Semen miRNAs Contained in Exosomes as Non-Invasive Biomarkers for Prostate Cancer Diagnosis

Although it is specific for prostatic tissue, serum prostate-specific antigen (PSA) screening has resulted in an over-diagnosis of prostate cancer (PCa) and many unnecessary biopsies of benign disease due to a well-documented low cancer specificity, thus improvement is required. We profiled the expression level of miRNAs contained in semen exosomes from men with moderately increased PSA levels to assess their usefulness, either alone or in addition to PSA marker, as non-invasive biomarkers, for the early efficient diagnosis and prognosis of PCa. An altered miRNA expression pattern was found by a high throughput profiling analysis in PCa when compared with healthy individuals (HCt) exosomal semen samples. The presence of vasectomy was taken into account for the interpretation of results. Fourteen miRNAs were selected for miRNA validation as PCa biomarkers in a subsequent set of semen samples. In this explorative study, we describe miRNA-based models, which included miRNA expression values together with PSA levels, that increased the classification function of the PSA screening test with diagnostic and/or prognostic potential: [PSA miR-142-3p miR-142-5p miR-223-3p] model (AUC:0,821) to discriminate PCa from BPH (Sn:91,7% Sp:42,9% vs Sn:100%Sp:14,3%); and [PSA miR-342-3p +/- miR-374b-5p] model (AUC: 0,891) to discriminate between GS >= 7 tumours and men presenting PSA >= 4 ng/ml with no cancer or GS6 tumours (Sn:81,8% Sp:95% vs Sn:54,5% Sp:90%). The pathway analysis of predicted miRNA target genes supports a role for these miRNAs in PCa aetiology and/or progression. Our study shows semen exosome miRNA-based models as molecular biomarkers with the potential to improve PCa diagnosis/prognosis efficiency. As the next step, further prospective studies on larger cohorts of patients are required to validate the diagnostic and/or prognostic role of the miRNA panel before it could be adopted into clinical practice

Impact of Extracellular Vesicle Isolation Methods on Downstream miRNA Analysis in Semen: A Comparative Study

Seminal plasma (SP) contains a unique concentration of miRNA, mostly contained in small extracellular vesicles (sEVs) such as exosomes, some of which could be clinically useful for diagnosis and/or prognosis of urogenital diseases such as prostate cancer (PCa). We optimized several exosome-EV isolation technologies for their use in semen, evaluating EV purifying effectiveness and impact on the downstream analysis of miRNAs against results from the standard ultracentrifugation (UC) method to implement the use of SP sEV_miRNAs as noninvasive biomarkers for PCa. Our results evidenced that commercial kits designed to isolate exosomes/EVs from blood or urine are mostly applicable to SP, but showed quantitative and qualitative variability between them. ExoGAG 3500× g and the miRCURY Cell/Urine/CSF 1500× g methods resulted as equivalent alternative procedures to UC for isolating exosomes/sEVs from semen for nanoparticle characteristics and quality of RNA contained in vesicles. Additionally, the expression profile of the altered semen sEV-miRNAs in PCa varies depending on the EV isolation method applied. This is possibly due to different extraction techniques yielding different proportions of sEV subtypes. This is evidence that the exosome-EV isolation method has a significant impact on the analysis of the miRNAs contained within, with important consequences for their use as clinical biomarkers. Therefore, miRNA analysis results for EVs cannot be directly extrapolated between different EV isolation methods until clear markers for delineation between microvesicles and exosomes are established. However, EV extraction methodology affects combined models (semen exosome miRNA signatures plus blood Prostate specific antigen (PSA) concentration for PCa diagnosis) less; specifically our previously described (miR-142-3p + miR-142-5p + miR-223-3p + PSA) model functions as molecular marker from EVs from any of the three isolation methods, potentially improving the efficiency of PSA PCa diagnosis

Adaptation and validation of the Spanish version of the patient-oriented prostate utility scale (PORPUS)

