Self-Normalized Detection of ANXA3 from Untreated Urine of Prostate Cancer Patients without Digital Rectal Examination.

A noninvasive quantitative assay that is capable of identifying prostate cancer biomarkers in untreated urine is an attractive diagnosis tool, but this method is subject to various obstacles. Difficulties presented by untreated urine include varying salt concentrations, and pH levels that may be different even though they are from the same patient. Untreated urine also presents interference from other biomolecules and possesses a fewer number of cancer biomarkers than can be found in serum. As a result, urine preconditioning processes and digital rectal examination (DRE) to increase biomarker secretion are mandatory in current urine assays. To address these challenges, an ion-responsive urine sensor (IRUS) that measures differential electrical signals is proposed as a self-normalized detection method. The proposed IRUS is based on a FET biosensor with a disposable sensing gate and has the capability to detect the prostate cancer antigen ANXA3 in untreated patient urine. The IRUS can detect ANXA3 at <1 fg mL-1 with high reliability. In addition, it is found that ANXA3 levels in urine show clinically significant correlation with real tumor volumes. This paper provides a guideline in developing a clinically ready accurate noninvasive platform, which is capable of predicting prostate cancer using untreated urine without DRE.

Prostate cancer biomarker annexin A3 detected in urines obtained following digital rectal examination presents antigenic variability.

OBJECTIVES: Annexin A3 (ANXA3) is a potential marker for prostate cancer (PCa). We aimed to develop robust immunoassays suitable for quantifying ANXA3 in urine samples obtained following digital rectal examination (DRE) in order to facilitate the diagnostic performance evaluation of this marker. DESIGN AND METHODS: Anti-ANXA3 monoclonal antibodies were generated and their epitopes mapped. Two different ANXA3 assay prototypes were established on the VIDAS® automated immunoanalyser and analytical validation was carried out using post-DRE urine samples obtained from patients with PCa (n=23) or benign prostate hyperplasia (n=31). RESULTS: The assays had the same capture antibody (TGC44) but different detection antibodies (13A12 or 5C5), recognizing novel distinct epitopes. Both had a lower limit of quantification <1ng/mL and were highly specific for ANXA3, not cross-reacting with other annexins. Interassay imprecision was ≤11% and ≤15% for 13A12 and 5C5 assays, respectively. Surprisingly, a total lack of correlation was observed between ANXA3 levels measured by these two assays in post-DRE urines, indicating detection of distinct antigenic variants. Two freeze-thaw cycles did not affect analyte stability in either assay, whereas a lack of stability of antigenic variants was observed when samples were stored at -80°C for 1month. CONCLUSIONS: Two different antigenic variants of ANXA3 are present in post-DRE urines and their clinical significance for diagnosis of prostate cancer should be further investigated. These variants are not stable over time in samples preserved at -80°C. Until this issue is resolved, ANXA3 should only be measured in freshly collected samples.

A panel of tumor markers, calreticulin, annexin A2, and annexin A3 in upper tract urothelial carcinoma identified by proteomic and immunological analysis.

BACKGROUND: Upper tract urothelial carcinoma (UTUC) is a tumor with sizable metastases and local recurrence. It has a worse prognosis than bladder cancer. This study was designed to investigate the urinary potential tumor markers of UTUC. METHODS: Between January 2008 and January 2009, urine was sampled from 13 patients with UTUC and 20 healthy adults. The current study identified biomarkers for UTUC using non-fixed volume stepwise weak anion exchange chromatography for fractionation of urine protein prior to two-dimensional gel electrophoresis. RESULTS: Fifty five differential proteins have been determined by comparing with the 2-DE maps of the urine of UTUC patients and those of healthy people. Western blotting analysis and immunohistochemistry of tumor tissues and normal tissues from patients with UTUC were carried out to further verify five possible UTUC biomarkers, including zinc-alpha-2-glycoprotein, calreticulin, annexin A2, annexin A3 and haptoglobin. The data of western blot and immunohistochemical analysis are consistent with the 2-DE data. Combined the experimental data in the urine and in tumor tissues collected from patients with UTUC, the crucial over-expressed proteins are calreticulin, annexin A2, and annexin A3. CONCLUSIONS: Calreticulin, annexin A2, and annexin A3 are very likely a panel of biomarkers with potential value for UTUC diagnosis.

