Biomarkers For CaP Research Articles

Reprogramming of Amino Acid Metabolism Differs between Community-Acquired Pneumonia and Infection-Associated Exacerbation of Chronic Obstructive Pulmonary Disease.

Amino acids and their metabolites are key regulators of immune responses, and plasma levels may change profoundly during acute disease states. Using targeted metabolomics, we evaluated concentration changes in plasma amino acids and related metabolites in community-acquired pneumonia (CAP, n = 29; compared against healthy controls, n = 33) from presentation to hospital through convalescence. We further aimed to identify biomarkers for acute CAP vs. the clinically potentially similar infection-triggered COPD exacerbation (n = 13). Amino acid metabolism was globally dysregulated in both CAP and COPD. Levels of most amino acids were markedly depressed in acute CAP, and total amino acid concentrations on admission were an accurate biomarker for the differentiation from COPD (AUC = 0.93), as were reduced asparagine and threonine levels (both AUC = 0.92). Reduced tryptophan and histidine levels constituted the most accurate biomarkers for acute CAP vs. controls (AUC = 0.96, 0.94). Only kynurenine, symmetric dimethyl arginine, and phenylalanine levels were increased in acute CAP, and the kynurenine/tryptophan ratio correlated best with clinical recovery and resolution of inflammation. Several amino acids did not reach normal levels by the 6-week follow-up. Glutamate levels were reduced on admission but rose during convalescence to 1.7-fold above levels measured in healthy control. Our data suggest that dysregulated amino acid metabolism in CAP partially persists through clinical recovery and that amino acid metabolism constitutes a source of promising biomarkers for CAP. In particular, total amino acids, asparagine, and threonine may constitute plasma biomarker candidates for the differentiation between CAP and infection-triggered COPD exacerbation and, perhaps, the detection of pneumonia in COPD.

RNA-based biomarkers for the diagnosis, prognosis, and therapeutic response monitoring of prostate cancer.

Prostate cancer (CaP) is the most common malignant neoplasm of the urinary tract. The current recommendations for CaP diagnosis rely on the prostate-specific antigen levels and a digital rectal examination for anatomical abnormalities. However, these diagnostic tools are not highly sensitive. In particular, prostate-specific antigen has a low positive predictive value (approximately 30%). Thus, there is a need to develop biomarkers to improve the early clinical detection of CaP. Several novel technologies enable the identification of biomarkers from diverse sources, including the urine, serum, and prostate tissues. Furthermore, advances in genomic techniques have enabled the analysis of novel biomarkers, such as deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), proteins, and circulating tumor cells. Previous studies have demonstrated that RNAs are potential diagnostic biomarkers for various cancers using high-throughput sequencing analysis. The sensitivity and specificity of RNA biomarkers are higher than those of protein biomarkers. Polymerase chain reaction enables the amplification of trace levels of RNAs with high sensitivity and specificity. RNA biomarkers provide dynamic insights into cellular states and regulatory processes when compared with DNA biomarkers. Additionally, multiple copies of various RNAs in a cell provide more information than DNA. The levels of specific RNAs in CaP tissues are upregulated when compared with those in non-cancerous tissues. Additionally, RNAs can be easily isolated from various body fluids. Thus, RNAs are potential non-invasive biomarkers for CaP. Moreover, the analysis of RNA levels adjusted for each stage of CaP enables the determination of prognostic individualized therapy for aggressive or progressive CaP. This review focused on the diagnostic and prognostic values of RNAs for CaP.

High level of phosphatidylcholines/lysophosphatidylcholine ratio in urine is associated with prostate cancer.

The altered levels of phospholipids (PLs) and lysophospholipids (LPLs) in prostate cancer (CaP) and benign tissues in our previous findings prompted us to explore PLs and LPLs as potential biomarkers for CaP. Urinary lipidomics has attracted increasing attention in clinical diagnostics and prognostics for CaP. In this study, 31 prostate tissues obtained from radical prostatectomy were assessed using high‐resolution matrix‐assisted laser desorption/ionization imaging mass spectrometry (HR‐MALDI‐IMS). Urine samples were collected after digital rectal examination (DRE), and urinary lipids were extracted using the acidified Bligh‐Dyer method. The discovery set comprised 75 patients with CaP and 44 with benign prostatic hyperplasia (BPH) at Kyoto University Hospital; the validation set comprised 74 patients with CaP and 59 with BPH at Osaka University Hospital. Urinary lipidomic screening was performed using MALDI time‐of‐flight MS (MALDI‐TOF/MS). The levels of urinary lysophosphatidylcholine (LPC) and phosphatidylcholines (PCs) were compared between the CaP and BPH groups. The (PC [34:2] + PC [34:1])/LPC (16:0) ratio was significantly higher (P < .001) in CaP tissues than in benign epithelial tissues. The urinary PCs/LPC ratio was significantly higher (P < .001) in the CaP group than in the BPH group in the discovery and validation sets.

