Urine Extracellular Vesicle GATA2 mRNA Discriminates Biopsy Result in Men with Suspicion of Prostate Cancer.

PURPOSE: Prostate specific antigen has limited performance in detecting prostate cancer. The transcription factor GATA2 is expressed in aggressive prostate cancer. We analyzed the predictive value of urine extracellular vesicle GATA2 mRNA alone and in combination with a multigene panel to improve detection of prostate cancer and high risk disease. MATERIALS AND METHODS: GATA2 mRNA was analyzed in matched extracellular vesicles isolated from urines before and after prostatectomy (16) and paired urine and tissue prostatectomy samples (19). Extracellular vesicle GATA2 mRNA performance to distinguish prostate cancer and high grade disease was tested in training (52) and validation (165) cohorts. The predictive value of a multigene score including GATA2, PCA3 and TMPRSS2-ERG (GAPT-E) was tested in both cohorts. RESULTS: Confirming its prostate origin, urine extracellular vesicle GATA2 mRNA levels decreased significantly after prostatectomy and correlated with prostate cancer tissue GATA2 mRNA levels. In the training and validation cohort GATA2 discriminated prostate cancer (AUC 0.74 and 0.66) and high grade disease (AUC 0.78 and 0.65), respectively. Notably, the GAPT-E score improved discrimination of prostate cancer (AUC 0.84 and 0.72) and high grade cancer (AUC 0.85 and 0.71) in both cohorts when compared with each biomarker alone and PT-E (PCA3 and TMPRSS2-ERG). A GAPT-E score for high grade prostate cancer would avoid 92.1% of unnecessary prostate biopsies, compared to 61.9% when a PT-E score is used. CONCLUSIONS: Urine extracellular vesicle GATA2 mRNA analysis improves the detection of high risk prostate cancer and may reduce the number of unnecessary biopsies.

The lag time in initiating clinical testing of new drugs in combination with radiation therapy, a significant barrier to progress?

BACKGROUND: The clinical development of new drugs with radiation appears to be limited. We hypothesised that phase I clinical trials with radiation therapy (RT) are initiated too late into a new drug's lifetime, impeding the ability to complete RT-drug development programmes before patent expiration. METHODS: We identified novel drug-radiation phase I combination trials performed between 1980 and 2012 within the PubMed and ClinicalTrials.gov databases. Data gathered for each drug included: date the initial phase I trial with/without RT was opened/published, date of the published positive phase III trials, and patent expiration dates. Lag time was defined as the interval between opening of the phase I trial without RT and the opening of the phase I with RT. Linear regression was used to model how the lag time has changed over time. RESULTS: The median lag time was 6 years. The initial phase I trial with RT was typically published 2 years after the first published positive phase III trial and 11 years before patent expiration. Using a best-fit linear model, lag time decreased from 10 years for phase I trials published in 1990 to 5 years in 2005 (slope significantly non-zero, P<0.001). CONCLUSIONS: Clinical drug development with RT commences late in the life cycle of anti-cancer agents. Taking into account the additional time required for late-phase clinical trials, the delay in initiating clinical testing of drug-RT combinations discourages drug companies from further pursuing RT-based development. Encouragingly, lag time appears to be decreasing. Further reduction in lag time may accelerate RT-based drug development, potentially improving patient outcomes.

Neuropilin-1 is upregulated in the adaptive response of prostate tumors to androgen-targeted therapies and is prognostic of metastatic progression and patient mortality.

Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.

Improved quantitation of DNA curvature using ligation ladders.

