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1.  A Candidate Molecular Biomarker Panel for the Detection of Bladder Cancer 
Background
Bladder cancer (BCa) is among the five most common malignancies world-wide, and due to high rates of recurrence, one of the most prevalent. Improvements in non-invasive urine-based assays to detect BCa would benefit both patients and healthcare systems. In this study, the goal was to identify urothelial cell transcriptomic signatures associated with BCa.
Methods
Gene expression profiling (Affymetrix U133 Plus 2.0 arrays) was applied to exfoliated urothelia obtained from a cohort of 92 subjects with known bladder disease status. Computational analyses identified candidate biomarkers of BCa and an optimal predictive model was derived. Selected targets from the profiling analyses were monitored in an independent cohort of 81 subjects using quantitative real-time PCR (RT-PCR),
Results
Transcriptome profiling data analysis identified 52 genes associated with BCa (p≤0.001), and gene models that optimally predicted class label were derived. RT-PCR analysis of 48 selected targets in an independent cohort identified a 14-gene diagnostic signature that predicted the presence of BCa with high accuracy.
Conclusions
Exfoliated urothelia sampling provides a robust analyte for the evaluation of patients with suspected BCa. The refinement and validation of the multi-gene urothelial cell signatures identified in this preliminary study may lead to accurate, non-invasive assays for the detection of BCa.
Impact
The development of an accurate, non-invasive BCa detection assay would benefit both the patient and healthcare systems through better detection, monitoring and control of disease.
doi:10.1158/1055-9965.EPI-12-0428
PMCID: PMC3537330  PMID: 23097579
Genomic profiling; Bladder cancer; Urinalysis; Non-invasive detection
2.  VEGF, CA9 and Angiogenin as a Urinary Biomarker for Bladder Cancer Detection 
Urology  2012;79(5):1185.e1-1185.e6.
Objective
To investigate whether elevated urinary levels of vascular endothelial growth factor (VEGF), carbonic anhydrase 9 (CA9) and angiogenin are associated with BCa.
Methods
This is a case-control study in which voided urines from 127 patients: control subjects (n = 63) and tumor bearing subjects (n = 64) were analyzed. The urinary concentrations of VEGF, CA9, angiogenin and BTA were assessed by enzyme-linked immunosorbent assay (ELISA). We used the area under the curve (AUC) of receiver operating characteristic curves to determine the ability of VEGF, CA9, and angiogenin to detect BCa in voided urine samples. Data were also compared to a commercial ELISA-based BCa detection assay (BTA-Trak©). Sensitivity, specificity, positive and negative predictive values were calculated.
Results
Urinary concentrations of VEGF, CA9, angiogenin and BTA were significantly elevated in BCa. VEGF was the most accurate urinary biomarker (AUC: 0.886; 95% confidence interval [CI]: 0.8301–0.9418). Furthermore, multivariate regression analysis highlighted VEGF (OR: 5.90; 95% CI: 2.60–13.40, p < 0.0001) as an independent variable. The sensitivities and specificities for VEGF (sensitivity, 83% and specificity, 87%) outperformed BTA (sensitivity, 80% and specificity, 84%).
Conclusions
VEGF may be a valuable addition to voided urine sample analysis for the detection of BCa. Larger, prospective studies are needed to determine the clinical utility of urinary VEGF and angiogenin as biomarkers in the non-invasive evaluation of BCa patients.
doi:10.1016/j.urology.2012.01.016
PMCID: PMC3341520  PMID: 22386755
angiogenin; bladder cancer; biomarkers; diagnosis; VEGF
3.  Metabolomic Profiling Reveals Potential Markers and Bioprocesses Altered in Bladder Cancer Progression 
Cancer research  2011;71(24):7376-7386.
While alterations in xenobiotic metabolism are considered causal in the development of bladder cancer (BCa), the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in BCa. This metabolic signature distinguished both normal and benign bladder from BCa. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing BCa from controls, and also non-muscle from muscle-invasive BCa. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in BCa. In particular, we validated tumor-associated hypermethylation in the CYP1A1 and CYP1B1 promoters of BCa tissues by bisulfite sequence analysis and methylation-specific PCR, and also by in vitro treatment of T-24 BCa cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine. Further, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of BCa specimens compared to matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of BCa, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression.
doi:10.1158/0008-5472.CAN-11-1154
PMCID: PMC3249241  PMID: 21990318
4.  HYAL-1 Hyaluronidase: A Potential Prognostic Indicator for Progression to Muscle Invasion and Recurrence in Bladder Cancer 
European urology  2009;57(1):86-94.
Background
For bladder cancer (BCa) patients undergoing bladder-sparing treatments, molecular markers may aid in accurately predicting progression to muscle invasion and recurrence. Hyaluronic acid (HA) is a glycosaminoglycan that promotes tumor metastasis. Hyaluronoglucosaminidase 1 (HYAL-1)–type hyaluronidase (HAase) promotes tumor growth, invasion, and angiogenesis. Urinary HA and HAase levels are diagnostic markers for BCa.
Objective
We evaluated whether HA and HYAL-1 can predict progression to muscle invasion and recurrence among patients with non–muscle-invasive BCa.
