Molecular profiling of single circulating tumor cells with diagnostic intention

Abstract

Several hundred clinical trials currently explore the role of circulating tumor cell ( CTC ) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTC s with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non‐random whole genome amplification method for single cells and applied it to 510 single CTC s and 189 leukocytes of 66 CTC ‐positive breast cancer patients. We defined a genome integrity index ( GII ) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high‐quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTC s of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTC s. Microheterogeneity analysis among individual CTC s uncovered pre‐existing cells resistant to ERBB 2 ‐targeted therapies suggesting ongoing microevolution at late‐stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance. image A novel workflow enabling detection, isolation and characterization of single circulating tumors cells ( CTC s) from blood suggests that CTC s may harbor genetic alterations undetectable in the primary tumor and associated with therapy resistance. Single circulating tumor cells ( CTC s) are analyzed by a semi‐automated workflow combining CellSearch® enrichment, DEPA rray TM isolation and Ampli1 TM whole genome amplification ( WGA ). The WGA quality of single CTC s is assessed by a genome integrity index ( GII ). The GII predicts outcome of downstream sequence‐based molecular assays. Single cell analysis reveals the existence of rare potential therapy escape variants. The diagnostic precision of the workflow enables molecular monitoring of CTC s under iatrogenic selection.