It is often desirable to estimate accurately the local shape of DNA molecules. Such measurements are useful in understanding the intrinsic contribution of DNA sequence to curvature, as well as in assessing the effects of chemical modifications. We have been investigating the effects of asymmetric phosphate neutralization on DNA shape using the well-characterized ligation ladder approach developed by Crothers and co-workers [D.M. Crothers and J.Drak (1992) Meth. Enzymol.,212, 46-71]. This technique is remarkably sensitive to differences in DNA shape. We now report a general quantitative assay of DNA curvature that we have validated using a set of phased A(5)tract standards. This approach allows simultaneous estimation of helix axis deflection magnitude and direction when a test sequence is monitored in at least three phasings relative to a reference A(5-6)tract in short DNA duplexes. Analysis using this improved approach confirms our published data on DNA curvature due to electrostatic effects.

Efficacy of post-operative radiation in a prostatectomy cohort adjusted for clinical and genomic risk.

BACKGROUND: To date, there have been no published trials examining the impact of salvage radiation therapy (SRT) in the post-operative setting for prostate cancer (PCa). We conducted a retrospective, comparative study of post-operative radiation following radical prostatectomy (RP) for men with pT3 disease or positive margins (adverse pathological features, APF). METHODS: 422 PCa men treated at four institutions with RP and having APF were analyzed with a primary end point of metastasis. Adjuvant radiation treatment (ART, n=111), minimal residual disease (MRD) SRT (n=70) and SRT (n=83) were defined by PSA levels of <0.2, 0.2-0.49 and ⩾0.5 ng ml(-1), respectively, before radiation therapy (RT) initiation. Remaining 157 men who did not receive additional therapy before metastasis formed the no RT arm. Clinical-genomic risk was assessed by Cancer of the Prostate Risk Assessment Post-Surgical (CAPRA-S) and Decipher. Cox regression was used to evaluate the impact of treatment on outcome. RESULTS: During the study follow-up, 37 men developed metastasis with a median follow-up of 8 years. Both CAPRA-S and Decipher had independent predictive value on multivariable analysis for metastasis (P<0.05). Adjusting for clinical-genomic risk, SRT and no RT had hazard ratios of 4.31 (95% confidence interval, 1.20-15.47) and 5.42 (95% confidence interval, 1.59-18.44) for metastasis compared with ART, respectively. No significant difference was observed between MRD-SRT and ART (P=0.28). Men with low-to-intermediate CAPRA-S and low Decipher value have a low rate of metastatic events regardless of treatment selection. In contrast, men with high CAPRA-S and Decipher benefit from ART, however the cumulative incidence of metastasis remains high. CONCLUSIONS: The decision as to the timing and need for additional local therapy following RP is nuanced and requires providers and patients to balance risks of morbidity with improved oncological outcomes. Post-RP treatment can be safely avoided for men who are low risk by clinical-genomic risk, whereas those at high risk should favor enrollment in clinical trials.

Decipher correlation patterns post prostatectomy: initial experience from 2 342 prospective patients.

BACKGROUND: Currently, there are multiple commercially available RNA-based biomarkers that are Medicare approved and suggested for use by the National Comprehensive Cancer Network guidelines. There is uncertainty as to which patients benefit from genomic testing and for whom these tests should be ordered. Here, we examined the correlation patterns of Decipher assay to understand the relationship between the Decipher and patient tumor characteristics. METHODS: De-identified Decipher test results (including Decipher risk scores and clinicopathologic data) from 2 342 consecutive radical prostatectomy (RP) patients tested between January and September 2015 were analyzed. For clinical testing, tumor specimen from the highest Gleason grade was sampled using a 1.5 mm tissue punch. Decipher scores were calculated based on a previously locked model. Correlations between Decipher score and clinicopathologic variables were computed using Spearman's rank correlation. Mixed-effect linear models were used to study the association of practice type and Decipher score. The significance level was 0.05 for all tests. RESULTS: Decipher score had a positive correlation with pathologic Gleason score (PGS; r=0.37, 95% confidence interval (CI) 0.34-0.41), pathologic T-stage (r=0.31, 95% CI 0.28-0.35), CAPRA-S (r=0.32, 95% CI 0.28-0.37) and patient age (r=0.09, 95% CI 0.05-0.13). Decipher reclassified 52%, 76% and 40% of patients in CAPRA-S low-, intermediate- and high-risk groups, respectively. We detected a 28% incidence of high-risk disease through the Decipher score in pT2 patients and 7% low risk in pT3b/pT4, PGS 8-10 patients. There was no significant difference in the Decipher score between patients from community centers and those from academic centers (P=0.82). CONCLUSIONS: Although Decipher correlated with baseline tumor characteristics for over 2 000 patients, there was significant reclassification of tumor aggressiveness as compared to clinical parameters alone. Utilization of the Decipher genomic classifier can have major implications in assessment of postoperative risk that may impact physician-patient decision making and ultimately patient management.