Design, setting, and participants
Based on tissue availability, tissue microarrays were prepared from a cohort of 178 BCa specimens (144 non–muscle invasive, 34 muscle invasive). Follow-up information was available on 111 patients with non–muscle-invasive BCa (mean follow-up: 69.5 mo); 58 patients recurred and 25 progressed to muscle invasion (mean time to progress: 22.3 mo).
Measurements
HA and HYAL-1 expression was evaluated by immunohistochemistry and graded for intensity and area of staining. Association of HA and HYAL-1 staining with BCa recurrence and muscle invasion was evaluated by univariate and multivariate models.
Results and limitations
HA and HYAL-1 expression correlated with tumor grade, stage, and multifocality (p < 0.05). In non–muscle-invasive BCa specimens, HYAL-1 staining was higher (234.3 ± 52.2; 200.6 ± 61.4) if patients experienced progression to muscle invasion or recurrence when compared with no progression or recurrence (164.1 ± 48.2; 172.1 ± 57; p < 0.001). HA staining correlated with muscle invasion (p < 0.001). In univariate analysis, age (p = 0.014), multifocality (p = 0.023), and HYAL-1 staining (p < 0.001) correlated with muscle invasion, whereas only HYAL-1 correlated with recurrence (p = 0.013). In multivariate analysis, significantly associated with muscle invasion (p < 0.001; 76.8% accuracy) and recurrence (p = 0.01; 67.8% accuracy).
Conclusions
HYAL-1 is a potential prognostic marker for predicting progression to muscle invasion and recurrence.
doi:10.1016/j.eururo.2009.03.057
PMCID: PMC2828527  PMID: 19345473
Bladder cancer; Hyaluronic acid; Hyaluronidase; HYAL-1; non-muscle invasive bladder cancer; Prognostic markers; Tissue microarray
5.  CCL18 in a Multiplex Urine-Based Assay for the Detection of Bladder Cancer 
PLoS ONE  2012;7(5):e37797.
The early detection of bladder cancer (BCa) is pivotal for successful patient treatment and management. Through genomic and proteomic studies, we have identified a number of bladder cancer-associated biomarkers that have potential clinical utility. In a case-control study, we examined voided urines from 127 subjects: 64 tumor-bearing subjects and 63 controls. The urine concentrations of the following proteins were assessed by enzyme-linked immunosorbent assay (ELISA); C-C motif chemokine 18 (CCL18), Plasminogen Activator Inhibitor 1 (PAI-1) and CD44. Data were compared to a commercial ELISA-based BCa detection assay (BTA-Trak©) and voided urinary cytology. We used analysis of the area under the curve of receiver operating characteristic curves to compare the ability of CCL18, PAI-1, CD44, and BTA to detect BCa in voided urine samples. Urinary concentrations of CCL18, PAI-1, and BTA were significantly elevated in subjects with BCa. CCL18 was the most accurate biomarker (AUC; 0.919; 95% confidence interval [CI], 0.8704-0.9674). Multivariate regression analysis highlighted CCL18 (OR; 18.31; 95% CI, 4.95-67.70, p<0.0001) and BTA (OR; 6.43; 95% CI, 1.86-22.21, p = 0.0033) as independent predictors of BCa in voided urine samples. The combination of CCL18, PAI-1 and CD44 improved the area under the curve to0.938. Preliminary results indicate that CCL18 was a highly accurate biomarker for BCa detection in this cohort. Monitoring CCL18 in voided urine samples has the potential to improve non-invasive tests for BCa diagnosis. Furthermore using the combination of CCL18, PAI-1 and CD44 may make the model more robust to errors to detect BCa over the individual biomarkers or BTA.
doi:10.1371/journal.pone.0037797
PMCID: PMC3357344  PMID: 22629457
6.  ASSOCIATION OF HYALURONIC ACID FAMILY MEMBERS (HAS1, HAS2 and HYAL-1) WITH BLADDER CANCER DIAGNOSIS AND PROGNOSIS 
Cancer  2010;117(6):1197-1209.
Background
Cancer biomarkers are the backbone for the implementation of individualized approaches to bladder cancer (BCa). Hyaluronic acid (HA) and all seven members of the HA-family i.e., HA-synthases (1,2,3), HYAL-1 hyaluronidase and HA-receptors (CD44s, CD44v and RHAMM) function in tumor growth and progression. However, the diagnostic and prognostic potential of these seven HA family members has not been compared simultaneously in any cancer. We evaluated the diagnostic and prognostic potential of HA-family members in BCa.
Methods
Using quantitative-PCR and immunohistochemistry, the expression of HA family members was evaluated in prospectively collected bladder tissues (n=72); mean and median follow-up: 29.6 ± 5.3; and 24 months, respectively. Transcript levels were also measured in exfoliated urothelial cells from urine specimens (n=148).
Results
Among HA family members, HA-synthase(s), HYAL-1, CD44v and RHAMM transcript levels were 4–16-fold elevated in BCa tissues when compared to normal tissues (P<0.0001), however, CD44s levels were lower. In univariate and multivariate analyses, tumor-stage (P=0.003), lymph node invasion (P=0.033), HYAL-1 (P=0.019) and HAS1 (P=0.027) transcript levels and HYAL-1 staining (P=0.021) independently associated with metastasis. Tumor-stage (p=0.019) and HYAL-1 (p=0.046) transcript levels also associated with disease specific mortality. While HA-synthase and HYAL-1 transcript levels were elevated in exfoliated urothelial cells from BCa patients, the combined HAS2-HYAL-1 expression detected BCa with overall 85.4% sensitivity and 79.5% specificity and predicted BCa recurrence within 6-months (P=0.004; RR=6.7).
