Serum Of Prostate Cancer Patients Research Articles

Abstract 1844: Reduced expression of miR-342-3p in prostate cancer.

Abstract Patients with bone metastasis of the prostate have a 5-year survival rate of less than 30%. Due to inconsistencies in the presentation, prognosis, and treatment options of metastatic prostate cancer (PCa), biomarkers are urgently needed for personalized treatment of this disease. MicroRNAs (miRNAs) are small, non-coding, single-stranded endogenous RNAs that regulate the expression of multiple messenger RNA targets. Studies have demonstrated that these non-coding RNAs are significantly expressed in lung, breast and prostate cancer tumors. We hypothesize that miRNAs are differentially expressed between non-cancerous and aggressive tumor phenotypes in PCa, e.g. bone-specific metastasis. To test this hypothesis, we evaluated 384 miRNAs in the serum of stage-matched patients with stage I (n = 5), stage III (n = 5), stage IV (n = 5) PCa compared to non-cancerous age and stage-matched controls (n = 5). Total RNA was isolated from 500 μl of serum (Bioserve Biotechnologies, Ltd). The expression profiles of miRNAs were measured using TLDA cards and statistical analyses were performed using Expression Suite Software version 1.0. MiRNAs (miR-106b/17, -302b, -342-3p) were differentially expressed in the serum of PCa patients with (stage I, III, IV) when compared to controls. Mir-342-3p was significantly reduced in expression (P = 0.018-0.028) in all PCa patients relative to non-cancerous controls, after adjusting multiple hypothesis testing. However, other serum miRNAs were differentially expressed for only stage III PCa (P=0.001-0.018). Validation of these differentially expressed miRNAs in large sample sizes will lead to the identification of pre- and metastatic biomarkers. Ultimately, the investigation of these biomarkers will improve personalized treatment of PCa patients. Citation Format: Dominique Jones, Divine Anene, April Aloway, Praise Anene, Diana Avila, Leila Gobejishvili, Shirish Barve, Lacey McNally, LaCreis Kidd. Reduced expression of miR-342-3p in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1844. doi:10.1158/1538-7445.AM2013-1844

Abstract 5063: Matrilysin cleavage of perlecan produces bioactive fragments acting as a molecular switch to alter prostate cancer cell behavior.

Abstract Perlecan/HSPG2[PLN], a large heparan sulfate (HS) proteoglycan, is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including PLN. After proteolysis, fragments of PLN can be released into circulation. A sandwich ELISA with antibodies generated across PLN detected fragments of PLN from IG-repeat domain IV in PCa patient serum. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for PLN proteolysis both in silico and in vitro. Purified PLN showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when embedded with other BM proteins. MMP-7 fragments of PLN were detected as limit peptides by western blot. While not predicted in silico, MMP-7 cleaved every subpart of recombinantly generated PLN domain IV. The resultant MMP-7 generated PLN Dm IV fragments were identified by mass spectrometry (LC-MS/MS and MALDI) and Edman degradation N-terminal sequencing. Other PCa relevant enzymes had limited ability to cleave PLN including prostate specific antigen, hepsin and fibroblast activation protein. To determine if PLN digestion affects metastatic PCa cells, C4-2B cells were seeded on plates coated with different forms of PLN. Cells on intact PLN formed large clusters of poorly adherent cells, while those on PLN digested with MMP-7 or heparitinase to remove the HS chains attached and spread within 30 minutes. Immunohistochemistry of PCa patient tissue indicates MMP-7 and PLN are in direct contact at interfaces in the stroma. We conclude that enzymatic processing of PLN in the BM and stroma as occurs in the tumor microenvironment can alter PCa cell behavior and favor cell dispersion and invasiveness. Citation Format: Brian J. Grindel. Matrilysin cleavage of perlecan produces bioactive fragments acting as a molecular switch to alter prostate cancer cell behavior. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5063. doi:10.1158/1538-7445.AM2013-5063

Development of an ELISA detecting Tumor Protein 53-Induced Nuclear Protein 1 in serum of prostate cancer patients

Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) plays an important role during cell stress response in synergy with the potent “genome-keeper” p53. In human, the gene encoding TP53INP1 is expressed at very high level in some pathological situations, such as inflammation and prostate cancer (PC). TP53INP1 overexpression in PC seems to be a worse prognostic factor, particularly predictive of biological cancer relapse, making TP53INP1 a relevant specific target for molecular therapy of Castration Resistant (CR) PC. In that context, detection of TP53INP1 in patient biological fluids is a promising diagnostic avenue. We report here successful development of a new Enzyme-Linked Immunosorbent Assay (ELISA) detecting TP53INP1, taking advantage of molecular tools (monoclonal antibodies (mAbs) and recombinant proteins) generated in the laboratory during the course of basic functional investigations devoted to TP53INP1. The ELISA principle is based on a sandwich immunoenzymatic system, TP53INP1 protein being trapped by a first specific mAb coated on microplate then recognized by a second specific mAb. This new assay allows specific detection of TP53INP1 in serum of several PC patients. This breakthrough paves the way towards investigation of a large cohort of patients and assessment of clinical applications of TP53INP1 dosage.

Abstract A53: Crosstalk between IGFR and MET in prostate cancer

Abstract Background: Ligand independent receptor tyrosine kinase activation can be mediated by activation through other growth factor receptors. Understanding this cross-talk has implications in the use of combination therapies, as growth factor receptors often stimulate overlapping signaling pathways. Purpose: To examine crosstalk between IGF-R1 and MET in prostate cancer (PCa) cells. Experimental procedures: IGF was added to PC3 prostate cancer cells and phosphorylation of c-Met and activation of signaling proteins was examined by immunoblotting various times thereafter. Activation of MET was determined by immunoblotting with antibodies to the tyrosine phosphorylation sites, Y1234/Y1235 and Y1349. To determine the requirement of transcription for c-Met activation, experiments were performed in the presence of actinomycin D. To determine the requirement of IGFR and/or Src in c-Met phosphorylation, IGF was added to PC3 cells with stable knockdown of IGFR or Src by expression of an shIGFR or shSrc construct. Rescue experiments were performed by expressing an sh-insensitive mouse c-Src. To determine the biological effect of MET activation, PC3 cells and PC3 cells with stable knockdown of c-Met were stimulated with IGF and allowed to migrate in Boyden chambers for various times thereafter. Results: We demonstrate that addition of 100ng/ml of IGF to culture media of PC3 cells (IGF was chosen because it is abundant in the sera of prostate cancer patients), induces MET tyrosine kinase activation beginning 18 hours after addition and sustained for a minimum of 6 hrs thereafter. Multiple sites on Y1234/Y1235 and Y1349 are phosphorylated, suggesting full MET activation, a conclusion supported by increased migration of cells after c-Met phosphorylation. Phosphorylation of MET is not due to the presence of HGF, which was undetectable in cells by quantitative RT-PCR and in media by ELISA, but requires IGF-1R activation, as IGFR stable knock down or the addition of the IGFR-1R inhibitor BMS754807 inhibited MET phosphorylation. We demonstrated that transcription is also required for MET activation, as MET was not tyrosine phosphorylated in cells grown in the presence of actinomycin D. As other groups have shown delayed activation of MET by EGF required the tyrosine kinase, Src, we performed the experiments in the presence of the pan Src Family Kinase inhibitor, dasatinib, and demonstrated that MET phosphorylation was abolished. We demonstrate that Src itself is required for MET phosphorylation, as a stable shSrc knockdown cell line inhibited IGF-induced MET activation, whereas expression of mouse Src (insensitive to sh knockdown) restores MET phosphorylation in the presence of IGF. Conclusion: We demonstrate that IGF induces MET activation through a ligand-independent mechanism requiring gene transcription following activation of IGF-1R and Src. Citation Format: Andreas S. Varkaris, Sanchaika Gaur, Nila Parikh, Christopher Logothetis, Gary E. Gallick. Crosstalk between IGFR and MET in prostate cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A53.

