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Background and Objective: Diagnosis of primary and relapsed bladder carcinomas is accomplished by urethrocystoscopy, an invasive procedure, combined with urinary cytology, with limited sensitivity, resulting in a substantial burden. Thus, noninvasive biomarkers have been investigated, among which DNA methylation has shown promise. This systematic review and meta-analysis sought to assess the diagnostic accuracy of DNA methylation biomarkers reported in the literature for bladder cancer detection, pinpointing the most informative one., Methods: The search for this systematic review and meta-analysis was conducted on PubMed, Scopus, and Cochrane Library for relevant studies published until December 31, 2022. A meta-analysis was performed using a random-effect model, to compute the pooled sensitivity and specificity of the markers. PROSPERO's registration ID for the study is CRD42023397703., Key Findings and Limitations: Out of the 2297 studies retrieved, 68 were included in the final analysis, despite considerable heterogeneity. These involved 12 696 participants, of whom 5557 were diagnosed with bladder cancer. Using diagnostic odds ratio (DOR) as a comparative measure, the five most promising markers (pooled sensitivity, specificity, and DOR) were SALL3 (61%, 97%, and 55.67, respectively), PENK (77%, 93%, and 47.90, respectively), ZNF154 (87%, 90%, and 45.07, respectively), VIM (82%, 90%, and 44.81, respectively), and POU4F2 (81%, 89%, and 34.89, respectively). Urinary cytology identified bladder cancer with 55% sensitivity, 92% specificity, and 14.37 DOR., Conclusions and Clinical Implications: DNA methylation biomarkers disclose high accuracy for bladder cancer detection in urine. Nonetheless, validation studies in different clinical settings are scarce, hampering clinical use. The identified biomarkers should be prioritized in future validation studies., Patient Summary: In this meta-analysis, we include previously published studies that used urine samples of bladder cancer patients' from all around the globe. We were able to compare the diagnostic accuracy of noninvasive markers across different populations. We were able to conclude on the most promising DNA methylation markers to detect bladder cancer using urine., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time., (© 2024. The Author(s).)

The long non-coding RNA HOX transcript antisense intergenic RNA ( HOTAIR ) is associated with oncogenic features in bladder cancer and is predictive of poor clinical outcomes in patients diagnosed with this disease. In this study, we evaluated the impact of the HOTAIR single nucleotide polymorphisms rs920778 and rs12826786 on bladder cancer risk and survival. This case-control study included 106 bladder cancer patients and 199 cancer-free controls. Polymorphisms were evaluated through PCR-restriction fragment length polymorphism. The odds ratio and 95% confidence intervals were tested using univariable and multivariable logistic regressions. The effects on patient survival were evaluated using the log-rank test and Cox regression models. Our data showed that the HOTAIR rs920778 and rs12826786 genetic variants are not associated with the risk of developing bladder cancer. Nevertheless, survival analyses suggested that the HOTAIR rs920778 TT genotype and rs12826786 CC genotype are associated with increased survival in male bladder cancer patients and in patients, both male and female, who have primary tumors with a pathological stage of pT2. Together, these results suggest that, despite not being associated with bladder cancer risk, HOTAIR rs920778 and rs12826786 polymorphisms might represent new prognostic factors in this type of cancer. This is particularly important as these polymorphisms might be easily evaluated in bladder cancer patients in a minimally invasive manner to better predict their clinical outcomes.

Bladder cancer (BlCa), specifically urothelial carcinomas, is a heterogeneous disease that derives from the urothelial lining. Two main classes of BlCa are acknowledged: the non-muscle invasive BlCa and the muscle-invasive BlCa; the latter constituting an aggressive disease which invades locally and metastasizes systemically. Distinguishing the specific microenvironment that cancer cells experience between mucosa and muscularis propria layers can help elucidate how these cells acquire invasive capacities. In this work, we propose to measure the micromechanical properties of both mucosa and muscularis propria layers of the bladder wall of BlCa patients, using atomic force microscopy (AFM). To do that, two cross-sections of both the macroscopically normal urinary bladder wall and the bladder wall adjacent to the tumor were collected and immediately frozen, prior to AFM samples analysis. The respective "twin" formalin-fixed paraffin-embedded tissue fragments were processed and later evaluated for histopathological examination. H&E staining suggested that tumors promoted the development of muscle-like structures in the mucosa surrounding the neoplastic region. The average Young's modulus (cell stiffness) in tumor-adjacent specimens was significantly higher in the muscularis propria than in the mucosa. Similarly, the tumor-free specimens had significantly higher Young's moduli in the muscularis propria than in the urothelium. Young's moduli were higher in all layers of tumor-adjacent tissues when compared with tumor-free samples. Here we provide insights into the stiffness of the bladder wall layers, and we show that the presence of tumor in the surrounding mucosa leads to an alteration of its smooth muscle content. The quantitative assessment of stiffness range here presented provides essential data for future research on BlCa and for understanding how the biomechanical stimuli can modulate cancer cells' capacity to invade through the different bladder layers.