Conclusion
HYAL-1 and HAS1 expression predicted BCa metastasis and HYAL-1 expression also predicted disease-specific survival. Furthermore, the combined HAS2-HYAL-1 biomarker detected BCa and significantly predicted its recurrence.
doi:10.1002/cncr.25565
PMCID: PMC3025265  PMID: 20960509
Prognostic makers; hyaluronic acid; HA-synthase; HYAL-1; HA-receptors; hyaluronidase; diagnosis; recurrence
7.  Long Interspersed Nuclear Element-1 Hypomethylation and Oxidative Stress: Correlation and Bladder Cancer Diagnostic Potential 
PLoS ONE  2012;7(5):e37009.
Although, increased oxidative stress and hypomethylation of long interspersed nuclear element-1 (LINE-1) associate with bladder cancer (BCa) development, the relationship between these alterations is unknown. We evaluated the oxidative stress and hypomethylation of the LINE-1 in 61 BCa patients and 45 normal individuals. To measure the methylation levels and to differentiate the LINE-1 loci into hypermethylated, partially methylated and hypomethylated, peripheral blood cells, urinary exfoliated cells and cancerous tissues were evaluated by combined bisulfite restriction analysis PCR. The urinary total antioxidant status (TAS) and plasma protein carbonyl content were determined. The LINE-1 methylation levels and patterns, especially hypomethylated loci, in the blood and urine cells of the BCa patients were different from the levels and patterns in the healthy controls. The urinary TAS was decreased, whereas the plasma protein carbonyl content was increased in the BCa patients relative to the controls. A positive correlation between the methylation of LINE-1 in the blood-derived DNA and urinary TAS was found in both the BCa and control groups. The urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content provided the best diagnostic potential for BCa prediction. Based on post-diagnostic samples, the combination test improved the diagnostic power to a sensitivity of 96% and a specificity of 96%. In conclusion, decreased LINE-1 methylation is associated with increased oxidative stress both in healthy and BCa subjects across the various tissue types, implying a dose-response association. Increases in the LINE-1 hypomethylation levels and the number of hypomethylated loci in both the blood- and urine-derived cells and increase in the oxidative stress were found in the BCa patients. The combination test of the urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content may be useful for BCa screening and monitoring of treatment.
doi:10.1371/journal.pone.0037009
PMCID: PMC3352860  PMID: 22615872
8.  A Multi-Analyte Assay for the Non-Invasive Detection of Bladder Cancer 
PLoS ONE  2012;7(10):e47469.
Accurate urinary assays for bladder cancer (BCa) detection would benefit both patients and healthcare systems. Through genomic and proteomic profiling of urine components, we have previously identified a panel of biomarkers that can outperform current urine-based biomarkers for the non-invasive detection of BCa. Herein, we report the diagnostic utility of various multivariate combinations of these biomarkers. We performed a case-controlled validation study in which voided urines from 127 patients (64 tumor bearing subjects) were analyzed. The urinary concentrations of 14 biomarkers (IL-8, MMP-9, MMP-10, SDC1, CCL18, PAI-1, CD44, VEGF, ANG, CA9, A1AT, OPN, PTX3, and APOE) were assessed by enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of each biomarker and multivariate models were compared using receiver operating characteristic curves and the chi-square test. An 8-biomarker model achieved the most accurate BCa diagnosis (sensitivity 92%, specificity 97%), but a combination of 3 of the 8 biomarkers (IL-8, VEGF, and APOE) was also highly accurate (sensitivity 90%, specificity 97%). For comparison, the commercial BTA-Trak ELISA test achieved a sensitivity of 79% and a specificity of 83%, and voided urine cytology detected only 33% of BCa cases in the same cohort. These datashow that a multivariate urine-based assay can markedly improve the accuracy of non-invasive BCa detection. Further validation studies are under way to investigate the clinical utility of this panel of biomarkers for BCa diagnosis and disease monitoring.
doi:10.1371/journal.pone.0047469
PMCID: PMC3477150  PMID: 23094052
9.  CXCR7: A Functionally Associated Molecular Marker for Bladder Cancer 
Cancer  2012;119(1):61-71.
Background
CXCR4 and CXCR7 are seven transmembrane chemokine receptors of the stroma-derived factor (SDF-1). CXCR4, but not CXCR7, has been examined in bladder cancer (BCa). We examined the functional and clinical significance of CXCR7 in BCa.
Methods
CXCR4 and CXCR7 levels were measured in BCa cell lines, tissues (normal = 25; BCa = 44) and urine specimens (n=186) by quantitative PCR and/or immunohistochemistry. CXCR7 function in BCa cells were examined by transient transfections using a CXCR7 expression vector or siRNA.