Autoantibody signatures as biomarkers to distinguish prostate cancer from benign prostatic hyperplasia in patients with increased serum prostate specific antigen

BackgroundSerum prostate specific antigen (PSA) concentrations lack the specificity to differentiate prostate cancer from benign prostate hyperplasia (BPH), resulting in unnecessary biopsies. We identified 5 autoantibody signatures to specific cancer targets which might be able to differentiate prostate cancer from BPH in patients with increased serum PSA. MethodsTo identify autoantibody signatures as biomarkers, a native antigen reverse capture microarray platform was used. Briefly, well-characterized monoclonal antibodies were arrayed onto nanoparticle slides to capture native antigens from prostate cancer cells. Prostate cancer patient serum samples (n=41) and BPH patient samples (collected starting at the time of initial diagnosis) with a mean follow-up of 6.56y without the diagnosis of cancer (n=39) were obtained. One hundred micrograms of IgGs were purified and labeled with a Cy3 dye and incubated on the arrays. The arrays were scanned for fluorescence and the intensity was quantified. Receiver operating characteristic curves were produced and the area under the curve (AUC) was determined. ResultsUsing our microarray platform, we identified autoantibody signatures capable of distinguishing between prostate cancer and BPH. The top 5 autoantibody signatures were TARDBP, TLN1, PARK7, LEDGF/PSIP1, and CALD1. Combining these signatures resulted in an AUC of 0.95 (sensitivity of 95% at 80% specificity) compared to AUC of 0.5 for serum concentration PSA (sensitivity of 12.2% at 80% specificity). ConclusionOur preliminary results showed that we were able to identify specific autoantibody signatures that can differentiate prostate cancer from BPH, and may result in the reduction of unnecessary biopsies in patients with increased serum PSA.

Abstract C7: Analysis of high-risk prostate cancer markers at RNA and protein level.

Abstract Background: In many developed countries, prostate cancer is the second leading cause of cancer-related deaths among men. Current diagnostic possibilities do not differentiate aggressive forms of prostate cancer from latent tumors. Therefore, having reliable and easy-to-detect marker is needed to treat this disease successfully. We determined potential novel tumor markers of high-risk prostate cancer (A) at the cell level, represented by cell lines 22Rv1 and PNT1A and (B) in the serum of 82 histologicaly -verified cases and 51 controls. Material and Methods: 22Rv1 cell line derived from primary prostate tumor and PNT1A cell line derived from normal prostate epithelium were used in our study. The real time PCR, electrophoretical separation technique, immunochemical detection and novel electrochemical analysis were used in tumor markers determination. Findings and Interpretation: Cell line 22Rv1 represents Gleason sum 9 primary tumor, compared to widely used cell lines PC-3, LNCaP and PD-145, representing metastasis, therefore, is more related to real tumor tissue. We determined RNA and protein level of genes alpha-methyl acyl-CoA racemase (AMACR), caveolin-1, metalothionein, p53, NF-kB, FOS, JUN, Ki-67, ZIP1 and ZnT-1. At the RNA level in cell lines we found significant (p < 0.01) down-regulation of Cav-1 (50-fold), NF-kB (11-fold) and others and significant (p < 0.01) up-regulation of metalothionein (2.4-fold), AMACR (8.4-fold), Ki-67 (2-fold) and zinc transporters ZnT-1 (25-fold) and ZIP1 (45-fold). Conversely, we observed down-regulation of metallothionein at protein level. In the serum of prostate cancer patients we found no significant differences in AMACR and caveolin-1 level and significantly (p < 0.001) higher metallothionein level suggesting metalothionein as a complementary marker to PSA. We found weak correlation of PSA on caveolin-1 (r = 0.41 at p = 0.002) and, interestingly, significant elevation of caveolin-1 in high-grade tumors (tumor stage 4 (2.3-fold elevation), Gleason sum 9, correlation at r = 0,29 at p = 0,028) suggesting caveolin-1 as potential high-risk prostate cancer marker. This study provides important new information about selected genes connected with prostate cancer and describes new electrochemical approaches for their determination in lower concentrations. Financial support from IGA MH of Czech republic no. NS10200–3 is greatly acknowledged. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C7.

