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Diagnostic Leukapheresis for transcriptomic profiling of single CTCs: Characterization of inter CTC heterogeneity in terms of endocrine resistance
Reinhardt F.1, Franken A.1, Meier-Stiegen F.1, Driemel C.2, Stoecklein N.H.2, Fischer J.C.3, Niederacher D.1, Ruckhaeberle E.1, Neubauer H.1, Fehm T.1
1Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, Düsseldorf, Deutschland, 2Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, Düsseldorf, Deutschland, 3Institute for Transplantation Diagnostics and Cell Therapeutics, University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, Düsseldorf, Deutschland

Introduction: CTCs hold great promise with regard to prognosis, monitoring and treatment optimization of breast cancer patients. Single CTC transcriptomic profiling might help reveal valuable information concerning intra-patient heterogeneity relevant to therapeutic interventions. In this study, we combined Diagnostic Leukapheresis (DLA), microfluidic enrichment with the Parsortix™ system, micromanipulation with the CellCelector™ and subsequent single cell multi-marker transcriptomic profiling.
Material and methods: A PCR panel consisting of 30 different phenotypic and endocrine resistance markers was validated for single cell profiling by using different breast cancer cell lines. This panel was applied to characterize uncultured and cultured CTCs, which were enriched from a cryopreserved DLA product obtained from a patient suffering from metastatic breast cancer resistant to endocrine therapy.
Results: Gene expression profiles of both CTC populations uncovered inter CTC heterogeneity for transcripts, which are associated with response or resistance to endocrine therapy (e.g., ESR1, HER2, FGFR1). Hierarchical clustering revealed CTC subpopulations with different expressions of transcripts regarding the CTCs' differential phenotypes (EpCAM, CD44, CD24, MYC, MUC1) and of transcripts involved in endocrine signaling pathways (FOXO, PTEN). ER-positive CTCs exhibited significant higher expression of Cyclin D1, which might be relevant for CDK4/6 inhibitor therapies. Overall, gene expression profiles of uncultured and cultured CTCs resulted in a partly combined grouping.
Conclusion: Multi-marker RNA profiling of enriched single uncultured CTCs and cultured CTCs form cryopreserved DLA samples may provide important insights into intra-patient heterogeneity relevant for targeted therapies and therapy resistance.