Results
In BCa cell lines CXCR7 mRNA levels were 5–37-fold higher than CXCR4 levels. Transient overexpression of CXCR7 in BCa cell lines promoted growth and chemotactic motility. CXCR7 co-localized and formed a functional complex with EGF-receptor, PI3-kinase/Akt, Erk and src and induced their phosphorylation. CXCR7 also induced upregulation of cyclin-D1 and bcl-2. Suppression of CXCR7 expression reversed these effects and induced apoptosis. CXCR7 mRNA levels and CXCR7 staining scores were significantly (5–10-fold) higher in BCa tissues than in normal tissues (P<0.001). CXCR7 expression independently associated with metastasis (P=0.019) and disease specific mortality (P=0.03). CXCR7 was highly expressed in endothelial cells in high-grade BCa tissues when compare to low-grade BCa and normal bladder. CXCR7 levels were elevated in exfoliated urothelial cells from high-grade BCa patients (P=0.0001; 90% sensitivity; 75% specificity); CXCR4 levels were unaltered.
Conclusion
CXCR7 promotes BCa cell proliferation and motility plausibly through EGF-receptor and Akt-signaling. CXCR7 expression is elevated in BCa tissues and exfoliated cells and is associated with high-grade and metastasis.
doi:10.1002/cncr.27661
PMCID: PMC3461116  PMID: 22736438
CXCR7; bladder cancer; molecular marker; metastasis; CXCR4
10.  IL-8 as a urinary biomarker for the detection of bladder cancer 
BMC Urology  2012;12:12.
Background
Current urine-based assays for bladder cancer (BCa) diagnosis lack accuracy, so the search for improved biomarkers continues. Through genomic and proteomic profiling of urine, we have identified a panel of biomarkers associated with the presence of BCa. In this study, we evaluated the utility of three of these biomarkers, interleukin 8 (IL-8), Matrix metallopeptidase 9 (MMP-9) and Syndecan in the diagnosis of BCa through urinalysis.
Methods
Voided urines from 127 subjects, cancer subjects (n = 64), non-cancer subjects (n = 63) were analyzed. The protein concentrations of IL-8, MMP-9, and Syndecan were assessed by enzyme-linked immunosorbent assay (ELISA). Data were also compared to a commercial ELISA-based BCa detection assay (BTA-Trak©) and urinary cytology. We used the area under the curve of a receiver operating characteristic (AUROC) to compare the performance of each biomarker.
Results
Urinary protein concentrations of IL-8, MMP-9 and BTA were significantly elevated in BCa subjects. Of the experimental markers compared to BTA-Trak©, IL-8 was the most prominent marker (AUC; 0.79; 95% confidence interval [CI], 0.72-0.86). Multivariate regression analysis revealed that only IL-8 (OR; 1.51; 95% CI, 1.16-1.97, p = 0.002) was an independent factor for the detection of BCa.
Conclusions
These results suggest that the measurement of IL-8 in voided urinary samples may have utility for urine-based detection of BCa. These findings need to be confirmed in a larger, prospective cohort.
doi:10.1186/1471-2490-12-12
PMCID: PMC3404900  PMID: 22559832
IL-8; Biomarkers; Diagnosis; Bladder cancer
11.  1 alpha, 25-dihydroxylvitamin D3 promotes Bacillus Calmette-Guérin immunotherapy of bladder cancer 
Oncotarget  2013;4(12):2397-2406.
Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis(TB), has been used and proven to be one of the most effective treatments for non-muscle invasive bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, thereby limiting the improvement of BCG therapy. Vitamin D deficiency has been associated with a high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy. To test this hypothesis, we examined the impact of 1alpha, 25-dihydroxyvitamin D3 (1,25-VD) on BCG-induced response in BCa cells and macrophage cells. Our data revealed that 1,25-VD promotes BCG-induced interleukin 8 (IL-8) secretion by BCa cells, consequently inducing the migration of macrophage, THP-1. This THP-1 cell migration promoted by 1,25-VD can be blocked by IL-8 neutralized antibody. Furthermore, 1,25-VD increased BCG-induced expression of macrophage markers in THP-1 cell, and enhanced the BCG-induced THP-1 cytotoxicity against low-grade BCa cells. Importantly, a pre-clinical trial using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model revealed that intravesical co-treatment of 1,25-VD with BCG can prolong mice survival. These data demonstrate a novel mechanism by which 1,25-VD promotes BCG-mediated anti-BCa pathways and provides a platform for improving BCG efficacy with combination of 1,25-VD.
PMCID: PMC3926835  PMID: 24353168
vitamin D; Bacillus Calmette-Guérin; bladder cancer; immunotherapy; interleukin 8
12.  Therapeutic potential for phenytoin: targeting Nav1.5 sodium channels to reduce migration and invasion in metastatic breast cancer 
Voltage-gated Na+ channels (VGSCs) are heteromeric membrane protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. VGSCs are classically expressed in excitable cells, including neurons and muscle cells, where they mediate action potential firing, neurite outgrowth, pathfinding, and migration. VGSCs are also expressed in metastatic cells from a number of cancers. The Nav1.5 α subunit (encoded by SCN5A) is expressed in breast cancer (BCa) cell lines, where it enhances migration and invasion. We studied the expression of SCN5A in BCa array data, and tested the effect of the VGSC-blocking anticonvulsant phenytoin (5,5-diphenylhydantoin) on Na+ current, migration, and invasion in BCa cells. SCN5A was up-regulated in BCa samples in several datasets, and was more highly expressed in samples from patients who had a recurrence, metastasis, or died within 5 years. SCN5A was also overexpressed as an outlier in a subset of samples, and associated with increased odds of developing metastasis. Phenytoin inhibited transient and persistent Na+ current recorded from strongly metastatic MDA-MB-231 cells, and this effect was more potent at depolarized holding voltages. It may thus be an effective VGSC-blocking drug in cancer cells, which typically have depolarized membrane potentials. At a concentration within the therapeutic range used to treat epilepsy, phenytoin significantly inhibited the migration and invasion of MDA-MB-231 cells, but had no effect on weakly metastatic MCF-7 cells, which do not express Na+ currents. We conclude that phenytoin suppresses Na+ current in VGSC-expressing metastatic BCa cells, thus inhibiting VGSC-dependent migration and invasion. Together, our data support the hypothesis that SCN5A is up-regulated in BCa, favoring an invasive/metastatic phenotype. We therefore propose that repurposing existing VGSC-blocking therapeutic drugs should be further investigated as a potential new strategy to improve patient outcomes in metastatic BCa.