Serum‐autoantibodies for discovery of prostate cancer specific biomarkers

The currently used prostate cancer serum marker has a low cancer specificity and improved diagnostics are needed. Here we evaluated whether autoantibodies are present in sera of prostate cancer patients and whether they are useful diagnostic markers for prostate cancer. Sera from 20 prostate cancer patients and 20 healthy controls were incubated on expression clone arrays containing more than 37,000 recombinant human proteins. Functional annotation clustering of the identified autoantigens was performed using the DAVID database. Autoantigens identified in the prostate cancer group were validated on microarrays using sera of 40 prostate cancer patients, 40 patients with elevated PSA levels but prostate cancer negative biopsies (benign disease), and 40 healthy controls. We detected autoantibodies against 408 different antigens in sera of prostate cancer patients. One hundred seventy-four of these were exclusively detected in the cancer group compared to the healthy control group. Functional annotation clustering revealed an enrichment of RNA-associated, cytoskeleton, and nuclear proteins. The autoantibody panel was validated in serum samples of independent prostate cancer patients. Autoantibody profiles discriminated between prostate cancer patients and benign disease patients with an ROC curve AUC of 0.71. TTLL12, a protein recently described to be over-expressed in prostate cancer, was the highest ranked discrimination autoantigen. A variety of autoantibodies were identified in sera of prostate cancer patients and provide a first step towards autoantibody diagnostics. Serum autoantibodies reflect the disease and represent valuable tools not only for prostate cancer, but also for other diseases affecting the immune response.

Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum

A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL −1 levels. MPs were conjugated with ∼90,000 antibodies and ∼200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL −1 for PSA and 0.30 pg mL −1 for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.