DNA bending by asymmetrically tethered cations: influence of tether flexibility.

BACKGROUND: We have been studying the proposal that laterally asymmetric charge neutralization along the DNA double helix can induce collapse toward the neutralized surface. Results of previous experiments implied that such a phenomenon can occur, suggesting a role for local interphosphate repulsive forces in DNA shape and rigidity. RESULTS: We now show that, whereas six ammonium ions tethered to one DNA face on flexible propyl chains can induce detectable DNA curvature, tethering of ammonium ions on rigid propynyl tethers does not induce DNA curvature. Molecular modeling indicates differing propensities for phosphate salt bridge formation between propyl- and propynyl-tethered ammonium ions. CONCLUSIONS: Ammonium ion localization is suggested as a key factor in induced bending. Rigidification of the double helix by stacking of propyne groups cannot be excluded.

Uncertainties encountered in implementation of adaptive planning with in vivo dosimeters.

Under a previously approved institutional review board protocol for prostate cancer patients, implanted metal-oxide semiconductor field-effect transistor dosimeters (dose verification system, Sicel Technologies) were used for measurement of the in vivo delivered daily dose. This dosimetric information provided the ability to adapt the plan if the measured doses did not match the dose expected from the planning system. Due to the inherent uncertainty in the dosimeters, the decision to adapt the treatment plan was made only if readings differed by more than 7 % for three consecutive days. To validate this method, we acquired daily cone beam computed tomography images for five patients, and the dose delivered to the dosimeters was calculated by use of (1) an automated procedure (MIM Maestro, MIM Software) and (2) the treatment planning system (XIO, Elekta). 72 % of the doses calculated automatically fell within 1 % of the doses calculated in the planning system, and 99 % agreed within 2 %. When compared to the calculated dose, 53 % of the in vivo measurements fell within 3 % of the calculated dose, 80 % fell within 5 %, and 9.8 % were greater than 7 %, but never on three consecutive days. The measured doses agreed reasonably well with the calculated doses, supporting the decision to adapt the plan only if there were discrepancies of more than 7 % over three consecutive days. Even with the inherent uncertainty in the dosimeters, this adaptive planning method can detect delivery inaccuracies that would not otherwise be caught with the use of only daily image guidance or other dose calculation surrogates.

High-throughput transcriptomic analysis nominates proteasomal genes as age-specific biomarkers and therapeutic targets in prostate cancer.

BACKGROUND: Although prostate cancer (PCa) is hypothesized to differ in nature between younger versus older patients, the underlying molecular distinctions are poorly understood. We hypothesized that high-throughput transcriptomic analysis would elucidate biological differences in PCas arising in younger versus older men, and would nominate potential age-specific biomarkers and therapeutic targets. METHODS: The high-density Affymetrix GeneChip platform, encompassing >1 million genomic loci, was utilized to assess gene expression in 1090 radical prostatectomy samples from patients with long-term follow-up. We identified genes associated with metastatic progression by 10 years post-treatment in younger (age<65) versus older (age⩾65) patients, and ranked these genes by their prognostic value. We performed Gene Set Enrichment Analysis (GSEA) to nominate biological concepts that demonstrated age-specific effects, and validated a target by treating with a clinically available drug in three PCa cell lines derived from younger men. RESULTS: Over 80% of the top 1000 prognostic genes in younger and older men were specific to that age group. GSEA nominated the proteasome pathway as the most differentially prognostic in younger versus older patients. High expression of proteasomal genes conferred worse prognosis in younger but not older men on univariate and multivariate analysis. Bortezomib, a Food and Drug Administration approved proteasome inhibitor, decreased proliferation in three PCa cell lines derived from younger patients. CONCLUSIONS: Our data show significant global differences in prognostic genes between older versus younger men. We nominate proteasomeal gene expression as an age-specific biomarker and potential therapeutic target specifically in younger men. Limitations of our study include clinical differences between cohorts, and increased comorbidities and lower survival in older patients. These intriguing findings suggest that current models of PCa biology do not adequately represent genetic heterogeneity of PCa related to age, and future clinical trials would benefit from stratification based on age.