Abstract A111: Urine-based gene expression panels with focus on prostate cancer in African Americans

Abstract Introduction: Prostate cancer (CaP) affects 1 in 7 men in their lifetime. African American (AA) men have significantly higher incidence and mortality from CaP compared to Caucasian American (CA) men. We and others have also noted that the genes commonly overexpressed in CaP (e.g., ERG) and currently used as diagnostic markers exhibit much lower frequency and more heterogeneity in AA men. The goals of this study are to develop CaP diagnostic/prognostic marker panels that takes into account race-associated differences in molecular alterations. Here we evaluated a panel of CaP associated genes using regular (non-DRE) urine from AA and CA CaP for discriminating between high-grade (Gleason score [GS], 7-10) and low-grade disease (GS, 6) and cancer negative biopsy. Methods: The quantitative expression assay protocols have been developed for noninvasive detection of candidate genes in exosomal RNAs from regular urine. The selected genes were reverse transcribed in a single reaction using gene-specific primer pool (GSP) followed by preamplification and PCR-based assay of all target genes. Results: Regular urine exosomal RNA-based assays were developed and optimized for PSGR, DLX1, HOXC4, NKX2-3, COL10A1, HOXC6, PCA3, PCGEM1, PCAT1, CTBP1, SChLAP1, ERG and SPDEF. All markers have been evaluated in a pilot study of 72 subjects undergoing diagnostic biopsies. For AA patients a sensitivity of 88.2%, specificity of 83.3%, and AUC of 0.81 were achieved for the panel (PCA3, PCGEM1, HOXC6 and SChLAP1). Consistent with numerous studies, ERG and PCA3 panel was optimal for CA patients. Preliminary analysis showed that 3 markers were found to be associated with improved discrimination between GS7 or greater and GS6 (AUC 0.78 for ERG; AUC 0.72 for PSGR and AUC 0.72 for SChLaP1) in AA CaP. In CA patients, PCA3 gave an AUC of 0.76 for predicting high-grade from low-grade disease. Analysis of an additional 100 patients is in progress. Conclusions: Given the genomic difference of prostate cancer among different race/ethnicity, this study highlights the potential for developing CaP biomarker panels in the context of race for enhancing precision medicine through improved CaP diagnosis/prognosis Citation Format: Indu Kohaar, Sreedatta Banerjee, Yongmei Chen, Amina Ali, Jacob Kagan, Sudhir Srivastava, Jennifer Cullen, Inger Rosner, Shiv Srivastava, Gyorgy Petrovics. Urine-based gene expression panels with focus on prostate cancer in African Americans [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A111.

Extensive serum cytokine analysis in patients with prostate cancer

Prostate cancer (CaP) is a common male malignancy. Using prostate specific antigen (PSA) and prostate cancer antigen 3 (PCA3) in the diagnosis of prostate cancer, sensitivity and specificity still require improvement. Additional targets are urgently needed for the diagnosis, prognosis, and prediction of therapeutic response, leading to better treatments in order to reduce the mortality of CaP. Here, we utilized a solid-phase antibody array, which can simultaneously detect 200 proteins, for the screening of novel blood-based biomarkers. The proteins differentially expressed in the pathogenesis of CaP were further analyzed using bioinformatics methods. The identified targets were further validated by the enzyme-linked immunosorbent assay (ELISA). A total of 38 proteins were identified with significantly differential levels in CaP serum compared to healthy control serum, including 21 up-regulated and 17 down-regulated cytokines. ELISA result showed that validated six ones of these differential cytokines were significantly differential between CaP and control, consistent with the antibody array result. The protein-protein interaction (PPI) analysis for these differentially expressed cytokines showed the top five cytokines interacting with most other cytokines were insulin, SDF-1a, CD40L, IL-18 and NCAM-1, suggesting these five targets are important in the pathogenesis of CaP, and more sensitive for the early diagnosis and prognosis of CaP. Targeting these cytokines may be more effective therapies against CaP. Among these differentially expressed cytokines, it was found that AR, BTC, IL-1 F8, IL-31, Marapsin, b-NGF, EDA-A2, MCP-3, MCP-4, MIP-3a, PIGF, and TECK decreased, while Fas, Flt-3L, and NCAM-1 increased in CaP when compared to the controls. Taken together, those 38 differentially expressed cytokines may service as novel serum biomarkers for CaP, which will be further validated with more clinical samples.