127 DIFFERENTIAL EXPRESSION OF RANKL AND OSTEOPROTEGERIN IN PATIENTS WITH CLINICALLY LOCALIZED PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011127 DIFFERENTIAL EXPRESSION OF RANKL AND OSTEOPROTEGERIN IN PATIENTS WITH CLINICALLY LOCALIZED PROSTATE CANCER Tilman Todenhoefer, Joerg Hennenlotter, Andrea Hohneder, Ursula Kuehs, David Schilling, Stefan Aufderklamm, Saladin Alloussi, Georgios Gakis, Arnulf Stenzl, and Christian Schwentner Tilman TodenhoeferTilman Todenhoefer Tuebingen, Germany More articles by this author , Joerg HennenlotterJoerg Hennenlotter Tuebingen, Germany More articles by this author , Andrea HohnederAndrea Hohneder Tuebingen, Germany More articles by this author , Ursula KuehsUrsula Kuehs Tuebingen, Germany More articles by this author , David SchillingDavid Schilling Tuebingen, Germany More articles by this author , Stefan AufderklammStefan Aufderklamm Tuebingen, Germany More articles by this author , Saladin AlloussiSaladin Alloussi Tuebingen, Germany More articles by this author , Georgios GakisGeorgios Gakis Tuebingen, Germany More articles by this author , Arnulf StenzlArnulf Stenzl Tuebingen, Germany More articles by this author , and Christian SchwentnerChristian Schwentner Tuebingen, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.194AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The receptor activator of NFkappaB Ligand (RANKL) pathway mediates interactions of tumor cells and bone microenvironment. RANKL is secreted by osteoblasts and bone marrow stromal cells and promotes osteolysis and development of bone metastases via activation of the RANK receptor on osteoclasts RANKL and osteoprotegerin (OPG) are also expressed by tumor cells. Our aim was to determine, if changes of RANKL and OPG are notable in the serum and bone marrow of patients with clinically localized prostate cancer (PC) without manifest bone metastases. METHODS Concentrations of total sRANKL (free and OPG-bound soluble RANKL) and OPG were determined in serum and bone marrow specimens of 20 patients undergoing radical prostatectomy for localized PC (Median Gleason score 7, Median PSA value 5,98 ng/ml, Median Age 66,5) and 9 individuals without PC (benign prostate hyperplasia or stone disease, Median Age 64,0) by Enzyme linked immunosorbent assay. Ratios of sRANKL/OPG were calculated. Disseminated tumor cells (DTC) in bone marrow were determined by cytokeratine immunocytochemistry. Values were compared and correlated to clinical data using the Wilcoxon-Kruskall-Wallis tests and linear regression analyses. RESULTS Median values of serum sRANKL, OPG and ratio for PC and non-PC patients were 33515, 62, 840 and 131, 63, 2 (p=0.015, 0.81 and 0.030), respectively. Serum and corresponding bone marrow values of all 3 investigated parameters correlated significantly (p<0.0001, p= 0,001 and p=0,008 for sRANKL, OPG and ratio, respectively). Concentrations of sRANKL and OPG in bone marrow and serum were independent from Gleason score and PSA value. Interestingly, patients with locally advanced prostate cancer (>pT2) showed lower serum sRANKL levels than those with organ-confined disease (p=0.04). The presence of DTCs did not significantly affect sRANKL and OPG in bone marrow and serum. CONCLUSIONS This is the first study analyzing sRANKL/OPG in bone marrow and corresponding serum of PC patients. Increased activity of RANKL with an elevated sRANKL/OPG ratio indicates osteolytic activity in these patients before bone metastases manifestation. As concentrations of total sRANKL and OPG in bone marrow and serum correlate significantly, measurement of sRANKL and OPG in serum reliably reflects the status of sRANKL/OPG in the bone marrow. These alterations are even notable in the absence of DTCs. These preliminary results give new insights in tumor-induced changes of RANKL activity and implicate a potential benefit of RANKL inhibition for patients with clinically localized PC. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e53 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tilman Todenhoefer Tuebingen, Germany More articles by this author Joerg Hennenlotter Tuebingen, Germany More articles by this author Andrea Hohneder Tuebingen, Germany More articles by this author Ursula Kuehs Tuebingen, Germany More articles by this author David Schilling Tuebingen, Germany More articles by this author Stefan Aufderklamm Tuebingen, Germany More articles by this author Saladin Alloussi Tuebingen, Germany More articles by this author Georgios Gakis Tuebingen, Germany More articles by this author Arnulf Stenzl Tuebingen, Germany More articles by this author Christian Schwentner Tuebingen, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The study of DJ-1 protein in tissue specimens, cultured cells and serum of prostate cancer patients

The study of DJ-1 protein in tissue specimens, cultured cells and serum of prostate cancer patients

Cancer genetics-guided discovery of serum biomarker signatures for diagnosis and prognosis of prostate cancer

A key barrier to the realization of personalized medicine for cancer is the identification of biomarkers. Here we describe a two-stage strategy for the discovery of serum biomarker signatures corresponding to specific cancer-causing mutations and its application to prostate cancer (PCa) in the context of the commonly occurring phosphatase and tensin homolog (PTEN) tumor-suppressor gene inactivation. In the first stage of our approach, we identified 775 N-linked glycoproteins from sera and prostate tissue of wild-type and Pten-null mice. Using label-free quantitative proteomics, we showed that Pten inactivation leads to measurable perturbations in the murine prostate and serum glycoproteome. Following bioinformatic prioritization, in a second stage we applied targeted proteomics to detect and quantify 39 human ortholog candidate biomarkers in the sera of PCa patients and control individuals. The resulting proteomic profiles were analyzed by machine learning to build predictive regression models for tissue PTEN status and diagnosis and grading of PCa. Our approach suggests a general path to rational cancer biomarker discovery and initial validation guided by cancer genetics and based on the integration of experimental mouse models, proteomics-based technologies, and computational modeling.

Microfluidic-Based Multiplex qRT-PCR Identifies Diagnostic and Prognostic microRNA Signatures in the Sera of Prostate Cancer Patients

Recent prostate-specific antigen-based screening trials indicate an urgent need for novel and noninvasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Noncoding microRNAs (miRNA) in the serum and plasma have been shown to have potential as noninvasive markers for physiologic and pathologic conditions. To identify serum miRNAs that diagnose and correlate with the prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR method involving the purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA-deficient) mouse ES cells as the benchmark, we confirmed the validity of our technique and uncovered a considerable lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA scores, we identified miRNA signatures that allow us to diagnose cancer patients and correlate with a prognosis. These serum signatures include oncogenic and tumor-suppressive miRNAs, suggesting functional roles in prostate cancer progression.

Study of Dj-1 protein in tissue specimens, cultured cells and serum of prostate cancer patients

Two isoforms of Dj-1 protein were identified using a proteomic study in tissue specimens from two groups of patients with confirmed benign prostate hyperplasia (BPH) and prostate cancer (PCa). Dj-1 was also found in the cell lines PC-3, DU-145, LNCaP, BPH-1, and the lowest level of Dj-1 was found in BPH-1. Immunochemical study (ELISA) of serum levels of Dj-1, Bcl-2, IGF-1 and IGFBP-3 proteins revealed statistically significant distinctions between two groups of patients (p=0,004, Mann-Whitney test) only for Dj-1. Taken together, these data suggest that Dj-1 protein is a perspective biomarker candidate for PCa.

Serum Free Circulating DNA Is a Useful Biomarker to Distinguish Benign versus Malignant Prostate Disease

Free circulating DNA (fcDNA) has been shown to be elevated in serum of prostate cancer patients compared with benign controls. However, studies evaluating the role of fcDNA as a biomarker in a "representative" patient group who have undergone prostate cancer screening are lacking. Our study examined the use of serum fcDNA levels as a biomarker of prostate cancer in such a setting. The study included 252 men, with prostate-specific antigen (PSA) levels >4 ng/mL and/or abnormal digital rectal exam. fcDNA levels in serum before prostate biopsy were quantitated by real-time PCR amplification of the glutathione S-transferase, pi, gene. Patients with PSA < or = 10 ng/mL with fcDNA > 180 ng/mL were at increased risk for prostate cancer compared with those with fcDNA < or =180 ng/mL (odds ratio, 4.27; 95% confidence interval, 2.05-8.88; P < 0.001; area under the curve, 0.742). The multivariate model including age, race, PSA, fcDNA, and interaction between fcDNA and PSA yielded a high negative predictive value of 93.1% and increased specificity of 33.1% compared with negative predictive value of 73.3% and specificity of 6.7% in the model excluding fcDNA. Our results indicate that fcDNA may improve the specificity of prostate cancer screening. Our study shows that adding fcDNA to prostate cancer screening can reduce the number of unnecessary prostate biopsies.

Beta‐2‐microglobulin expression correlates with high‐grade prostate cancer and specific defects in androgen signaling

Previously, we identified Beta-2-microglobulin (beta2M) as an androgen-regulated secreted protein elevated in the serum of prostate cancer patients. In this study, we explore an interaction between beta2M expression, prostate cancer tissue, and the androgen signaling axis. beta2M expression in relation to clinical and pathologic variables was examined in a tissue microarray representing specimens obtained at the time of radical prostatectomy. Viral vectors were designed to down-regulate beta2M expression, and the effects on androgen-dependent growth, transcriptional regulation, and androgen receptor recruitment was investigated in human prostate cancer cell lines. Variation in beta2M expression in human prostate cancer is associated with characteristics of clinically aggressive disease such as high tumor grade. Knockdown of beta2M expression in human prostate cancer cells resulted in selective defects in androgen-dependent events including growth, gene regulation, and chromatin assembly. beta2M expression may provide prognostic information in patients treated with surgery for prostate cancer. Targeting beta2M expression or activity may represent a new and important mechanism to manipulate the androgen signaling axis in patients with prostate cancer.

