To evaluate the effectiveness of Sterile water injections (SWIs) for acute pain relief in renal colic. This study was conducted in accordance with the Preferred Reporting items for Systematic Reviews and Meta-Analyses. MEDLINE, CINAHL, Web of Science and Cochrane were searched to identify randomised controlled trials (RCTs) comparing SWIs with placebo or other analgesics in renal colic patients. Data were pooled and analysed using random effects modelling with 95

Effective renal colic management relies on optimal pain relief. Each medication has its distinct side effects and contraindications. Sterile water injections (SWIs) have emerged as a potential alternative to standard treatment. This systematic review and meta-analysis of randomised controlled trials (RCTs) aimed to evaluate the effectiveness of SWIs analgesia in renal colic. This study was conducted in accordance with the Preferred Reporting items for Systematic Reviews and Meta-Analyses. MEDLINE, CINALH, Web of Science and Cochrane were searched to identify RCTs comparing SWIs with placebo or other analgesics in renal colic patients. Data were pooled and analysed using random-effects modelling with 95% confidence intervals (CIs). Six RCTs including 1,322 renal colic patients were included, with 466 (35.2%) receiving SWIs. Patients receiving SWIs had comparable demographics and presenting features to the control groups, with control medications ranging from placebos, non-steroidal anti-inflammatories, and opioids. Self-reported pain scores at 30 minutes following intervention were lower in SWI than placebo (MD=-4.63, 95% CI:-5.16, -4.10, P < 0.00001) and other analgesics (MD = -0.36, 95% CI: -0.52, -0.21, P < 0.00001). The use of rescue analgesia was lower in those receiving SWIs compared to placebo (OR = 0.24, 95% CI: 0.10, 0.59, P = 0.002) and other analgesics (OR = 0.46, 95% CI: 0.29, 0.74, P = 0.001). No significant side effects were attributed to SWI use. SWIs demonstrated superior pain relief and reduced rescue analgesia requirements, compared to placebo and standard treatment, offering a promising alternative for patients where traditional options are unsuitable.

Aims. Polycystic ovarian syndrome (PCOS) is a common endocrine disorder characterised by ovarian morphological, systemic biochemical, and menstrual changes. Women with PCOS are at significantly increased risk of raised fasting glucose, impaired glucose tolerance, and diabetes. Recognition of these complications and early intervention are key to good health outcomes. We sought to identify DNA methylation patterns that may predict future diabetes onset in this high-risk PCOS population. Patients and Methods. Peripheral blood samples from women with PCOS and women with PCOS who later developed diabetes, were analysed by Illumina HumanMethylation450 BeadChip-arrays. Bisulphite-Pyrosequencing™ was used to validate and confirm array methylation data. Results. Array analyses identified 273 differentially methylated CpG loci (≥ 0.2 β-value change) at initial diagnosis of PCOS, between women who did or did not later develop diabetes. 19 of these sites demonstrated differential methylation in the same direction in ≥ five sample pairs. Methylation in three of the candidates (cg11897887, cg02819655, and cg25542007) showed the best concordance with corresponding array β-values, and, most clearly differentiated ‘cases’ from ‘controls’. Conclusions. We have identified novel methylation biomarkers that could predict future onset of diabetes in this high-risk population. Use of methylation analyses to identify women who are likely to develop diabetes at diagnosis of PCOS may facilitate timely lifestyle interventions to reduce future morbidity.

Objective: To calculate the re-admission rate with haematuria within 30 days of elective transurethral resection of bladder tumour (TURBT), and identify factors associated with this. Materials and Methods: This was a multicentre, retrospective audit, identifying all adult patients over the age of 16 who underwent elective TURBT between 1 September and 30 November 2019. Data were collected from medical records and operation notes on patient demographics, intra-operative factors and post-operative management. Primary outcome measure was the proportion of patients emergently re-admitted with haematuria. Secondary outcome measures were the re-operation rate for haematuria, and the rate of new acute thrombotic event (TE). Fisher’s exact test was used to calculate p values within subgroups for re-admission rates. Results: 443 patients from 12 hospitals were included. Median age was 75 years (17–99). 15 patients (3.4%) were re-admitted with haematuria. Subgroup analysis demonstrated higher rate of re-admission for pre-existing antithrombotic agents (ATAs) (2.0% vs. 6.1%, p = 0.046), increased for non-Aspirin ATAs (10.5%, p = 0.0015). 52% of non-Aspirin ATAs were restarted within 48 hours of surgery; post-operative plan for restarting was not documented in 22.1%. One patient (0.23%) developed acute TE (pulmonary embolus). Conclusion: Pre-existing use of non-Aspirin ATAs is associated with increased risk of post-TURBT haematuria, with variable practice in post-operative recommencement. Level of evidence: Level 3