Cellular dose conversion factors for alpha-particle--emitting radionuclides of interest in radionuclide therapy.

UNLABELLED: alpha-Particle--emitting radionuclides are of increasing interest in radionuclide therapy. The decay scheme of alpha-emitting radionuclides typically includes a chain of unstable progeny. It is generally assumed that alpha-particle emission by the parent radionuclide will break the chemical bond with its carrier molecule and that the resulting daughter atom will no longer be associated with the carrier molecule. If the daughter is very short lived, it will not have enough time to be carried any significant distance from the site of parent decay and a cellular, absorbed dose estimate must consider the energy deposited by the daughter as well as the parent. Depending on the site of parent decay and the expected removal rate of daughter atoms from this site, the contribution of emissions from longer-lived daughters may also be warranted. In this study, dose conversion factors (DCFs) for cellular dimensions that incorporate the fate of daughter radionuclides were derived for (225)Ac, (213)Bi, (211)At, and (223)Ra, the alpha-particle--emitting radionuclides of interest in radionuclide therapy. METHODS: The dose contribution of daughter radionuclides at the site of parent decay was made dependent on a cutoff time parameter, which was used to estimate the fraction of daughter decays expected at the site of parent decay. Previously tabulated S values (cell-surface to nucleus and cell-surface to cell) for each daughter in the decay scheme were scaled by this fraction and a sum over all daughters was performed to yield a cutoff time--dependent set of corresponding DCF values for each radionuclide. RESULTS: DCF values for the absorbed dose to the nuclear or cellular volume from cell-surface decays are presented as a function of the cutoff time for 4 different cellular and nuclear dimensions. CONCLUSION: In contrast to the cellular S values that account only for parent decay, the DCF values provided in this study make it possible to easily include the contribution of daughter decays in cellular alpha-particle emitter dose calculations.

Relating independent measures of DNA curvature: electrophoretic anomaly and cyclization efficiency.

Electrophoretic methods are often used to measure DNA curvature and protein-induced DNA bending. Though convenient and widely-applied, quantitative analyses are generally limited to assays for which empirical calibration standards have been developed. Alternatively, solution-based cyclization of short DNA duplexes allows analysis of DNA curvature and bending from first principles, but a detailed understanding of this assay is still lacking. In this work, we demonstrate that calibration with an independent electrophoretic assay of DNA curvature permits interpretation of cyclization assay results in a quantitatively meaningful way. We systematically measure intrinsic DNA curvature in short duplexes using a well-established empirical ligation ladder assay. We then compare the results to those obtained from the analysis of the distribution of circular products obtained in simple enzymatic cyclization assays of the same duplexes when polymerized. A strong correlation between DNA curvature estimates from these two assays is obtained for DNA fragments between 150-300 bp in length. We discuss how this result might be used to improve quantitative analysis of protein-mediated bending events evaluated by cyclization methods. Our results suggest that measurements of DNA curvature obtained under similar conditions, in solution and in an acrylamide gel matrix, can be compared directly. The ability to correlate results of these simple assays may prove convenient in monitoring DNA curvature and flexibility.