Profiling the Urinary Microbiome in Men with Positive versus Negative Biopsies for Prostate Cancer.

PURPOSE: Studies demonstrating bacterial DNA and cultivable bacteria in urine samples have challenged the clinical dogma that urine is sterile. Furthermore, studies now indicate that dysbiosis of the urinary microbiome is associated with pathological conditions. We propose that the urinary microbiome may influence chronic inflammation observed in the prostate, leading to prostate cancer development and progression. Therefore, we profiled the urinary microbiome in men with positive vs negative biopsies for prostate cancer. MATERIALS AND METHODS: Urine was collected from men prior to biopsy for prostate cancer. DNA was extracted from urine pellet samples and subjected to bacterial 16S rDNA Illumina® sequencing and 16S rDNA quantitative polymerase chain reaction. We determined the association between bacterial species and the presence or absence of cancer, cancer grade, and type and degree of prostate inflammation. RESULTS: Urine samples revealed diverse bacterial populations. There were no significant differences in α or ß diversity and no clear hierarchical clustering of benign or cancer samples. We identified a cluster of pro-inflammatory bacteria previously implicated in urogenital infections in a subset of samples. Many species, including known uropathogens, were significantly and differentially abundant among cancer and benign samples, in low vs higher grade cancers and in relation to prostate inflammation type and degree. CONCLUSIONS: To our knowledge we report the most comprehensive study to date of the male urinary microbiome and its relationship to prostate cancer. Our results suggest a prevalence of pro-inflammatory bacteria and uropathogens in the urinary tract of men with prostate cancer.

Clinical validation of an epigenetic assay to predict negative histopathological results in repeat prostate biopsies.

PURPOSE: The DOCUMENT multicenter trial in the United States validated the performance of an epigenetic test as an independent predictor of prostate cancer risk to guide decision making for repeat biopsy. Confirming an increased negative predictive value could help avoid unnecessary repeat biopsies. MATERIALS AND METHODS: We evaluated the archived, cancer negative prostate biopsy core tissue samples of 350 subjects from a total of 5 urological centers in the United States. All subjects underwent repeat biopsy within 24 months with a negative (controls) or positive (cases) histopathological result. Centralized blinded pathology evaluation of the 2 biopsy series was performed in all available subjects from each site. Biopsies were epigenetically profiled for GSTP1, APC and RASSF1 relative to the ACTB reference gene using quantitative methylation specific polymerase chain reaction. Predetermined analytical marker cutoffs were used to determine assay performance. Multivariate logistic regression was used to evaluate all risk factors. RESULTS: The epigenetic assay resulted in a negative predictive value of 88% (95% CI 85-91). In multivariate models correcting for age, prostate specific antigen, digital rectal examination, first biopsy histopathological characteristics and race the test proved to be the most significant independent predictor of patient outcome (OR 2.69, 95% CI 1.60-4.51). CONCLUSIONS: The DOCUMENT study validated that the epigenetic assay was a significant, independent predictor of prostate cancer detection in a repeat biopsy collected an average of 13 months after an initial negative result. Due to its 88% negative predictive value adding this epigenetic assay to other known risk factors may help decrease unnecessary repeat prostate biopsies.

Cyr61 is regulated by cAMP-dependent protein kinase with serum levels correlating with prostate cancer aggressiveness.

BACKGROUND: Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. Previously, we demonstrated that Cyr61 was highly expressed in prostate cancer (PCa) but that the expression levels were associated with a lower risk of PCa recurrence. In the present study, we demonstrate that serum Cyr61 is a potential biomarker that correlates with PCa aggressiveness. Furthermore, we also explore the potential mechanism underlying the changes in Cyr61 expression during PCa progression. METHODS: Cyr61 concentrations in the medium from PCa cell lines and in serum samples obtained from PCa patients were measured by sandwich ELISA. Serum Cyr61 levels were correlated with disease characteristics and the association between Cyr61 expression changes by several types of stimulation or stress and cAMP/cAMP-dependent protein kinase (PKA) pathway were examined. RESULTS: There was a positive correlation between Cyr61 levels in cell supernatants and mRNA expression in these cell lines. Serum Cyr61 levels were significantly higher in non-organ-confined PCa patients (116.3 ± 140.2 ng/ml) than in organ-confined PCa patients (79.7 ± 56.1 ng/ml) (P = 0.031). Cyr61 expression was up-regulated in response to both lysophosphatidic acid and androgen treatments which promoted PCa cell invasion. Serum starvation and phosphoinositide-3-kinase inhibition also resulted in Cyr61 up-regulation; however, they suppressed cell proliferation. Cyr61 up-regulation was correlated with an increase in cAMP and suppressed by PKA inhibition. CONCLUSIONS: These findings suggest that Cyr61 expression in PCa is regulated by the cAMP/PKA pathway and that circulating Cyr61 levels are a potential serum-based biomarker for characterizing PCa.