Label-free electrochemical quantification of microRNA-375 in prostate cancer cells

Clinical applicability of a label-free electrochemical method was investigated, where prostate cancer (CaP) biomarkers miR-107 and miR-375 were examined in two different groups of prostate cell lines, metastatic CaP cells (PC-3, DU145, and LNCaP) and immortalized prostate epithelial cells (267B1, X/267B1, and Ki/267B1). First, the expression levels of miR-107 and miR-375 in these cells were analyzed by quantitative real-time polymerase chain reaction (PCR), resulting in useful discernable expression patterns from miR-375. Choosing miR-375 as a target CaP biomarker, a label-free electrochemical analysis was performed based on the optimized conditions of 100nmol/mL miR-375 capture probe for surface immobilization and 30-minute hybridization. Standard curves were prepared both in buffer and in 100-fold diluted serum resulting in lower detection limit of 11.7aM and 12.4aM respectively. The regression equations for buffer and diluted serum were I=−15.1349 log c+250.9735 and I=−11.8179 log c+193.1639, respectively, where I is the peak current in μA and c is the concentration of miR-375 in fM. The selectivity of the sensor was confirmed by a one-base mismatch test in 3 different concentrations of miR-375 under diluted serum, 10aM, 10fM, and 10pM. Finally, miR-375 expression in the metastatic CaP cells was quantified to range between 10 and 30fM, while its expression in the prostate epithelial cells was negligible, demonstrating close agreement with the results from quantitative real-time PCR, and thereby potential viability of the electrochemical evaluation method in clinical environment.

Abstract 5765: Immunobiomarkers: Structural and functional characterization of single chain fragment variable (scFv) to ERG from a mouse monoclonal antibody

Abstract Introduction: Biomarkers for early detection, disease stratification as well as treatment strategies are a major area of focus in prostate cancer (CaP) research. The gene fusion involving ERG and the TMPRSS2 gene promoter contributes to expression of ERG in CaP, serving as a biomarker for CaP. This led to the development of monoclonal or polyclonal antibodies against ERG protein for diagnostic or therapeutic purposes. As the use of intact monoclonal antibody is limited due to large size and low cell penetration, we hypothesized that single chain fragment variable (scFv) and single domain antibody (sdAb) known as minibodies will be valuable as therapeutic agents targeting ERG protein. Towards this goal, the present study addresses the following: i) Characterization of variable heavy (VH) and variable light (VL) chain sequences and generation of expression vectors; ii) Assessing the specificity of binding of scFv and sdAb to ERG protein; iii) Functional evaluation of scFv and sdAb in cellsMethods: We generated VH and VL encoding sequences using RNA derived from hybridoma cells secreting anti-ERG monoclonal antibody designated 9FY. scFv-9FY and sdAb-9FY fragments represent scFv and sdAb , respectively were cloned in an eukaryotic and prokaryotic expression vectors. The proteins (scFv and sdAb) were analyzed by western blot and ELISA. The binding of scFv and sdAb to ERG protein in live cells was evaluated by bimolecular fluorescence complementation (BiFC) assay. Biological effects of scFv and sdAb were assessed in HEK293, VCaP and LNCaP cells. Results: Analysis was carried out to determine the primary DNA sequence of scFv fragments. Using the predicted amino acid sequence, we identified CDR1, CDR2 and CDR3 regions. scFv expressed in prokaryotic vector was purified and used for the evaluation of immunoreactivity to ERG by ELISA. The affinity of scFv and sdAb to ERG was demonstrated through their binding to ERG protein in HEK293 cells by using BiFC assay. This involved the generation of chimeric proteins in which scFv or sdAb coding sequences were fused in-frame to the Venus N- or C-terminal segment separated by a flexible linker of 10 amino acids. Minibodies scFv and sdAb, upon introduction into VCaP cells through transfection, showed cytotoxic effects similar to the observations noted with siRNA against ERG.Conclusions: Here we showed that scFv and sdAb minibodies exhibit high affinity for ERG protein. This property enables them to disrupt the functions mediated by ERG in cells. Given the prevalence of ERG overexpression in prostate cancer, the novel agents based on scFv and sdAb have the potential to interfere with the effect or functions of ERG and are likely to be beneficial in the treatment of CaP. Source of Funding: Center for Prostate Disease Research, USU Grant HU0001-10-2-0002, NCI/EDRN Grant ACN12011-001-0, and the NCI Grant R01CA162383. Citation Format: Shyh-Han Tan, Anshu Rastogi, Sreedatta Banerjee, Annie Bagga, Charles Xavier, Ahmed Mohamed, Denise Young, Gyorgy Petrovics, Albert Dobi, Isabell A. Sesterhenn, Jacob Kagan, Sudhir Srivastava, David G. David, Inger L. Rosner, Shiv Srivastava, Alagarsamy Srinivasan. Immunobiomarkers: Structural and functional characterization of single chain fragment variable (scFv) to ERG from a mouse monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5765.

