Background: Advanced transitional cell carcinoma (TCC) of the bladder is a lethal malignancy with few available biomarkers to guide therapy. Recent evidence suggests that circulating tumor cells (CTCs) are detectable in TCC and may have a prognostic role; however, most studies to date have focused on CTC enumeration. We leveraged novel enrichment and capture technologies to isolate and recover ultra-pure CTC (upCTC) that can be used for single cell molecular analysis.
Methods: Patients with metastatic bladder cancer were enrolled under an institutional review board approved pilot study. Blood samples (7.5 ml each) were drawn into EDTA or Cell-Free DNA blood collection tubes (Streck), and preliminary CTC enrichment was performed using one of 3 microfluidic platforms: i) EpCAM-based LiquidBiopsy system (Cynvenio); ii) size/deformability-based ClearCell FX system (Clearbridge); or iii) size/deformability-based Parsortix system (Angle). Enriched CTCs were labeled with Cytokeratin (CK), EpCAM, and CD45 immunofluorescent antibodies and Hoechst nuclear stain. Identification and recovery of upCTCs was performed on a DEPArray v2 system (Silicon BioSystems). Additional experiments using cell lines were performed to gauge the recovery of mRNA from rare cancer cells collected in EDTA vs. preservative tubes.
Results: To date, samples were collected from 8 patients of whom 5 (∼63%) had detectable CTCs. All 3 enrichment platforms successfully yielded CTC fractions from which Hoechst+/CK+/CD45- upCTCs were subsequently identified and recovered on the DEPArray v2 system. Additional cells that were Hoechst+/CK-/CD45- were identified and collected, and cells also were gated by integral Hoechst intensity and collected for correlation to aneuploidy. Comparison of mRNA recovery from 10 cancer cells collected into EDTA vs. fixative tubes (CellSave, Cynvenio, Streck Cell-free RNA) demonstrated that gene expression readings by qPCR from rare fixed cells was feasible but associated with variable results, likely due to variable proteinase K reversal of mRNA-protein cross-linkages.
Conclusion: We leveraged new techniques to develop and optimize a workflow for identification and collection of upCTCs from bladder TCC patients, and we established that gene expression profiling is also feasible, albeit currently is still best achieved using EDTA collection and not any of the available preservative tubes. Single upCTC molecular profiles (DNA alterations, gene expression) from patient samples generated using these protocols may help to elucidate mechanisms of disease progression and ultimately may advance the management of advanced bladder cancer.
Citation Format: Gareth Morrison, Cory Hugen, Tong Xu, Yucheng Xu, Dorff Tanya, David Quinn, Sarmad Sadeghi, Amir Goldkorn. Circulating tumor cell enrichment and dielectric manipulation for ultra-pure cell recovery in advanced bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3160.