Assays for prostate cancer : changing the screening paradigm?

Prostate cancer (PCa) screening and detection have changed dramatically since the introduction of serum prostate-specific antigen (PSA) testing. Despite the resulting improvement in early PCa detection and stage migration, in clinical practice the use of PSA testing may cause overdetection and ultimately overtreatment. As a consequence, novel biomarkers are needed to increase the specificity of PCa detection. The aim of this article is to present an overview of novel blood- and urine-based biomarkers that may optimize PCa detection, with improved identification of patients with significant PCa and avoidance of unnecessary prostate biopsies. A systematic and comprehensive PubMed search was performed using the MeSH search terms 'prostate cancer', 'biomarker', 'marker', and 'detection'. Results were restricted to the English language. Several blood- and urine-based biomarkers have the potential to improve prediction of the presence and/or significance of PCa. Ideally, biomarkers should be used in combination within multivariate models, leading to superior accuracy for prediction of any PCa or clinically significant PCa, compared with the use of a single marker.

Urine markers in monitoring for prostate cancer.

The major advantages of urine-based assays are their noninvasive character and ability to monitor prostate cancer with heterogeneous foci. Almost all urine-detectable prostate-specific markers have been recently reviewed. For this reason, we focus here on only a few promising markers which have been independently evaluated (in particular PCA3, fusion genes, TERT, AMACR, GSTP1, MMP9 and VEGF) and very recent ones (ANXA3 and sarcosine). The emphasis is also on multiplex biomarker analysis and on microarray-based analysis of fusion genes. A combination of multiple urine biomarkers may be valuable in the case of men with persistently elevated serum prostate-specific antigen and a history of negative biopsies. The emerging urine tests should help in both early diagnosis of prostate cancer and identifying aggressive tumors for radical treatment.

Annexin A3 in urine: a highly specific noninvasive marker for prostate cancer early detection.

PURPOSE: In prostate cancer cases the early diagnosis of tumors carrying a high risk of progression is of the utmost importance. There is an urgent clinical need to avoid unnecessary biopsies and subsequent overtreatment. We validated annexin A3 as a diagnostic marker for prostatic disease in typical clinical populations and relevant segments, such as patients with a negative digital rectal examination and low prostate specific antigen. MATERIALS AND METHODS: We performed a blinded clinical study (ClinicalTrials.gov Identifier NCT00400894) from September 2005 to January 2007 in 591 patients who were continuously recruited from 4 European urological clinics. Urine was obtained directly after digital rectal examination and the annexin A3 concentration in urine was quantified by Western blot. Statistical analysis included combinations of annexin A3 with total, percent free, complexed and percent complexed prostate specific antigen. RESULTS: Combined readouts of prostate specific antigen and urinary annexin A3 were superior to all others with an area under the ROC curve of 0.82 for a total prostate specific antigen range of 2 to 6 ng/ml, 0.83 for a total prostate specific antigen range of 4 to 10 ng/ml and 0.81 in all patients. The best performing prostate specific antigen derivative was percent free prostate specific antigen with an area under the ROC curve of 0.68 for a total prostate specific antigen range of 2 to 6 ng/ml, 0.72 for a total prostate specific antigen range of 4 to 10 ng/ml and 0.73 in all patients. Annexin A3 has an inverse relationship to cancer and, therefore, its specificity was much better than that of prostate specific antigen. CONCLUSIONS: Annexin A3 quantification in urine provides a novel noninvasive biomarker with high specificity. Annexin A3 is complementary to prostate specific antigen or to any other cancer marker. It has a huge potential to avoid unnecessary biopsies with a particular strength in the clinically relevant large group of patients who have a negative digital rectal examination and prostate specific antigen in the lower range of values (2 to 10 ng/ml).