Evaluation of GSTP1 and APC methylation as indicators for repeat biopsy in a high-risk cohort of men with negative initial prostate biopsies.

UNLABELLED: Study Type - Diagnostic (exploratory cohort) Level of Evidence 2b What's known on the subject? and What does the study add? Hypermethylation of genes such as glutathione-S-transferase P1 (GSTP1) and adenomatous polyposis coli (APC) occurs with high frequency in prostate tumour tissue but is much less common in the benign prostate; however, the potential value of gene methylation biomarkers as an adjunct to biopsy histopathology has had little study. When measured in histologically benign prostate biopsy tissue, APC gene hypermethylation was found to have high negative predictive value and high sensitivity. GSTP1 hypermethylation was found to have lower performance than APC. OBJECTIVE: To evaluate the performance of DNA methylation biomarkers in the setting of repeat biopsy in men with an initially negative prostate biopsy but a high index of suspicion for missed prostate cancer. PATIENTS AND METHODS: We prospectively evaluated 86 men with an initial histologically negative prostate biopsy and high-risk features. All men underwent repeat 12-core ultrasonography-guided biopsy. DNA methylation of glutathione-S-transferase P1 (GSTP1) and adenomatous polyposis coli (APC) was determined using tissue from the initially negative biopsy and compared with histology of the repeat biopsy. The primary outcome was the relative negative predictive value (NPV) of APC compared with GSTP1, and its 95% confidence interval (CI). RESULTS: On repeat biopsy, 21/86 (24%) men had prostate cancer. APC and GSTP1 methylation ratios below the threshold (predicting no cancer) produced a NPV of 0.96 and 0.80, respectively. The relative NPV was 1.2 (95% CI: 1.06-1.36), indicating APC has significantly higher NPV. Methylation ratios above the threshold yielded a sensitivity of 0.95 for APC and 0.43 for GSTP1. Combining both methylation markers produced a performance similar to that of APC alone. APC methylation patterns were consistent with a possible field effect or occurrence early in carcinogenesis. CONCLUSIONS: APC methylation provided a very high NPV with a low percentage of false-negatives, in the first prospective study to evaluate performance of DNA methylation markers in a clinical cohort of men undergoing repeat biopsy. The potential of APC methylation to reduce unnecessary repeat biopsies warrants validation in a larger prospective cohort.

AIM1 promoter hypermethylation as a predictor of decreased risk of recurrence following radical prostatectomy.

PURPOSE: To evaluate the prognostic significance of six epigenetic biomarkers (AIM1, CDH1, KIF1A, MT1G, PAK3, and RBM6 promoter hypermethlation) in a homogeneous group of prostate cancer patients, following radical prostatectomy (RP). PATIENTS AND METHODS: Biomarker analyses were performed retrospectively on tumors from 95 prostate cancer patients all with a Gleason score of 3 + 4 = 7 and a minimum follow-up period of 8 years. Using Quantitative Methylation Specific PCR (QMSP), we analyzed the promoter region of six genes in primary prostate tumor tissues. Time to any progression was the primary endpoint and development of metastatic disease and/or death from prostate cancer was a secondary endpoint. The association of clinicopathological and biomolecular risk factors to recurrence was performed using the Log-rank test and Cox proportional hazards model for multivariate analysis. To identify independent prognostic factors, a stepwise selection method was used. RESULTS: At a median follow-up time of 10 years, 48 patients (50.5%) had evidence of recurrence: Biochemical/PSA relapse, metastases, or death from prostate cancer. In the final multivariate analysis for time to progression, the significant factors were: Older age, HR = 0.95 (95% CI: 0.91, 1.0) (P = 0.03), positive lymph nodes HR = 2.11 (95% CI: 1.05, 4.26) (P = 0.04), and decreased hypermethylation of AIM1 HR = 0.45 (95% CI: 0.2, 1.0) (P = 0.05). CONCLUSIONS: Methylation status of AIM1 in the prostate cancer specimen may predict for time to recurrence in Gleason 3 + 4 = 7 patients undergoing prostatectomy. These results should be validated in a larger and unselected cohort.

Small integrin-binding proteins as serum markers for prostate cancer detection.

PURPOSE: The small integrin-binding ligand N-linked glycoprotein (SIBLING) gene family includes bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE), and osteopontin (OPN). Previous studies have separately reported elevated expression of BSP, OPN, or DSPP in prostate tumor paraffin sections. We hypothesized that SIBLINGs may be informative serum markers for subjects with prostate cancer. METHODS: Expression levels of SIBLINGs in biopsies of normal tissue and tumors from prostate were determined by cDNA array and by immunohistochemical staining with monoclonal antibodies. Competitive ELISAs for measuring total BSP, DSPP, MEPE, and OPN were applied to a test group of 102 subjects with prostate cancer and 110 normal subjects and a validation group of 90 subjects. RESULTS: BSP, DMP1, DSPP, and OPN exhibited elevated mRNA expression and protein levels in biopsies. BSP, DSPP, and OPN were elevated in serum from prostate cancer subjects, with serum DSPP exhibiting the greatest difference, yielding an area under the receiver operator characteristic curve value of 0.98. Serum BSP and OPN levels were significantly elevated only in late stages, whereas DSPP was significantly elevated at all stages. Optimal serum value cutoff points derived for BSP, OPN, and DSPP were applied as a validation test to a new group of 90 subjects and DSPP yielded a sensitivity of 90% and a specificity of 100%. CONCLUSION: Of the SIBLING gene family members, DSPP appears to be a strong candidate for use in serum assays for prostate cancer detection.

DNA Ploidy as surrogate for biopsy gleason score for preoperative organ versus nonorgan-confined prostate cancer prediction.

OBJECTIVES: Transformation of normal epithelium into cancer cells involves epigenetic and genetic changes and modifications in nuclear structure and tissue architecture. To evaluate nuclear morphometric alterations and clinicopathologic features for organ- vs nonorgan-confined prostate carcinoma (PCa) prediction. METHODS: Of 557 prospectively enrolled patients, 370 had complete information and sufficient tumor area for all evaluated parameters (281 organ-confined and 89 nonorgan-confined PCa cases). Digital images of Feulgen DNA-stained nuclei were captured from biopsies using the AutoCyte imaging system, and the nuclear morphometric alterations were calculated. Logistic regression analysis with bootstrap resampling was used to determine the factors important for differentiation of the 2 groups and to generate models for organ- vs nonorgan-confined PCa prediction. RESULTS: Several nuclear morphometric features were significantly altered and could differentiate organ- and nonorgan-confined disease. DNA ploidy was the most important factor among the significant nuclear morphometric features and was the second most important factor for organ- vs nonorgan-confined PCa prediction when considered with total prostate-specific antigen (PSA), complexed PSA, free/total PSA, biopsy Gleason score, and clinical stage. The combination of DNA ploidy with clinical stage, total PSA, and biopsy Gleason score showed an improvement of 1.5% in the area under the receiver operator characteristic curves compared with the combination of clinical stage, total PSA, and biopsy Gleason (73.97% vs 72.43%). The use of DNA ploidy in lieu of the biopsy Gleason score in each preoperative model evaluated resulted in equivalent or improved organ- vs nonorgan-confined PCa prediction. CONCLUSIONS: The results of our study have shown that DNA ploidy can serve as a surrogate biomarker that has the potential to replace biopsy Gleason scores for organ- vs nonorgan-confined PCa prediction.

Predicting the outcome of prostate biopsy: comparison of a novel logistic regression-based model, the prostate cancer risk calculator, and prostate-specific antigen level alone.

OBJECTIVES: To develop a logistic regression-based model to predict prostate cancer biopsy at, and compare its performance to the risk calculator developed by the Prostate Cancer Prevention Trial (PCPT), which was based on age, race, prostate-specific antigen (PSA) level, a digital rectal examination (DRE), family history, and history of a previous negative biopsy, and to PSA level alone. PATIENTS AND METHODS: We retrospectively analysed the data of 1280 men who had a biopsy while enrolled in a prospective, multicentre clinical trial. Of these, 1108 had all relevant clinical and pathological data available, and no previous diagnosis of prostate cancer. Using the PCPT risk calculator, we calculated the risks of prostate cancer and of high-grade disease (Gleason score > or =7) for each man. Receiver operating characteristic (ROC) curves for the risk calculator, PSA level and the novel regression-based model were compared. RESULTS: Prostate cancer was detected in 394 (35.6%) men, and 155 (14.0%) had Gleason > or =7 disease. For cancer prediction, the area under the ROC curve (AUC) for the risk calculator was 66.7%, statistically greater than the AUC for PSA level of 61.9% (P < 0.001). For predicting high-grade disease, the AUCs were 74.1% and 70.7% for the risk calculator and PSA level, respectively (P = 0.024). The AUCs increased to 71.2% (P < 0.001) and 78.7% (P = 0.001) for detection and high-grade disease, respectively, with our novel regression-based models. CONCLUSIONS: ROC analyses show that the PCPT risk calculator modestly improves the performance of PSA level alone in predicting an individual's risk of prostate cancer or high-grade disease on biopsy. This predictive tool might be enhanced by including percentage free PSA and the number of biopsy cores.

Endoglin (CD105) as a urinary and serum marker of prostate cancer.

We have previously shown that endoglin (CD105) is upregulated in prostatic fluid of men with large volume prostate cancer. We chose to assess endoglin levels in urine and serum from men with prostate cancer or at increased risk for the disease: Urine samples were collected after digital rectal examination (DRE) from 99 men whose cancer status was confirmed by biopsy, and serum samples were collected from 20 men without prostate cancer at low risk for the disease and from 69 men diagnosed with prostate cancer that subsequently underwent radical prostatectomy (30 pT2, 39 pT3). Endoglin levels were assessed by ELISA. Urinary endoglin was elevated in men with biopsy-positive prostate cancer compared to biopsy-negative men (p=0.0014). Urinary endoglin levels in men with prostate cancer correlated with radical prostatectomy tumor volume. The area under the receiver operating characteristic (ROC) curve was 0.72 for urinary endoglin and 0.50 for serum prostate-specific antigen (PSA; sensitivity for cancer detection 73%, specificity 63%). There were no differences in serum endoglin between normal and cancer cases, but there were increases in serum endoglin in non-organ confined (NOC, pT3+) versus organ-confined (OC, pT2) cases (p=0.0004). The area under the ROC curve was 0.75 for serum endoglin and 0.63 for PSA for predicting NOC status, with a sensitivity of 67% and a specificity of 80%. In conclusion, elevations in post-DRE urinary endoglin suggest there may be value in further studying endoglin as a urinary biomarker of prostate cancer. Endoglin levels in both urine and serum may aid in prostate cancer detection and prognostication.

Long-term assessment of prostate cancer progression free survival: evaluation of pathological parameters, nuclear shape and molecular biomarkers of pathogenesis.

BACKGROUND: Molecular pathways of proliferation, angiogenesis, neuroendocrine differentiation, apoptosis and alterations in nuclear structure of cancer epithelial cells are important in the pathogenesis of prostate cancer (PCa). Therefore, we evaluated the prognostic value of these parameters in 105 clinically localized PCa tumors with long-term follow-up after radical prostatectomy for progression-free survival (PFS). METHOD: Nuclear roundness variance (NRV) was calculated for tumor nuclei using the graphic tracing DynaCELL system. Immunohistochemistry assessed expression of Ki67, PCNA (proliferation), Chromogranin A (neuroendocrine differentiation), CD31 (angiogenesis), BCL2 (apoptosis), and Her-2/neu (oncogene) in the tumors. Cox proportional hazards regression, Spearman's rank correlation, and Kaplan-Meier plots were employed to analyze the data. RESULTS: Gleason score, focal vs. non-focal extra-prostatic extension, organ confined status, NRV, Her-2/neu, CD-31 and Ki67 were univariately significant predictors of PFS. NRV was the most significant prognostic indicator with the highest concordance index (0.7) for PFS. Gleason score, NRV and Her-2/neu were multivariately significant and yielded a concordance index of 0.77. CONCLUSION: Her-2/neu oncogene and NRV were shown to be significant in the prediction of PFS. The assessment of alterations in nuclear structure using NRV proved to be the most significant factor in the prediction of PFS. Integration of image analysis-based NRV and molecular biomarkers with pathologic parameters should be considered for validation in the prediction of PFS.

Prognostic value of Her-2/neu and DNA index for progression, metastasis and prostate cancer-specific death in men with long-term follow-up after radical prostatectomy.

Abnormal DNA content in tumor cells represents large scale chromosomal alterations and reflects later changes of genetic instability. Her-2/neu oncogene is amplified in 20-30% of breast and ovarian cancer patients and is associated with poor prognosis. Therefore, we evaluated prognostic value of Her-2/neu expression and DNA content measurements in 252 clinically localized PCa patients with long-term follow-up after radical prostatectomy for progression, metastasis and PCa-specific death. Her-2/neu expression was determined by immunohistochemistry and DNA content measurements employed Feulgen-stained cancer nuclei captured using static image cytometry system. Cox proportional hazard regression and Kaplan-Meir plots were used to identify significant prognostic factors for progression, metastasis and PCa-specific death. The proportions of Her-2/neu positive and high %DNA index tumors significantly increased from nonprogressor to progressors without metastasis to progressors with metastasis (p < 0.0001; <0.0001). Further, the proportions of Her-2/neu positive and high %DNA index tumors significantly increased from patients who died from another cause without progression to those who died from another cause with progression to those died with PCa-specific death (p = 0.027; <0.0001). Her-2/neu expression and %DNA index were significant prognosticators for progression (p

Effect of DNA methylation on identification of aggressive prostate cancer.

OBJECTIVES: Biochemical (prostate-specific antigen) recurrence of prostate cancer after radical prostatectomy remains a major problem. Better biomarkers are needed to identify high-risk patients. DNA methylation of promoter regions leads to gene silencing in many cancers. In this study, we assessed the effect of DNA methylation on the identification of recurrent prostate cancer. METHODS: We studied the methylation status of 15 pre-specified genes using methylation-specific polymerase chain reaction on tissue samples from 151 patients with localized prostate cancer and at least 5 years of follow-up after prostatectomy. RESULTS: On multivariate logistic regression analysis, a high Gleason score and involvement of the capsule, lymph nodes, seminal vesicles, or surgical margin were associated with an increased risk of biochemical recurrence. Methylation of CDH13 by itself (odds ratio 5.50, 95% confidence interval [CI] 1.34 to 22.67; P = 0.02) or combined with methylation of ASC (odds ratio 5.64, 95% CI 1.47 to 21.7; P = 0.01) was also associated with an increased risk of biochemical recurrence. The presence of methylation of ASC and/or CDH13 yielded a sensitivity of 72.3% (95% CI 57% to 84.4%) and negative predictive value of 79% (95% CI 66.8% to 88.3%), similar to the weighted risk of recurrence (determined from the lymph node status, seminal vesicle status, surgical margin status, and postoperative Gleason score), a powerful clinicopathologic prognostic score. However, 34% (95% CI 21% to 49%) of the patients with recurrence were identified by the methylation profile of ASC and CDH13 rather than the weighted risk of recurrence. CONCLUSIONS: The results of our study have shown that methylation of CDH13 alone or combined with methylation of ASC is independently associated with an increased risk of biochemical recurrence after radical prostatectomy even considering the weighted risk of recurrence score. These findings should be validated in an independent, larger cohort of patients with prostate cancer who have undergone radical prostatectomy.

External validation of University of California, San Francisco, Cancer of the Prostate Risk Assessment score.

OBJECTIVES: In 2005, the University of California, San Francisco, proposed the Cancer of the Prostate Risk Assessment (UCSF-CAPRA) score to predict the risk of biochemical recurrence (BR) after radical prostatectomy. This study provides external validation and a modified version of the model using a large cohort of men treated with radical prostatectomy at a high-volume, tertiary referral center. METHODS: From 1984 to 2006, 6737 men underwent radical prostatectomy at our institution for clinical Stage T1c-T3a prostate cancer with available follow-up information and no neoadjuvant or adjuvant therapy before BR. The BR-free survival was estimated using the Kaplan-Meier method and compared by UCSF-CAPRA score using the log-rank statistic. Performance of the UCSF-CAPRA was evaluated using Cox proportional hazards regression analysis and Harrell's concordance (c) index and compared with the Kattan nomogram. The UCSF-CAPRA score and final pathologic findings were assessed by odds ratios. RESULTS: The 5-year BR-free survival rate was 83.1% overall and decreased from 94.4% for men with a UCSF-CAPRA score of 1 or less to 25.8% for those with a score of 7 or more (P <0.0001). The hazards ratio approximately doubled for each UCSF-CAPRA point until a score of 4, when the hazards ratio increased at a slower rate. The c-index of the UCSF-CAPRA and Kattan nomogram was 0.76 and 0.78, respectively. A greater UCSF-CAPRA score correlated with the final pathologic findings. CONCLUSIONS: The UCSF-CAPRA performed well in this tertiary, referral-based cohort with a c-index similar to that of the Kattan nomogram. It remains an effective prognostic instrument for predicting the risk of biochemical recurrence after radical prostatectomy.

Progression after radical prostatectomy for men in their thirties compared to older men.

OBJECTIVE: To assess the biochemical outcome after radical prostatectomy (RP) specifically for men aged 30-39 years, as previous studies suggest that prostate cancer in young men might be more aggressive. PATIENTS AND METHODS: From a large (15 899) database of RPs (1975-2007) we identified 42 men aged 30-39, 893 aged 40-49, 4085 aged 50-59, 3766 aged 60-69, and 182 men aged > or =70 years old. The clinical characteristics and treatment outcomes were compared between men aged 30-39 years and older men. RESULTS: Among the men in their thirties, 81% had organ-confined disease in the RP specimen, vs 62% of men aged > or =40 years. At a mean follow-up of 5 years, there was biochemical progression in 4.8% of men in their thirties and 16.1% of men age > or =40 years (P = 0.055). The corresponding 5-year biochemical progression-free survival estimates were 95% for men in their thirties and 83% for men aged > or =40 years (P = 0.045). On multivariate analysis, increasing age was a significant independent predictor of biochemical progression. CONCLUSION: Contrary to earlier reports, in the present study men in their thirties did not have more aggressive disease. Instead, they had more favourable pathological features and progression-free survival rates than their older counterparts. After controlling for other prognostic variables on multivariate analysis, being in the fourth decade was independently associated with a lower risk of biochemical progression. These results suggest that early aggressive treatment for these patients with a long life-expectancy is associated with favourable long-term biochemical outcomes.

CpG island hypermethylation profile in the serum of men with clinically localized and hormone refractory metastatic prostate cancer.

PURPOSE: We have noted that hypermethylation at GSTP1 in the preoperative serum of men with localized prostate cancer predicts early prostate specific antigen failure following surgical treatment. In this study we investigated the hypermethylation profile of several genes in the serum of men with localized and hormone refractory prostate cancer. MATERIALS AND METHODS: We assayed the serum of 192 men with clinically localized prostate cancer and 18 with hormone refractory metastatic disease. A total of 35 serum samples from patients with negative prostate biopsy served as a negative control. CpG Island hypermethylation status of certain genes was assessed, including MDR1, EDNRB, CD44, NEP, PTGS2, RASSF1A, RAR-beta and ESR1. The results of hypermethylation at GSTP1 were included from a previous study. RESULTS: CpG island hypermethylation at MDR1 was positive in 38.2% of cases without PSA recurrence and in 16.1% of those with biochemical recurrence after radical prostatectomy. DNA hypermethylation at the remaining 7 gene loci was not detected in the serum of patients with localized prostate cancer. In serum from metastatic prostate cancer cases CpG island hypermethylation was detected at MDR1 in 15 (83.3%), EDNRB in 9 (50%), RAR-beta in 7 (38.9%), GTSP1 in 5 (27.8%) and NEP or RASSF1A in 3 (16.7%). CpG island hypermethylation at CD44, PTGS2 or ESR was not detected in any samples. All histologically normal cases were negative for CpG island hypermethylation. CONCLUSIONS: DNA hypermethylation at MDR1 was detected in cases of localized prostate cancer. CpG island hypermethylation at several gene loci was detected in men with advanced disease. No single gene was consistently observed to be hypermethylated in men with hormone refractory disease. These results suggest that the CpG island hypermethylation status of a defined panel of genes may be a useful biomarker in men with hormone refractory prostate cancer.

The natural history of men treated with deferred androgen deprivation therapy in whom metastatic prostate cancer developed following radical prostatectomy.

PURPOSE: We report on the natural history and factors influencing the prognosis of a cohort of hormone naïve, prostate specific antigen era patients in whom metastatic prostate cancer developed after radical prostatectomy who were followed closely and treated with deferred androgen deprivation therapy at the time of metastasis. MATERIALS AND METHODS: A total of 3,096 men underwent radical prostatectomy performed by a single surgeon at Johns Hopkins Hospital between 1987 and 2005. Of these men 422 had prostate specific antigen failure. Distant metastasis developed in 123 patients, of whom 91 with complete data formed the study cohort initially treated during the prostate specific antigen era (1987 to 2005) and receiving androgen deprivation therapy after documented metastasis. A total of 41 men died of prostate cancer. Median survival times were estimated by Kaplan-Meier analysis. Prognostic impact was estimated as the hazard ratio derived from the Cox proportional hazards model. RESULTS: Median followup from radical prostatectomy was 120 months (range 24 to 216). Kaplan-Meier median (range) times to failure were 24 months (12 to 144) from radical prostatectomy to prostate specific antigen failure, 36 months (0 to 132) from prostate specific antigen failure to metastasis, 84 months (12 to 180) from metastasis to death and 168 months (24 to 216) from radical prostatectomy to death. Statistically significant univariate risk factors for prostate cancer specific mortality at the time of metastasis were pain at diagnosis of metastases (p = 0.002), time from radical prostatectomy to metastasis (p = 0.024) and prostate specific antigen doubling time less than 3 months during the 24 months before metastasis (p = 0.016). Multivariable analysis demonstrated independent predictors of prostate cancer specific mortality at the time of metastasis, namely pain (HR 3.5, p = 0.003) and prostate specific antigen doubling time less than 3 months (HR 3.4, p = 0.017). CONCLUSIONS: Men treated with deferred androgen deprivation therapy for the development of metastasis after radical prostatectomy may have a long life span, 169 months after radical prostatectomy (range 24 to 216). The presence of pain and short prostate specific antigen doubling time predicted an unfavorable outcome.

Association between two unlinked loci at 8q24 and prostate cancer risk among European Americans.

BACKGROUND: Recent studies have provided evidence of associations between genetic markers at human chromosome 8q24 and an increased risk of prostate cancer. We examined whether multiple independent risk variants exist in this region and whether the strength of observed associations differs as a function of disease aggressiveness. METHODS: We evaluated associations between 18 single-nucleotide polymorphisms (SNPs) in a 1-Mb interval at 8q24 and the risk of prostate cancer among 1563 case patients (1017 of whom had high-grade [Gleason score > or = 7] and/or non-organ-confined disease) and 576 control subjects of European American ancestry. Differences in genotype frequencies between case and control subjects were compared using logistic regression analysis, with adjustment for age, and the Wald chi-square test. All statistical tests were two-sided. RESULTS: We identified multiple SNPs in a 50-kb region (referred to as locus 1) that are in linkage disequilibrium with a previously reported risk-associated SNP at 8q24, rs1447295, but were more strongly associated with prostate cancer risk in our study population. We also identified a novel susceptibility SNP, rs6983267, at a second locus (locus 2) that is approximately 70 kb centromeric of rs1447295 and in linkage equilibrium with, and independent of, locus 1. Risk alleles at locus 2 were common in our study population (minor allele frequency approximately 50%, 25% homozygous for risk-associated allele). Analysis of the National Cancer Institute's Cancer Genetic Markers of Susceptibility (CGEMS) prostate cancer association study database alone and in combination with our data provided further evidence for this second prostate cancer risk locus; in the combined analysis, the allele frequencies for rs6983267 differed statistically significantly between case patients and control subjects (P = 1.61 x 10(-9)). We also identified a third locus at 8q24, approximately 400 kb centromeric to locus 2, that was statistically significantly associated with prostate cancer risk in a combined analysis of our data and CGEMS study data (P = 6.8 x 10(-4)). A joint analysis of loci 1 and 2 indicated that 35% of the control subjects carried risk genotypes at one or both these loci; compared with men with the non-risk genotype at both loci, men with risk genotypes at both loci had an odds ratio of prostate cancer of 2.68 (95% confidence interval [CI] = 1.62 to 4.43) and men with risk genotypes at either locus had an odds ratio of prostate cancer of 1.70 (95% CI = 1.39 to 2.07). CONCLUSIONS: Three loci at 8q24 are independent genetic risk factors for prostate cancer.

Prognostic value of preoperative serum cell-free circulating DNA in men with prostate cancer undergoing radical prostatectomy.

PURPOSE: We evaluated the association of preoperative serum cell-free circulating DNA concentration in men with clinically localized prostate cancer who underwent radical prostatectomy with prostate-specific antigen (PSA) recurrence. EXPERIMENTAL DESIGN: One hundred and ninety-two men with clinically localized prostate cancer, who underwent radical prostatectomy at the Johns Hopkins Hospital and had preoperative serum available for analyses constituted our study population. All serum samples were collected before prostate biopsy or at least 4 months after prostate biopsy. The total amount of serum cell-free circulating DNA from each sample was calculated using a standard curve generated via quantitative real-time PCR. PSA recurrence was defined as a single postoperative PSA level of > or =0.2. The natural logarithm (ln) of the DNA concentration was used for statistical analyses. RESULTS: Of the 192 men in our study, 56 (29%) experienced PSA recurrence within the study period (median time to PSA recurrence 2 years). The median follow-up time for men free of disease at last follow-up was 3 years. The median serum cell-free DNA concentration of all men in the study was 5.3 ng/mL (mean 18.05 ng/mL; range 0.2-320 ng/mL). The mean serum DNA concentration for men who recurred and for those who did not was 3.8 +/- 34.1 and 13.7 +/- 33.6 ng/mL, respectively (P = 0.001). In a univariate analysis, ln DNA concentration was significantly associated with PSA recurrence (hazard ratio, 1.49; 95% confidence interval, 1.3-1.8; P < 0.001). In the multivariate model, ln DNA concentration was significantly associated with PSA recurrence (hazard ratio, 2.56; 95% confidence interval, 1.1-1.6; P = 0.003). Using bootstrap analyses, serum cell-free DNA concentrations > or =5.75 ng/mL were associated with an increased risk of PSA recurrence within 2 years of radical prostatectomy. CONCLUSION: Our study suggests that preoperative serum cell-free DNA concentration may be a useful prognostic biomarker for men with clinically localized prostate cancer treated with radical prostatectomy.

Updated nomogram to predict pathologic stage of prostate cancer given prostate-specific antigen level, clinical stage, and biopsy Gleason score (Partin tables) based on cases from 2000 to 2005.

OBJECTIVES: To update the 2001 "Partin tables" with a contemporary patient cohort and revised variable categorization, correcting for the effects of stage migration. METHODS: We analyzed 5730 men treated with prostatectomy (without neoadjuvant therapy) between 2000 and 2005 at the Johns Hopkins Hospital. Average age was 57 years. Multivariable logistic regression was used to estimate the probability of organ-confined disease, extraprostatic extension, seminal vesicle involvement, or lymph node involvement. Predictor variables included preoperative prostate-specific antigen (PSA) level (0 to 2.5, 2.6 to 4.0, 4.1 to 6.0, 6.1 to 10.0, and greater than 10.0 ng/mL), clinical stage (T1c, T2a, and T2b/T2c), and biopsy Gleason score (5 to 6, 3 + 4 = 7, 4 + 3 = 7, or 8 to 10). Bootstrap resampling was used to generate 95% confidence intervals for predicted probabilities. RESULTS: Seventy-seven percent of patients had T1c, 76% had Gleason score 5 to 6, 80% had a PSA level between 2.5 and 10.0 ng/mL, and 73% had organ-confined disease. Nomograms were developed for the predicted probability of pathologically organ-confined disease, extraprostatic extension, seminal vesicle invasion, or lymph node involvement. The risk of non-organ-confined disease increased with increases in any individual prognostic factor. The dramatic decrease in clinical stage T2c compared with the patient series used in the previous models resulted in T2b and T2c being combined as a single predictor in the nomogram. CONCLUSIONS: These updated "Partin tables" were generated to reflect trends in presentation and pathologic stage for men diagnosed with clinically localized prostate cancer at our institution. Clinicians and patients can use these nomograms to help make important decisions regarding management of prostate cancer.

Do racial differences in prostate size explain higher serum prostate-specific antigen concentrations among black men?

OBJECTIVES: To determine whether elevated serum prostate-specific antigen (PSA) values in black men are due, at least partially, to larger prostate size among black men. METHODS: The study population consisted of two cohorts: (1) 1410 men undergoing radical prostatectomy between 1988 and 2005 at five equal-access medical centers comprising the Shared Equal Access Regional Cancer Hospital (SEARCH) Database; and (2) 9601 men undergoing radical prostatectomy between 1988 and 2004 at the Johns Hopkins Hospital. We evaluated the association between race and serum PSA value and prostate weight using multivariable linear regression while adjusting for demographic and clinicopathologic cancer characteristics. RESULTS: In both cohorts, black men had higher serum PSA values (P < or = 0.001). After adjusting for either demographic characteristics or demographic and cancer-specific characteristics, there were no significant associations between race and prostate size in either cohort. After adjusting for multiple demographic, clinical, and pathologic cancer-specific characteristics, black men had 15% higher serum PSA values relative to white men in both the SEARCH (P = 0.001) and Hopkins cohorts (P < 0.001). CONCLUSIONS: In this study of patients undergoing radical prostatectomy in two very different practice settings, black men in both cohorts had higher serum PSA values relative to white men, despite adjustment for demographic and cancer-specific characteristics, including prostate weight. The lack of significant association between race and prostate size suggests that alternative reasons are needed to explain higher serum PSA values in black men.