Cancer of the urinary bladder is among the five most common malignancies worldwide. Urothelial cell carcinomas constitute approximately 95% of all bladder cancer cases, [11
] and associated with tobacco exposure [12
]. At presentation, more than 80% of bladder tumors are non-muscle invasive papillary tumors (Ta or T1), which harbor a 5-year survival rate of approximately 94%, [13
] 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 (in America it is second only to colorectal cancer) and is, therefore, a great burden to healthcare systems [13
]. Once BCa is detected and treated, patients will routinely get frequent surveillance cystoscopy to monitor for tumor recurrence [14
]. If left untreated these initially non-invasive tumors can progress to muscle-invasive tumors which have significantly reduced 5-year survival rate [15
]. Thus, early detection, ideally through non-invasive urine-based analysis, remains one of the most urgent issues in BCa research.
In the current report, we describe the analysis of IL-8, MMP-9 and Syndecan in a validation phase cohort of 127 subjects using ELISA assays. Urinary protein concentrations of IL-8, MMP-9 and BTA were significantly associated with BCa. IL-8 outperformed the other experimental biomarkers (specificity 97% and positive predictive value 95%). Multivariate logistic regression analysis highlighted only IL-8’s (OR: 1.51; 95% CI: 1.16-1.97) association with BCa.
In performing studies discovering urinary biomarkers, frozen (banked) urinary samples are critical. In the current study, the cellular component of voided urine was removed from the supernatant to prevent cellular lysis, which may skew results upon thawing analyzing. Upon reviewing available commercial assays to detect BCa in urine samples, only BTA has been confirm on frozen banked samples (see product insert) and thus BTA-Trak served as one of our controls. BTA-Trak© was the initial assay that was used to measure BTA in the urines of BCa subjects in a large multicenter trial [16
]. Subsequently, BTA-Trak© was modified and incorporated into a point of care assay (BTA-stat©) which received FDA approval in 1997 and has become a standard tool in the non-invasive diagnosis ofBCa. BTA-Trak©, median sensitivity is quoted as 71% (range: 60–83%) with improved sensitivity in high-grade tumors. Median specificity is 66% (range: 60–79%), significantly lower than VUC [17
Previous reports have implicated both IL-8 in bladder tumor biology and for use as biomarkers of BCa. IL-8 is an angiogenic factor associated with inflammation and carcinogenesis, and previous reports have documented elevated urinary protein levels of IL-8 in subjects with urothelial cell carcinoma [18
]. Studies have indicated that elevated urinary levels are associated with increased stage of disease, [18
] disease recurrence [19
] and lack of efficacy of intravesical therapies, including bacillus Calmette-Guérin and mitomycin C [21
]. As an initial diagnostic indicator, urinary IL-8 achieved a sensitivity of 59%, and a specificity of 90% in a study of 140 subjects [18
] and 50%/90% in a study of 79 subjects, [20
] results very much in line with our findings. In biological 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. Inhibition of tumor growth in mouse xenograft models by anti-IL-8 antibodies was shown to act via down-regulation of nuclear factor kappa-B [22
Matrix metalloproteinase 9 (MMP-9) has been associated with tumor cell invasion and metastasis in many human cancers, including BCa. As a marker for BCa, studies have reported that elevated urinary protein levels of MMP-9 are associated with cancer. In a study of 188 subjects, high MMP-9 levels were significantly correlated with large tumor size and poor malignancy grade, and increasing levels were associated with poor overall survival [23
]. Studies that measured urinary MMP-2 and MMP-9 by both ELISA and zymography have suggested that MMP-9 levels may be useful as an adjunct to cytology, [24
] or as a diagnostic with good sensitivity (80%) [25
]. Though we demonstrated that urinary protein MMP-9 levels were significantly elevated in BCa subjects by univariate analysis, this did not hold true in logistic regression analysis, and the sensitivity in our cohort was only 56%.
Similar to the study by Aaboe et al.,
] we previously identified Syndecan, a transmembrane heparin sulfate proteoglycan, as a potential biomarker using gene expression profiling [27
]. We went on to confirm that it was more highly expressed in urine samples from BCa subjects via quantitative PCR of shed urothelia cells, however, we were unable to confirm that Syndecan protein was significantly elevated in the urines from BCa subjects. This may be due to the fact that Syndecan is membrane-bound and so is less likely to be present in the soluble fraction of the urine, but a study has shown elevated serum Syndecan levels to be an independent prognostic marker for multiple myeloma [28
In order to monitor whether experimental biomarkers were associated with hematuria, urinary hemoglobin levels were quantitated in each sample using ELISA. As expected, hemoglobin was revealed to be significantly elevated in the urines of subjects with BCa. Interestingly, the levels of urinary BTA had the highest correlation with urinary hemoglobin, raising the possibility that an appreciable source of BTA is the blood routinely found in BCa patients’ urine. This was previously suggested by Oge et al.
] We are currently investigating this possibility in additional experiments. The accurate quantitation of hematuria using a hemoglobin ELISA provides valuable insight into the potential clinical utility of a biomarker prior to development of new assays designed to detect BCa in voided urine samples [30
We recognize that our study has several limitations. First, processed, banked urines were analyzed. Urines were centrifuged and separated into cellular pellet and supernatant prior to storage at -800
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. Second, 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. Next, we are a tertiary care facility that is preferentially referred high grade, higher stage disease, which is reflected in our cohort. To confirm the robustness, subsequent studies will assess more urines from subjects with low-grade, low-stage disease in community urologic practices. Subsequently, the sensitivity of VUC in our cohort of predominantly high-grade (grade 3) disease (28%) 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. Lastly, our cohort was comprised of two cohorts: active cancer or control cases with no active cancer, no history of cancer, no urinary tract infection, no urolithiasis, and no gross hematuria. This may account for favorable detection levels with IL-8. Currently, we are assessing our panel of validated biomarkers in a large diverse cohort to further validate the robustness of this diagnostic signature.