Detection of aggressive prostate cancer associated glycoproteins in urine using glycoproteomics and mass spectrometry

Clinical management of prostate cancer remains a significant challenge due to the lack of available tests for guiding treatment decisions. The blood prostate‐specific antigen test has facilitated early detection and intervention of prostate cancer. However, blood prostate‐specific antigen levels are less effective in distinguishing aggressive from indolent prostate cancers and other benign prostatic diseases. Thus, the development of novel approaches specific for prostate cancer that can differentiate aggressive from indolent disease remains an urgent medical need. In the current study, we evaluated urine specimens from prostate cancer patients using LC‐MS/MS, with the aim of identifying effective urinary prostate cancer biomarkers. Glycoproteins from urine samples of prostate cancer patients with different Gleason scores were characterized via solid phase extraction of N‐linked glycosite‐containing peptides and LC‐MS/MS. A total of 2923 unique glycosite‐containing peptides were identified. Glycoproteomic comparison on urine and tissues from aggressive and non‐aggressive prostate cancers as well as sera from prostate cancer patients revealed that the majority of AG prostate cancer associated glycoproteins were more readily detected in patient's urine than serum samples. Our data collectively indicate that urine provides a potential source for biomarker testing in patients with AG prostate cancer.     

Proteomic analysis of conditioned media from the PC3, LNCaP, and 22Rv1 prostate cancer cell lines: discovery and validation of candidate prostate cancer biomarkers

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen (PSA) test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from three human prostate cancer cell lines of differing origin [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)] to identify secreted proteins that could serve as novel prostate cancer biomarkers. Each cell line was cultured in triplicate, followed by a bottom-up analysis of the peptides by two-dimensional chromatography and tandem mass spectrometry. Approximately, 12% (329) of the proteins identified were classified as extracellular and 18% (504) as membrane-bound among which were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. On the basis of this, four novel candidates, follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2, were validated in the serum of patients with and without prostate cancer. The proteins presented in this study represent a comprehensive sampling of the secreted and shed proteins expressed by prostate cancer cells, which may be useful as diagnostic, prognostic or predictive serological markers for prostate cancer.     

Newer potential biomarkers in prostate cancer

Prostate-specific antigen (PSA) screening has led to a significant rise in the number of men diagnosed with prostate cancer and an associated increase in biopsies performed. Despite its limitations, including a positive predictive value of only 25%-40%, PSA remains the only generally accepted biomarker for prostate cancer. There is a need for better tools to not only identify men with prostate cancer, but also to recognize those with potentially lethal disease who will benefit from intervention. A great deal of work has been done worldwide to improve our knowledge of the genetics behind prostate cancer and the specificity of PSA by developing assays for different PSA isoforms. Common genetic alterations in prostate cancer patients have been identified, including CpG hypermethylation of GSPT1 and TMPRSS2:ERG gene fusion. Serum and urine detection of RNA biomarkers (eg, PCA3) and prostate cancer tissue protein antibodies (eg, EPCA) are being evaluated for detection and prognostic tools. This article reviews some of the promising developments in biomarkers.     

Prostate cancer: risk assessment and diagnostic approaches

The successful treatment of prostate cancer relies on detection of the disease at its earliest stages. Although prostate-specific antigen (PSA)-based screening has been a significant advance in the early diagnosis of prostate cancer, identifying specific genetic alterations in a given family or patient will allow more appropriate screening for early disease. Mapping and identification of specific prostate cancer susceptibility genes is slowly becoming a reality. Other prostate cancer risks include a family history, race, and possibly serum markers such as insulin-like growth factor-I (IGF-I). Once a high-risk man is identified, transrectal ultrasound (TRUS)-guided biopsies are the standard to diagnose prostate cancer. Although TRUS is an advance over traditional digitally directed biopsies, it represents a random sampling of the prostate since most lesions cannot be visualized. Newer modalities such as ultrasound contrast agents, pattern recognition, and artificial neural networks (ANNs), applied to TRUS images, may improve diagnostic accuracy. If a man at risk for prostate cancer has undergone a negative TRUS biopsy, the decision for the need for additional biopsies is problematic. Use of PSA derivatives such as free and total PSA and the initial biopsy abnormalities such as atypia or high-grade prostatic intraepithelial neoplasia may define those patients in need of follow-up biopsy.     

