The non-invasive detection and monitoring of bladder cancer (BCa) remains a challenge. Voided urinary cytology (VUC) remains the most established urine-based assay for this purpose, but while VUC has a specificity of >93%, the assay suffers from low sensitivity (25–40%) and observer-dependent variability [1].Commercial tests measuring nuclear matrix protein (NMP-22) and bladder tumor antigen (BTA) have emerged as diagnostic urinary protein tests for BCa, but these single-marker assays lack the specificity of VUC [2], [3]. The concept that the presence or absence of one molecular marker will aid clinical evaluation has not proved to be the case. This is not surprising when one considers the variation between individuals, the cross-talk between molecular pathways, and the heterogeneity of solid tumors.

The advent of high-throughput techniques has enabled an evolution from single-marker research to a more global assessment strategy, and we have used these to identify promising novel biomarkers of BCa. Using genomics [4] and proteomics [5] approaches to profile the soluble and cellular components of voided urine, we have identified a panel of biomarkers that show promise for development into accurate assays for non-invasive BCa detection. We have performed a series of validation studies [6]–[9] to evaluate the potential clinical utility of a number of these biomarkers. In this study, we combined data on 14 of our candidate biomarkers in a cohort of 127 subjects in order to derive an accurate and robust multivariate assay for the non-invasive detection of BCa.

Demographic, clinical and pathologic characteristics of the 127 subjects who comprised our study group are illustrated in Table 1. Five subjects (3 in control group, 2 in cancer group) with missing biomarker data were excluded from final analysis.No subjects in the control cohort had an abnormal cystoscopy or axial imaging. Furthermore in follow-up, none of the control subjects were noted to develop BCa or even gross hematuria. In the cancer cohort, 41% of subjects had non-muscle invasive disease and 19% of subjects had low-grade disease. The median tumor size was 4.5 cm. In line with our previous experience [4], voided urinary cytology (VUC) in the cohort achieved 98% specificity, but only 33% sensitivity.

The mean and median levels of the 14 biomarkers in urine are presented in Table 2. The levels of 10 of the biomarkers were significantly elevated in subjects with BCa, relative to subjects without BCa. CD44 and OPN were significantly, but negatively, associated with BCa, and SDC1 and PTX3 were not significantly associated with BCa. Given the fact that the majority of patients present at the urology clinic with hematuria, one has to consider whether specific biomarker assays are detecting a tumor antigen or a serum protein introduced into the test sample via bleeding. To control for this possibility, we quantitated the hemoglobin level in all samples and evaluated the correlation of all 14 biomarkers with this measure of hematuria. This analysis revealed that levels of CCL18 and A1AT had a high correlation to urinary hemoglobin (Spearman’s correlation coefficient >0.8), and so these were not included in the multivariate models described below.

Figure 1 illustrates the diagnostic performance of various combinations of biomarkers. When all 8 positively associated BCa biomarkers were combined, the overall diagnostic accuracy was 94%, with a sensitivity of 92% and a specificity of 97%. Logistic regression and a 1000 Bootstrap sample strategywere performed to determine optimal diagnostic biomarker combinations, i.e. high accuracy with as few biomarkers as possible.For each Bootstrap sample, candidate subsets were ranked using Bayesian information criterion [15]. Analyses found that the combination of IL-8, VEGF, and APOE were ranked number one 410 times (41%), the combination of VEGF and APOE ranked number one 379 times (38%), and all other combinations ranked number one less than 100 times (<10%). The combination of IL-8, VEGF, and APOE achieved a performance almost as good as all 8 biomarkers combined (Figure 1). The 3-biomarker assay had an AUC of 0.968 (95% confidence interval [CI], 0.942–0.992), a sensitivity of 90% and specificity of 97% (overall accuracy 93%). The combination of VEGF and APOE had a reduced overall accuracy (89%). The 2-biomarker assay had an AUC of 0.957 (95% confidence interval [CI], 0.927–0.987), a sensitivity of 81% and a specificity of 97%. For comparison, the BTA-Trak commercial assay achieved a sensitivity of 79%, specificity of 83%, and overall accuracy of 81% (Figure 1).

Cancer of the urinary bladder is among the five most common malignancies worldwide. At presentation, more than 80% of bladder tumors are non-muscle invasive papillary tumors which have a 5-year survival rate of >90%, however, approximately 70% of patients with these lesions develop tumor recurrence within two years of initial diagnosis. The recurrence phenomenon of non-muscle invasive BCa makes it one of the most prevalent cancers world-wide. Once BCa is detected and treated, patients will routinely get frequent surveillance cystoscopy to monitor for tumor recurrence. If left untreated, initially non-invasive tumors can progress to muscle-invasive tumors which have a significantly reduced 5-year survival rate. Thus, accurate detection of BCa, ideally through non-invasive urine-based analysis, remains an urgent goal.