Bladder cancer (BlCa) is the ninth most common cancer worldwide, associated with significant morbidity and mortality. Thus, understand the biological mechanisms underlying tumour progression is of great clinical significance. Vimentin (VIM) is (over)expressed in several carcinomas, putatively in association with EMT. We have previously found that VIM promoter methylation accurately identified BlCa and VIM expression associated with unfavourable prognosis. Herein, we sought to investigate VIM expression regulation and its role in malignant transformation of BlCa. Analysis of tissue samples disclosed higher VIM transcript, protein, and methylation levels in BlCa compared with normal urothelium. VIM protein and transcript levels significantly increased from non-muscle invasive (NMIBC) to muscle-invasive (MIBC) cases and to BlCa metastases. Inverse correlation between epithelial CDH1 and VIM , and a positive correlation between mesenchymal CDH2 and VIM were also observed. In BlCa cell lines, exposure to demethylating agent increased VIM protein, with concomitant decrease in VIM methylation. Moreover, exposure to histone deacetylases pan-inhibitor increased the deposit of active post-translational marks (PTMs) across VIM promoter. In primary normal urothelium cells, lower levels of active PTMs with concomitant higher levels of repressive marks deposit were observed. Finally, VIM knockdown in UMUC3 cell line increased epithelial-like features and decreased migration and invasion in vitro , decreasing tumour size and angiogenesis in vivo . We demonstrated that VIM promoter is epigenetically regulated in normal and neoplastic urothelium, which determine a VIM switch associated with EMT and acquisition of invasive and metastatic properties. These findings might allow for development of new, epigenetic-based, therapeutic strategies for BlCa., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Cystoscopy is the gold standard for bladder cancer detection, but is costly, invasive and has imperfect diagnostic accuracy. We aimed to identify novel and accurate DNA methylation biomarkers for non-invasive detection of bladder cancer in urine, with the potential to reduce the number of cystoscopies among hematuria patients., Results: Biomarker candidates (n = 32) were identified from methylome sequencing of urological cancer cell lines (n = 16) and subjected to targeted methylation analysis in tissue samples (n = 60). The most promising biomarkers (n = 8) were combined into a panel named BladMetrix. The performance of BladMetrix in urine was assessed in a discovery series (n = 112), consisting of bladder cancer patients, patients with other urological cancers and healthy individuals, resulting in 95.7% sensitivity and 94.7% specificity. BladMetrix was furthermore evaluated in an independent prospective and blinded series of urine from patients with gross hematuria (n = 273), achieving 92.1% sensitivity, 93.3% specificity and a negative predictive value of 98.1%, with the potential to reduce the number of cystoscopies by 56.4%., Conclusions: We here present BladMetrix, a novel DNA methylation urine test for non-invasive detection of bladder cancer, with high accuracy across tumor grades and stages, and the ability to spare a significant number of cystoscopies among patients with gross hematuria., (© 2022. The Author(s).)

N6-methyladenosine (m 6 A) and its regulatory proteins have been associated with tumorigenesis in several cancer types. However, knowledge on the mechanistic network related to m 6 A in bladder cancer (BlCa) is rather limited, requiring further investigation of its functional role. We aimed to uncover the biological role of m 6 A and related proteins in BlCa and understand how this influences tumor aggressiveness. N6-adenosine-methyltransferase catalytic subunit (METTL3), N6-adenosine-methyltransferase noncatalytic subunit (METTL14), protein virilizer homolog (VIRMA), and RNA demethylase ALKBH5 (ALKBH5) had significantly lower expression levels in BlCa compared to that in normal urothelium. METTL14 knockdown led to disruption of the remaining methyltransferase complex and a decrease in m 6 A abundance, as well as overall reduced tumor aggressiveness (decreased cell invasion and migration capacity and increased apoptosis). Furthermore, in vivo, METTL14 knockdown caused tumor size reduction. Collectively, we propose methyltransferase METTL14 as a key component for m 6 A RNA deposit and that it is closely related to BlCa progression, playing an important role in tumor aggressiveness. These data contribute to a better understanding of the m 6 A writer complex, which might constitute an appealing therapeutic target., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Mindfulness-based interventions (MBIs) have been used in oncology contexts as a promising tool with numerous benefits for various health-related and psychosocial outcomes. Despite the increasing popularity of MBIs, few randomized controlled trials (RCTs) have examined their effects upon biological parameters. Specifically, no previous study has examined the effects of MBIs on extracellular vesicles (EVs), which are potentially important markers of health, disease, and stress. Moreover, the lack of RCTs is even more limited within the context of technology-mediated MBIs and long-term effects., Methods: The current study protocol presents a two-arm, parallel, randomized controlled study investigating the effects of internet-supported mindfulness-based cognitive therapy (MBCT) compared with treatment as usual (TAU). Primary outcomes are psychological distress and EV cargo of distressed participants with previous breast, colorectal, or prostate cancer diagnoses. Secondary outcomes are self-reported psychosocial and health-related measures, and additional biological markers. Outcomes will be assessed at baseline, 4 weeks after baseline (mid-point of the intervention), 8 weeks after baseline (immediately post-intervention), 24 weeks after baseline (after booster sessions), and 52 weeks after baseline. Our goal is to recruit at least 111 participants who have been diagnosed with breast, prostate, or colorectal cancer (cancer stage I to III), are between 18 and 65 years old, and have had primary cancer treatments completed between 3 months and 5 years ago. Half of the participants will be randomized to the TAU group, and the other half will participate in an 8-week online MBCT intervention with weekly group sessions via videoconference. The intervention also includes asynchronous homework, an online retreat after the fifth week, and 4 monthly booster sessions after completion of the 8-week programme., Discussion: This study will allow characterizing the effects of internet-based MBCT on psychosocial and biological indicators in the context of cancer. The effects on circulating EVs will also be investigated, as a possible neurobiological pathway underlying mind-body intervention effects., Trial Registration: ClinicalTrials.gov NCT04727593 (date of registration: 27 January 2021; date of record verification: 6 October 2021)., (© 2022. The Author(s).)

Purpose: Non-muscle invasive bladder cancer (NMIBC) is a highly recurrent disease that progresses to muscle-invasive bladder cancer (MIBC) in 5-25% of the cases. Epithelial-mesenchymal transition (EMT) has been associated with features of disease progression. Thus, we aimed to characterize the cadherin switch (CS), an EMT hallmark, and its regulatory mechanisms in bladder cancer (BlCa) progression, as well as the biological role of RCAD, a lesser-known cadherin, in bladder carcinogenesis., Methods: Cadherin mRNA and promoter methylation levels were retrieved from The Cancer Genome Atlas (TCGA). Validation was performed in an independent set of 121 primary BlCa (NMIBC and MIBC) and 40 normal bladder samples from IPO Porto, using RT-qPCR and qMSP. Immunohistochemistry was performed in these samples and in 14 additional sarcomatoid BlCa. CRISPR-Cas9 was performed to explore the potential in vitro impact of RCAD on BlCa cell migration and invasion., Results: In both the TCGA and IPO Porto BlCa cohorts, cadherin gene deregulation was observed compared to normal tissue samples, independent of promoter methylation. At the protein level, decreased E-cadherin and increased P- and R-cadherin expression was noted in BlCa tissues. In sarcomatoid BlCa the same trend was observed, with a more intense staining compared to that in conventional MIBCs. RCAD knockout considerably reduced the malignant properties of BlCa cells., Conclusions: Our data indicate that E-, P- and R-cadherin switches occur in BlCa, being associated with tumor progression. Promoter methylation is not the likely mechanism underlying cadherin expression deregulation. Our findings suggest an oncogenic role of RCAD in BlCa progression., (© 2022. Springer Nature Switzerland AG.)

Objective: A large number of studies have been conducted exploring the effects of mindfulness programs on health outcomes, such as psychological and biological outcomes. However, there is substantial heterogeneity among studies and, consequently, in the systematic reviews/meta-analyses. Since clinical practice is massively informed by evidence on review studies, our main objective was to summarize the reported evidence regarding the effects of structured mindfulness-based programs on psychological, biological, and quality-of-life outcomes in cancer patients., Methods: We conducted a meta-review, using a literature search from inception to June 2020 in several electronic databases using a combination of keywords including MBSR, MBCT, cancer, and meta-analysis OR "systematic review" (PROSPERO registration CRD42020186511)., Results: Ten studies met the eligibility criteria and were included. The main findings were beneficial small to medium effect sizes of Mindfulness-Based Stress Reduction (MBSR)/Mindfulness-Based Cognitive Therapy (MBCT)/Mindfulness-Based Cancer Recovery (MBCR) on psychological health, such as anxiety, depression, stress, and quality of life. A beneficial effect was found for biological outcomes, albeit based on a reduced number of studies. Studies were moderate homogenous regarding the intervention, population, and outcomes explored. Results on long-term follow-up seem to indicate that the effects tend not to be maintained, namely in shorter follow-ups (6 months)., Conclusions: This meta-review brings a broad perspective on the actual evidence regarding MBSR/MBCT/MBCR. We expect to contribute to future project design, focused on developing high-quality studies and exploring the moderating effects that might contribute to biased results, as well as exploring who might benefit more from MBSR/MBCT/MBCT interventions., (© 2021 The Authors. Psycho-Oncology published by John Wiley & Sons Ltd.)

Secreted extracellular vesicles (EVs) are heterogeneous cell-derived membranous granules which carry a large diversity of molecules and participate in intercellular communication by transferring these molecules to target cells by endocytosis. In the last decade, EVs' role in several pathological conditions, from etiology to disease progression or therapy evasion, has been consolidated, including in central nervous system (CNS)-related disorders. For this review, we performed a systematic search of original works published, reporting the presence of molecular components expressed in the CNS via EVs, which have been purified from plasma, serum or cerebrospinal fluid. Our aim is to provide a list of molecular EV components that have been identified from both nonpathological conditions and the most common CNS-related disorders. We discuss the methods used to isolate and enrich EVs from specific CNS-cells and the relevance of its components in each disease context.

Somatic mutations in the telomerase reverse transcriptase ( TERT ) promoter regions are frequent events in urothelial cancer (UC) and their detection in urine (supernatant cell-free DNA or DNA from exfoliated cells) could serve as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumor-borne mutations in urine requires highly sensitive methods, capable of measuring low-level mutations. In this study, we developed sensitive droplet digital PCR (ddPCR) assays for detecting TERT promoter mutations (C228T, C228A, CC242-243TT, and C250T). We tested the C228T and C250T ddPCR assays on all samples with sufficient quantity of urinary DNA (urine supernatant cell-free DNA (US cfDNA) or urine pellet cellular DNA (UP cellDNA)) from the DIAGURO ( n = 89/93 cases and n = 92/94 controls) and from the IPO-PORTO ( n = 49/50 cases and n = 50/50 controls) series that were previously screened with the UroMuTERT assay and compared the performance of the two approaches. In the DIAGURO series, the sensitivity and specificity of the ddPCR assays for detecting UC using either US cfDNA or UP cellDNA were 86.8% and 92.4%. The sensitivity was slightly higher than that of the UroMuTERT assay in the IPO-PORTO series (67.4% vs. 65.3%, respectively), but not in the DIAGURO series (86.8% vs. 90.7%). The specificity was 100% in the IPO-PORTO controls for both the UroMuTERT and ddPCR assays, whereas in the DIAGURO series, the specificity dropped for ddPCR (92.4% versus 95.6%). Overall, an almost perfect agreement between the two methods was observed for both US cfDNA ( n = 164; kappa coefficient of 0.91) and UP cellDNA ( n = 280; kappa coefficient of 0.94). In a large independent series of serial urine samples from DIAGURO follow-up BC cases ( n = 394), the agreement between ddPCR and UroMuTERT was (i) strong (kappa coefficient of 0.87), regardless of urine DNA types (kappa coefficient 0.89 for US cfDNA and 0.85 for UP cellDNA), (ii) the highest for samples with mutant allelic fractions (MAFs) > 2% (kappa coefficient of 0.99) and (iii) only minimal for the samples with the lowest MAFs (< 0.5%; kappa coefficient 0.32). Altogether, our results indicate that the two methods (ddPCR and UroMuTERT) for detecting urinary TERT promoter mutations are comparable and that the discrepancies relate to the detection of low-allelic fraction mutations. The simplicity of the ddPCR assays makes them suitable for implementation in clinical settings.

Aim: Characterize DNA methyltransferases/demethylases expression in testicular germ cell tumors (TGCTs). Methods: In silico analysis of TCGA database, assessment of transcript levels of most relevant enzymes in four TGCT cell lines and validation in patient cohort (real-time quantitative polymerase chain reaction; immunohistochemistry). Results: DNMT3A , DNMT3B and TET2 were the most differentially expressed between seminomas (SEs) and nonseminomas (NSs). DNMT3B was significantly overexpressed in NS-related cell lines, and the opposite was found for TET2 . Significantly higher DNMT3A/B mRNA expression was observed in NS, indicating a role for de novo methylation in reprogramming. Significantly higher TET2 protein expression was observed in SEs, suggesting active demethylation contributes for SE hypomethylated state. More differentiated histologies disclosed distinct expression patterns. Conclusion: DNA-modifying enzymes are differentially expressed between TGCT subtypes, influencing reprogramming and differentiation.

Background: Bladder cancer (BlCa) taxonomy has proved its impact in patient outcome and selection for targeted therapies, but such transcriptomic-based classification has not yet translated to routine practice. Moreover, epithelial-to-mesenchymal transition (EMT) has shown relevance in acquisition of more aggressive BlCa phenotype. We aimed to test the usefulness of the molecular classification, as defined by immunohistochemistry (a routinely performed and easy-to-implement technique), in a well-defined BlCa cohort of both non-muscle invasive (NMIBC) and muscle invasive (MIBC) disease. Also, we aimed to assess the additional prognostic value of the mesenchymal marker vimentin to the stratification strategy., Methods: A total of 186 samples were available. Immunohistochemistry/RT-qPCR for luminal markers GATA3/FOXA1, basal markers KRT5/KRT6A and vimentin were performed., Results: mRNA expression levels of the markers positively correlated with immunoexpression scores. We found substantial overlapping in immunoexpression of luminal and basal markers, evidencing tumor heterogeneity. In MIBC, basal tumors developed recurrence more frequently. NMIBC patients with higher vimentin immunoexpression endured poorer disease-free survival, and increased expression was observed from normal bladder-NMIBC-MIBC-metastases., Conclusions: The classification has the potential to be implemented in routine, but further adjustments in practical scoring should be defined; focusing on additional markers, including those related to EMT, may further refine BlCa molecular taxonomy.

Sirtuins are emerging players in cancer biology and other age-related disorders, and their putative role in bladder cancer (BlCa) remains elusive. Further understanding of disease biology may allow for generation of more effective pathway-based biomarkers and targeted therapies. Herein, we aimed to illuminate the role of sirtuins' family in BlCa and evaluate their potential as disease biomarkers and therapeutic targets. SIRT1-7 transcripts and protein levels were evaluated in a series of primary BlCa and normal bladder mucosa tissues. SIRT7 knockdown was performed through lentiviral transduction in MGHU3, 5637 and J82 cells and its functional role was assessed. SIRT1, 2, 4 and 5 expression levels were significantly lower in BlCa, whereas SIRT6 and 7 were overexpressed, and these results were corroborated by TCGA cohort analysis. SIRT7 transcript levels were significantly decreased in muscle-invasive vs. papillary BlCa. In vitro studies showed that SIRT7 downregulation promoted cells migration and invasion. Accordingly, increased EMT markers expression and decreased E-Cadherin (CDH1) was observed in those BlCa cells. Moreover, increased EZH2 expression and H3K27 me3 deposition in E-Cadherin promoter was found in sh-SIRT7 cells. We demonstrated that sirtuins are globally deregulated in BlCa, and specifically SIRT7 downregulation is implicated in EMT, fostering BlCa invasiveness through EZH2-CDH1 axis.

Bladder cancer (BlCa) is a common malignancy with significant morbidity and mortality. Current diagnostic methods are invasive and costly, showing the need for newer biomarkers. Although several epigenetic-based biomarkers have been proposed, their ability to discriminate BlCa from common benign conditions of the urinary tract, especially inflammatory diseases, has not been adequately explored. Herein, we sought to determine whether VIM me and miR663a me might accurately discriminate those two conditions, using a multiplex test. Performance of VIM me and miR663a me in tissue samples and urines in testing set confirmed previous results (96.3% sensitivity, 88.2% specificity, area under de curve (AUC) 0.98 and 92.6% sensitivity, 75% specificity, AUC 0.83, respectively). In the validation sets, VIM me -miR663a me multiplex test in urine discriminated BlCa patients from healthy donors or patients with inflammatory conditions, with 87% sensitivity, 86% specificity and 80% sensitivity, 75% specificity, respectively. Furthermore, positive likelihood ratio (LR) of 2.41 and negative LR of 0.21 were also disclosed. Compared to urinary cytology, VIM me -miR663a me multiplex panel correctly detected 87% of the analysed cases, whereas cytology only forecasted 41%. Furthermore, high miR663a me independently predicted worse clinical outcome, especially in patients with invasive BlCa. We concluded that the implementation of this panel might better stratify patients for confirmatory, invasive examinations, ultimately improving the cost-effectiveness of BlCa diagnosis and management. Moreover, miR663a me analysis might provide relevant information for patient monitoring, identifying patients at higher risk for cancer progression.

Background: Recurrent mutations in the promoter of the telomerase reverse transcriptase (TERT) gene (C228T and C250T) detected in tumours and cells shed into urine of urothelial cancer (UC) patients are putative biomarkers for UC detection and monitoring. However, the possibility of detecting these mutations in cell-free circulating DNA (cfDNA) in blood and urine, or DNA from urinary exfoliated cells (cellDNA) with a single-gene sensitive assay has never been tested in a case-control setting., Methods: We developed a single-plex assay (UroMuTERT) for the detection of low-abundance TERT promoter mutations. We tested 93 primary and recurrent UC cases and 94 controls recruited in France (blood, urine samples and tumours for the cases), and 50 primary UC cases and 50 controls recruited in Portugal (urinary exfoliated cell samples). We compared our assay with urine cytology., Findings: In the French series, C228T or C250T were detected in urinary cfDNA or cellDNA in 81 cases (87·1%; 95% CI 78·6-93·2), and five controls (Specificity 94·7%; 95%CI 88·0-98·3), with 98·6% (95% CI 92·5-99·96) concordance in matched tumours. Detection rate in plasma cfDNA among cases was 7·1%. The UroMuTERT sensitivity was (i) highest for urinary cfDNA and cellDNA combined, (ii) consistent across primary and recurrent cases, tumour stages and grades, (iii) higher for low-risk non-muscle invasive UC (86·1%) than urine cytology (23·0%) (P < 0·0001) and (iv) 93·9% when combined with cytology. In the Portuguese series - the sensitivity and specificity for detection of UC with urinary cellDNA was 68·0% (95% CI 53·3-80·5) and 98·0% (95% CI 89·3-100·0)., Interpretation: TERT promoter mutations detected by the UroMuTERT assay in urinary DNA (cfDNA or cellDNA) show excellent sensitivity and specificity for the detection of UC, significantly outperforming that of urine cytology notably for detection of low-grade early stages UC. FUND: French Cancer League; French Foster Research in Molecular Biology and European Commission FP7 Marie Curie COFUND., (Copyright © 2019. Published by Elsevier B.V.)

Background: Prostate cancer (PCa), a major cause of cancer-related morbidity and mortality worldwide and mostly asymptomatic at earliest stages, is characterized by disruption of genetic and epigenetic balance. A better understanding of how those mechanisms orchestrate disease might improve diagnostic and prognostic tools, allowing for improvements in treatment efficacy. Replacement of canonical histones, an epigenetic mechanism, is highly conserved among species and altered expression of histones variants (e.g., MacroH2A1) has been associated with tumorigenesis. H2AFY gene encodes two isoforms of H2A histone variant MacroH2A1: MacroH2A1.1 and MacroH2A1.2. Specifically, MacroH2A1.1 isoform inhibits cell proliferation and promotes cellular differentiation. Because the contribution of this histone variant to carcinogenesis has been reported in several cancer types, but not for PCa, we aimed to investigate the contribution of MacroH2A1 for prostate carcinogenesis., Methods: MacroH2A1, MacroH2A1.1 and MacroH2A1.2 isoforms and the corresponding splicing regulators transcript levels were evaluated by RT-qPCR, in a tissue cohort composed by PCa, prostatic intraepithelial neoplasia (PIN) and normal prostate cases. Knockdown for MacroH2A1 and MacroH2A1.1 was performed through lentiviral transduction in DU145 cells, and MacroH2A1.1 overexpression was achieved in LNCaP cells by plasmid transfection, followed by functional assays. Biological and/or experimental replicates were performed when necessary, and specific statistical tests were applied to perform data analysis., Results: MacroH2A1.1 transcript levels were downregulated in PIN and primary PCa compared to normal prostate tissues. The same was found for QKI, a MacroH2A1.1's splicing regulator. Moreover, lower MacroH2A1.1 and QKI expression levels associated with less differentiated tumors (Gleason score ≥ 7). Interestingly, MacroH2A1.1, but more impressively DDX17 (AUC = 0.93; p < 0.0001) and QKI (AUC = 0.94; p < 0.0001), accurately discriminated cancerous from noncancerous prostate tissues. Furthermore, in PCa cell lines, total MacroH2A1 knockdown augmented malignant features, whereas MacroH2A1.1 overexpression impressively attenuated the malignant phenotype., Conclusions: Overall, our data, derived from primary PCa tissues and cell lines, anticipate a tumor suppressive role for MacroH2A1, particularly for the MacroH2A1.1 isoform, in prostate carcinogenesis., Competing Interests: The authors declare that they have no competing interests.

Bladder cancer is one of the most incident neoplasms worldwide, and its treatment remains a significant challenge, since the mechanisms underlying disease progression are still poorly understood. The epithelial to mesenchymal transition (EMT) has been proven to play an important role in the tumorigenic process, particularly in cancer cell invasiveness and metastatic potential. Several studies have reported the importance of epigenetic mechanisms and enzymes, which orchestrate them in several features of cancer cells and, specifically, in EMT. In this paper, we discuss the epigenetic enzymes, protein-coding and non-coding genes, and mechanisms altered in the EMT process occurring in bladder cancer cells, as well as its implications, which allows for improved understanding of bladder cancer biology and for the development of novel targeted therapies.

Purpose: Studies on the performance of epigenetic-based biomarkers in colorectal cancer (CRC) are scarce and have shown contradictory results. Thus, we sought to examine the prognostic value of histone-modifying enzymes (EZH2, SETDB1 and LSD-1) and histone post-translational marks (H3K27me3 and H3K9me3) in CRC., Methods: A retrospective series of 207 CRC patients primarily submitted to surgery in a cancer center was included in this study. Clinicopathological data were retrieved. One representative paraffin block per case was selected for immunohistochemistry, including normal and CRC tissues whenever possible. The percentage of positive nuclear staining (digital image analysis) was used to classify patients into "low" and "high" expression groups for each biomarker. Correlations between immunoexpression levels, clinicopathological features and clinical outcomes [disease-specific (DSS) and disease-free (DFS) survival] were examined. Statistical significance was set at p < 0.05., Results: CRC tissues showed significantly lower expression of SETDB1 and higher expression of the remainder four biomarkers compared to normal mucosa. High EZH2 expression correlated with disease recurrence/progression, whereas low LSD1 expression and high H3K9me3 and H3K27me3 expression were associated with more advanced stage. In multivariable analysis, cases with high LSD1 expression displayed significantly better DSS and DFS (HR 0.477, 95% confidence interval: 0.247-0.923) adjusted for pathological TNM stage., Conclusion: EZH2, SETDB1, LSD1, H3K9me3 and H3K27me3 expression are altered in CRC and may play a role in colorectal carcinogenesis. LSD1 immunoexpression levels independently predicted patient outcome in this cohort. Further investigations, using larger series, are warranted to confirm its potential clinical value and unravel underlying molecular mechanisms.

Introduction: Recent studies provide a convincing support that the presence of cancer cells in the body leads to the alteration of volatile organic compounds (VOCs) emanating from biological samples, particularly of those closely related with tumoral tissues. Thus, a great interest emerged for the study of cancer volatilome and subsequent attempts to confirm VOCs as potential diagnostic biomarkers., Objectives: The aim of this study was to determine the volatile metabolomic signature of bladder cancer (BC) cell lines and provide an in vitro proof-of-principle that VOCs emanated into the extracellular medium may discriminate BC cells from normal bladder epithelial cells., Methods: VOCs in the culture media of three BC cell lines (Scaber, J82, 5637) and one normal bladder cell line (SV-HUC-1) were extracted by headspace-solid phase microextraction and analysed by gas chromatography-mass spectrometry (HS-SPME/GC-MS). Two different pH (pH 2 and 7) were used for VOCs extraction to infer the best pH to be used in in vitro metabolomic studies., Results: Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal bladder cells, at both pHs, although a higher number of discriminative VOCs was obtained at neutral pH. Most of the altered metabolites were ketones and alkanes, which were generally increased in BC compared to normal cells, and alcohols, which were significantly decreased in BC cells. Among them, three metabolites, namely 2-pentadecanone, dodecanal and γ-dodecalactone (the latter only tentatively identified), stood out as particularly important metabolites and promising volatile biomarkers for BC detection. Furthermore, our results also showed the potential of VOCs in discriminating BC cell lines according to tumour grade and histological subtype., Conclusions: We demonstrate that a GC-MS metabolomics-based approach for analysis of VOCs is a valuable strategy for identifying new and specific biomarkers that may improve BC diagnosis. Future studies should entail the validation of volatile signature found for BC cell lines in biofluids from BC patients.

Background: Urothelial carcinoma (UC) is the most common cancer affecting the urinary system, worldwide. Lack of accurate early detection tools entails delayed diagnosis, precluding more efficient and timely treatment. In a previous study, we found that miR-129-2 and miR-663a were differentially methylated in UC compared with other genitourinary tract malignancies. Here, we evaluated the diagnostic performance of those microRNAs in urine., Methods: Promoter methylation levels of miR-129-2 and miR-663a were assessed, using real-time quantitative methylation-specific PCR, in UC tissue samples (using normal urothelium as control) and, subsequently, in urine samples from UC and other genitourinary malignancies. Diagnostic and prognostic performances were evaluated by receiver operator characteristics curve and survival analyses, respectively., Results: Promoter methylation levels of both microRNAs were significantly higher in UC tissue samples compared with normal urothelium. In urine, the assay was able to distinguish UC from other genitourinary tract carcinomas with 87.7% sensitivity and 84% specificity, resulting in 85.85% overall accuracy., Conclusions: This panel of miRNAs promoter methylation accurately detects UC in urine, comparing well with other promising epigenetic-based biomarkers. This may constitute the basis for a non-invasive assay to detect UC.

Sirtuins participate in hormone imbalance, metabolism and aging, which are important processes for endometrial cancer (EC) development. Sirtuins mRNA expression (SIRT1 to 7) was determined in 76 ECs (63 Type I, 12 Type II and one mixed EC), and 30 non-neoplastic endometria (NNE) by quantitative real-time PCR. SIRT1 and SIRT7 protein expression was evaluated by immunohistochemistry using Allred score. Compared to NNE, ECs showed SIRT7 (p < 0.001) mRNA overexpression, whereas SIRT1 (p < 0.001), SIRT2 (p < 0.001), SIRT4 (p < 0.001) and SIRT5 (p < 0.001) were underexpressed. No significant differences were observed for SIRT3 and SIRT6. Type II ECs displayed lower SIRT1 (p = 0.032) and SIRT3 (p = 0.016) transcript levels than Type I ECs. Concerning protein expression, SIRT1 immunostaining median score was higher in ECs compared to NNE epithelium (EC = 5 vs. NNE = 2, p < 0.001), while SIRT7 was lower in ECs (EC = 6 vs. NNE = 7, p < 0.001). No significant associations were found between SIRT1/7 immunoexpression and histological subtype, grade, lymphovascular invasion or stage. Our data shows that sirtuins are deregulated in EC. The diversity of expression patterns observed suggests that sirtuins may have distinctive roles in endometrial cancer similarly to what has been described in other cancer models.

Patients with endometrial endometrioid carcinoma (EEC) that present with advanced primary disease and develop recurrences have a poor outcome. The phenotype of EEC metastases and recurrences is poorly studied. We evaluated the morphological features and ER-alpha/PRA/p53 immunohistochemical expression of a sample of 45 EEC metastases compared to matched primary tumors. Additionally, we studied methylation levels of ER-alpha/PRA gene promoters. The distribution of histological FIGO grade was significantly different in metastases, which disclosed higher grade than primary tumors (p = 0.005). Mitotic index was significantly lower in metastases compared to matched primary tumors (p<0.001). ER-alpha (p = 0.002) and PRA (p<0.001) median H-scores were significantly lower in metastases than in matched primary EECs, but there was no significant difference concerning p53 expression (p = 0.056). ER-alpha/PRA expression differences did not correlate with differences in metastases morphology. ER-alpha/PRA gene promoter levels were globally low (range: 0% to 11.9%). One case showed higher ER-alpha gene promoter methylation in metastasis compared to matched EEC primary tumor. Regarding PRA, there was a significant higher frequency of its promotor methylation in metastases compared to primary tumors (51.6% vs. 22.7%, p = 0.022). In conclusion, EEC metastatic disease displays phenotypic changes along with ER-alpha and PRA decreased expression compared to primary tumors. ER-alpha and PRA gene promoter methylation seems to play a limited role in the etiology of these alterations. PR expression assessment for hormonal treatment decision of patients with advanced tumors, may be more adequate in metastases than in EEC primary tumors.

Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.

Aim of the Study: Upper tract urothelial carcinoma (UTUC) accounts for 5-10% of all urothelial tumours. It is mostly diagnosed at advanced stages, entailing a worse prognosis, owing to the lack of early and specific symptoms as well as of effective diagnostic tools. We previously identified a panel of epigenetic biomarkers (GDF15, TMEFF2 and VIM promoter methylation) that accurately identifies bladder cancer in urine. Herein, we assessed the performance of the same panel for UTUC detection and prognosis, in tissue and urine., Material and Methods: Methylation levels of reference and target genes were determined using real-time quantitative methylation-specific polymerase chain reaction (MSP) in bisulphite-modified DNA of 57 UTUC tissues, 36 normal upper tract urothelium (NUTUs), 22 urines from UTUC suspects and 20 urines from controls. Receiver operator characteristics (ROC)-curve analysis was performed to determine the performance of the biomarker panel and survival analyses were conducted to evaluate their prognostic value., Results: Methylation levels of GDF15, TMEFF2 and VIM were significantly higher in UTUC compared to NUTUs (P=0.022; P<0.001; P<0.001, respectively). The panel accurately identified UTUC with 100% and 91% sensitivity, corresponding to an area under the curve of 1.000 and 0.923 in tissue and urines, respectively, with 100% specificity. Low VIM promoter methylation levels independently predicted poor disease-specific survival., Conclusions: GDF15, TMEFF2 and VIM promoter methylation allows for accurate identification of UTUC, in tissue and urine and VIM methylation provides relevant prognostic information, especially in high-stage disease. This assay may improve the clinical management of UTUC patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)