Advances in proteomic prostate cancer biomarker discovery

Prostate cancer is the most common non-cutaneous cancer in men in the United States. For reasons largely unknown, the incidence of prostate cancer has increased in the last two decades, in spite or perhaps because of a concomitant increase in serum prostate-specific antigen (PSA) screening. While PSA is acknowledged not to be an ideal biomarker for prostate cancer detection, it is however widely used by physicians due to lack of an alternative. Thus, the identification of a biomarker(s) that can complement or replace PSA represents a major goal for prostate cancer research. Screening complex biological specimens such as blood, urine, and tissue to identify protein biomarkers has become increasingly popular over the last decade thanks to advances in proteomic discovery methods. The completion of human genome sequence together with new development in mass spectrometry instrumentation and bioinformatics has been a major driving force in biomarker discovery research. Here we review the current state of proteomic applications as applied to various sample sources including blood, urine, tissue, and “secretome” for the purpose of prostate cancer biomarker discovery. Additionally, we review recent developments in validation of putative markers, efforts at systems biology approach, and current challenges of proteomics in biomarker discovery.     

The application of the RT-PCR method for the staging of the prostate cancer progression

Molecular biology seems to bring more convincing markers for the detection of prostate cancer as well as the development of metastases than immunohistochemistry. The main goal of present work was to detect the expression of prostate specific antigen (PSA) and prostate-specific membrane antigen (PSM) genes in the micrometastases by the RT-PCR to assess the progression of prostate cancer. We analyzed 50 patients: 28 patients with clinically localized or locally advanced prostate cancer who underwent radical prostatectomy, 7 patients with clinically proven metastases, 8 patients with benign prostatic hyperplasia, and 7 healthy young men. The results of RT-PCR in the first group of 28 patients varied, however, they were in good correlation with the health status of the patients. Positive results of PSA and notably for PSM were good predictors of beginning metastasing process. Seven patients with metastatic disease had positive RT-PCR results both for PSA and PSM. All of the patients with benign prostatic hyperplasia and healthy young men had negative RT-PCR results for PSA and PSM. The study showed that positive RT-PCR results for PSA and especially for PSM correlated well with the progression of the disease and negative results reflected good health status of the patients.     

Prostate cancer diagnostics: Clinical challenges and the ongoing need for disruptive and effective diagnostic tools

The increased incidence and the significant health burden associated with carcinoma of the prostate have led to substantial changes in its diagnosis over the past century. Despite technological advancements, the management of prostate cancer has become progressively more complex and controversial for both early and late-stage disease. The limitations and potential harms associated with the use of prostate-specific antigen (PSA) as a diagnostic marker have stimulated significant investigation of numerous novel biomarkers that demonstrate varying capacities to detect prostate cancer and can decrease unnecessary biopsies. However, only a few of these markers have been approved for specific clinical settings while the others have not been adequately validated for use. This review systematically and critically assesses ongoing issues and emerging challenges in the current state of prostate cancer diagnostic tools and the need for disruptive next generation tools based on analysis of combinations of these biomarkers to enhance predictive accuracy which will benefit clinical diagnostics and patient welfare.     

Détection des différentes formes du prostate-specific antigen et autres kallicréines dans le cancer de la prostate

Prostate cancer is the most common cancer in men over 50 years old and the second leading cause of cancer death. Prostate-specific antigen (PSA) is widely used for the diagnosis and follow-up of prostate cancer. PSA, also called kallikrein 3, is a member of the human kallikrein-type serine protease family and circulates in the blood stream in the form of complexes with serum protease inhibitors and in free form. However, free PSA is also a heterogeneous mixture of different molecular PSA forms: proPSA, intact and clived mature forms. The clinical significance of these different forms is still unclear but their specific measurement in serum could improve the specificity of PSA for detecting cancer or predicting treatment outcome. Others kallikreins including kallikrein 2, 4, 11, 14 and 15 are also emerging as complementary markers to PSA for prostate cancer. Multiple detection of the different molecular forms of PSA, as well as of these kallikreins, in addition to total PSA, could significantly increase the diagnostic utility of PSA and may add prognostic value by bringing clinical information on the cancer progression.     

Cancer de la prostate : les niveaux de preuve des biomarqueurs de la détection précoce

As part of an overall review on the role of PSA and other emerging biomarkers in the detection, diagnosis and treatment of prostate cancer, we present here a part of a review of the literature made by the working group Biologie de la Prostate (AFU, CNBH, SFBC, SFMN) on the use of PSA and other biomarkers in the early detection of prostate cancer with an assessment of the level of evidence.     