Introduction Non-muscle-invasive bladder cancer (NMIBC) is a common and heterogeneous disease; many patients develop recurrent or progress to muscle-invasive disease. Intravesical drug therapy is a pillar in the current management of NMIBC; notwithstanding, Mitomycin C (MMC) and Bacillus Calmette-Guérin (BCG) have numerous limitations including international supply issues, and local and systemic toxicity. Here we review novel intravesical therapeutic options and drug delivery devices with potential for clinical use in the treatment of NMIBC. Methods PubMed, ClinicalTrials.gov and Cochrane Library searches were undertaken. Systematic reviews, meta-analyses, randomised controlled trials, single-arm clinical trials and national/international conference proceedings were included. Results Novel intravesical drugs, including chemotherapeutic agents, immune checkpoint inhibitors, monoclonal antibodies and gene therapies, have demonstrated varying efficacy in the treatment of NMIBC. Current evidence for the majority of treatments is mostly limited to single-arm trials in patients with recurrent NMIBC. Various novel methods of drug delivery have also been investigated, with encouraging preliminary results supporting the intravesical delivery of hyperthermic MMC and MMC hydrogel formulations. Conclusions Novel therapeutic agents and drug delivery systems will be important in the future intravesical management of NMIBC. As our understanding of the molecular diversity of NMIBC develops, molecular subtyping will become fundamental in the personalisation of intravesical treatments. Further randomised studies are urgently required to investigate the efficacy of novel intravesical treatments and novel regimens, in comparison to current standards-of-care, particularly in the context of international BCG shortages.

Radical cystectomy (RC) is commonly performed with curative intent for primary or recurrent high-risk non-muscle-invasive and muscle-invasive bladder cancers. Urethral recurrence (UR) within the residual urethra, often proximally where the epithelial lining comprises urothelial cells, is a rare but well-described occurrence associated with adverse clinical outcomes. Current national guidelines therefore suggest that male patients with a defunctioned urethra should undergo annual endoscopic or urethral washing surveillance for 5 years following RC, to identify UR early, where local disease management (e.g., urethrectomy) may still be possible. Anecdotally, however, urethroscopy and urethral washing cytology appear to be infrequently performed. Our regional trainee-led research collaborative evaluated the frequency and tim-ing of urethral surveillance in the West Midlands in comparison to National Institute for Health and Care Excellence (NICE) guidelines. Patients and methodsOur 10-year cross-sectional retrospective regional analysis included 495 patients from 2008–2018. Clinical and demographic data were collected alongside cross-sectional staging and imaging, and timings and frequency of urethral endoscopic surveillance or urethral washing cytology. ResultsOverall, 159 (35.2%) patients received one (or more) surveillance urethroscopy. A minority of surveillance urethroscopies were annual, with hugely variable frequency or intervals ranging from every 4–50 months. Only 81 (19.6%) patients had urethral surveillance in keeping with the frequency suggested by NICE guidelines. At 10 years, disease-specific mortality was 42.0%, and overall or all-cause mortality was 44.7%. The overall UR rate (as detected by staging CT and/or urethroscopy) was 1.0% (n = 4); all four cases of UR were found in patients with positive urethral margins after RC who did not undergo immediate urethrectomy. ConclusionsOur regional urethral surveillance practice following RC is heterogeneous and suboptimal in comparison to NICE guidelines. Our UR rate was so low that we are unable to assert whether early detection has any clinical benefit, and therefore we cannot advocate routine urethral surveillance, but suggest that patients with positive urethral margins should be offered immediate urethrectomy post RC. In addition, we encourage collaborative urological research and data collection to generate higher volume series, more representative and generalisable data, and more meaningful conclusions.