doi:10.1007/s10549-012-2102-9
PMCID: PMC3401508  PMID: 22678159
Electrophysiology; Invasion; Metastasis; Migration; Phenytoin; Voltage-gated Na+ channel
13.  Therapeutic potential for phenytoin: targeting Nav1.5 sodium channels to reduce migration and invasion in metastatic breast cancer 
Voltage-gated Na+ channels (VGSCs) are heteromeric membrane protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. VGSCs are classically expressed in excitable cells, including neurons and muscle cells, where they mediate action potential firing, neurite outgrowth, pathfinding, and migration. VGSCs are also expressed in metastatic cells from a number of cancers. The Nav1.5 α subunit (encoded by SCN5A) is expressed in breast cancer (BCa) cell lines, where it enhances migration and invasion. We studied the expression of SCN5A in BCa array data, and tested the effect of the VGSC-blocking anticonvulsant phenytoin (5,5-diphenylhydantoin) on Na+ current, migration, and invasion in BCa cells. SCN5A was up-regulated in BCa samples in several datasets, and was more highly expressed in samples from patients who had a recurrence, metastasis, or died within 5 years. SCN5A was also overexpressed as an outlier in a subset of samples, and associated with increased odds of developing metastasis. Phenytoin inhibited transient and persistent Na+ current recorded from strongly metastatic MDA-MB-231 cells, and this effect was more potent at depolarized holding voltages. It may thus be an effective VGSC-blocking drug in cancer cells, which typically have depolarized membrane potentials. At a concentration within the therapeutic range used to treat epilepsy, phenytoin significantly inhibited the migration and invasion of MDA-MB-231 cells, but had no effect on weakly metastatic MCF-7 cells, which do not express Na+ currents. We conclude that phenytoin suppresses Na+ current in VGSC-expressing metastatic BCa cells, thus inhibiting VGSC-dependent migration and invasion. Together, our data support the hypothesis that SCN5A is up-regulated in BCa, favoring an invasive/metastatic phenotype. We therefore propose that repurposing existing VGSC-blocking therapeutic drugs should be further investigated as a potential new strategy to improve patient outcomes in metastatic BCa.
doi:10.1007/s10549-012-2102-9
PMCID: PMC3401508  PMID: 22678159
Electrophysiology; Invasion; Metastasis; Migration; Phenytoin; Voltage-gated Na+ channel
14.  Urinary BTA: Indicator of Bladder Cancer or of Hematuria 
World journal of urology  2012;30(6):869-873.
Background
In this study, we investigated the influence of hematuria on the performance of the BTA tests in a clinical cohort and in an experimental model.
Materials and Methods
Analysis of 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 1μl/10ml was enough to produce positive a BTA test. High levels of BTA were found equally in the serum of subjects with, or without BCa (mean BTA levels 355,159 U/ml 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.
doi:10.1007/s00345-012-0935-9
PMCID: PMC3537326  PMID: 22932760
complement factor H; hematuria; BTA; bladder cancer; diagnosis
15.  Detection of Pancreatic Carcinomas by Imaging Lactose-Binding Protein Expression in Peritumoral Pancreas Using [18F]Fluoroethyl-Deoxylactose PET/CT 
PLoS ONE  2009;4(11):e7977.
Background
Early diagnosis of pancreatic carcinoma with highly sensitive diagnostic imaging methods could save lives of many thousands of patients, because early detection increases resectability and survival rates. Current non-invasive diagnostic imaging techniques have inadequate resolution and sensitivity for detection of small size (∼2–3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with β-O-D-galactopyranosyl-(1,4′)-2′-deoxy-2′-[18F]fluoroethyl-D-glucopyranose ([18F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [18F]FEDL is a novel radioligand of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which is overexpressed in peritumoral pancreatic acinar cells.
Methodology/Principal Findings
Dynamic PET/CT imaging demonstrated rapid accumulation of [18F]FEDL in peritumoral pancreatic tissue (4.04±2.06%ID/g), bi-exponential blood clearance with half-lives of 1.65±0.50 min and 14.14±3.60 min, and rapid elimination from other organs and tissues, predominantly by renal clearance. Using model-independent graphical analysis of dynamic PET data, the average distribution volume ratio (DVR) for [18F]FEDL in peritumoral pancreatic tissue was estimated as 3.57±0.60 and 0.94±0.72 in sham-operated control pancreas. Comparative analysis of quantitative autoradiographic images and densitometry of immunohistochemically stained and co-registered adjacent tissue sections demonstrated a strong linear correlation between the magnitude of [18F]FEDL binding and HIP/PAP expression in corresponding regions (r = 0.88). The in situ analysis demonstrated that at least a 2–4 fold apparent lesion size amplification was achieved for submillimeter tumors and to nearly half a murine pancreas for tumors larger than 3 mm.