Urinary biomarkers in prostate cancer detection and monitoring progression.

Prostate cancer (CaP) is the most common cancer in men and the second leading cause of cancer deaths in males in Australia. Although serum prostate-specific antigen (PSA) has been the most widely used biomarker in CaP detection for decades, PSA screening has limitations such as low specificity and potential association with over-diagnosis. Current biomarkers used in the clinic are not useful for the early detection of CaP, or monitoring its progression, and have limited value in predicting response to treatment. Urine is an ideal body fluid for the detection of protein markers of CaP and is emerging as a potential source for biomarker discovery. Gene-based biomarkers in urine such as prostate cancer antigen-3 (PCA3), and genes for transmembrane protease serine-2 (TMPRSS2), and glutathione S-transferase P (GSTP1) have been developed and evaluated in the past decades. Among these biomarkers, urinary PCA3 is the only one approved by the FDA in the USA for clinical use. The study of urine microRNAs (miRNAs) is another burgeoning area for investigating biomarkers to achieve a pre-biopsy prediction of CaP to contribute to early detection. The development of mass spectrometry (MS)-based proteomic techniques has sparked new searches for novel protein markers for many diseases including CaP. Urinary biomarkers for CaP represent a promising alternative or an addition to traditional biomarkers. Future success in biomarker discovery will rely on collaboration between clinics and laboratories. In addition, research efforts need to be moved from biomarker discovery to validation in a large cohort or separate population of patients and translation of these findings to clinical practice. In this review, we discuss urine as a potential source for CaP biomarker discovery, summarise important genetic urine biomarkers in CaP and focus on MS-based proteomic approaches as well as other recent developments in quantitative techniques for CaP urine biomarker discovery.

Abstract 2830: Secreted protein acidic and rich in cysteine antigen and autoantibodies in sera of prostate cancer patients: Potential use in diagnosis /prognosis

Abstract Objectives: Improvements in blood based biomarkers for detecting clinically significant prostate cancer (CaP) or for distinguishing between indolent and aggressive CaP are critical in enhancing the management of this most common non-skin cancer of men in the US. These biomarkers include tumor associated antigens (TAAs) and autoantibodies (AAbs) against TAAs in patient sera. SPARC/Osteonectin is highly expressed in metastatic cancers (glioblastoma, melanoma, breast cancer and prostate cancer) and promotes bone metastasis and epithelial-mesemchymal transition. Our comparative transcriptome analyses of well/moderately differentiated CaP with poorly differentiated CaP, defined SPARC as central node in the network of genes with common regulatory elements (NKXH_NKXH_HOX) associating with poorly differentiated CaP. Further, we reported associations of high levels of SPARC mRNA or protein with Gleason 8-10 or poorly differentiated primary tumors and association with metastatic progression. The aims of this study are: i) Quantify SPARC antigen in the sera CaP patients; ii) Are AAbs against SPARC present in the serum of CaP patients?; iii) Is there a correlation between SPARC AAb level in patient sera and disease status? iii) Does SPARC AAb level vary according to distinct ethnic groups? Methods: Sera from CaP patients and healthy controls were evaluated for SPARC antigen using a commercial enzyme-linked immunosorbent assay (ELISA) kit. AAbs against SPARC were determined by ELISA utilizing recombinant full-length human SPARC protein as a substrate. For evaluation we used Caucasian American (CA, n=117) and African American (AA, n=111) CaP patients comprising Gleason 6-10, and healthy controls (CA, n=52; AA, n=45). Results: SPARC antigen levels in the sera showed a difference in the overall case versus control groups (p=0.0016) and this trend was observed only in AA (p=0.0015) in comparison to CA patients (p=0.1709). SPARC AAbs were detected in the sera and the values were significant in the overall case versus control (p&amp;lt; .0001) and also in CA (p&amp;lt;.0001) and AA (p&amp;lt;.0001) CaP patients with levels significantly lower in patients compared to controls. AAb reactivity for CaP patients was similar between CA and AA groups. Conclusions: This study demonstrated the presence of AAbs against SPARC in CaP patient serum for the first time. Of note, highly significant differences were noted between CaP patient (low) and controls (high) sera, across different ethnic groups. These data suggest further evaluation of SPARC AAbs as a promising serum biomarker for CaP. Source of Funding: This research was supported by the Center for Prostate Disease Research, Uniformed Services University Grant HU0001-10-2-0002. Citation Format: Anshu Rastogi, Andy Martinez, Amina Ali, Shyh-Han Tan, Jennifer Cullen, Yongmei Chen, Gyorgy Petrovics, Albert Dobi, Lakshmi Ravindranath, Denise Young, Isabell Sesterhenn, Jacob Kagan, Sudhir Srivastava, David McLeod, Inger Rosner, Shiv Srivastava, Alagarsamy Srinivasan. Secreted protein acidic and rich in cysteine antigen and autoantibodies in sera of prostate cancer patients: Potential use in diagnosis /prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2830. doi:10.1158/1538-7445.AM2017-2830