Objective: the Patient-Oriented Prostate Utility Scale (PORPUS) is a combined profile and utility-based quality of life measure for prostate cancer patients. Our objectives were to adapt the PORPUS into Spanish and to assess its acceptability, reliability, and validity. Methods: the PORPUS was adapted into Spanish using forward and back translations and cognitive debriefing. PORPUS was administered jointly with the SF-36 and the Expanded Prostate Index Composite (EPIC) to 480 Spanish prostate cancer patients treated with radical prostatectomy or radiotherapy. The Spanish PORPUS scores' distribution and reliability were examined and compared with the original instrument. To evaluate construct validity, relationships were assessed between PORPUS and other instruments (testing hypotheses of the original PORPUS study), and among known groups defined by side effect severity. Results: reliability coefficient was 0.76 (similar to the original PORPUS' 0.81). Spanish PORPUS items presented correlations ranging 0.57-0.88 with the corresponding EPIC domains, as in the original PORPUS study (0.60-0.83). Both PORPUS-P and PORPUS-U showed significant differences and large effect sizes (0.94-1.90) when comparing severe versus no problem groups on urinary, bowel, sexual and hormonal side effects defined by EPIC. Conclusions: a conceptually equivalent Spanish version was obtained, with high reliability and good construct validity, similar to the original Canadian PORPUS version. It can therefore be used to measure health-related quality of life and utilities in Spanish prostate cancer patients

Early experience and quality of life in SBRT prostate cancer boost of 9 Gy in a phase II trial

Purpose or ObjectiveExtracranial stereotactic body radiation therapy (SBRT) allows delivering high doses per fraction with high accuracy to the prostatic gland in a low number of fractions.Dose escalation in normofractionaded radiation prostate cancer trials showed an increased toxicity.In order to evaluate the feasibility and toxicity of a regimen of a single dose hypofractionated prostate stereotactic boost a phase II study was undertaken. Self-reported quality of life (QOL) measures were also obtained in order to better define the possible deleterious effect of treatment

Single nucleotide polymorphisms in DNA repair genes as risk factors associated to prostate cancer progression

Background Besides serum levels of PSA, there is a lack of prostate cancer specific biomarkers. It is need to develop new biological markers associated with the tumor behavior which would be valuable to better individualize treatment. The aim of this study was to elucidate the relationship between single nucleotide polymorphisms (SNPs) in genes involved in DNA repair and prostate cancer progression.Methods A total of 494 prostate cancer patients from a Spanish multicenter study were genotyped for 10 SNPs in XRCC1, ERCC2, ERCC1, LIG4, ATM and TP53 genes. The SNP genotyping was made in a Biotrove OpenArray® NT Cycler. Clinical tumor stage, diagnostic PSA serum levels, and Gleason score at diagnosis were obtained for all participants. Genotypic and allelic frequencies were determined using the web-based environment SNPator.Results SNPs rs11615 (ERCC1) and rs17503908 (ATM) appeared as risk factors for prostate cancer aggressiveness. Patients wild homozygous for these SNPs (AA and TT, respectively) were at higher risk for developing cT2b – cT4 (OR = 2.21 (confidence interval (CI) 95% 1.47 – 3.31), p < 0.001) and Gleason scores ≥ 7 (OR = 2.22 (CI 95% 1.38 – 3.57), p < 0.001), respectively. Moreover, those patients wild homozygous for both SNPs had the greatest risk of presenting D’Amico high-risk tumors (OR = 2.57 (CI 95% 1.28 – 5.16)).Conclusions Genetic variants at DNA repair genes are associated with prostate cancer progression, and would be taken into account when assessing the malignancy of prostate cancer.This work was subsidized by a grant from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad from Spain), ID: PI12/01867. Almudena Valenciano has a grant from the Instituto Canario de Investigación del Cáncer (ICIC)

Polymorphisms in DNA-repair genes in a cohort of prostate cancer patients from different areas in Spain: heterogeneity between populations as a confounding factor in association studies

Background: Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. Objective: To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. Design, Setting, and Participants: A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArrayH NT Cycler. Outcome Measurements and Statistical Analysis: Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. Results and Limitations: We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. Conclusion: Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out

Intraethnic variation in steroid-5-alpha-reductase polymorphismsin prostate cancer patients: a potential factor implicated in 5-alpha-reductase inhibitor treatment.

Intraethnic variation in steroid-5-alpha-reductase polymorphismsin prostate cancer patients: a potential factor implicated in 5-alpha-reductase inhibitor treatment.This work was subsidized by the grant from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad from Spain), ID: PI12/01867. Almudena Valenciano has a grant from the Instituto Canario de Investigación del Cáncer (ICIC)

Polymorphisms in DNA-repair genes in a cohort of prostate cancer patients from different areas in Spain: heterogeneity between populations as a confounding factor in association studies

Background: Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. Objective: To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. Design, Setting, and Participants: A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArrayH NT Cycler. Outcome Measurements and Statistical Analysis: Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. Results and Limitations: We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. Conclusion: Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out

Genetic variations in genes involved in testosterone metabolism are associated with prostate cancer progression: A Spanish multicenter study.