Conclusion/Significance
We have demonstrated the feasibility of detection of early pancreatic tumors by non-invasive imaging with [18F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [18F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.
doi:10.1371/journal.pone.0007977
PMCID: PMC2776527  PMID: 19956730
16.  Burkholderia cenocepacia ShvR-Regulated Genes That Influence Colony Morphology, Biofilm Formation, and Virulence ▿  
Infection and Immunity  2011;79(8):2984-2997.
Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn5 mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates, l-arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia.
doi:10.1128/IAI.00170-11
PMCID: PMC3147549  PMID: 21690240
17.  Regulation of breast cancer metastasis by Runx2 and estrogen signaling: the role of SNAI2 
Breast Cancer Research : BCR  2011;13(6):R127.
Introduction
In contrast to its role in breast cancer (BCa) initiation, estrogen signaling has a protective effect in later stages, where estrogen receptor (ER)α loss associates with aggressive metastatic disease. We asked whether the beneficial effect of estrogen signaling in late-stage BCa is attributable to the recently reported estrogen-mediated antagonism of the pro-metastatic transcription factor Runx2.
Methods
MCF7/Rx2dox breast cancer cells were engineered with a lentivirus expressing Runx2 in response to doxycycline (dox). Cells treated with dox and/or estradiol (E2) were subjected to genome-wide expression profiling, RT-qPCR analysis of specific genes, and Matrigel™ invasion assays. Knockdown of genes of interest was performed using lentiviruses expressing appropriate shRNAs, either constitutively or in response to dox. Gene expression in BCa tumors was investigated using a cohort of 557 patients compiled from publicly available datasets. Association of gene expression with clinical metastasis was assessed by dichotomizing patients into those expressing genes of interest at either high or low levels, and comparing the respective Kaplan-Meier curves of metastasis-free survival.
Results
Runx2 induced epithelial-mesenchymal transition (EMT) evidenced by acquisition of a fibroblastic morphology, decreased expression of E-cadherin, increased expression of vimentin and invasiveness. Runx2 stimulated SNAI2 expression in a WNT- and transforming growth factor (TGF)β-dependent manner, and knockdown of SNAI2 abrogated the pro-metastatic activities of Runx2. E2 antagonized the pro-metastatic activities of Runx2, including SNAI2 upregulation. In primary BCa tumors, Runx2 activity, SNAI2 expression, and metastasis were positively correlated, and SNAI2 expression was negatively correlated with ERα. However, the negative correlation between SNAI2 and ERα in bone-seeking BCa cells was weaker than the respective negative correlation in tumors seeking lung. Furthermore, the absence of ERα in primary tumors was associated with lung- and brain- but not with bone metastasis, and tumor biopsies from bone metastatic sites displayed the unusual combination of high Runx2/SNAI2 and high ERα expression.
Conclusions
E2 antagonizes Runx2-induced EMT and invasiveness of BCa cells, partly through attenuating expression of SNAI2, a Runx2 target required for mediating its pro-metastatic property. That ERα loss promotes non-osseous metastasis by unleashing Runx2/SNAI2 is supported by the negative correlation observed in corresponding tumors. Unknown mechanisms in bone-seeking BCa allow high Runx2/SNAI2 expression despite high ERα level
doi:10.1186/bcr3073
PMCID: PMC3326569  PMID: 22151997
18.  Pharmacokinetics and bioavailability of the isoflavone biochanin A in rats 
The AAPS Journal  2006;8(3):E433-E442.
Biochanin A(BCA) is a dietary isoflavone present in legumes, most notably red clover, and in many herbal dietary supplements. BCA has been reported to have chemopreventive properties and is metabolized to the isoflavone genistein (GEN), BCA conjugates, and GEN conjugates. The metabolites may contribute to the chemopreventive effects of BCA. The absorption, metabolism, and disposition of BCA have not been determined in rats. Our objective was to evaluate the pharmacokinetics and metabolism of BCA in rats. Male Sprague-Dawley rats were administered BCA by intravenous injection (1 and 5 mg/kg), by intraperitoneal injection (5 and 50 mg/kg), and orally (5 and 50 mg/kg). Plasma and bile samples were enzymatically hydrolyzed in vitro to determine conjugate concentrations for BCA and GEN. Equilibrium dialysis was used to determine protein binding. The BCA and GEN concentrations in plasma, urine, and bile were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The pharmacokinetic parameters of BCA were analyzed by noncompartmental analysis. Significant levels of BCA conjugates and GEN conjugates were detected in plasma and bile. Both BCA and GEN were found to have a high clearance and a large apparent volume of distribution; the bioavailability of both was poor (<4%). Reentry peaks were evident after oral administration of both BCA and GEN, suggesting enterohepatic cycling. The free fraction of BCA in rat plasma was 1.5%. A2-compartment model that included both linear and nonlinear clearance terms and enterohepatic recirculation best described the plasma data. This represents the first evaluation of the dose-dependent pharmacokinetics and metabolism of BCA in rats.
doi:10.1208/aapsj080351
PMCID: PMC2761049  PMID: 17025260
Biochanin A; pharmacokinetics; intraperitoneal administration; enterohepatic recirculation; rat; genistein
19.  MMP7 and MMP8 genetic polymorphisms in bladder cancer patients 
Introduction
Breakdown of the extracellular matrix by matrix metalloproteinases (MMPs), as we know, is one of mechanisms involved and required in tumor invasion. MMP7 is a negative prognostic factor of various malignances, while MMP8 exhibits an inhibitory effect on tumorigenesis and metastasis. We evaluated the potential association of functional polymorphisms in the promoter of the MMP7 (rs11568818) and MMP8 (rs11225395) genes and bladder cancer (BCa) risk.
