Circulating tumor cells (CTCs) offer an opportunity for non-invasive characterization of metastatic cancer, including the ability to follow subtype evolution, development of resistance, tissue homing biomarkers, and changes in cell signaling mechanisms required for metastasis and continued growth and survival of the tumor. Analysis of CTCs and translation of a CTC assay into a clinical test has been difficult due to issues of obtaining high purity, high yield preparations and/or isolating individual CTCs. This is further compounded by the heterogeneity of CTCs, hurdles in profiling their molecular signatures, and difficulty correlating CTC number/subtype to actionable therapy. We pursued a detailed analysis of CTC gene expression using whole transcriptome TempO-Seq targeted sequencing, a highly sensitive, direct lysis, addition only assay. As this assay does not require purified CTC isolation or RNA extraction, there is no risk of CTC nor RNA loss. We exploited the single cell sensitivity of TempO-Seq to profile enriched CTC preparations prepared by RosetteSep gradient centrifugation, allowing us to characterize the phenotypes of CTCs within a high background of normal blood cells. To handle this large and potentially overwhelming background we developed and exploited a proprietary method of sample normalization that increased the sensitivity and consistency of CTC gene signature measurements. We demonstrated that the TempO-Seq assay could detect a single MCF7 (EpCAM+) Luminal-like cell in a background of 1,000 MDA MB 231 (EpCAM-) Basal-like cells (positive for an Endothelial Mesenchymal Transition, EMT), and vice versa. Next, we prepared mixtures of different proportions of these cultured cells spiked into normal blood, enriched the model “CTCs” using RosetteSep, and profiled the bulk preparation. Proportional discrimination of cell-specific signatures were obtained with as few as 40 spike-in cells. Finally, we profiled enriched CTC preparations from the blood of patients with metastatic breast cancer. The results demonstrated that the expression of HER2, ER, and EpCAM could be measured as well as biomarkers of drug resistance, bone/brain homing, EMT, and all 50 Hallmark cell signaling pathways. TempO-Seq EpCAM gene level correlated with the percent EpCAM+ cells measured by flow analysis while EMT signature levels correlated inversely. Patients with HER2- primary tumor and HER2+ CTCs could be identified, as could patients with elevated chemotherapy resistance pathways. These data demonstrate that it is possible to profile CTC phenotypes that are important in metastases and useful for selecting therapy using a bulk CTC preparation combined with the sensitivity of the TempO-Seq whole transcriptome assay. This suggests the opportunity for the development of a predictive test using currently available purification platforms without need for expensive specialized equipment or separation into single cells.

Citation Format: Bruce Seligmann, Marilyn Marron, Milos Babic, Elliot Imler, Peter Shepard, Pavani Chalasani, Alison Stopeck, Joanne Yeakley. Detection of gene expression biomarkers from enriched CTC preparations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1729. doi:10.1158/1538-7445.AM2017-1729