Diagnostic significance of urinary long non-coding PCA3 RNA in prostate cancer.

Prostate cancer antigen 3(PCA3) is a long non-coding RNA, which was found increased expression in CaP patients than healthy individual. In this study, the individual nucleic acid of PCA3 and PSA was recombinant expressed as a reference reagent, and a quantitative RT-PCR with TaqMan assay was developed to examine the copies of PCA3 and PSA gene in urine. The results showed that the area under the receiver operating characteristic curve (AUROC) was 0.717, 0.444 and 0.916 for the number of PCA3 copy, PSA copy and for the score of PCA3/PSA RNA, respectively. Additionally, the AUROC for serum tPSA was 0.674 with a low specificity of 12.07%. Finally, the algorithm of PCA3 RNA versus PSA RNA was evaluated and corroborated as CaP biomarker by conducting a multicentric clinical trial. This study not only validated the developed technique of qRT-PCR with TaqMan assay for examination of urinary PCA3 and PSA RNA, it also demonstrated that the score of the PCA/PSA RNA was a reliable signature for CaP diagnosis.

Autoantibodies against oncogenic ERG protein in prostate cancer: potential use in diagnosis and prognosis in a panel with C-MYC, AMACR and HERV-K Gag.

Overdiagnosis and overtreatment of prostate cancer (CaP) is attributable to widespread reliance on PSA screening in the US. This has prompted us and others to search for improved biomarkers for CaP, to facilitate early detection and disease stratification. In this regard, autoantibodies (AAbs) against tumor antigens could serve as potential candidates for diagnosis and prognosis of CaP. Towards this, our goals were: i) To investigate whether AAbs against ERG oncoprotein (overexpressed in 25-50% of Caucasian American and African American CaP) are present in the sera of CaP patients; ii) To evaluate an AAb panel to enhance CaP detection. The results using an enzyme-linked immunosorbent assay (ELISA) showed that anti-ERG AAbs are present in a significantly higher proportion in the sera of CaP patients compared to healthy controls (p = 0.0001). Furthermore, a panel of AAbs against ERG, AMACR and human endogenous retrovirus-K Gag successfully differentiated CaP patient sera from healthy controls (AUC = 0.791). These results demonstrate for the first time that anti-ERG AAbs are present in the sera of CaP patients. In addition, the data also suggest that AAbs against ERG together with AMACR and HERV-K Gag may be a useful panel of biomarkers for diagnosis and prognosis of CaP.

Correlation of p53 immunoexpression with DNA ploidy and apoptotic index in subsets of prostate cancer: A marker reiterated in progression and recurrence of prostate cancer.

Background:Prediction of biological behavior in patients of prostate cancer (CaP) is a major challenge as current parameters only partially meet the need for prognostication. p53 as a prognostic indicator has been studied in several human cancers, including breast, lung, and colorectal carcinoma. However, its significance as a predictive biomarker for CaP is less well-studied.Materials and Methods:This study included 125 cases of CaP, 27 cases of prostatic intraepithelial neoplasia and 25 cases of benign prostatic hyperplasia. Immunohistochemical assessment for p53 nuclear protein was performed. Assessment for apoptotic index and DNA ploidy status by flow cytometry were also done.Results:p53 immunoreactivity was low in organ confined CaP cases having Gleason score ≤3 (P < 0.003). More hormone resistant cases 37 (83%) were aneuploid when compared with hormone sensitive cases 26 (33%) (P < 0.005). 93% of p53 positive cases and none of the p53 negative patient were aneuploid suggesting a significant relation between p53 immunoreactivity and aneuploidy. p53 positivity and DNA aneuploidy, independently, were also predictors of progression and relapse.Conclusion:DNA ploidy and p53 positivity go hand in hand and together yield additional prognostic information in CaP. p53 positivity is possibly a late event in carcinogenesis in CaP and a marker of change in biological behavior of CaP.

