Chien-Lun Chen

Multiplexed quantification of 63 proteins in human urine by multiple reaction monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers.

Three common urological diseases are bladder cancer, urinary tract infection, and hematuria. Seventeen bladder cancer biomarkers were previously discovered using iTRAQ - these findings were verified by MRM-MS in this current study. Urine samples from 156 patients with hernia (n=57, control), bladder cancer (n=76), or urinary tract infection/hematuria (n=23) were collected and subjected to multiplexed LC-MRM/MS to determine the concentrations of 63 proteins that are normally considered to be plasma proteins, but which include proteins found in our earlier iTRAQ study. Sixty-five stable isotope-labeled standard proteotypic peptides were used as internal standards for 63 targeted proteins. Twelve proteins showed higher concentrations in the bladder cancer group than in the hernia and the urinary tract infection/hematuria groups, and thus represent potential urinary biomarkers for detection of bladder cancer. Prothrombin had the highest AUC (0.796), with 71.1% sensitivity and 75.0% specificity for differentiating bladder cancer (n=76) from non-cancerous (n=80) patients. The multiplexed MRM-MS data was used to generate a six-peptide marker panel. This six-peptide panel (afamin, adiponectin, complement C4 gamma chain, apolipoprotein A-II precursor, ceruloplasmin, and prothrombin) can discriminate bladder cancer subjects from non-cancerous subjects with an AUC of 0.814, with a 76.3% positive predictive value, and a 77.5% negative predictive value. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

Comparative and Targeted Proteomic Analyses of Urinary Microparticles from Bladder Cancer and Hernia Patients

Bladder cancer is a common urologic cancer whose incidence continues to rise annually. Urinary microparticles are an attractive material for noninvasive bladder cancer biomarker discovery. In this study, we applied isotopic dimethylation labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to discover bladder cancer biomarkers in urinary microparticles isolated from hernia (control) and bladder cancer patients. This approach identified 2964 proteins based on more than two distinct peptides, of which 2058 had not previously been reported as constituents of human urine exosomes/microparticles. A total of 107 differentially expressed proteins were identified as candidate biomarkers. Differences in the concentrations of 29 proteins (41 signature peptides) were precisely quantified by LC-MRM/MS in 48 urine samples of bladder cancer, hernia, and urinary tract infection/hematuria. Concentrations of 24 proteins changed significantly (p<0.05) between bladder cancer (n=28) and hernia (n=12), with area-under-the-curve values ranging from 0.702 to 0.896. Finally, we quantified tumor-associated calcium-signal transducer 2 (TACSTD2) in raw urine specimens (n=221) using a commercial ELISA and confirmed its potential value for diagnosis of bladder cancer. Our study reveals a strong association of TACSTD2 with bladder cancer and highlights the potential of human urinary microparticles in the noninvasive diagnosis of bladder cancer.

Discovery of novel bladder cancer biomarkers by comparative urine proteomics using iTRAQ technology.

A urine sample preparation workflow for the iTRAQ (isobaric tag for relative and absolute quantitation) technique was established. The reproducibility of this platform was evaluated and applied to discover proteins with differential levels between pooled urine samples from nontumor controls and three bladder cancer patient subgroups with different grades/stages (a total of 14 controls and 23 cancer cases in two multiplex iTRAQ runs). Combining the results of two independent clinical sample sets, a total of 638 urine proteins were identified. Among them, 55 proteins consistently showed >2-fold differences in both sample sets. Western blot analyses of individual urine samples confirmed that the levels of apolipoprotein A-I (APOA1), apolipoprotein A-II, heparin cofactor 2 precursor and peroxiredoxin-2 were significantly elevated in bladder cancer urine specimens (n = 25-74). Finally, we quantified APOA1 in a number of urine samples using a commercial ELISA and confirmed again its potential value for diagnosis (n = 126, 94.6% sensitivity and 92.0% specificity at a cutoff value of 11.16 ng/mL) and early detection (n = 71, 83.8% sensitivity and 94.0% specificity). Collectively, our results provide the first iTRAQ-based quantitative profile of bladder cancer urine proteins and represent a valuable resource for the discovery of bladder cancer markers.

Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics.

UNLABELLED In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. Six apolipoproteins (APOA1, APOA2, APOB, APOC2, APOC3, and APOE) were able to differentiate bladder cancer from hernia. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity (AUC=0.80 and p<0.001) in discriminating bladder cancer from hernia than either marker alone. Using MetaCore software to interpret global changes of the urine proteome caused by bladder cancer, we found that the most notable alterations were in immune-response/alternative complement and blood-coagulation pathways. This study confirmed the clinical significance of the urine proteome in the development of non-invasive biomarkers for the detection of bladder cancer. BIOLOGICAL SIGNIFICANCE In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity in discriminating bladder cancer from hernia than either marker alone. A marker panel composed by two novel biomarker candidates, SAA4 and proEGF, was first discovered and verified successfully using Western blotting. To the best of our knowledge, the associations of urinary SAA4 and proEGF with bladder tumor and kidney cancer have not been mentioned before. In the present study, we discovered and verified SAA4 and proEGF as potential bladder cancer biomarker for the first time.

Development of a Universal Metabolome-Standard Method for Long-Term LC–MS Metabolome Profiling and Its Application for Bladder Cancer Urine-Metabolite-Biomarker Discovery

Large-scale metabolomics study requires a quantitative method to generate metabolome data over an extended period with high technical reproducibility. We report a universal metabolome-standard (UMS) method, in conjunction with chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS), to provide long-term analytical reproducibility and facilitate metabolome comparison among different data sets. In this method, UMS of a specific type of sample labeled by an isotope reagent is prepared a priori. The UMS is spiked into any individual samples labeled by another form of the isotope reagent in a metabolomics study. The resultant mixture is analyzed by LC-MS to provide relative quantification of the individual sample metabolome to UMS. UMS is independent of a study undertaking as well as the time of analysis and useful for profiling the same type of samples in multiple studies. In this work, the UMS method was developed and applied for a urine metabolomics study of bladder cancer. UMS of human urine was prepared by (13)C2-dansyl labeling of a pooled sample from 20 healthy individuals. This method was first used to profile the discovery samples to generate a list of putative biomarkers potentially useful for bladder cancer detection and then used to analyze the verification samples about one year later. Within the discovery sample set, three-month technical reproducibility was examined using a quality control sample and found a mean CV of 13.9% and median CV of 9.4% for all the quantified metabolites. Statistical analysis of the urine metabolome data showed a clear separation between the bladder cancer group and the control group from the discovery samples, which was confirmed by the verification samples. Receiver operating characteristic (ROC) test showed that the area under the curve (AUC) was 0.956 in the discovery data set and 0.935 in the verification data set. These results demonstrated the utility of the UMS method for long-term metabolomics and discovering potential metabolite biomarkers for diagnosis of bladder cancer.

Association of nucleophosmin/B23 with bladder cancer recurrence based on immunohistochemical assessment in clinical samples.

AbstractAim:To investigate the possible correlation of nucleophosmin/B23 expression with bladder carcinoma recurrence.Methods:Surgically-resected bladder tumors staged pTa to pT4 were examined for nucleophosmin/B23 expression by immuno-histochemistry. The study group consisted of 132 consecutive patients surgically treated at Chang Gung Memorial Hospital between December 1998 and November 1999. The mean follow up was 72 months (range: 48-84 months).Results:Nuclear nucleophosmin/B23 staining was detected in 96% of advanced stage and poorly-differentiated tumors. Higher nucleophosmin/B23 levels were linked to more advanced tumor stages, grades, poor prognosis, and likelihood of recurrence (P<0.05). The Cox multivariate analysis indicated the nucleophosmin/B23 expression as an independent indicator for tumor recurrence (P=0.009).Conclusion:The results suggest that nucleophosmin/B23 is a favorable prognostic indicator for bladder cancer. Nucleophosmin/B23 could be a useful molecular tumor marker for predicting bladder cancer recurrence.

Cardiac output derived from arterial pressure waveform analysis in patients undergoing liver transplantation: validity of a third-generation device.

