Heterogeneity Of Circulating Tumor Cells Isolated From Bladder Cancer Patients Using Apostream And Biomarkers Of Epithelial-Mesenchymal Transition.

349 Background: Elucidating the molecular pathways that drive the progression of metastatic bladder cancer (BC) from local disease to metastatic may provide insight into prognosis and potentially treatment. The frequency of detection of circulating tumor cells (CTCs) in metastatic BC patients is only 30% using EpCAM based methods. Reports have shown that EpCAM based methods detect only a fraction of CTCs and miss the heterogeneous subpopulations of CTCs related to epithelial to mesenchymal transition (EMT). EMT is a hallmark of cellular invasion and metastasis and CTCs undergoing EMT may have prognostic value in BC if reliable detection and characterization methods were developed. Here we used ApoStream, a novel antibody-free CTC isolation device to isolate CTCs and perform molecular characterization. Methods: Blood samples from 13 early stage or metastatic BC patients were collected and processed using ApoStream. Isolated cells were immunophenotyped using a multiplexed immunofluorescence assay for CK, CD45, DAPI, uroplakin, vimentin and Twist. Laser scanning cytometry analysis was applied to identify subsets of CK + CD45 - DAPI + or CK - CD45 - DAPI + cells for the expression and distribution of uroplakin, vimentin and Twist. Urovysion FISH analysis was performed on CTCs from 6 BC patients. Results: CK + CD45 - DAPI + cells were detected in 4/13 (31%) of patients with vimentin detected in this subset. CK - CD45 - DAPI + cells were detected in 9/12 (75%) of patients with Twist expression detected in this subset. Uroplakin expression was not detected. Chromosomal abnormalities were detected in CK - CD45 - DAPI + cells isolated from the blood of 3/6 (50%) BC patients. Conclusions: ApoStream isolated cells from the blood of BC patients with phenotypic and genotypic characteristics of CTCs. The CK +/- CD45 - DAPI + Twist and vimentin phenotype indicates a population of circulating cells with relevant biomarkers of EMT and may represent an important population of CTCs mediating disease progression. Detection of chromosomal abnormalities on non-canonical CTCs highlights heterogeneity and underscores the need for expanded definitions of CTCs in BC.