Validation and clinicopathologic associations of a urine-based bladder cancer biomarker signature.

BACKGROUND: To validate the expression of a urine-based bladder cancer associated diagnostic signature comprised of 10 targets; ANG, CA9, MMP9, MMP10, SERPINA1, APOE, SDC1, VEGFA, SERPINE1 and IL8 in bladder tumor tissues. METHODS: Immunohistochemical analyses were performed on tumor specimens from 213 bladder cancer patients (transitional cell carcinoma only) and 74 controls. Staining patterns were digitally captured and quantitated (Aperio, Vista, CA), and expression was correlated with tumor stage, tumor grade and outcome measures. RESULTS: We revealed a positive association of 9 of the 10 proteins (excluding VEGF) in bladder cancer. Relative to control cases, a reduction in SDC1 and overexpression of MMP9, MMP10, SERPINE1, IL8, APOE, SERPINA1, ANG were associated with high stage bladder cancer. Reduced VEGF and increased SERPINA1 were associated with high-grade bladder cancer. Disease-specific survival was significantly reduced in tumors with high expression of SERPINE1 and/or IL8. CONCLUSIONS: These findings confirm that the proteins in a urine-based diagnostic signature are aberrantly expressed in bladder tumor tissues, and support the potential additional utility of selected biomarkers for the clinicopathological evaluation of excised tissue or biopsy material. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_200.

Matrix metalloproteinase-10 promotes tumor progression through regulation of angiogenic and apoptotic pathways in cervical tumors.

BACKGROUND: Cancer invasion and metastasis develops through a series of steps that involve the loss of cell to cell and cell to matrix adhesion, degradation of extracellular matrix and induction of angiogenesis. Different protease systems (e.g., matrix metalloproteinases, MMPs) are involved in these steps. MMP-10, one of the lesser studied MMPs, is limited to epithelial cells and can facilitate tumor cell invasion by targeting collagen, elastin and laminin. Enhanced MMP-10 expression has been linked to poor clinical prognosis in some cancers, however, mechanisms underlying a role for MMP-10 in tumorigenesis and progression remain largely unknown. Here, we report that MMP-10 expression is positively correlated with the invasiveness of human cervical and bladder cancers. METHODS: Using commercial tissue microarray (TMA) of cervical and bladder tissues, MMP-10 immunohistochemical staining was performed. Furthermore using a panel of human cells (HeLa and UROtsa), in vitro and in vivo experiments were performed in which MMP-10 was overexpressed or silenced and we noted phenotypic and genotypic changes. RESULTS: Experimentally, we showed that MMP-10 can regulate tumor cell migration and invasion, and endothelial cell tube formation, and that MMP-10 effects are associated with a resistance to apoptosis. Further investigation revealed that increasing MMP-10 expression stimulates the expression of HIF-1α and MMP-2 (pro-angiogenic factors) and PAI-1 and CXCR2 (pro-metastatic factors), and accordingly, targeting MMP-10 with siRNA in vivo resulted in diminution of xenograft tumor growth with a concomitant reduction of angiogenesis and a stimulation of apoptosis. CONCLUSION: Taken together, our findings show that MMP-10 can play a significant role in tumor growth and progression, and that MMP-10 perturbation may represent a rational strategy for cancer treatment.

Clinical implications in the shift of syndecan-1 expression from the cell membrane to the cytoplasm in bladder cancer.

