External validation of a multiplex urinary protein panel for the detection of bladder cancer in a multicenter cohort.

BACKGROUND: Because of the faltering sensitivity and/or specificity, urine-based assays currently have a limited role in the management of patients with bladder cancer. The aim of this study was to externally validate our previously reported protein biomarker panel from multiple sites in the United States and Europe. METHODS: This multicenter external validation study included a total of 320 subjects (bladder cancer = 183). The 10 biomarkers (IL8, MMP9, MMP10, SERPINA1, VEGFA, ANG, CA9, APOE, SDC1, and SERPINE1) were measured using commercial ELISA assays in an external laboratory. The diagnostic performance of the biomarker panel was assessed using receiver operator curves (ROC) and descriptive statistical values. RESULTS: Utilizing the combination of all 10 biomarkers, the area under the ROC for the diagnostic panel was noted to be 0.847 (95% confidence interval, 0.796-0.899), outperforming any single biomarker. The multiplex assay at optimal cutoff value achieved an overall sensitivity of 0.79, specificity of 0.79, positive prediction value of 0.73, and negative prediction value of 0.84 for bladder cancer classification. Sensitivity values of the diagnostic panel for high-grade bladder cancer, low-grade bladder cancer, muscle invasive bladder cancer, and non-muscle invasive bladder cancer were 0.81, 0.90, 0.95, and 0.77, respectively. CONCLUSIONS: Urinary levels of the biomarker panel enabled discrimination of patients with bladder cancer and controls, and the levels of biomarker subsets were associated with advancing tumor grade and stage. IMPACT: If proven to be reliable, urinary diagnostic biomarker assays can detect bladder cancer in a timely manner such that the patient can expect improvements in overall survival and quality of life.

Urinary protein biomarker panel for the detection of recurrent bladder cancer.

BACKGROUND: Up to 70% of patients with non-muscle-invasive bladder cancer (NMIBC) experience disease recurrence, making it one of the most prevalent cancers in the United States. The purpose of this study was to test the performance of a multiplex urinary biomarker assay for the monitoring of voided urine for recurrent bladder cancer. METHODS: This retrospective, multicenter study included a total of 125 subjects with a history of bladder cancer. Voided urine specimens were collected before procedure from these subjects (53 with confirmed tumor recurrence and 72 with confirmed non-tumor recurrence) for analysis. A prediction rule generated from the performance characteristics of 10 single biomarkers (IL8, MMP9, MMP10, SERPINA1, VEGFA, ANG, CA9, APOE, SERPINE1, and SDC1) was measured using ELISA. The diagnostic performance of the biomarker panel was assessed using receiver operator curves (ROC) and descriptive statistical values (e.g., sensitivity and specificity). RESULTS: The combination of all 10 biomarkers outperformed any single biomarker with a calculated AUROC for the diagnostic panel of 0.904 [95% confidence interval (CI), 0.853-0.956]. The multiplex assay achieved an overall sensitivity of 79% and specificity of 88% for recurrent bladder cancer and significantly outperformed the Urovysion cytogenetic assay (sensitivity 42%, specificity 94%) and voided urinary cytology (sensitivity 33%, specificity 90%). CONCLUSIONS: A diagnostic panel of 10 urinary biomarkers that accurately detects primary bladder cancer also performs well for the detection of recurrent bladder cancer. IMPACT: The identification of a reliable urine-based surveillance and detection assay would be of benefit to both patients and the healthcare system.

Investigation of CCL18 and A1AT as potential urinary biomarkers for bladder cancer detection.

BACKGROUND: In this study, we further investigated the association of two biomarkers, CCL18 and A1AT, with bladder cancer (BCa) and evaluated the influence of potentially confounding factors in an experimental model. METHODS: In a cohort of 308 subjects (102 with BCa), urinary concentrations of CCL18 and A1AT were assessed by enzyme-linked immunosorbent assay (ELISA). In an experimental model, benign or cancerous cells, in addition to blood, were added to urines from healthy controls and analyzed by ELISA. Lastly, immunohistochemical staining for CCL18 and A1AT in human bladder tumors was performed. RESULTS: Median urinary protein concentrations of CCL18 (52.84 pg/ml vs. 11.13 pg/ml, p < 0.0001) and A1AT (606.4 ng/ml vs. 120.0 ng/ml, p < 0.0001) were significantly elevated in BCa subjects compared to controls. Furthermore, the addition of whole blood to pooled normal urine resulted in a significant increase in both CCL18 and A1AT. IHC staining of bladder tumors revealed CCL18 immunoreactivity in inflammatory cells only, and there was no significant increase in these immunoreactive cells within benign and cancerous tissue and no association with BCa grade nor stage was noted. A1AT immunoreactivity was observed in the cytoplasm of epithelia cells and intensity of immunostaining increased with tumor grade, but not tumor stage. CONCLUSIONS: Further development of A1AT as a diagnostic biomarker for BCa is warranted.

