Matthew Truong

Toward the detection of prostate cancer in urine: a critical analysis.

PURPOSE Prostate specific antigen and digital rectal examination have low specificity for detecting prostate cancer and they poorly predict the presence of aggressive disease. Urine is readily available and noninvasive, and it represents a promising source of biomarkers for the early detection and prediction of prostate cancer prognosis. We identified promising biomarkers for urine based prostate cancer, examined trends and outlined potential pitfalls. MATERIALS AND METHODS We performed PubMed® and Web of Science® database searches of the peer reviewed literature on urine based testing for prostate cancer. Original studies of this subject as well as a small number of reviews were analyzed, including the strengths and weaknesses. We provide a comprehensive review of urine based testing for prostate cancer that covers the technical aspects, including the methodology of urine collection, as well as recent developments in biomarkers spanning the fields of genomics, epigenetics, transcriptomics, proteomics and metabolomics. RESULTS The process of urine collection is subject to variability, which may result in conflicting clinical results. Detecting prostate cancer in urine is technically feasible, as demonstrated by numerous proof of principle studies, but few markers have been validated in multiple large sample sets. Biomarker development using urine has been accelerating in recent years with numerous studies identifying DNA, RNA, protein and metabolite based biomarkers in urine. Advanced clinical studies have identified PCA3 and TMPRSS2:ERG fusion transcripts as promising RNA markers for cancer detection and possibly prognosis. DNA methylation analysis of multiple genes improves specificity and represents a promising platform for developing clinical grade assays. CONCLUSIONS Urine based testing is noninvasive and represents a rich source of novel biomarkers for prostate cancer. Although urine shows promise for detecting cancer, the ability to identify aggressive subsets of prostate cancer needs further development.

Development and multi‐institutional validation of an upgrading risk tool for Gleason 6 prostate cancer

Many patients with low‐risk prostate cancer (PC) who are diagnosed with Gleason score 6 at biopsy are ultimately found to harbor higher grade PC (Gleason ≥ 7) at radical prostatectomy. This finding increases risk of recurrence and cancer‐specific mortality. Validated clinical tools that are available preoperatively are needed to improve the ability to recognize likelihood of upgrading in patients with low‐risk PC.

Resveratrol Induces Notch2-mediated Apoptosis and Suppression of Neuroendocrine Markers in Medullary Thyroid Cancer

BackgroundCurrently, complete surgical resection is the only curative option for medullary thyroid cancer (MTC). Previous work has shown the Notch pathway is a potent tumor suppressor in MTC and that resveratrol activates the Notch pathway in carcinoid cancer, a related neuroedocrine malignancy. In this study, we hypothesized that the effects observed on carcinoid cells could be extended to MTC.MethodsMTC cells treated with varying doses of resveratrol were assayed for viability by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Western blot analysis for achaete-scute complex-like 1 (ASCL1), chromogranin A (CgA), full-length and cleaved caspase 3, and poly-ADP ribose polymerase (PARP) was performed. Quantitative real-time polymerase chain reaction (qPCR) was used to measure relative mRNA expression.ResultsTreatment with resveratrol resulted in growth suppression and an increase in the cleavage of caspase-3 and PARP. A dose-dependent inhibition of ASCL1, a neuroedocrine transcription factor, was observed at the protein and mRNA levels. Protein levels of CgA, a marker of hormone secretion, were also reduced after treatment with resveratrol. A dose-dependent induction of Notch2 mRNA was observed by qPCR.ConclusionsResveratrol suppresses in vitro growth, likely through apoptosis, as demonstrated by cleavage of caspase-3 and PARP. Furthermore, resveratrol decreased neuroedocrine markers ASCL1 and chromogranin A. Induction of Notch2 mRNA suggests that this pathway may be central in the anti-MTC effects observed.

Using the Epigenetic Field Defect to Detect Prostate Cancer in Biopsy Negative Patients

PURPOSE We determined whether a novel combination of field defect DNA methylation markers could predict the presence of prostate cancer using histologically normal transrectal ultrasound guided biopsy cores. MATERIALS AND METHODS Methylation was assessed using quantitative Pyrosequencing® in a training set consisting of 65 nontumor and tumor associated prostate tissues from University of Wisconsin. A multiplex model was generated using multivariate logistic regression and externally validated in blinded fashion in a set of 47 nontumor and tumor associated biopsy specimens from University of Washington. RESULTS We observed robust methylation differences in all genes at all CpGs assayed (p <0.0001). Regression models incorporating individual genes (EVX1, CAV1 and FGF1) and a gene combination (EVX1 and FGF1) discriminated nontumor from tumor associated tissues in the original training set (AUC 0.796-0.898, p <0.001). On external validation uniplex models incorporating EVX1, CAV1 or FGF1 discriminated tumor from nontumor associated biopsy negative specimens (AUC 0.702, 0.696 and 0.658, respectively, p <0.05). A multiplex model (EVX1 and FGF1) identified patients with prostate cancer (AUC 0.774, p = 0.001) and had a negative predictive value of 0.909. Comparison between 2 separate cores in patients in this validation set revealed similar methylation defects, indicating detection of a widespread field defect. CONCLUSIONS A widespread epigenetic field defect can be used to detect prostate cancer in patients with histologically negative biopsies. To our knowledge this assay is unique, in that it detects alterations in nontumor cells. With further validation this marker combination (EVX1 and FGF1) has the potential to decrease the need for repeat prostate biopsies, a procedure associated with cost and complications.

HP1γ expression is elevated in prostate cancer and is superior to Gleason score as a predictor of biochemical recurrence after radical prostatectomy

BackgroundAberrant chromatin structure in cancer cells results from altered proteins involved in its packaging. Heterochromatin protein 1 gamma (HP1γ) is a non-histone heterochromatic protein that functions to maintain chromatin stability and is important in embryonic development. Given an interest in the role developmental genes play in cancer, we investigated HP1γ expression in prostate cancer (PCa) and its prognostic associations.MethodsTissue microarrays consisting of benign (N = 96), localized cancer (N = 146), metastatic PCa (N = 44), and HGPIN (N = 50) were immunoflourescently stained for HP1γ and Ki-67. Using a novel, automated quantitative imaging system, VECTRA™, epithelial staining in both the nucleus and cytoplasm was quantified and compared against clinicopathologic variables.ResultsHP1γ is significantly elevated in HGPIN (80%), localized PCa (76%), and metastatic PCa (98%) compared to benign tissues from both the nuclear and cytoplasmic compartments (P < 0.0001). Increased nuclear and total HP1γ expression was associated with Gleason score (P = 0.02 and P = 0.04 respectively). Given known binding to the C-terminus of Ki-67, a co-expression analysis was performed that revealed a correlation between nuclear and cytoplasmic HP1γ and Ki-67 (Pearson Coefficient 0.321 and 0.562 respectively, P < 0.0001). Cox survival analysis demonstrated that cytoplasmic HP1γ expression was an independent prognostic marker and out-performed pathological Gleason score for predicting PSA-recurrence after radical prostatectomy.ConclusionsIn this first detailed analysis of HP1γ expression in cancer, VECTRA™ demonstrates compartmentalized and total HP1γ protein expression is increased in PCa and that expression correlates with clinical outcomes better than Gleason score. Given the critical role HP1γ plays in chromatin organization and gene expression, it represents a novel prognostic and therapeutic target.

Overexpression of the novel senescence marker β-galactosidase (GLB1) in prostate cancer predicts reduced PSA recurrence.

Purpose Senescence is a terminal growth arrest that functions as a tumor suppressor in aging and precancerous cells and is a response to selected anticancer compounds. Lysosomal-β-galactosidase (GLB1) hydrolyzes β-galactose from glycoconjugates and is the origin of senescence-associated β-gal activity (SA-β-gal). Using a new GLB1 antibody, senescence biology was investigated in prostate cancer (PCa) tissues. Experimental Design In vitro characterization of GLB1 was determined in primary prostate epithelial cell cultures passaged to replicative senescence and in therapy-induced senescence in PCa lines using chemotherapeutic agents. FFPE tissue microarrays were subjected to immunofluorescent staining for GLB1, Ki67 and HP1γ and automated quantitative imaging initially using AQUA in exploratory samples and Vectra in a validation series. Results GLB1 expression accumulates in replicative and induced senescence and correlates with senescent morphology and P16 (CDKN2) expression. In tissue arrays, quantitative imaging detects increased GLB1 expression in high-grade prostatic intraepithelial neoplasia (HGPIN), known to contain senescent cells, and cancer compared to benign prostate tissues (p<0.01) and senescent cells contain low Ki67 and elevated HP1γ. Within primary tumors, elevated GLB1 associates with lower T stage (p=0.01), localized versus metastatic disease (p=0.0003) and improved PSA-free survival (p=0.03). Increased GLB1 stratifies better PSA-free survival in intermediate grade PCa (0.01). Tissues that elaborate higher GLB1 display increased uniformity of expression. Conclusion Increased GLB1 is a valuable marker in formalin-fixed paraffin-embedded (FFPE) tissues for the senescence-like phenotype and associates with improved cancer outcomes. This protein addresses a lack of senescence markers and should be applicable to study the biologic role of senescence in other cancers.

Analysis of Promoter Non-CG Methylation in Prostate Cancer

BACKGROUND In vertebrates, DNA methylation occurs primarily at CG dinucleotides but recently, non-CG methylation has been found at appreciable levels in embryonic stem cells. MATERIALS & METHODS To assess non-CG methylation in cancer, we compared the extent of non-CG methylation at several biologically important CG islands in prostate cancer and normal cell lines. An assessment of the promoter CG islands EVX1 and FILIP1L demonstrates a fourfold higher rate of non-CG methylation at EVX1 compared with FILIP1L across all cell lines. These loci are densely methylated at CG sites in cancer. RESULTS No significant difference in non-CG methylation was demonstrated between cancer and normal. Treatment of cancer cell lines with 5-azacytidine significantly reduced methylation within EVX1 at CG and CC sites, preferentially. CONCLUSION Non-CG methylation does not correlate with CG methylation at hypermethylated promoter regions in cancer. Furthermore, global inhibition of DNA methyltransferases does not affect all methylated cytosines uniformly.

Analysis of urological procedures in men who died from prostate cancer using a population‐based approach

Whats known on the subject? and What does the study add?

MP08-12 MULTI-INSTITUTIONAL EVALUATION OF MRI AND FUSION BIOPSY IN CONFIRMATORY BIOPSY FOR ACTIVE SURVEILLANCE

INTRODUCTION AND OBJECTIVES: To provide standardization as prostate MRI becomes increasingly utilized, the Prostate Imaging-Reporting and Data System (PIRADS) was developed and has been modified to its latest version (v2). Using biopsy outcome as the standard, we examined the predictive accuracy of a PIRADS 4 or 5 read for clinically significant (Gleason 7+) PCa in a blinded fashion. METHODS: We reviewed our prospectively maintained database of consecutive men who underwent prostate MRI prior to biopsy between September 2014 and December 2015. A proportionally representative sample (based on the original clinical PIRADS v2 interpretation) was selected for re-examination (n1⁄432). The prostate MRIs for these patients were de-identified and were loaded by a blinded third party. Four radiologists of varying levels of experience independently interpreted all prostate MRI, blinded to all clinical information. An 00overread00 was defined as a PIRADS 4 or 5 read with biopsy result of benign prostate or Gleason 6 PCa. An 00under-read00 was defined as a PIRADS 1-3 read with resulting biopsy result of Gleason 7+ PCa. RESULTS: The distribution of accuracy is provided in Table 1. Accurate interpretation ranged from 56% (18/32) to 75% (24/32), and the differences among the radiologists were not significant (p1⁄40.48). The improvement of accuracy with a 00majority read00, as defined by two or more accurate radiologists0 blinded interpretations, over the original clinical read trends toward significance (p1⁄40.16). No clinical variable was predictive of an incorrect 00majority read00, including age, PSA, family history, use of 5-alpha reductase inhibitors, prostate volume, or previous biopsy history. CONCLUSIONS: In a blinded assessment of radiologists at our institution, we find that the predictive accuracy of PIRADS 4 or 5 for clinically significant PCa varies among radiologists independent of experience level. A 00majority read00 performed better than the original clinical interpretation, suggesting that consensus interpretation of prostate MRI may improve predictive accuracy.

MP65-12 TARGETING ESTROGEN/ESTROGEN RECEPTOR SIGNAL PATHWAYS TO ENHANCE THE EFFICACY OF BACILLUS CALMETTE-GUÉRIN TREATMENT IN BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Although Bacillus Calmette-Guerin (BCG) is the most effective agent for non-muscleinvasive bladder cancers, approximately 30% of patients treated with intravesical BCG fail to respond to this agent. Previous studies from our lab showed the potential linkage of estrogen/estrogen receptor signaling with the efficacy of BCG, yet the detailed mechanisms remain unclear. Our new data showed the combination of BCG and the anti-estrogen ICI 182,780 (ICI) or tamoxifen could lead to a better suppression of bladder cancer (BCa) than BCG alone. METHODS: We first applied PCR to detect BCG internalization in two ERa positive BCa cell lines to investigate the potential effect of anti-estrogen ICI. Then, we used Q-PCR and western blot and examined the E2/ER effects on the integrin-a5b1 expression and the BCG attachment/internalization to BCa cells. To examine whether ICI can help the recruitment of macrophages toward BCa cells, we applied the transwell migration assay and in vivo mouse BCG model. Q-PCR, Elisa assay and MTT assay were used to detect the cytokine profile changes and BCa cell viability. For our in vivo studies, we applied the BBN-induced mouse BCa model, HE staining, BrdU and F4/80 staining to show the changes of macrophage infiltration and to prove the better efficacy of combining BCG plus anti-estrogen. RESULTS: We found treatment with either 1 mM ICI or tamoxifen significantly increased the BCG attachment/internalization, and the neutralization of integrin-a5b1 could reduce the ability of the ICI enhanced BCG attachment/internalization to BCa cells (Figure 1). Mechanism dissection revealed ICI could promote BCG attachment/ internalization to the BCa cells through targeting ERa and increased the integrin-a5b1 expression and IL-6 secretion. The increased cytokine production may enhance BCG-mediated suppression of BCa cell growth and TNF-a production via recruiting more monocytes/macrophages to BCa cells (Figure 2-3). Consistently, in vivo studies found ICI could potentiate the anti-BCa effects of BCG in the carcinogen-induced mouse BCa models (Figure 4). CONCLUSIONS: Taken together, these in vitro and in vivo results suggest that combining BCG with the anti-estrogen may become a new therapeutic approach with better efficacy to suppress BCa progression and recurrence. Source of Funding: none