BACKGROUND: Prostate cancer (PCa) is an androgen-dependent disease. Nonetheless, the role of single nucleotide polymorphisms (SNPs) in genes encoding androgen metabolism remains an unexplored area. PURPOSE: To investigate the role of germline variations in cytochrome P450 17A1 (CYP17A1) and steroid-5α-reductase, α-polypeptides 1 and 2 (SRD5A1 and SRD5A2) genes in PCa. PATIENTS AND METHODS: In total, 494 consecutive Spanish patients diagnosed with nonmetastatic localized PCa were included in this multicenter study and were genotyped for 32 SNPs in SRD5A1, SRD5A2, and CYP17A1 genes using a Biotrove OpenArray NT Cycler. Clinical data were available. Genotypic and allelic frequencies, as well as haplotype analyses, were determined using the web-based environment SNPator. All additional statistical analyses comparing clinical data and SNPs were performed using PASW Statistics 15. RESULTS: The call rate obtained (determined as the percentage of successful determinations) was 97.3% of detection. A total of 2 SNPs in SRD5A1-rs3822430 and rs1691053-were associated with prostate-specific antigen level at diagnosis. Moreover, G carriers for both SNPs were at higher risk of presenting initial prostate-specific antigen levels>20ng/ml (Exp(B) = 2.812, 95% CI: 1.397-5.657, P = 0.004) than those who are AA-AA carriers. Haplotype analyses showed that patients with PCa nonhomozygous for the haplotype GCTTGTAGTA were at an elevated risk of presenting bigger clinical tumor size (Exp(B) = 3.823, 95% CI: 1.280-11.416, P = 0.016), and higher Gleason score (Exp(B) = 2.808, 95% CI: 1.134-6.953, P = 0.026). CONCLUSIONS: SNPs in SRD5A1 seem to affect the clinical characteristics of Spanish patients with PCa. Copyright © 2015 Elsevier Inc. All rights reserved.This work was subsidized by Grants from the Instituto de Salud Carlos III, Madrid, Spain (Ministerio de Economía y Competitividad, Spain), ID: PI12/01867 and/nPI13/00412; and Instituto Canario de Investigación del Cáncer, Canary Islands, Spain (ID: ICIC-GR-F-14/11). Almudena Valenciano received a Grant from the/nInstituto Canario de Investigación del Cáncer (ICIC)

Epigenetic landscape in the kick-and-kill therapeutic vaccine BCN02 clinical trial is associated with antiretroviral treatment interruption (ATI) outcome.

BackgroundThe BCN02-trial combined therapeutic vaccination with a viral latency reversing agent (romidepsin, RMD) in HIV-1-infected individuals and included a monitored antiretroviral pause (MAP) as an efficacy read-out identifying individuals with an early or late (&lt; or &gt; 4weeks) viral-rebound. Integrated -omics analyses were applied prior treatment interruption to identify markers of virus control during MAP.MethodsPBMC, whole-genome DNA methylation and transcriptomics were assessed in 14 BCN02 participants, including 8 Early and 4 Late viral-rebound individuals. Chromatin state, histone marks and integration analysis (histone-3 acetylation (H3Ac), viral load, proviral levels and HIV-specific T cells responses) were included. REDUC-trial samples (n&nbsp;=&nbsp;5) were included as a control group for RMD administration alone.FindingsDNA methylation imprints after receiving the complete intervention discriminated Early versus Late viral-rebound individuals before MAP. Also, differential chromatin accessibility and histone marks at DNA methylation level were detected. Importantly, the differential DNA methylation positions (DMPs) between Early and Late rebounders before MAP were strongly associated with viral load, proviral levels as well as the HIV-specific T-cell responses. Most of these DMPs were already present prior to the intervention and accentuated after RMD infusion.InterpretationThis study identifies host DNA methylation profiles and epigenetic cascades that are predictive of subsequent virus control in a kick-and-kill HIV cure strategy.FundingEuropean Union Horizon 2020 Framework Programme for Research and Innovation under Grant Agreement N°681137-EAVI2020 and N°847943-MISTRAL, the Ministerio de Ciencia e Innovación (SAF2017_89726_R), and the National Institutes of Health-National Institute of Allergy and Infectious Diseases Program Grant P01-AI131568