Combining biomarkers to detect disease with application to prostate cancer

In early detection of disease, combinations of biomarkers promise improved discrimination over diagnostic tests based on single markers. An example of this is in prostate cancer screening, where additional markers have been sought to improve the specificity of the conventional Prostate-Specific Antigen (PSA) test. A marker of particular interest is the percent free PSA. Studies evaluating the benefits of percent free PSA reflect the need for a methodological approach that is statistically valid and useful in the clinical setting. This article presents methods that address this need. We focus on and-or combinations of biomarker results that we call logic rules and present novel definitions for the ROC curve and the area under the curve (AUC) that are applicable to this class of combination tests. Our estimates of the ROC and AUC are amenable to statistical inference including comparisons of tests and regression analysis. The methods are applied to data on free and total PSA levels among prostate cancer cases and matched controls enrolled in the Physicians' Health Study.     

Prostate cancer: epidemiology, screening, and biomarkers

Carcinoma of the prostate continues to be a major health problem in the United States. Beginning in 1988, a marked increase in detection of prostate cancer occurred due to the development of a test for prostate-specific antigen (PSA). Controversy exists, however, about the value of PSA as a tumor marker. Although it has prognostic significance both before and after definitive therapy for prostate cancer, it is unclear whether routine PSA screening will translate into a survival advantage for patients. Because of its limitations, PSA may not ultimately be a good enough marker to be used as a screening tool. However, molecular biology has led to a rapid rise in the number of potential new prostate tumor markers, which may eventually overcome the weaknesses of PSA. Considerable progress has occurred in the diagnosis and management of prostate cancer: more is understood about the risk factors for the disease, possible ways to prevent it, and new ways to diagnose and monitor it. These developments have already translated into better patient care, while also identifying where further improvements are needed.     

Reaction Pattern in Vertebrate Cells (RiV): Studies of the Course of Tumor Therapy using an anti‐RiV ELISA (Initial Results)

Detection of anti‐RiV antibodies by ELISA can be used to follow a patient's response to treatments such as cancer surgery or RiV therapy. The initial results of the authors demonstrated that it is necessary to optimize the sensitivity of the ELISA. Known tumor markers such as prostate specific antigen (PSA), especially the ratio of free PSA (fPSA)/total PSA (tPSA) or neurones‐specific enolase (NSE) detect the therapeutic effect of treatment with RiV particle preparation (RiV‐PP) more rapidly and with greater sensitivity than does the anti‐RiV antibody assay. However, a continuous decrease of anti‐RiV‐antibody titers seems to indicate a good prognosis. RiV therapy improved the quality of life and achieved an apparent prolongation of life of cancer patients. After treatment with 12 mL of RiV‐PP, a patient with a prostate hyperplasia of uncertain genesis became free of symptoms. This monotherapy with an adequate dose of RiV‐PP resulted in a decrease of tPSA and an increase of fPSA in the first 200 days. The general value of RiV ELISA is emphasized by the fact that it could detect RiV antigen in urinary samples offering a simple means of early diagnosis and monitoring. Since chemo‐ or/and radiotherapy, both of which are immunosuppressive, are frequently used to treat cancer patients, an ELISA for the detection of RiV (particles as) antigen may be more informative than one designed to detect anti RiV antibodies.     

Novel electrical detection of label-free disease marker proteins using piezoresistive self-sensing micro-cantilevers

We report an electro-mechanical biosensor for electrical detection of proteins with disease markers using self-sensing piezoresistive micro-cantilevers. Electrical detection, via surface stress changes, of antigen-antibody (Ag-Ab) specific binding was accomplished through a direct nano-mechanical response of micro-fabricated self-sensing micro-cantilevers. A piezoresistive sensor measures the film resistance variation with respect to surface stress caused by biomolecules specific binding. When specific binding occurred on a functionalized Au surface, surface stress was induced throughout the cantilever, resulting in cantilever bending and resistance change of the piezoresistive layer. The cantilever biosensors were used for the detection of prostate specific antigen (PSA) and C-reactive proteins (CRP), which are a specific marker of prostate cancer and cardiac disease. From the above experiment, it was revealed that the sensor output voltage was proportional to the injected antigen concentration (without antigen, 10 ng/ml, 100 ng/ml, 1 microg/ml). PSA and CRP antibodies were found to be very specific for their antigens, respectively. This indicated that the self-sensing micro-cantilever approach is beneficial for detecting disease markers, and our piezoresistive micro-cantilever sensor system is applicable to miniaturized biosensor systems.     