Materials and methods
The study included 241 BCa cases and 199 healthy population controls that were collected at the First Department of Urology, Medical University (Łódź, Poland) and at the Nofer Institute of Occupational Medicine (Łódź, Poland). Genomic DNA samples were isolated from venous blood and genetic polymorphisms were analyzed by real–time polymerase chain reaction using TaqMan fluorescent probes. Associations between genotype and allele status were estimated by logistic regression models adjusted for classic risk factors (e.g. age, gender and cigarette smoking).
Results
MMP7 and MMP8 genotypes were distributed similarly in BCa patients and in controls and at least one variant allele was not associated with BCa cancer risk (OR, 0.91; 95% CI, 0.60–1.39; p = 0.662 for MMP7 and OR, 0.96; 95% CI, 0.63–1.46; p = 0.836 for MMP8). We observed higher prevalence of MMP7 GG genotypes among BCa patients than in controls (OR, 1.54; 95% CI, 0.93–2.55; p = 0.093). Additionally, genetic polymorphisms in the MMP7 and MMP8 were not associated with the tumor grade or stage.
Conclusions
Our results suggest that genetic variations in two genes encoding members of the MMP7 and MMP8 are not associated with a risk of BCa in the Caucasian population.
doi:10.5173/ceju.2013.04.art3
PMCID: PMC3992442  PMID: 24757528
MMP; genetic polymorphism; case–control study; bladder cancer
20.  Experimental acute pancreatitis in PAP/HIP knock‐out mice 
Gut  2007;56(8):1091-1097.
Background and aims
PAP/HIP was first reported as an additional pancreatic secretory protein expressed during the acute phase of pancreatitis. It was shown in vitro to be anti‐apoptotic and anti‐inflammatory. This study aims to look at whether PAP/HIP plays the same role in vivo.
Methods
A model of caerulein‐induced pancreatitis was used to compare the outcome of pancreatitis in PAP/HIP−/− and wild‐type mice.
Results
PAP/HIP−/− mice showed the normal phenotype at birth and normal postnatal development. Caerulein‐induced pancreatic necrosis was, however, less severe in PAP/HIP−/− mice than in wild‐type mice, as judged by lower amylasemia and lipasemia levels and smaller areas of necrosis. On the contrary, pancreas from PAP/HIP−/− mice was more sensitive to apoptosis, in agreement with the anti‐apoptotic effect of PAP/HIP in vitro. Surprisingly, pancreatic inflammation was more extensive in PAP/HIP−/− mice, as judged from histological parameters, increased myeloperoxidase activity and increased pro‐inflammatory cytokine expression. This result, in apparent contradiction with the limited necrosis observed in these mice, is, however, in agreement with the anti‐inflammatory function previously reported in vitro for PAP/HIP. This is supported by the observation that activation of the STAT3/SOCS3 pathway was strongly decreased in the pancreas of PAP/HIP−/− mice and by the reversion of the apoptotic and inflammatory phenotypes upon administration of recombinant PAP/HIP to PAP/HIP−/− mice.
Conclusion
The anti‐apoptotic and anti‐inflammatory functions described in vitro for PAP/HIP have physiological relevance in the pancreas in vivo during caerulein‐induced pancreatitis.
doi:10.1136/gut.2006.116087
PMCID: PMC1955488  PMID: 17409121
21.  PET/CT and MRI in Bladder Cancer 
Journal of cancer science & therapy  2012;Suppl 14(1):7692.
Bladder Cancer (BCa) is the most common malignancy arising from the urinary tract. One of the mainstays of diagnosis, staging, and therapeutic decision-making for BCa is accurate and appropriate imaging. The ability to identify metastatic disease preoperatively is of utmost importance in determining treatment. Advances in standard cross sectional imaging techniques like Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) have improved imaging of bladder cancer. Over the last decade, 18F-fluorodeoxyglucose (FDG) Positron Emission Tomography (PET) in combination with CT (18F-FDG PET/CT) has become an important non-invasive imaging modality for the preoperative staging of various malignancies. 18F-FDG PET/CT is useful for detection of metastatic disease in BCa, but the ability to detect primary bladder wall lesions remains to be elucidated. To overcome the problem with urinary excretion of 18F-FDG, new PET tracers are being tested. MRI is an accurate technique for the local staging of BCa due to its superior spatial and contrast resolution. Anatomical MRI has a modest utility in NM-staging of BCa. However, incorporation of functional MR techniques, such as diffusion weighted MRI can improve the results for lesion detection and staging and multi-parametric MRI`s role is yet to be explored widely. The aim of this review is to present the recent advances in PET/CT and MRI in BCa, with particular focus on improvements in staging.
doi:10.4172/1948-5956.S14-001
PMCID: PMC3587689  PMID: 23471167
Positron Emission Tomography/Computed Tomography (PET/CT); Magnetic Resonance Imaging (MRI); Urothelial cancer; Bladder cancer
22.  Effects of partner proteins on BCA2 RING ligase activity 
BMC Cancer  2012;12:63.