In order to validate a panel of promising biomarkers discovered using genomics and proteomics approaches [4], [5], we have performed a series of studies using ELISA-based assays [6]–[9]. In this study, we investigated an additional target and combined all data -14 biomarkers in a cohort of 127 subjects- to derive the most accurate diagnostic multi-analyte assays. We also monitored the performance of the commercial BTA-Trak assay in the same cases for comparison, and we quantitated hemoglobin in each case in order to investigate potential correlations between biomarkers and hematuria. Two biomarkers, CCL18 and A1AT, were found to have a relatively high correlation with urinary hemoglobin, raising the possibility that the source of these proteins is, at least in part, serum introduced through bleeding. We will continue to investigate these two biomarkers in independent cohorts, but they were not included in the logistic regression analyses performed to identify optimal diagnostic biomarker combinations. An 8-biomarker panel (IL-8, MMP-9, PAI-1, VEGF, ANG, CA-9, APOE, MMP-10) proved to be the most accurate (overall accuracy of 94%) multi-analyte assay for the detection of BCa in the 127-subject cohort. With multiplex molecular signatures, it is usual that a few of the components will provide much of the predictive power, but additional markers will make the model more robust to errors [4]. By testing all possible combinations via a bootstrap strategy, we revealed that three of the 8 biomarker panel (IL-8, VEGF and APOE) contributed the most information. As a stand-alone assay the 3-biomarker panel achieved an overall accuracy of 93% and maintained both high sensitivity (90%) and specificity (97%). To achieve high accuracy with as few biomarkers as possible is optimal for practical reasons, but it is important that we monitor all of the promising biomarkers further in larger, more diverse cohorts, because different combinations may be more robust in specific conditions, such as the presence of infection, or be more accurate for recurrence versus initial diagnosis.

The components of the 3-biomarker panel (VEGF, IL-8, APOE) have been associated with a number of cancers, including bladder cancer, to varying degrees. In a study of 26 subjects, Crews et al. demonstrated that elevated urinary levels of VEGF correlated with levels in excised tissue [17]. In a study of 219 Middle East subjects, urinary levels of VEGF were significantly higher in patients with BCa. In their cohort measuring VEGF outperformed VUC, achieving a diagnostic sensitivity of 76% [18]. Similarly, Bian demonstrated an improved sensitivity of urinary VEGF compared to VUC, achieving 69% vs. 38%, respectively [19]. In another study, patients with elevated urinary VEGF levels had a higher risk of disease recurrence [20]. Previous reports have implicated IL-8 in bladder tumor biology and its use as a potential biomarker of BCa has been investigated in a few studies. IL-8 has been shown to have mitogenic and angiogenic properties, and high levels result in increased tumorigenicity, progression and metastasis in mouse models, reportedly via regulation of nuclear factor kappa-B [21]. Clinical studies have indicated that elevated urinary levels of IL-8 can be associated with the presence of bladder cancer. In a study of 140 subjects, an IL-8 assay achieved a sensitivity of 59%, and a specificity of 90% [22], and in a study of 79 subjects, a sensitivity of 50% and a specificity of 90% was reported [23]. These results are very much in line with our findings when assessing IL-8 as an individual biomarker [7]. Data is scarce on the role of APOE in cancers. Functions include enhancement of lipid transport into cells and mediation of signal transduction upon binding to lipoprotein receptors [24], [25]. APOE has been shown to interact with the transitional epithelial response gene (TERE1), a tumor suppressor gene, in bladder tumor cells, resulting in increased cell turnover and resistance to apoptosis [26], [27]. Recently, Lindén et al. reported that APOE was a component of a panel of biomarkers detected by mass spectroscopy, and may be associated with non-muscle invasive BCa [28].

We recognize that our study has several limitations.First as a tertiary care facility, we tend to see more high-grade, high-stage disease, which is reflected in our study cohort. To confirm the robustness of our signature, subsequent studies must assess larger cohorts that include subjects with low-grade, low-stage disease. Second, processed, banked urines were analyzed. Urines were centrifuged and separated into cellular pellet and supernatant prior to storage at −80°C. It is feasible that freshly voided urine samples may provide different results, and it is fresh urine that would be the material used for point-of-care assays. We are currently investigating the performance of selected biomarkers in urines processed via a number of different protocols, including freshly voided urines. Next, the sensitivity of VUC in our cohort of predominantly high-grade (grade 3) disease (33%) was lower than would be expected. This calls into question the known inter-observer variability of interpreting VUC. In subsequent studies, we will utilize two cytopathologists to interpret these results. Furthermore, it is uncertain how the protein composition of the urine supernatant may change during frozen storage. The number of freeze-thaw cycles was kept to 1–2 by dividing the urine supernatant into multiple small aliquots. Lastly, our sample size of 127 is small and the two groups that comprised the 127 subjects were relatively homogeneous, i.e. either active cancer, or control cases with no active cancer, no history of cancer, no urinary tract infection, no urolithiasis, and no gross hematuria. Thus we were not able to assess sensitivity/specificity of our biomarkers among different stages/grades.The specificity of promising biomarkers need to be tested in cohorts that are known to be problematic with other urine-based assays (e.g., hematuria, urinary tract infection, stones and voiding dysfunction).

The identification of robust BCa-associated biomarkers and the establishment of multiplex urine-based assays will have multiple short-term and long-term impacts. Many of the validated biomarkers in this study have not been associated with cancer previously. Analysis of the role of these proteins in tumor cell biology may elucidate mechanisms in tumor initiation or progression, and may reveal novel therapeutic targets. Clinically, accurate BCa assays will have a clear impact on initial diagnostic performance, and on the clinical management of patients post-treatment. If reliable urinary diagnostic biomarker assays can reduce the number of necessary invasive and uncomfortable cystoscopies, improvements in patient compliance and satisfaction will follow, and increased efficiency and cost-savings will benefit the healthcare system. The ultimate goal is to be able to detect BCa in a timely manner such that the patient can expect an improved quality of life and overall outcome.