Detection of Urothelial Bladder Carcinoma via Microfluidic Immunoassay and Single-Cell DNA Copy-Number Alteration Analysis of Captured Urinary-Exfoliated Tumor Cells

Abstract

The increasing incidence of bladder cancer 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 suffer from insufficient sensitivity or specificity. We developed a novel microfluidic approach for harvesting intact urinary-exfoliated tumor cells (UETC), either individually or in clusters, in a clean and segregated environment, which is crucial to minimize crosscontamination 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 patients with bladder cancer and 43 age-matched normal controls (NC) were enrolled in the study. We detected considerably higher UETC counts in patients with bladder cancer versus the NC group [53.3 (10.7-1001.9) vs. 0.0 (0-3.0) UETCs/10 mL; P < 0.0001]. For bladder cancer detection, a stratified 10-fold cross-validation of training data reveals an overall predictive accuracy of 0.84 [95% confidence interval (CI), 0.76-0.93] with an 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 compared with other techniques for the detection of bladder cancer.Significance: A unique and promising diagnostic assay for bladder cancer is proposed with potential clinical utility as a complement for cytology. Cancer Res; 78(14); 4073-85. Ó2018 AACR.

Phenotypic Analysis of Urothelial Exfoliated Cells in Bladder Cancer via Microfluidic Immunoassays: Sialyl-Tn as a Novel Biomarker in Liquid Biopsies

“…Spiking experiments demonstrated that, using the UriChip, the CE of BC cells spiked in leukocytes and urine from healthy donors achieved 53%, typical for size-based filtration (53,54). Higher efficiencies have recently been reported by Cheng et al, using a size-based microfluidic system, but with less complex model samples, i.e., with BC cells diluted in PBS which do not fully represent the cellular heterogeneity of biological samples (33). Accordingly, cells with different sizes and morphologies were found to be retained in the UriChip, with those originating from BC patients exhibiting higher shape deformation capacity and squeezing though narrower gaps in comparison to similar sized urothelial cells from urine of healthy donors.…”

“…Despite the prognostic significance of CTCs in BC, microfluidic analysis of putative cancer biomarkers in voided urine would be ideal, non-invasive and provide significant benefits for patient monitoring, particularly at early stages of the disease, when survival rates are higher and CTCs may not yet be present. Nevertheless, only a scarcity of studies have used microfluidic chips for urine-based BC detection, employing different detection principles, complex systems, and processing times (31)(32)(33)(34).…”

Interference From Haematuria Renders the Urinary BTA Test Not Specific Enough for Diagnosing Bladder Cancer: Evidence From Clinical Practice

“…This antigen has been recognized as a human complement factor H-related protein (hCFHrp) and inhibits the complement pathway to cause cytolysis in cells with a resulting selective advantage for the tumour [7]. Previous studies have suggested that the sensitivity of the BTA stat test is superior to urine cytology, and it has been approved by the US Food and Drug Administration for diagnosing and monitoring of BC [8,9]. However, the speci city of the BTA stat test is often disturbed by some factors, such as infection and intravesical treatment [10][11][12].…”

The influence of haematuria on the utility of the BTA stat test in the diagnosis of bladder cancer: a prospective study

“…The bladder tumour antigen (BTA) stat test is the most concerned BC marker detection method and it has been approved by the US Food and Drug Administration for diagnosing and monitoring of BC [7,8]. This antigen has been recognized as a human complement factor H-related protein (hCFHrp) and inhibits the complement pathway to cause cytolysis in cells with a resulting selective advantage for the tumour [9].…”