in the evaluable tissue biopsy
Conclusions: CTC recovered by EpCAM selection directly from blood can be sequenced by NGS. The predictive value of the CK+ phenotype as a surrogate marker for mutation bearing cells is not supported by these data. Even with a relatively narrow 50 gene target platform, cancer relevant SNVs were detected in &gt;30% of the patients, making targeted NGS of CTCs a promising approach for biomarker discovery and validation in MBC.
Citation Format: William Strauss, Jessamine Winer-Jones, Laura Austin, Paul W Dempsey, Massimo Cristofanilli. The LiquidBiopsy in metastatic breast cancer (MBC): A novel diagnostic platform for next generation sequencing (NGS) of circulating tumor cells (CTCs) [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 P5-10-07.