Pluripotent stem cells (PSC) possess unlimited proliferation, self-renewal, and a differentiation capacity spanning all germ layers. Appropriate culture conditions are important for the maintenance of self-renewal, pluripotency, proliferation, differentiation, and epigenetic states. Oxygen concentrations vary across different human tissues depending on precise cell location and proximity to vascularisation. The bulk of PSC culture-based research is performed in a physiologically hyperoxic, air oxygen (21% O2) environment, with numerous reports now detailing the impact of a physiologic normoxia (physoxia), low oxygen culture in the maintenance of stemness, survival, morphology, proliferation, differentiation potential, and epigenetic profiles. Epigenetic mechanisms affect multiple cellular characteristics including gene expression during development and cell-fate determination in differentiated cells. We hypothesized that epigenetic marks are responsive to a reduced oxygen microenvironment in PSCs and their differentiation progeny. Here, we evaluated the role of physoxia in PSC culture, the regulation of DNA methylation (5mC (5-methylcytosine) and 5hmC (5-hydroxymethylcytosine)), and the expression of regulatory enzyme DNMTs and TETs. Physoxia enhanced the functional profile of PSC including proliferation, metabolic activity, and stemness attributes. PSCs cultured in physoxia revealed the significant downregulation of DNMT3B, DNMT3L, TET1, and TET3 vs. air oxygen, accompanied by significantly reduced 5mC and 5hmC levels. The downregulation of DNMT3B was associated with an increase in its promoter methylation. Coupled with the above, we also noted decreased HIF1A but increased HIF2A expression in physoxia-cultured PSCs versus air oxygen. In conclusion, PSCs display oxygen-sensitive methylation patterns that correlate with the transcriptional and translational regulation of the de novo methylase DNMT3B.

Inappropriate DNA methylation of tumour suppressor genes can affect their expression and function, and as such, DNA methylation has been a promising target for anti-cancer therapy. In gynaecological malignancy, ovarian cancer has the highest associated rate of mortality. This study investigated the in vitro cytotoxicity of four sesquiterpene lactones (SLs), including dehydroleucodine (Deh), alantolactone (Ala), costunolide (Cos), and parthenolide (Pat), and their effect on DNA methylation in ovarian cancer. The binding of the SLs with DNA methyltransferases (DNMTs) were studied using molecular docking. The in vitro antiproliferative activity was evaluated in human ovarian cancer cell lines. SL-induced pro-apoptotic activity and effect on cell cycles was evaluated using enzymatic assays and flow cytometry. Global DNA methylation and DNMTs activity by SLs were measured using ELISA assays. DNA methylation and expression of tumour suppressor genes MHL1 and PTEN were quantified using pyrosequencing and RT-qPCR, respectively. SLs displayed strong binding to DNMT1, 3A and 3B in silico, with Deh exhibiting the strongest binding. SLs showed cytotoxicity in the order of Deh ≈ Ala > Pat > Cos, induced apoptosis, and arrested cell cycle at G2/M phase. Deh and Ala possessed DNMT inhibitory activity, reduced global and gene-specific DNA methylation, and increased expression of notable MHL1 and PTEN, which could contribute to the induction of apoptosis and cell death. In conclusion, Deh and Ala, possess strong cytotoxicity and hold the exciting potential as novel therapeutic agents in ovarian cancer.

Background: Historic evidence suggests up to 16% (approximately) of non-visible haematuria (NVH) referrals result in Urological cancer diagnosis. The majority are bladder cancers, for which flexible cystoscopy is regarded the “gold standard” diagnostic procedure. Recent changes to suspected cancer referral guidelines, public information campaigns and reduced smoking prevalence may have changed this percentage. We retrospectively calculated cancer detection rates from NVH referrals to assess whether flexible cystoscopy, an invasive and morbid procedure, remains necessary. Patients and methods: All patients referred to our University teaching hospital on a suspected (“twoweek”) cancer pathway with NVH over a 16-week period were included. Clinical and demographic data were collected for a series of 200 patients (96 male, age range 27–92, median 68). Results: Only eight patients had urological malignancy found (two renal and six bladder cancers). Both renal, and four bladder cancers, were identified on imaging prior to flexible cystoscopy. Only two bladder cancers were therefore detected by cystoscopy; one low-risk non-muscle invasive (patient has already been discharged) and one in a patient that was unfit for treatment (died of heart failure). Only seven (3.5%) of the patients were offered the option of not undergoing flexible cystoscopy. Conclusion: Our analyses suggest that flexible cystoscopy is rarely of benefit in patients with NVH. We suggest that patients should be given an accurate risk of bladder cancer diagnosis during the consent process. We advocate that flexible cystoscopy can be avoided for the majority of NVH referrals, particularly in patients without strong risk factors for urothelial cell carcinoma. Avoidance of flexible cystoscopy would reduce patient risks from procedural morbidity, reduce risks of acquiring coronavirus from hospital attendance, and there could be huge reductions in financial and service delivery demands in an overstretched secondary-care service.

Background Despite National Institute for Clinical Excellence (NICE) guidelines suggesting the use of urine cytology (UC) for the diagnosis of bladder cancer, its use is variable. Reasons for this include sub-optimal sensitivity, financial cost, availability of alternative tests, and uncertainty over interpretation of results. Anecdotally, however, suspicious or malignant UC when other investigations are normal, occasionally leads to a cancer diagnosis. Therefore, we retrospectively assessed a cohort of our haematuria patients to determine the value of UC in cancer diagnosis and the clinical significance of atypical UC (graded as C3). Patients and methods We identified 3018 patients with haematuria referred on the suspected cancer pathway (“two-week wait”) in 2015. We retrospectively analysed clinical, demographic, and follow-up/outcome data in a random cohort of 500 cases. Results Median follow up was 58 months. Urological malignancy was diagnosed in 61/500 patients; all were identified by cystoscopy or imaging, i.e., irrespective of UC result. No cases of atypical UC re-presented with a ‘missed’ cancer diagnosis within the five-year follow-up period. However, suspicious and malignant cytology was associated with high-grade/aggressive tumours and subsequent tumour recurrence. Conclusion Urine cytology did not identify any cancers that were not already found by imaging or cystoscopy. Atypical UC in the presence of negative haematuria investigations does not appear to be associated with malignancy, and therefore should not alter patient management nor prompt further investigation. Suspicious and malignant UC was associated with higher risk cancers and could therefore be used to prioritise waiting lists for transurethral resection of bladder tumour (TURBT), however, it is unclear whether this might benefit patient outcomes. We conclude therefore that UC has no role in haematuria investigations.

Supplies of intravesical Bacillus Calmette-Guérin (BCG), the first-line treatment for most intermediate- and high-risk non-muscle-invasive bladder cancers (NMIBC), have proven unreliable over the past decade. This review considers the evolution of BCG immunotherapy for NMIBC: from the discovery of the antitumour side effects of tuberculosis and subsequently the BCG vaccine, to recent advances in novel immunotherapeutic agents. We summarize the evidence for alternative options to standard intravesical BCG therapy regimens and describe the potential for immune response manipulating drugs in the treatment of NMIBC. These new agents, including immune checkpoint inhibitors, toll-like receptor agonists and recombinant viral vectors, may provide better options in the management of NMIBC in the future.

The application of physiological oxygen (physoxia) concentrations is becoming increasingly commonplace within a mammalian stem cell culture. Human mesenchymal stem cells (hMSCs) attract widespread interest for clinical application due to their unique immunomodulatory, multi-lineage potential, and regenerative capacities. Descriptions of the impact of physoxia on global DNA methylation patterns in hMSCs and the activity of enzymatic machinery responsible for its regulation remain limited. Human bone marrow-derived mesenchymal stem cells (BM-hMSCs, passage 1) isolated in reduced oxygen conditions displayed an upregulation of SOX2 in reduced oxygen conditions vs. air oxygen (21% O2, AO), while no change was noted for either OCT-4 or NANOG. DNA methylation marks 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) showed decreases in 2% O2 environment (workstation) (2% WKS). DNMT3B (DNA methyltransferase 3B) and TET1 (Ten-eleven translocation enzyme 1) displayed reduced transcription in physoxia. Consistent with transcriptional downregulation, we noted increased promoter methylation levels of DNMT3B in 2% WKS accompanied by reduced DNMT3B and TET1 protein expression. Finally, a decrease in HIF1A (Hypoxia-inducible factor 1A) gene expression in 2% WKS environment correlated with protein levels, while HIF2A was significantly higher in physoxia correlated with protein expression levels vs. AO. Together, these data have demonstrated, for the first time, that global 5mC, 5hmC, and DNMT3B are oxygen-sensitive in hMSCs. Further insights into the appropriate epigenetic regulation within hMSCs may enable increased safety and efficacy development within the therapeutic ambitions.

Skeletal muscle tissue demonstrates global hypermethylation with age. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 h, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alterations in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P. Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6–8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and HDMCs and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

Background and Objectives Bladder pain syndrome (BPS) is an uncommon and potentially debilitating spectrum of chronic pain typically accompanied by lower urinary tract symptoms. Intravesical hyaluronic acid (HA) is a commonly used treatment option, but requires multiple follow-up clinic appointments. We introduced a novel patient-led ‘at-home’ pathway of self-administered HA treatment to reduce the number hospital visits required by patients. We assessed and compared patient-reported outcome measures (PROMS) from patients receiving nurse-administered ‘in-hospital’ and patient-led self-administered ‘at-home’ intravesical HA (Cystistat® and Hyacyst®, respectively). Secondary outcome measures included differences between waiting times for treatment, frequency of treatments, number of clinician and nurse clinic appointments, and estimated financial costs. Patients and Methods Sixty consecutive patients commencing intravesical HA for BPS symptoms between 1st January 2016 and 31st March 2019 were included. O’Leary-Sant Interstitial Cystitis Symptom Index questionnaires were completed prior to, and following, six treatments. Relevant clinical and demographic data were also collected. Results Overall, 76.6% of the patients had improvement in symptoms after HA treatment. Mean O’Leary-Sant questionnaire symptom and problem scores were significantly improved following HA treatment (11.8 (range 6–17) to 8.5 (range 4–13) (p=0.00005) and 11.4 (range 4–16) to 7.9 (range 4–14) (p=0.0002), respectively. There were no significant differences in symptom improvements between patients on either pathway. Mean waiting time for treatment and number of treatments were lower in the patient-led pathway, and number of hospital visits was significantly lower in the patient-led at home pathway. The patient-led pathway could ‘save’ approximately 76 nurse clinic and 11 outpatient clinic appointments per year, and confer cost-savings of more than £1,500 per patient, per year. Conclusions Our case series analyses suggest that patient-led ‘at-home’ intravesical HA administration (Hyacyst®) is acceptable to patients and confers similar symptomatic benefit to ‘in-hospital’ nurse-led HA (Cystistat®). In addition, it appears that BPS sufferers established on the patient-led pathway require fewer HA treatments and fewer hospital visits, and as such, the patient-led pathway may also confer financial cost savings, and relieve some pressures on clinic appointment availability.

Several recent reports have described associations between gestational diabetes (GDM) and changes to the epigenomic landscape where the DNA samples were derived from either cord or placental sources. We employed genome-wide 450K array analysis to determine changes to the epigenome in a unique cohort of maternal blood DNA from 11 pregnant women prior to GDM development relative to matched controls. Hierarchical clustering segregated the samples into 2 distinct clusters comprising GDM and healthy pregnancies. Screening identified 100 CpGs with a mean β-value difference of ≥0.2 between cases and controls. Using stringent criteria, 5 CpGs (within COPS8, PIK3R5, HAAO, CCDC124, and C5orf34 genes) demonstrated potentials to be clinical biomarkers as revealed by differential methylation in 8 of 11 women who developed GDM relative to matched controls. We identified, for the first time, maternal methylation changes prior to the onset of GDM that may prove useful as biomarkers for early therapeutic intervention.

BACKGROUND:High-risk non-muscle invasive bladder cancer (HR-NMIBC) is a clinically unpredictable disease. Despite clinical risk estimation tools, many patients are undertreated with intra-vesical therapies alone, whereas others may be over-treated with early radical surgery. Molecular biomarkers, particularly DNA methylation, have been reported as predictive of tumour/patient outcomes in numerous solid organ and haematologic malignancies; however, there are few reports in HR-NMIBC and none using genome-wide array assessment. We therefore sought to identify novel DNA methylation markers of HR-NMIBC clinical outcomes that might predict tumour behaviour at initial diagnosis and help guide patient management. PATIENTS AND METHODS:A total of 21 primary initial diagnosis HR-NMIBC tumours were analysed by Illumina HumanMethylation450 BeadChip arrays and subsequently bisulphite Pyrosequencing. In all, 7 had not recurred at 1 year after resection and 14 had recurred and/or progressed despite intra-vesical BCG. A further independent cohort of 32 HR-NMIBC tumours (17 no recurrence and 15 recurrence and/or progression despite BCG) were also assessed by bisulphite Pyrosequencing. RESULTS:Array analyses identified 206 CpG loci that segregated non-recurrent HR-NMIBC tumours from clinically more aggressive recurrence/progression tumours. Hypermethylation of CpG cg11850659 and hypomethylation of CpG cg01149192 in combination predicted HR-NMIBC recurrence and/or progression within 1 year of diagnosis with 83% sensitivity, 79% specificity, and 83% positive and 79% negative predictive values. CONCLUSIONS:This is the first genome-wide DNA methylation analysis of a unique HR-NMIBC tumour cohort encompassing known 1-year clinical outcomes. Our analyses identified potential novel epigenetic markers that could help guide individual patient management in this clinically unpredictable disease.

High-grade non-muscle invasive bladder cancer (HG-NMIBC) is a clinically unpredictable disease with greater risks of recurrence and progression relative to their low-intermediate-grade counterparts. The molecular events, including those affecting the epigenome, that characterize this disease entity in the context of tumor development, recurrence, and progression, are incompletely understood. We therefore interrogated genome-wide DNA methylation using HumanMethylation450 BeadChip arrays in 21 primary HG-NMIBC tumors relative to normal bladder controls. Using strict inclusion-exclusion criteria we identified 1,057 hypermethylated CpGs within gene promoter-associated CpG islands, representing 256 genes. We validated the array data by bisulphite pyrosequencing and examined 25 array-identified candidate genes in an independent cohort of 30 HG-NMIBC and 18 low-intermediate-grade NMIBC. These analyses revealed significantly higher methylation frequencies in high-grade tumors relative to low-intermediate-grade tumors for the ATP5G2, IRX1 and VAX2 genes (P<0.05), and similarly significant increases in mean levels of methylation in high-grade tumors for the ATP5G2, VAX2, INSRR, PRDM14, VSX1, TFAP2b, PRRX1, and HIST1H4F genes (P<0.05). Although inappropriate promoter methylation was not invariantly associated with reduced transcript expression, a significant association was apparent for the ARHGEF4, PON3, STAT5a, and VAX2 gene transcripts (P<0.05). Herein, we present the first genome-wide DNA methylation analysis in a unique HG-NMIBC cohort, showing extensive and discrete methylation changes relative to normal bladder and low-intermediate-grade tumors. The genes we identified hold significant potential as targets for novel therapeutic intervention either alone, or in combination, with more conventional therapeutic options in the treatment of this clinically unpredictable disease.

Introduction Transgenic mice overexpressing the high mobility group A ( HMGA ) genes, Hmga1 or Hmga2 develop pituitary tumours and their overexpression is also a frequent finding in human pituitary adenomas. In some cases, increased expression of HMGA2 but not that of HMGA1 is consequent to genetic perturbations. However, recent studies show that down-regulation of microRNA (miRNA), that contemporaneously target the HMGA1 and HMGA2 transcripts, are associated with their overexpression. Results In a cohort of primary pituitary adenoma we determine the impact of epigenetic modifications on the expression of HMGA-targeting miRNA. For these miRNAs, chromatin immunoprecipitations showed that transcript down-regulation is correlated with histone tail modifications associated with condensed silenced genes. The functional impact of epigenetic modification on miRNA expression was determined in the rodent pituitary cell line, GH3. In these cells, histone tail, miRNA-associated, modifications were similar to those apparent in human adenoma and likely account for their repression. Indeed, challenge of GH3 cells with the epidrugs, zebularine and TSA, led to enrichment of the histone modification, H3K9Ac, associated with active genes, and depletion of the modification, H3K27me3, associated with silent genes and re-expression of HMGA-targeting miRNA. Moreover, epidrugs challenges were also associated with a concomitant decrease in hmga1 transcript and protein levels and concurrent increase in bmp-4 expression. Conclusions These findings show that the inverse relationship between HMGA expression and targeting miRNA is reversible through epidrug interventions. In addition to showing a mechanistic link between epigenetic modifications and miRNA expression these findings underscore their potential as therapeutic targets in this and other diseases.

Introduction Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis. Methods Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases. Results The ISL1 genes’ promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM). Conclusions In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.