Chemokine (C-X-C) ligand 1 (CXCL1) protein expression is increased in aggressive bladder cancers.

BACKGROUND: Chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), may regulate tumor epithelial-stromal interactions that facilitate tumor growth and invasion. Studies have linked CXCL1 expression to gastric, colon and skin cancers, but limited studies to date have described CXCL1 protein expression in human bladder cancer (BCa). METHODS: CXCL1 protein expression was examined in 152 bladder tissue specimens (142 BCa) by immunohistochemical staining. The expression of CXCL1 was scored by assigning a combined score based on the proportion of cells staining and intensity of staining. CXCL1 expression patterns were correlated with clinicopathological features and follow-up data. RESULTS: CXCL1 protein expression was present in cancerous tissues, but was entirely absent in benign tissue. CXCL1 combined immunostaining score was significantly higher in high-grade tumors relative to low-grade tumors (p = 0.012). Similarly, CXCL1 combined immunostaining score was higher in high stage tumors (T2-T4) than in low stage tumors (Ta-T1) (p < 0.0001). An increase in the combined immunostaining score of CXCL1 was also associated with reduced disease-specific survival. CONCLUSION: To date, this is the largest study describing increased CXCL1 protein expression in more aggressive phenotypes in human BCa. Further studies are warranted to define the role CXCL1 plays in bladder carcinogenesis and progression.

Multiplex protein signature for the detection of bladder cancer in voided urine samples.

PURPOSE: Accurate urine assays for bladder cancer detection would benefit patients and health care systems. Through extensive genomic and proteomic profiling of urine components we previously identified a panel of 8 biomarkers that can facilitate the detection of bladder cancer in voided urine samples. In this study we confirmed this diagnostic molecular signature in a diverse multicenter cohort. MATERIALS AND METHODS: We performed a case-control, phase II study in which we analyzed voided urine from 102 subjects with bladder cancer and 206 with varying urological disorders. The urinary concentration of 8 biomarkers (IL-8, MMP-9 and 10, PAI-1, VEGF, ANG, CA9 and APOE) was assessed by enzyme-linked immunosorbent assay. Diagnostic performance of the panel of tested biomarkers was evaluated using ROCs and descriptive statistical values, eg sensitivity and specificity. RESULTS: Seven of the 8 urine biomarkers were increased in subjects with bladder cancer relative to those without bladder cancer. The 7 biomarkers were assessed in a new model, which had an AUROC of 0.88 (95% CI 0.84-0.93), and 74% sensitivity and 90% specificity. In contrast, the sensitivity of voided urine cytology and the UroVysion® cytogenetic test in this cohort was 39% and 54%, respectively. Study limitations include analysis performed on banked urine samples and the lack of voided urine cytology and cytogenetic test data on controls. CONCLUSIONS: The study provides further evidence that the reported panel of diagnostic biomarkers can reliably achieve the noninvasive detection of bladder cancer with higher sensitivity than currently available urine based assays.

Diagnostic potential of urinary α1-antitrypsin and apolipoprotein E in the detection of bladder cancer.

PURPOSE: The ability to reliably diagnose bladder cancer in voided urine samples would be a major advance. Using high throughput technologies, we identified a panel of bladder cancer associated biomarkers with potential clinical usefulness. In this study we tested 4 potential biomarkers for the noninvasive detection of bladder cancer. MATERIALS AND METHODS: We examined voided urine specimens from 124 patients, including 63 newly diagnosed with bladder cancer and 61 controls. Concentrations of proteins were assessed by enzyme-linked immunosorbent assay, including α1-antitrypsin, apolipoprotein E, osteopontin and pentraxin 3. Data were compared to the results of urinary cytology and the BTA Trak® enzyme-linked immunosorbent assay based bladder cancer detection assay. We used the AUC of ROC curves to compare the usefulness of each biomarker to detect bladder cancer. RESULTS: Urinary levels of α1-antitrypsin, apolipoprotein E and bladder tumor antigen were significantly increased in subjects with bladder cancer. α1-Antitrypsin (AUC 0.9087, 95% CI 0.8555-0.9619) and apolipoprotein E (AUC 0.8987, 95% CI 0.8449-0.9525) were the most accurate biomarkers. The combination of α1-antitrypsin and apolipoprotein E (AUC 0.9399) achieved 91% sensitivity, 89% specificity, and a positive and negative predictive value of 89% and 90%, respectively. Multivariate regression analysis highlighted only apolipoprotein E as an independent predictor of bladder cancer (OR 24.9, 95% CI 4.22-146.7, p = 0.0004). CONCLUSIONS: Alone or in combination, α1-antitrypsin and apolipoprotein E show promise for the noninvasive detection of bladder cancer (OR 24.9, 95% CI 4.22-146.7, p = 0.0004). Larger, prospective studies including more low grade, low stage tumors are needed to confirm these results.

Urinary BTA: indicator of bladder cancer or of hematuria.

BACKGROUND: In this study, we investigated the influence of hematuria on the performance of the bladder tumor antigen (BTA) tests in a clinical cohort and in an experimental model. MATERIALS AND METHODS: Urine samples from a cohort of 126 subjects (64 with BCa and 62 controls) were analyzed by ELISA for hemoglobin and BTA. The experimental model involved the spiking of urine with blood from the same subject, and hemoglobin, red blood cell count, and BTA levels (BTA stat© and BTA-TRAK©). BTA-TRAK© analyses were also performed on serum samples obtained from 40 subjects (20 with confirmed with BCa). RESULTS: In the 126 subject cohort, correlation between hemoglobin and BTA was 0.732. Of the 64 BCa samples, 72 % had a positive BTA assay, but 47 % of controls were also positive. The sensitivity and specificity of BTA to detect BCa was 72 and 53 %, respectively. Hematuria, measured by urinary hemoglobin, was a better indicator of BCa with 75 % sensitivity and 90 % specificity. Spiking of BTA-negative urine samples with as little as 1 µl/10 ml was enough to produce a positive BTA test. High levels of BTA were found equally in the serum of subjects with or without BCa (mean BTA levels 355,159 vs. 332,329 U/ml, respectively). CONCLUSIONS: Rather than detecting a bladder tumor antigen, urinary BTA assays may be measuring serum cFH introduced by bleeding, a common presenting factor in BCa subjects. The presence of hematuria in subjects without malignant disease can result in false-positive BTA assays.

Influencing factors on the NMP-22 urine assay: an experimental model.

BACKGROUND: The commercial NMP-22 urine assays for bladder cancer (BCa) detect nuclear mitotic apparatus protein 1 (NUMA1) using monoclonal antibodies. It remains unclear whether these assays are monitoring a tumor antigen or some other phenomenon associated with the disease state. In this study, we investigated the influence of urinary cellular and protein concentration, and hematuria on the performance of the NMP-22 tests in an experimental model. METHODS: Pooled urine from healthy subjects were spiked with varying concentrations of benign (UROtsa) cells, cancer cells (RT4, T24, KU-7 and UM-UC-14), whole blood or serum, prior to analysis with both NMP22® Bladder Cancer ELISA test and the NMP22® BladderChek® point-of-care test. RESULTS: Urines from control subjects were negative for NMP-22. The addition of whole blood at 50ul/10 ml, but not serum, resulted in a false-positive result. Furthermore, the addition of a high concentration of benign urothelial cells (10(6)) or the cell lysate from these cells (306 µg protein) resulted in a false-positive result. High concentrations of pooled-cancer cells (10(6)) or cell lysate (30.6 µg and above) resulted in a positive NMP-22 assay. Concordance between the NMP-22 ELISA assay and the NMP-22 point of care assay was >90%. CONCLUSIONS: Rather than detecting a specific tumor antigen, urinary NMP-22 assays may be measuring the cellularity or amount of cell turnover that may be introduced into the urine by a variety of conditions, including surface shedding from bladder tumors. The absence of significant urinary cellularity in some cases due to lesion characteristics or the timing of sampling may result in false-negative NMP-2 assays.