Prognostic Value of Discs Large Homolog 7 Transcript Levels in Prostate Cancer

Hypoxia has been associated with malignant progression, metastasis and resistance to therapy. Hence, we studied expression of hypoxia–regulated genes in 100 prostate cancer (CaP) bulk tissues and 71 adjacent benign tissues. We found 24 transcripts significantly overexpressed (p≤0.02). Importantly, higher transcript levels of disc large (drosophila) homolog-associated protein 5 (DLGAP5)/discs large homolog 7 (DLG7)/hepatoma up-regulated protein (HURP), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with higher Gleason score and more advanced systemic progression. Since the products of HMMR and CCNB1 have been identified recently as molecular markers of CaP progression, we postulated that DLG7 has prognostic value too. To test this hypothesis, we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within six years of surgery) and controls (no progression within eight years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CI, 0.68 to 0.8). Overall, our data indicate that expression of DLG7, a hypoxia-controlled gene, holds prognostic potential in high-risk CaP; this also demonstrates that variation of oxygen tension may constitute a tool for identification of novel biomarkers for CaP.

BMI1, stem cell factor acting as novel serum-biomarker for Caucasian and African-American prostate cancer.

BackgroundLack of reliable predictive biomarkers is a stumbling block in the management of prostate cancer (CaP). Prostate-specific antigen (PSA) widely used in clinics has several caveats as a CaP biomarker. African-American CaP patients have poor prognosis than Caucasians, and notably the serum-PSA does not perform well in this group. Further, some men with low serum-PSA remain unnoticed for CaP until they develop disease. Thus, there is a need to identify a reliable diagnostic and predictive biomarker of CaP. Here, we show that BMI1 stem-cell protein is secretory and could be explored for biomarker use in CaP patients.Methodology/Principal FindingsSemi-quantitative analysis of BMI1 was performed in prostatic tissues of TRAMP (autochthonous transgenic mouse model), human CaP patients, and in cell-based models representing normal and different CaP phenotypes in African-American and Caucasian men, by employing immunohistochemistry, immunoblotting and Slot-blotting. Quantitative analysis of BMI1 and PSA were performed in blood and culture-media of siRNA-transfected and non-transfected cells by employing ELISA. BMI1 protein is (i) secreted by CaP cells, (ii) increased in the apical region of epithelial cells and stromal region in prostatic tumors, and (iii) detected in human blood. BMI1 is detectable in blood of CaP patients in an order of increasing tumor stage, exhibit a positive correlation with serum-PSA and importantly is detectable in patients which exhibit low serum-PSA. The clinical significance of BMI1 as a biomarker could be ascertained from observation that CaP cells secrete this protein in higher levels than cells representative of benign prostatic hyperplasia (BPH).Conclusions/SignificanceBMI1 could be developed as a dual bio-marker (serum and biopsy) for the diagnosis and prognosis of CaP in Caucasian and African-American men. Though compelling these data warrant further investigation in a cohort of African-American patients.

Abstract 116: Targeting hypoxia for the identification of novel biomarkers for prostate cancer

Abstract Tumor-associated hypoxia has been associated with malignant progression, metastasis, resistance to therapy, and poor clinical outcome in prostate cancer (CaP). Prompted by the evidence that hypoxia affects CaP, we studied global gene expression in 100 CaP tissues and 71 samples of adjacent benign tissues. RNA was extracted from cancer cells isolated by laser-capture microdissection (LCM) or without isolation (“bulk tissue”). We found 24 hypoxia-associated genes significantly overexpressed in CaP (p≤0.02), both in bulk tissue and LCM. Among hypoxia-associated genes, the disc large (drosophila) homolog-associated protein 5 (DLG7), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with Gleason score and systemic progression. Since the products of HMMR and CCNB1 have been recently identified as molecular markers of CaP progression, we postulated that DLG7 has prognostic value also. To test this hypothesis we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within five years of surgery) and controls (no progression within seven years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CL, 0.68 to 0.8), independent of the for ERG status. Overall, our data indicate that DLG7, a hypoxia-controlled gene have a prognostic value in high-risk CaP. To better define the value of DLG7 as a molecular predictor of CaP progression, we are currently assessing DLG7 protein levels by immunohistochemistry. Then we will analyze the association of DLG7 levels with clinical parameters. Introduction of oxygen tension as a variable may constitute a tool for the identification of novel biomarkers for CaP. Present address CRG: University of Mississippi Cancer Institute, Jackson, MS, USA. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG), Mayo Clinic Prostate SPORE 5P50CA091956 (SV-P); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 116. doi:1538-7445.AM2012-116

