Combined serum and EPS-urine proteomic analysis using iTRAQ technology for discovery of potential prostate cancer biomarkers.

Prostate cancer (PCa) is one of the most common malignant tumors and a major cause of cancer-related death for men worldwide. The aim of our study was to identify potential non-invasive serum and expressed prostatic secretion (EPS)-urine biomarkers for accurate diagnosis of PCa. Here, we performed a combined isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to compare protein profiles using pooled serum and EPS-urine samples from 4 groups of patients: benign prostate hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN), localized PCa and metastatic PCa. The differentially expressed proteins were rigorously selected and further validated in a large and independent cohort using classical ELISA and Western blot assays. Finally, we established a multiplex biomarker panel consisting of 3 proteins (serum PF4V1, PSA, and urinary CRISP3) with an excellent diagnostic capacity to differentiate PCa from BPH [area under the receiver operating characteristic curve (AUC) of 0.941], which showed an evidently greater discriminatory ability than PSA alone (AUC, 0.757) (P<0.001). Importantly, even when PSA level was in the gray zone (4-10 ng/mL), a combination of PF4V1 and CRISP3 could achieve a relatively high diagnostic efficacy (AUC, 0.895). Furthermore, their combination also had the potential to distinguish PCa from HGPIN (AUC, 0.934). Our results demonstrated that the combined application of serum and EPS-urine biomarkers can improve the diagnosis of PCa and provide a new prospect for non-invasive PCa detection.

In the search of novel urine biomarkers for the early diagnosis of prostate cancer. Intracellular or secreted proteins as the target group? Where and how to search for possible biomarkers useful in the everyday clinical practice.

OBJECTIVE: To search which category of proteins can be detected in urine in order to examine subsequently its ability to improve our accuracy for the diagnosis of Prostate Cancer (PCa) as biomarkers in clinical useful fluids like urine and serum. Material and method(s): Urine samples of 127 patients were obtained after a vigorous transrectal prostatic massage to both lobes. The patients were considered to have a high risk for PCa according to their PSA (> 4 ng/ml), their digital rectal examination (DRE) (positive for suspicious prostatic lesions) or to their abnormal PSA kinetics (PSA velocity (PSAV > 0.75 ng/mL). All patients subsequently were subjected to an extended 10-core per prostatic lobe TRUS-b (total 20 prostatic samples). The proteins that were chosen to be detected in the urine samples with Western-blot, as possible biomarkers, were Glutathione peroxidase 3 precursor (GPx3), Cofilin-1 (CFL1), Heat shock protein-90ß (HSP 90ß), Zinc alpha 2-glycoprotein (ZAG) and secreted protein acidic and rich in cysteine (SPARC).These proteins have been detected previously in the prostatic tissue by proteomics proving their discriminative ability between patients with prostate cancer and benign prostatic hyperplasia. RESULT(S): From the five proteins, only the secreted Zinc alpha 2-glycoprotein was detected in urine showing a promising ability in the improvement of our diagnostic accuracy for the early diagnosis of prostate cancer. CONCLUSIONS: From various categories of proteins that have already been detected in the tissue of prostate by proteomics, only secreted protein Zinc alpha 2-glycoprotein showed a clear signal in the urine, proving its discriminative potential for the early diagnosis of PCa.

Adipokine zinc-alpha-2-glycoprotein as a novel urinary biomarker presents earlier than microalbuminuria in diabetic nephropathy.

OBJECTIVE: To investigate the role of zinc-alpha-2-glycoprotein (ZAG) in the early stage of diabetic nephropathy, in patients with type 2 diabetes mellitus (T2DM). METHODS: This cross-sectional observational study recruited patients with longstanding T2DM and healthy control subjects. Patients with T2DM were further stratified based on their urine albumin-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR). Serum and urine concentrations of ZAG were determined using an enzyme-linked immunosorbent assay. RESULTS: Eighty patients with T2DM and 20 healthy control subjects were enrolled in the study. Mean ± SD concentrations of ZAG in serum and urine were both significantly higher in patients with T2DM (serum: 38.29 ± 22.72 mg/l; urine: 53.64 ± 29.48 mg/g) compared with concentrations in healthy control subjects (serum: 21.61 ± 8.83 mg/l; urine: 28.17 ± 10.64 mg/g). Serum ZAG concentration was positively correlated with serum creatinine and eGFR. Urine ZAG concentration was positively correlated with UACR. Urine concentration of ZAG in the higher eGFR group was higher than that in the normal eGFR group (41.26 ± 13.67 versus 32.05 ± 8.55 mg/g, respectively). CONCLUSION: These preliminary findings suggest that ZAG might be a potentially useful biomarker for early diagnosis of diabetic nephropathy in patients with T2DM.

Zinc α2-glycoprotein as a potential novel urine biomarker for the early diagnosis of prostate cancer.