Beyond prostate-specific antigen: new serologic biomarkers for improved diagnosis and management of prostate cancer

The use of total prostate-specific antigen (tPSA) measurement has dramatically improved the ability to detect prostate cancer at earlier stages. However, as the number of men presenting with advanced disease (and high tPSA levels) has decreased, and given the fact that tPSA is highly reflective of benign prostatic hyperplasia, the need has emerged for novel biomarkers specifically associated with prostate cancer in order to improve predictive models. Several new biomarkers have shown promise, and studies continue to investigate the role of these markers in the detection, staging, and prognosis of prostate cancer. As new useful biomarkers continue to emerge, guidelines for their employment, as well as coordination of further research studies, are needed; a systematic, phased, nomogram-based model is a rational way to manage these efforts.     

Identification of nuclear structural protein alterations associated with seminomas

Currently, there are no specific markers available for the early detection and for monitoring testicular cancer. Based upon an approach that targets nuclear structure, we have identified a set of proteins that are specific for seminomas, which may then have clinical utility for the disease. Utilizing samples obtained from men with no evidence of testicular cancer (n = 5) as well as those with seminomas (n = 6), nuclear matrix proteins were extracted and separated using a high‐resolution two‐dimensional electrophoresis gel system. The proteins were identified by mass spectrometry analysis. These analyses revealed seven nuclear matrix proteins associated with the normal testes, which did not appear in the seminomas. In the seminomas, four nuclear matrix proteins were identified to be associated with the disease that were absent in the normal testes. Mass spectrometric and immunoblot analyses of these proteins revealed that one of the proteins identified in the normal testes appears to be StAR‐related lipid transfer protein 7 (StARD7). In the non‐seminoma tissues, one of the identified proteins appears to be cell division protein kinase 10 (CDK10). Both StarD7 and CDK10 could potentially be involved in cell differentiation and growth, and thus may serve as potential targets for therapy of prognostication of seminomas. This is the first study to examine the role of nuclear structural proteins as potential biomarkers in testicular cancer. We are currently examining the roles of some of the identified proteins as potential biomarkers for the disease. J. Cell. Biochem. 108: 1274–1279, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular insights into substrate specificity of prostate specific antigen through structural modeling

Prostate Specific Antigen's (PSA) role as a biomarker for prostate cancer is well established but the physiological role of its serine protease activity in the pathobiology of normal prostate and prostate carcinogenesis remains largely unknown. In light of recent studies that implicate PSA's enzymatic activity in the initiation and/or progression of prostate cancer, we performed a molecular modeling study of substrate binding at the catalytic site of PSA wherein a PSA‐selective substrate (HSSKLQ) was docked in an acyl‐enzyme conformation to a three‐dimensional homology model of PSA. Additionally, virtual positional scanning studies were conducted to gain mechanistic insights into substrate recognition of PSA. Subsequently, 13 novel peptide substrates of 6‐aa length and four peptide substrates with varying length were synthesized and assayed for PSA hydrolysis to evaluate the experimental validity of docking insights. Additionally, six novel aldehyde‐containing transition state analog inhibitors were synthesized and tested for their inhibitory potencies. The experimental data on the hydrolysis rates of the newly synthesized substrates and inhibitory potencies of the aldehyde peptides agreed with the docking predictions, providing validation of the docking methodology and demonstrating its utility towards the design of substrate‐mimetic inhibitors that can be used to explore PSA's role in the pathobiology of prostate cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Imaging in the diagnosis and management of prostate cancer

The current diagnosis and management of prostate cancer is largely based on the use of serum prostate-specific antigen (PSA) and pathologic risk factors such as Gleason score and clinical stage. The use of serum PSA in clinical practice has resulted in significant stage migration and, as such, imaging modalities historically utilized to stage prostate cancer are no longer able to reliably identify the small amounts of prostate cancer most often found at presentation. Molecular imaging techniques have focused on improving sensitivity and specificity for cancer detection through knowledge of specific attributes of disease biology. The evolution of imaging techniques has created a new role for imaging in the management of prostate cancer.