Abstract B54: miR-125b and miR-17-3p: Newly identified tumor suppressors for prostate cancer

Abstract MicroRNAs (miRs) are small molecules that post-transcriptionally regulate expression of target mRNAs and are thought to play a major role in prostate cancer (CaP) tumorigenesis. We compared miR expression in a progressive prostate cancer cell model consisting of the immortalized p69 human prostate cell line compared to M12, a highly tumorigenic, metastatic subline, and F6, a weakly tumorigenic variant using Exiqon's miRCURY LNA™ miR human panels. Most notable was a decrease in expression of miR-125b and miR-17-3p, which was confirmed by single miR analysis via RT-qPCR. Previously, miR array screens have been done on RNA extracted from a variety of whole tumors. However, due to the heterogeneous nature of prostate tumors, it is impossible to determine which cell-type is contributing to differential miR expression. To address this concern, we have developed laser capture microdissection (LCM) to validate miR expression in RNA extracted from the individual cell-types (normal stroma, normal epithelial, PIN, and tumor cell of increasing Gleason score) that comprise human prostate tumors. This analysis validated that the expression of miR-17-3p and miR-125b is reduced in the prostate tumor cell compared to normal epithelium. Previously, we have shown that miR-17-3p targets vimentin and IGF1R. Restoration of miR-17-3p expression in the M12 cell line resulted in decreased cell motility and invasion in transwell (Boyden) chamber assays as well as decreased growth of subcutaneous and orthotopic tumors in male, athymic nude mice confirming miR-17-3p is behaving as a tumor suppressor. Previously, data from miRNA array screens coupled to gene arrays have been used to predict mRNA targets. This approach assumes that all miRNAs regulate gene expression by degrading their respective targets, which is not always the case. Since the end result of miRNA targeting is reduced protein synthesis, we propose that proteomics using RPMA, Reverse Phase Protein MicroArrays, is a better approach for deducing targets of miR regulation. Proteomic analysis of these same cell lines confirmed a correlative increase in pErB2/ErB3 and vimentin protein levels in cell lines where miR-125b and miR-17-3p expression are low. MiR-125b is proposed to target ErbB2/ErbB3. Interestingly, a network analysis of oncogenic miRNA targets in the prostate revealed that the EGFR family of which ErB2/ErB3 is thought to be a major driver of CaP was one of the most highly connected proteins. Currently, we are using a variety of in vitro and in vivo experiments to prove that miR-125b can function as a tumor suppressor as we have shown for miR-17-3p. Altogether this multi-factorial approach of miRNA array analysis coupled to proteomics, validated by LCM analysis of human tumor samples and additional experiments has identified two new tumor suppressors, miR-17-3p and miR-125b, which could be relevant biomarkers for CaP. Restoration of miR-17-3p and/or miR-125 expression could provide new targets/treatments to block prostate cancer progression. Citation Format: William T. Budd, Danielle E. Weaver, Valerie Calvert, Emanuel F. Petricoin, III, Joy L. Ware, Zendra E. Zehner. miR-125b and miR-17-3p: Newly identified tumor suppressors for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B54.

A panel of five circulating microRNAs as potential biomarkers for prostate cancer

Circulating microRNA (miRNAs) have been shown to have the potential as noninvasive diagnosis markers in several types of cancers. In this study, we investigated whether circulating miRNAs could be used in the diagnosis of prostate cancer (CaP) in a Chinese patient population. Illumina's Human v2 miRNA microarray was used to analyze miRNAs levels in a small set of patients [25 CaP, 17 benign prostatic hyperplasia (BPH)] in an effort to identify CaP-specific miRNAs. The identified miRNAs were further examined by quantitative real-time PCR (qRT-PCR) in the same small set of patients. After the training phase of screening and selecting, the candidate miRNAs were validated in a larger independent cohort (80 CaP, 44 BPH, and 54 healthy controls) with qRT-PCR in the verification phase. Five miRNAs were confirmed by qRT-PCR analysis in validation sets. Receiver operating characteristic (ROC) curve analysis showed all 5 miRNAs had diagnostic value. More importantly, further principal component analysis indicated component 1 extracted from expression data of the 5 miRNAs could differentiate CaP from BPH and healthy controls with high diagnosis performance, with an AUC of 0.924 and 0.860, respectively. Our data suggested that circulating miRNAs could serve as biomarkers for CaP, and compared to single miRNA, the 5 miRNAs panel can accurately discriminate CaP from BPH and healthy controls with high sensitivity and specificity, and therefore, combined with routine PSA test, these 5 CaP-specific miRNAs may help improve CaP diagnosis in clinical application.