BACKGROUND: To determine the diagnostic and prognostic capability of urinary and tumoral syndecan-1 (SDC-1) levels in patients with cancer of the urinary bladder. METHODS: SDC-1 levels were quantitated by enzyme-linked immunosorbent assay (ELISA) in 308 subjects (102 cancer subjects and 206 non-cancer subjects) to assess its diagnostic capabilities in voided urine. The performance of SDC-1 was evaluated using the area under the curve of a receiver operating characteristic curve. In addition, immunohistochemical (IHC) staining assessed SDC-1 protein expression in 193 bladder specimens (185 cancer subjects and 8 non-cancer subjects). Outcomes were correlated to SDC-1 levels. RESULTS: Mean urinary levels of SDC-1 did not differ between the cancer subjects and the non-cancer subjects, however, the mean urinary levels of SDC-1 were reduced in high-grade compared to low-grade disease (p < 0.0001), and in muscle invasive bladder cancer (MIBC) compared to non-muscle invasive bladder cancer (NMIBC) (p = 0.005). Correspondingly, preliminary data note a shift from a membranous cellular localization of SDC-1 in normal tissue, low-grade tumors and NMIBC, to a distinctly cytoplasmic localization in high-grade tumors and MIBC was observed in tissue specimens. CONCLUSION: Alone urinary SDC-1 may not be a diagnostic biomarker for bladder cancer, but its urinary levels and cellular localization were associated with the differentiation status of patients with bladder tumors. Further studies are warranted to define the potential role for SDC-1 in bladder cancer progression.

PAI-1 leads to G1-phase cell-cycle progression through cyclin D3/cdk4/6 upregulation.

UNLABELLED: The canonical function of plasminogen activator inhibitor-1 (PAI-1/SERPINE1) is as an inhibitor of urokinase-type plasminogen activator for blood clot maintenance, but it is now also considered a pleiotropic factor that can exert diverse cellular and tumorigenic effects. However, the mechanism controlling its pleiotropic effects is far from being understood. To elucidate the tumorigenic role of PAI-1, we tested the effects of PAI-1 after manipulation of its expression or through the use of a small-molecule inhibitor, tiplaxtinin. Downregulation of PAI-1 significantly reduced cellular proliferation through an inability to progress from the G(0-G1) phase of the cell cycle. Accordingly, overexpression of PAI-1 augmented proliferation by encouraging S-phase entry. Biochemically, cell-cycle arrest was associated with the depletion of the G(1)-phase transition complexes, cyclin D3/cdk4/6 and cyclin E/cdk2, in parallel with the upregulation of the cell-cycle inhibitors p53, p21Cip1/Waf1, and p27Kip1. PAI-1 depletion significantly decreased the tumor size of urothelial T24 and UM-UC-14 xenografts, and overexpression of PAI-1 substantially increased the tumor size of HeLa xenografts. Finally, immunohistochemical analysis of human bladder and cervical tumor tissue microarrays revealed increased expression of PAI-1 in cancerous tissue, specifically in aggressive tumors, supporting the relevance of this molecule in human tumor biology. IMPLICATIONS: Targeting PAI-1 has beneficial antitumoral effects and should be further investigated clinically.

Chemokine (C-X-C motif) ligand 1 (CXCL1) protein expression is increased in high-grade prostate cancer.

Chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), may enhance tumor epithelial-stromal interactions facilitating tumor growth and invasion. Studies have linked CXCL1 expression to gastric, colon and skin cancers, however, no study to date has been reported describing CXCL1 in human prostate tumors. Herein, we set out to describe the expression pattern of CXCL1 in human prostate tumors. Utilizing a commercial tissue microarray, immunohistochemical staining was used to monitor CXCL1 protein expression in 90 primary prostate tumors and 20 benign prostate tissues. CXCL1 protein expression was noted to be predominantly in the cytoplasm of both the benign epithelia glands and cancerous epithelia glands) with >75% of benign or cancerous glands demonstrating immunoreactivity. However, staining intensity was noted to be significantly different between benign and cancerous tissue with 84% of cancerous tissue staining moderate (++) to strong (+++) compared to only 30% of benign prostate samples staining moderate (++) to strong (+++) (p<0.0001). Increased CXCL1 protein levels were associated with higher-grade tumors (Gleason≤6 vs. Gleason score 7-10, p=0.038). An increase in CXCL1 protein was present in of high-grade malignancy. Further studies are warranted to clearly define the role of CXCL1 in prostate cancer.

Erythropoietin is a JAK2 and ERK1/2 effector that can promote renal tumor cell proliferation under hypoxic conditions.

BACKGROUND: Erythropoietin (EPO) provides an alternative to transfusion for increasing red blood cell mass and treating anemia in cancer patients. However, recent studies have reported increased adverse events and/or reduced survival in patients receiving both EPO and chemotherapy, potentially related to EPO-induced cancer progression. Additional preclinical studies that elucidate the possible mechanism underlying EPO cellular growth stimulation are needed. METHODS: Using commercial tissue microarray (TMA) of a variety of cancers and benign tissues, EPO and EPO receptor immunohistochemical staining was performed. Furthermore using a panel of human renal cells (Caki-1, 786-O, 769-P, RPTEC), in vitro and in vivo experiments were performed with the addition of EPO in normoxic and hypoxic states to note phenotypic and genotypic changes. RESULTS: EPO expression score was significantly elevated in lung cancer and lymphoma (compared to benign tissues), while EPOR expression score was significantly elevated in lymphoma, thyroid, uterine, lung and prostate cancers (compared to benign tissues). EPO and EPOR expression scores in RCC and benign renal tissue were not significantly different. Experimentally, we show that exposure of human renal cells to recombinant EPO (rhEPO) induces cellular proliferation, which we report for the first time, is further enhanced in a hypoxic state. Mechanistic investigations revealed that EPO stimulates the expression of cyclin D1 while inhibiting the expression of p21cip1 and p27kip1 through the phosphorylation of JAK2 and ERK1/2, leading to a more rapid progression through the cell cycle. We also demonstrate an increase in the growth of renal cell carcinoma xenograft tumors when systemic rhEPO is administered. CONCLUSIONS: In summary, we elucidated a previously unidentified mechanism by which EPO administration regulates progression through the cell cycle, and show that EPO effects are significantly enhanced under hypoxic conditions.

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.

Pheochromocytoma of the urinary bladder: a systematic review of the contemporary literature.

BACKGROUND: Pheochromocytoma (paraganglioma) of the urinary bladder is a rare tumor. Herein we sought to review the contemporary literature on pheochromocytomas of the urinary bladder in order to further illustrate the presentation, treatment options and outcomes of patients diagnosed with these tumors. METHODS: A comprehensive review of the current literature was conducted according to the PRISMA guidelines by accessing the NCBI PubMed database and using the search terms "paraganglioma, pheochromocytoma, bladder." This search resulted in the identification of 186 articles published between January 1980 and April 2012 of which 80 articles were ultimately included in our analysis. RESULTS: Pheochromocytomas usually occurred in young adult Caucasians (mean age, 43.3 years; range,11-84 years). According to the literature, the most common symptoms and signs of pheochromocytomas of the urinary bladder were hypertension, headache, and hematuria. Of the 77 cases that commented on catecholamine production, 65 patients had biochemically functional tumors. Approximately 20% of patients were treated by transurethral resection alone, 70% by partial cystectomy and 10% by radical cystectomy. The 75 patients with follow-up information had a mean follow-up of 35 months. At the time of last follow-up, 15 (14.2%) had disease recurrence, 10 (9.4%) had metastasis, and 65 (61.3%) were alive. CONCLUSIONS: Pheochromocytomas of the urinary bladder tend to be functional and occur mostly in young adult Caucasians. Patients with localized tumors have an extremely favorable prognosis and may be managed by less aggressive modalities, whereas patients with metastatic disease have a significant reduction in survival rates despite aggressive treatment.

Locally advanced paraganglioma of the urinary bladder: a case report.

BACKGROUND: Paraganglioma of the urinary bladder is a rare tumor. Herein we sought to describe a case of locally advanced paraganglioma of the urinary bladder managed by partial cystectomy and extended pelvic lymph node dissection. CASE PRESENTATION: The case of a 43-year old Haitian male with locally advanced paraganglioma of the urinary bladder is presented in detail. Through surgical extirpation, our patient was rendered disease-free. Eighteen months later the patient is doing well without symptoms but is noted to have subcentimeter bilateral pulmonary nodules and retroperitoneal lymph nodes. No further therapy has been initiated at this time. CONCLUSIONS: Patients with localized tumors have an extremely favorable prognosis and may be managed by less aggressive modalities, whereas patients with metastatic disease have a significant reduced survival rate despite aggressive treatment.