BACKGROUND Hemodynamic monitoring is essential to a successful liver transplantation procedure. FloTrac, a hemodynamic monitor that uses arterial-waveform-based pulse contour analysis for cardiac output (CO) measurement, has proven useful in many clinical settings. One of the primary foci of FloTracs recent third-generation software upgrade was improving its accuracy in low systemic vascular resistance status. We evaluated the accuracy of the upgraded FloTrac monitor during liver transplantation. MATERIALS AND METHODS Twenty-eight patients undergoing liver transplantation were enrolled in the study. Two sets of CO were measured with a radial arterial line connected to a FloTrac monitor (COFT) and a pulmonary artery catheter connected to a continuous cardiac output Vigilence monitor (COPAC). Simultaneous CO measurement was performed and recorded every 5 minutes throughout the surgery. Bland-Altman analysis was used to estimate the accuracy. The comparative method and reference method were considered interchangeable if the limits of agreement did not exceed a threshold set a priori at the greater of ±1 L/min, or a percentage error of lesser than 30%. RESULTS In all, 3234 paired data were collected. The bias was -0.8 L/min and the limits of agreements were -5.6 to 4.0 L/min. Percentage error was 75%. Regression analysis of the systemic vascular resistance index (SVRI) and the bias between COPAC and COFT showed that the bias was inversely related to the SVRI [r2=0.49; P<.001, y=-32.1983+9.9978 Log(x)]. CONCLUSIONS Despite a software upgrade, the effectiveness of the FloTrac artery-derived cardiac output monitor for CO measurement during liver transplantation remains limited.

Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer

More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins—SLC3A2, STMN1, and TAGLN2—in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant overexpression in individual bladder cancer tissues and urine specimens, and thus represents a potential biomarker for noninvasive screening for bladder cancer. Our findings highlight the value of bladder tissue proteome in providing valuable information for future validation studies of potential biomarkers in urothelial carcinoma.

A sensitive and selective magnetic graphene composite-modified polycrystalline-silicon nanowire field-effect transistor for bladder cancer diagnosis

In this study, we describe the urinary quantification of apolipoprotein A II protein (APOA2 protein), a biomarker for the diagnosis of bladder cancer, using an n-type polycrystalline silicon nanowire field-effect transistor (poly-SiNW-FET). The modification of poly-SiNW-FET by magnetic graphene with long-chain acid groups (MGLA) synthesized via Friedel-Crafts acylation was compared with that obtained using short-chain acid groups (MGSA). Compared with MGSA, the MGLA showed a higher immobilization degree and bioactivity to the anti-APOA2 antibody (Ab) due to its lower steric hindrance. In addition, the magnetic properties enabled rapid separation and purification during Ab immobilization, ultimately preserving its bioactivity. The Ab-MGLA/poly-SiNW-FET exhibited a linear dependence of relative response to the logarithmical concentration in a range between 19.5pgmL(-1) and 1.95µgmL(-1), with a limit of detection (LOD) of 6.7pgmL(-1). An additional washing step before measurement aimed at excluding the interfering biocomponents ensured the reliability of the assay. We conclude that our biosensor efficiently distinguishes mean values of urinary APOA2 protein concentrations between patients with bladder cancer (29-344ngmL(-1)) and those with hernia (0.425-9.47ngmL(-1)).

Prostatectomy using different lasers for the treatment of benign prostate hyperplasia in aging males

Purpose Endoscopic lasers have become a treatment option for benign prostate hyperplasia (BPH). The study reported here sought to elucidate the benefits and drawbacks of different laser systems in the treatment of patients with BPH. Methods The study enrolled 741 patients diagnosed with lower urinary tract symptoms secondary to BPH during the period January 2005 to December 2011. The techniques used in the study were photoselective vaporization of the prostate, thulium laser prostatectomy, and diode laser prostatectomy. Patients were assigned to one of three groups according to the type of laser treatment they received. Outcomes were evaluated using the International Prostate Symptom Score (IPSS), quality of life, maximal urinary flow rate, post-voiding residual urine volume, and prostate-specific antigen (PSA) level. Results The baseline characteristics of patients who received diode laser prostatectomy show a significant elevated risk and high American Society of Anesthesiology score (P=0.001). Operative time and catheter removal time differed significantly between the three groups (P=0.001). No cases were converted to transurethral resection of the prostate intraoperatively due to bleeding (P=0.142). Among the three groups, there were no significant differences in maximal flow rate, lower post-void residual urine, and postoperative PSA level during the entire follow-up period (P<0.05). Further, no significant differences in postoperative IPSS, quality of life, or bladder neck contracture (P=0.23) were observed. However, a significant difference was observed with regard to prolonged use of Foley catheters and prolonged hospital stay among patients in the diode laser group (P=0.001). Conclusion Laser prostatectomies are effective in dealing with lower urinary tract symptoms. Early subjective functional results (maximal flow rate, IPSS, and post-void residual urine) appeared the same as those obtained following laser prostatectomy. Thus, it appears that lasers are safe and effective as long as the patients are carefully selected for treatment.