Differential expression of protease activity in serum samples of prostate carcinoma patients with metastases

We present here the results from MS peptide profiling experiments of prostate carcinoma patients and controls with a specific focus on protease activity-related protein fragments. After purification with surface-active magnetic beads, MALDI-TOF profiling experiments were performed on tryptic digests of serum samples of prostate cancer patients with metastases (n=27) and controls (n=30). This resulted in the reproducible detection of eight differentially expressed peptides, which were then identified by nanoLC-MALDI-TOF/TOF and confirmed by MALDI-FTMS exact mass measurements. All differentially expressed peptides are derived from two homologous parts of human serum albumin; two of the eight peptides were tryptic and six were nontryptic. The presence of the nontryptic fragments indicates that a proteolysis process occurs which is not mediated by trypsin. Since the nontryptic fragments were found at significantly higher levels in control samples compared with metastases samples, it is proposed that a specific proteolytic inhibition process is in effect in the serum of prostate cancer patients. Experiments using synthetic peptides showed that this proteolytic activity occurs ex vivo and is sequence specific. Importantly, the observed prostate carcinoma-related inhibition of the proteolysis was reproduced ex vivo using synthetic peptides.

Clinical and imaging correlations of bone turnover markers in prostate cancer patients with bone only metastases

To correlate serum levels of bone markers with pain levels and extent of skeletal disease (EOD), in patients suffering from prostate cancer with bone only metastases. Thirty-six males with hormone-refractory prostate carcinoma, bone only metastases and no history of therapies, drugs, or diseases that affect bone metabolism were studied. Karnofsky performance status, pain scoring, EOD, osteocalcin (OC), prostate-specific antigen, bone alkaline phosphatase amino-terminal and carboxy-terminal propeptides and telopeptides of type I collagen were analysed. Twenty-four healthy controls of the same age were also established. With only the exception of OC, bone marker values of patients were significantly increased compared with the upper reference limits (P<0.0001 for bone alkaline phosphatase and amino-terminal telopeptide of type I collagen, 0.012 for amino-terminal propeptide of type I collagen, 0.0023 for carboxy-terminal propeptide of type I collagen, and 0.04 for carboxy-terminal telopeptide of type I collagen). All bone markers and prostate-specific antigen also showed significant paired correlations (P < or = 0.019) and linear increases with advancing EOD (P < or = 0.032). Finally, none of the measured markers correlated significantly with pain levels. Bone markers are remarkably elevated in the serum of prostate cancer patients with metastatic bone disease and correlate with EOD. Paired correlations also suggest an accelerated but proportional (coupled) bone metabolism.

Aberrant promoter methylation in serum of prostate cancer patients and controls

e16046 Background: The current screening method for prostate cancer (PC), which is based on prostate specific antigen (PSA) level lacks specificity and sensitivity. Previous studies have reported that increased levels of free circulating DNA (fcDNA) and hypermethylation of genes are associated with several cancers. In this study we evaluated the use of fcDNA level and aberrant promoter methylation of 4 genes in serum as biomarkers for PC. Methods: 89 PC patients, 59 with prostatitis (PT) and 104 with benign prostatic hyperplasia (BPH) were enrolled. Serum DNA was analyzed for methylation of GSTPi, RARB, RASSF1A and ECAD by methylation-specific PCR. FcDNA level was analyzed by quantitative PCR. We examined the effect of methylation and fcDNA on PC risk by fitting two logistic regression models, one comparing PC vs BPH and PT vs BPH and the other comparing PC vs PT+BPH. Models were adjusted for age, race, and log2PSA, and tested for variation in effect of methylation by race. Similar analyses assessed the effect of fcDNA as continuous variable, after log2 transformation, and as dichotomous variable with cutpoint near the upper quartile for BPH controls. Results: When adjusted for age and log2PSA, positive GSTPi methylation was associated with increased risk of PC in whites (OR = 6.11, p &lt; 0.001, PC vs BPH; OR = 3.93, p = 0.001, PC vs BPH + PT). Methylation of RARB was associated with PT independent of race (OR = 2.85, p = 0.032, PT vs BPH). Patients with fcDNA ≥180ng/ml were at twice the risk of PC compared to those with levels &lt;180ng/ml, and this result did not vary by race. Models including both GSTPi and fcDNA confirmed the independent effect of these two factors in whites (high fcDNA OR = 3.77, p = 0.002, positive GSTPi methylation OR = 5.41, p &lt; 0.001, PC v. BPH + PT). Although high fcDNA also increased risk of PC in blacks, the effect was not statistically significant (OR = 1.91, p = 0.189, PC vs BPH + PT). Conclusions: Our results suggest that aberrant promoter methylation of GSTPi in serum is a potential biomarker for prostate cancer in whites. Level of fcDNA in serum also appears to be an independent diagnostic marker in whites. No significant financial relationships to disclose.