UNLABELLED: What's known on the subject? and What does the study add? The use of biomarkers to detect a cancer early, especially prostate cancer, is not a new idea and PSA has been proved to be the best biomarker for the early diagnosis of prostate cancer. Since the introduction and wide use of PSA various efforts have been made to find novel biomarkers in both serum and urine of individuals at high risk for prostate cancer. The best example of a biomarker detected in the urine after a vigorous digital rectal examination is PCA3, which is used mainly in the subgroup of patients with PSA 4-10 ng/mL whose prostate biopsy was repeatedly negative for prostate cancer in order to decide the performance or not of a new biopsy. Proteomics is a state of the art new biotechnology used to identify the proteome of a certain tissue meaning the whole group of proteins related to the anatomy and biochemistry of the tissue. Using proteomics can effectively and more specifically identify proteins that can be used as potential biomarkers for the early diagnosis of prostate cancer. Zinc α2-glycoprotein has been studied in the past as a protein related to cancer cachexia and it has been measured in both prostate tissue and serum in patients with prostate cancer. Zinc α2-glycoprotein has also been recently identified by proteomics in prostate tissue showing different values in patients with prostate cancer and benign prostate hyperplasia. It is the first time that zinc α2-glycoprotein has been systematically measured and studied in an easily obtained biological fluid such as urine showing a very optimistic potential both as a novel solo biomarker and as an adjunct to PSA for the early diagnosis of prostate cancer. PSA has revolutionized the way we approximate prostate cancer diagnosis. Even though PSA is still the best biomarker for the diagnosis of prostate cancer it constitutes an organ-specific and not a disease-specific biomarker and diagnostic dilemmas are often raised concerning the performance or not of a prostate biopsy. Thus novel biomarkers are required in order to improve the diagnostic ability of PSA. Increasingly in the literature it is stated that the future of prostate cancer diagnosis could be not a single biomarker but a band of different biomarkers that as a total could give the possibility of an individual having prostate cancer. By detecting and measuring zinc α2-glycoprotein in the urine we believe that interesting conclusions can be made: first that proteomics is the way to detect with accuracy proteins that could be proved to be valuable novel biomarkers; second that zinc α2-glycoprotein detected in the urine could be used both as a solo biomarker and as an adjunct to PSA for the early diagnosis of prostate cancer. OBJECTIVE: • To examine the potential utility as a novel biomarker in the urine of zinc α2-glygoprotein (ZAG) for the early diagnosis of prostate cancer. PATIENTS AND METHODS: • The urine of 127 consecutive candidates for a transrectal ultrasound prostatic biopsy with a mean age of 65.7 ± 8.7 years and mean PSA 9.1 ± 5.3 ng/mL was collected. • Western blot analysis and immunohistochemistry for ZAG were performed. • Receiver operating characteristic curves and logistic regression models were used to estimate the predictive ability of ZAG and to determine the optimal sensitivity and specificity by using various cut-off values for the prediction of prostate cancer. RESULTS: • In all, 42 patients had prostate cancer, 29 showed high grade prostatic intraepithelial neoplasia and 56 were negative. • Receiver operating characteristic curve analysis showed a significant predictive ability of ZAG for prostate cancer. The area under the curve (AUC) for the prediction of prostate cancer was 0.68 (95% CI 0.59-0.78). • The combination of ZAG with PSA showed a significant improvement in the predictive ability (P= 0.010), with AUC equal to 0.75 (95% CI 0.66-0.85). Separate analysis in patients with PSA levels of 4-10 ng/mL (70.1%) showed that ZAG had a discriminative power with AUC equal to 0.68. • The optimal cut-off was 1.13 for ZAG, which corresponded to 6.88 times greater odds for prostate cancer. CONCLUSIONS: • Urine detected ZAG showed promising results in the prediction of prostate cancer. • Further validation is required to establish ZAG as a novel biomarker.

Identification of zinc-alpha-2-glycoprotein binding to clone AE7A5 antihuman EPO antibody by means of nano-HPLC and high-resolution high-mass accuracy ESI-MS/MS.

The detection of doping with recombinant erythropoietins (Epo) by isoelectric focusing (IEF) and Western double blotting strongly relies on the specificity of the detection antibody used. Currently a monoclonal mouse antibody (clone AE7A5) is used for that purpose. Despite its excellent sensitivity (amol range) the antibody shows some nonspecific binding behavior. However, the binding occurs outside the currently used pH range for evaluating erythropoietin IEF profiles. A shotgun proteomics approach is described consisting of preparative IEF on large-sized carrier ampholyte gels (pH 3-5), SDS-PAGE, Western single and double blotting, on-membrane elution of intact proteins, on-membrane and in-solution tryptic digestions, as well as nano-HPLC peptide separation and high-resolution high-mass accuracy ESI-MS/MS peptide sequencing. The nonspecifically interacting protein could be identified as zinc-alpha-2-glycoprotein (ZAG). Confirmation analyses were performed using recombinant ZAG (rhZAG) and a monoclonal anti-ZAG antibody. It could be demonstrated that the binding of the monoclonal antihuman EPO antibody (clone AE7A5) to ZAG occurs in a highly concentration-dependant manner and that only samples containing increased amounts of urinary ZAG lead to a detectable interaction of the AE7A5 antibody on Epo-IEF gels.

Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer.

Identification and characterization of biomarkers in body fluids such as serum or urine serve as a basis for early detection of diseases, particularly of cancer. Performing 2-DE with subsequent MS analyses, conventional immunoblotting and immunohistochemistry we identified two proteins, orosomucoid (ORM) and human zinc-alpha(2)-glycoprotein (ZAG), which were increased in the urine samples of patients with bladder cancer in comparison to the urine samples of healthy volunteers. The highest amount of both proteins was found in invasive bladder cancer stages such as pT2-3. Immunohistochemical studies showed ORM in inflammatory cells but also in endothelial cells of blood vessels within or adjacent to the tumor area and in part of the tumor cells. ZAG was prominent in tumor cells at the tumor invasion front. Additionally, ZAG was localized at the luminal surface of normal urothelium, which switches to the basal side when a superficial papillary tumor was observed. These results show that we have been able to identify two new proteins that may be related to the development of superficial bladder cancer and to its switch to an invasive phenotype.