Background
BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2.
Methods
Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays.
Results
Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways.
Conclusions
The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression.
doi:10.1186/1471-2407-12-63
PMCID: PMC3298473  PMID: 22315970
23.  Investigation of CCL18 and A1AT as potential urinary biomarkers for bladder cancer detection 
BMC Urology  2013;13:42.
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.
doi:10.1186/1471-2490-13-42
PMCID: PMC3846766  PMID: 24011266
Biomarkers; Bladder cancer; Specificity; Urine
24.  Highly sensitive proximity mediated immunoassay reveals HER2 status conversion in the circulating tumor cells of metastatic breast cancer patients 
Proteome Science  2011;9:75.
Background
The clinical benefits associated with targeted oncology agents are generally limited to subsets of patients. Even with favorable biomarker profiles, many patients do not respond or acquire resistance. Existing technologies are ineffective for treatment monitoring as they provide only static and limited information and require substantial amounts of tissue. Therefore, there is an urgent need to develop methods that can profile potential therapeutic targets with limited clinical specimens during the course of treatment.
Methods
We have developed a novel proteomics-based assay, Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) that can be used for analyzing clinical samples. CEER utilizes the formation of unique immuno-complex between capture-antibodies and two additional detector-Abs on a microarray surface. One of the detector-Abs is conjugated to glucose oxidase (GO), and the other is conjugated to Horse Radish Peroxidase (HRP). Target detection requires the presence of both detector-Abs because the enzyme channeling event between GO and HRP will not occur unless both Abs are in close proximity.
Results
CEER was able to detect single-cell level expression and phosphorylation of human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 1 (HER1) in breast cancer (BCa) systems. The shift in phosphorylation profiles of receptor tyrosine kinases (RTKs) and other signal transduction proteins upon differential ligand stimulation further demonstrated extreme assay specificity in a multiplexed array format. HER2 analysis by CEER in 227 BCa tissues showed superior accuracy when compared to the outcome from immunohistochemistry (IHC) (83% vs. 96%). A significant incidence of HER2 status alteration with recurrent disease was observed via circulating tumor cell (CTC) analysis, suggesting an evolving and dynamic disease progression. HER2-positive CTCs were found in 41% (7/17) while CTCs with significant HER2-activation without apparent over-expression were found in 18% (3/17) of relapsed BCa patients with HER2-negative primary tumors. The apparent 'HER2 status conversion' observed in recurrent BCa may have significant implications on understanding breast cancer metastasis and associated therapeutic development.
Conclusion
CEER can be multiplexed to analyze pathway proteins in a comprehensive manner with extreme specificity and sensitivity. This format is ideal for analyzing clinical samples with limited availability.
doi:10.1186/1477-5956-9-75
PMCID: PMC3271991  PMID: 22172159
Companion diagnostics; Collaborative enzyme enhanced reactive-immunoassay; Metastatic breast cancer, Circulating tumor cells; HER2 conversion
25.  Syndecan Binding Protein (SDCBP) Is Overexpressed in Estrogen Receptor Negative Breast Cancers, and Is a Potential Promoter for Tumor Proliferation 
PLoS ONE  2013;8(3):e60046.
Background
Syndecan binding protein (SDCBP), an adapter protein containing PDZ domains, contributes to the tumorigenicity and metastasis of many malignant tumors, such as malignant melanoma. Our study aimed in revealing the expression profile of SDCBP in breast cancer (BCa) and its role in tumor cell proliferation, and then exploring its value in the targeted treatment of BCa.
Methodology/Principal Findings
We first evaluated the SDCBP expression by immunohistochemistry in normal breast and BCa tissues. Then we explored the expression profile of SDCBP in different BCa cell lines. By constructing SDCBP-silenced BCa cell clones, we further assessed the effects of SDCBP suppression on tumor cells in vitro by cell culture and in vivo by tumorigenicity. SDCBP expression was detected in 80.6% (n = 160) of BCa tissues, in contrast to its expression in 13% (n = 23) of normal breast tissues (P<0.001). Among the tumors, the level of its expression was positively correlated with histological grade and tumor staging while negatively correlated with the estrogen receptor (ER) expression. Higher expression of SDCBP was also noted in ER-negative BCa cell lines. It was also identified that SDCBP expression was down-regulated in a dose-dependent mode by 17-β estradiol in estrogen-responsive MCF-7. Furthermore, SDCBP silence inhibited ER-negative tumor cell growth in vivo and in vitro. Cell cycle studies showed that SDCBP silence increased G1 cell population and resulted in related cell-cycle-regulator changes: up-regulation of p21 and p27 while down-regulation of cyclin E.
Conclusion/Significance
Our results suggested that SDCBP played an important role in tumor growth of ER-negative BCas. In these tumors where the estrogen signaling pathway is not available, SDCBP probably contribute to tumor growth through an alternative signaling pathway by promoting tumor cells passing the G1/S checkpoint into the cell cycle. Suppression of SDCBP expression may have its potential to become a targeted therapy for ER-negative BCas.
doi:10.1371/journal.pone.0060046
PMCID: PMC3606191  PMID: 23533663

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