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Τρίτη 22 Μαΐου 2018

Detection of Bladder Cancer Via Microfluidic Immunoassay and Single-Cell DNA Copy Number Alteration Analysis of Captured Urinary Exfoliated Tumor Cells

The increasing incidence of bladder cancer (BC) and its high rate of recurrence over a 5-year period necessitate the need for diagnosis and surveillance amelioration. Cystoscopy and urinary cytology are the current tools, and molecular techniques such as BTA stat, NMP22, survivin mRNA, and urovysion FISH have attracted attention, however, they suf-fer from insufficient sensitivity or specificity. We developed a novel microfluidic approach for harvesting intact urinary-exfoliated tumor cells (UETCs), either individually or in clus-ters, in a clean and segregated environment, which is crucial to minimize cross-contamination and misreads. To reliably and accurately identify UETC, our quantitative immunoassay involved concurrent use of two oncoproteins CK20 and CD44v6 antigen. CK20 is an intermediate filament protein overexpressed in urothelial tumors, and CD44v6 is a membrane adhesion molecule closely associated with cell invasion, tumor progression and metastatic spread. Single-cell whole-genome sequencing on 12 captured UETCs and copy number alteration analysis showed that 11/12 (91.7%) of the immunofluorescence-identified UETCs possessed genomic instability. A total of 79 BC patients and 43 age-matched normal controls (NC) were enrolled in the study. We detected considerably high-er UETC counts in BC patients versus the NC group [53.3 (10.7-1001.9) vs. 0.0 (0-3.0) UETCs/10 mL; p < 0.0001]. For BC detection, a stratified 10-fold cross-validation of train-ing data reveals an overall predictive accuracy of 0.84 (95%CI: 0.76-0.93) with a 89.8% (95%CI: 71.5-86.4%) for sensitivity and 71.5% (95%CI: 59.7-83.3%) for specificity. Overall, the microfluidic immunoassay demonstrates increased sensitivity and specificity com-pared to other techniques for the detection of bladder cancer.

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