Abstract 4173: Prostate derived Ets (E-twenty six) transcription factor (PDEF) as a prognostic biomarker for metastatic prostate cancer

Abstract While early stage prostate cancer (CaP) is unique in that many patients will take decades to progress to symptomatic disease; there exists a subset of patients whose disease may quickly become life-threatening. Unfortunately, due to the high incidence of CaP and clinicians’ inability to differentiate the majority of patients with disease that will never become symptomatic from the minority with aggressive life-threatening tumors, patients with CaP are often over-treated. Prognostic biomarkers, such as prostate specific antigen (PSA), have shown great efficacy in improving patients’ chances of surviving cancer by allowing physicians to identify the disease at an earlier stage, though there does not exist a biomarker for CaP that adequately differentiates disease that will quickly become metastatic. In order to facilitate prognosis, the expression of prostate derived Ets transcription factor (PDEF), a protein of significance to the progression of CaP, was studied. PDEF is expressed in normal epithelial cells and negatively regulates transcriptional networks controlling migration and invasion. Previous findings in breast cancer have also demonstrated that loss of PDEF expression results in upregulation of the antiapoptotic protein survivin, indicating a possible connection to growth and survival. Loss of PDEF expression has also been shown to increase the metastatic potential of several cancers. Patients PDEF expression was determined via immunohistochemistry staining of tissue microarray in set of over 700 archival CaP tissues taken at radical prostatectomy; and patients were subsequently stratified based on their tumor PDEF expression. The stratified CaP patients were then analyzed for the association of PDEF expression status with clinical outcomes. Univariate analysis showed that low PDEF expression is correlated with incidence of biochemical failure as defined by American Urological Association (AUA; OR=1.30, p=0.0493) or National Comprehensive Cancer Network (NCCN; OR=1.36, p=0.0164) definitions; and with metastatic disease incidence (OR=3.61, p=0.0047). Multivariate analysis also showed that low PDEF expression was associated with metastatic disease incidence (OR = 4.02, p=0.0146). Furthermore, Kaplan-Meier survival analysis showed that stratification of patients into subgroups based on PDEF expression (high, middle, low) was able to significantly predict relative time to biochemical failure (NCCN, p=0.0241) or metastatic disease (p=0.0084). This study demonstrates the potential usefulness of PDEF as a prognostic biomarker to differentiate those patients at the highest risk of developing life-threatening disease. We propose that the status of PDEF expression in CaP tissues could be used in conjunction with PSA to distinguish patients who are suitable for watchful waiting and patients who require aggressive treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2011-4173

α-Methylacyl-CoA Racemase Spliced Variants and Their Expression in Normal and Malignant Prostate Tissues

To evaluate the possibility of using α-methylacyl-CoA racemase (AMACR) variants to improve the specificity of prostate cancer (CaP) detection. AMACR has been used as a diagnostic biomarker for CaP and is now a standard biomarker for needle biopsy specimens with ambiguous lesions. We used in silico analysis and molecular cloning to discover new AMACR variants and quantitative reverse transcription-polymerase chain reaction (RT-PCR) to measure the transcript levels of AMACR and its variants in 4 prostate cell lines and in 23 pairs of CaP and adjacent normal tissue. We found 4 novel variants, IAs, IBL, IBLd, and IBLi. Transcript levels of most AMACR variants were significantly upregulated in CaP compared with its adjacent normal counterparts. A variants, the functional variants based on bioinformatic analysis, showed levels of transcript expression in CaP in this order: IA≫IAd=IIA≫IIAs>IAs. In contrast, the expression of the B variants, which appear to be nonfunctional due to the absence of exon 3, was lower than that of the A variants. IB was the most abundant form of B variant; and expression of IIB was negligible. More important, the difference between levels of variant IA, IAd, IIA, IIAs, IB, and IBLi in CaP and normal tissue was significantly higher than the difference in levels of total AMACR. Our data suggest that AMACR variants have better power than total AMACR in discriminating between CaP and adjacent normal tissue. These findings may be useful for the development of future diagnostic assays.

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening.