Cancer of the urinary bladder is a common neoplastic disease with high rates of recurrence and progression. The rate of recurrence makes it one of the most prevalent cancers worldwide [27]. Disease detection currently relies upon invasive cystoscopic examination of the bladder. The only urinary assay in routine use is voided urine cytology (VUC), but as it lacks sensitivity, it is typically deployed as an adjunct to cystoscopy rather than a stand-alone test. The development of accurate, non-invasive urinary tests would benefit both patients and health care systems. A robust test could avoid unnecessary invasive patient evaluation and improve patient compliance on clinical surveillance and follow-up regimes. The development of multiplex assays that reflect the complexity of molecular events involved in neoplasia can provide a more accurate assessment with broad clinical utility.
Multiplex assay advantages include reduced cost through lower labor needs and reagent consumption, and the generation of more data with less sample, but the major advantage is the potential to significantly improve clinical test sensitivity and specificity by a combination of multiple biomarkers. Many tissue-based analyses focus on multiplexing nucleic acid targets, but for liquid biopsy settings protein multiplexing may be more appropriate as the test is relatively straightforward with minimal sample processing, fast and economical throughput, and can achieve direct quantitation without requiring molecular target amplification. Notably, one multiplex protein cancer diagnostic test is FDA approved, OVA1, which is being employed for the early detection of ovarian cancer [28]. The test measures absolute serum levels of CA125, apolipoprotein A1, beta 2 microglobulin, prealbumin, and transferrin to determine the risk for malignancy. The test has a reported overall sensitivity of > 90% as a stand-alone test and can provide a valuable adjunct to ultrasound imaging and physical examination [28]. Coupling the advantages of a multiplex protein test with non-invasive urine sampling could provide a highly accurate bladder cancer diagnostic test as well as providing data for monitoring disease progression and response to therapy. The development of the Oncuria™ test has been reported from transcriptomic and proteomic profiling discovery [8–11], to refinement and validation of candidate biomarkers [12–15], to custom multiplex design and analytical validation [29, 30]. In this study, the test was applied to 348 naturally micturated urine samples prospectively obtained from patients visiting urology clinics at three institutions.
The 10 biomarkers associated with Oncuria™ were reliably detect in the 362 urine samples; MMP9 in 64.3%, IL8 in 84.4%, VEGFA in 88.6%, CA9 in 40.6%, SDC1 in 99.3%, PAI1 in 71.7%, ApoE in 95.7%, A1AT in 93.2%, ANG in 81.8% and MMP10 in 57.7%. Further, these 10 biomarkers were present at higher levels in voided urines from bladder cancer subjects compared to controls with significance being reached for IL8, VEGFA, PAI1, ApoE, A1AT and ANG. SDC1 had only slightly elevated mean levels in cancer compared to controls; 9,461 pg/mL vs. 8,707 pg/mL. Previously we reported that while SDC1 is not always elevated in bladder cancer urine samples relative to controls it can provide prognostic information based on its association with tumor grade and stage [31]. Single biomarkers were noted to have lower sensitivity and/or specificity; best response PAI1 AUROC of 0.89 (95% confidence interval, 0.83–0.95) with a sensitivity of 78% and a specificity of 90% and ApoE AUROC of 0.89 (95% confidence interval, 0.84–0.94), with a sensitivity of 73% and a specificity of 90%. A combinatorial analysis of all ten biomarkers noted an AUROC of 0.93 (95% confidence interval, 0.87–0.98), with a sensitivity of 87% and a specificity of 92%. These parameters were noted to improve with the addition of the three demographic factors (age, sex and race) to the hybrid signature: AUROC of 0.95 (95% confidence interval, 0.90–1.00), with a sensitivity of 93% and a specificity of 93%. Lastly, we noted that urine samples from patients with history of renal cell carcinoma or renal cell carcinoma and urine samples from patients with history of prostate cancer or prostate cancer did not result in positive Oncuria™ test (data not shown). This finding confirms our previous report in that thus attesting to its specificity. We were able to confirm the clinical utility of monitoring a diagnostic biomarker signature for the detection of bladder cancer in non-invasively obtained urine samples. The Oncuria™ test achieved encouraging values of sensitivity and specificity and NPV.
Recently, several groups have begun to identify panels of diagnostic biomarkers for potential bladder cancer application. For example, through analysis of nine gene promoters, Hoque et al. found that 69% of bladder cancer patients had methylation in at least one of four genes (CDKN2A, ARF, MGMT, GSTP1), whereas the controls had no such methylation detectable [32]. By combining the data from all nine genes, a logistic prediction model was derived that achieved a sensitivity of 82% and specificity of 96%. Chung et al. selected 10 candidate hypermethylated genes from data collected from tumor tissue and tested these 10 genes in voided urine samples by quantitative methylation-specific RT-PCR and identified a multigene predictive model comprised of five target genes (MYO3A, CA10, NKX6-2, DBC1, and SOX11). Sensitivity and specificity of this model were 85% and 95%, respectively [33]. Further examples include RNA signatures proposed by Hanke et al. [34] and Mengual et al. [35] possessing sensitivities ranging from 80%-92% and specificities ranging from 85%-99%. To date, these studies have had small sample size, with limited populations analyzed (i.e., few benign confounding conditions included) and have not undergone extensive validation. Only Holyoake et al. from New Zealand have reported on the discovery [36] and validation of a multiplexed RNA signature comprised of CDC2, MDK, IGFBP5 and HOXA13 (Cxbladder™), with a reported sensitivity of 82% and specificity of 85% [37, 38].
We recognize that the study has several limitations. First, as tertiary-care facilities, we tend to see more high-grade, high-stage disease, which is reflected in our study cohort. To further confirm the robustness of the multiplex assay, subsequent studies must assess larger cohorts that include more subjects with low-grade, low-stage disease. Second, we did not have complete smoking data for all subjects in the cohort and, therefore, an association with smoking history was not possible. Third, processed, banked urines were analyzed. Urines were centrifuged and separated into cellular pellet and supernatant before storage at -800C. It is feasible that freshly voided urine samples may provide different results. We are currently investigating the performance of the test in urines processed via a number of different protocols, including freshly voided urines. To address these issues, the Oncuria™ test is currently being evaluated in three large multicenter, international prospective clinical trials (NCT 03193515, 03193528, and 03193541). These trials will include first-event diagnosis and disease recurrence monitoring.