Serum CXCL13 positively correlates with prostatic disease, prostate-specific antigen and mediates prostate cancer cell invasion, integrin clustering and cell adhesion

Chemokines and their corresponding receptor interactions have been shown to be involved in prostate cancer (PCa) progression and organ-specific metastasis. We have recently shown that PCa cell lines and primary prostate tumors express CXCR5, which correlates with PCa grade. In this study, we present the first evidence that CXCL13, the only ligand for CXCR5, and IL-6 were significantly elevated in PCa patient serum compared to serum from subjects with benign prostatic hyperplasia (BPH), or high-grade prostatic intraepithelial neoplasia (HGPIN) as well as normal healthy donors (NHD). Serum CXCL13 levels significantly ( p < 0.0001) correlated with serum prostate-specific antigen (PSA), whereas serum IL-6 levels significantly ( p < 0.0003) correlated with CXCL13 serum levels. CXCL13 was found to be a better predictor of PCa than PSA. CXCL13 was highly expressed by human bone marrow endothelial (HBME) cells and osteoblasts (OBs), but not osteoclasts (OCs), following treatment with physiologically relevant levels of interleukin-6 (IL-6). We further demonstrate that CXCL13, produced by IL-6-treated HBME cells, was able to induce PCa cell invasion in a CXCR5-dependent manner. CXCL13-mediated PCa cell adhesion to HBME cells and α vβ 3-integrin clustering was abrogated by CXCR5 blockade. These results demonstrate that the CXCL13-CXCR5 axis is significantly associated with PCa progression.

Associations of IgG N‐linked oligosaccharide chains and proteases in sera of prostate cancer patients with and without α2‐macroglobulin deficiency

We previously reported on a number of cases of metastatic prostate cancer (PCa) in which serum alpha2-macroglobulin (alpha2M) levels were markedly decreased to less than 20 mg/dl (alpha2M deficiency). All PCa patients with alpha2M deficiency had multiple bone metastases. Proteases in ten PCa patients with and without alpha2M deficiency were studied and compared against ten healthy controls in order to elucidate the relationships between changes in sugar chain structure and neoplasia. We assessed the relationship between ratios of Fr4 to Fr1 and Fr2 (Fr4/Fr1+Fr2 ratios) of oligosaccharide chains, and ratios of free prostate-specific antigen (PSA) to total PSA (F/T ratios), and serum levels of matrix-metalloproteinase-2 (MMP-2) in PCa progression. Measurement of serum alpha2M concentration was performed by laser nephelometry. Serum PSA and MMP-2 levels were determined by enzyme immunoassay and free PSA by radioimmunoassay. N-linked oligosaccharides of human serum immunoglobulin G were analyzed using fluorophore-associated carbohydrate electrophoresis. In those PCa patients with alpha2M deficiency: (a) serum alpha2M and F/T ratios were lower (P<0.05) and (b) Fr4/Fr1+Fr2 ratios and serum MMP-2 levels were higher when compared with those PCa patients without alpha2M deficiency. There was a significant correlation between Fr4/Fr1+Fr2 ratios and F/T ratios or serum MMP-2 levels in PCa with alpha2M deficiency (P<0.05). Therefore, these markers may serve as an auxiliary serum tumor marker for monitoring of the bone metastases or progression of disease in PCa.