Immunotherapy for non-muscle-invasive bladder cancer: from the origins of BCG to novel therapies.

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.

Lay abstract Many patients with non-invasive bladder cancers may need treatments into the bladder, including one called Bacillus Calmette-Guérin (BCG). Unfortunately, the supplies of BCG have been interrupted and somewhat unreliable since 2012. Because of this, we have been forced to look at other means of treating our patients using drugs similar to BCG. This has made us think about how BCG treatment was first developed more than 40 years ago and how it has evolved as a treatment for bladder cancer. In this article, we review the current uses of BCG and other treatments for bladder cancer and explore what the future may hold for bladder cancer treatment.

Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells.

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.

Novel intravesical therapeutics in the treatment of non-muscle invasive bladder cancer: Horizon scanning.

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.

Maternal genome-wide DNA methylation profiling in gestational diabetes shows distinctive disease-associated changes relative to matched healthy pregnancies.

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 ß-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.

HumanMethylation450K Array-Identified Biomarkers Predict Tumour Recurrence/Progression at Initial Diagnosis of High-risk Non-muscle Invasive Bladder Cancer.

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.

Quantitative genome-wide methylation analysis of high-grade non-muscle invasive bladder cancer.

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.

Methylation of HOXA9 and ISL1 Predicts Patient Outcome in High-Grade Non-Invasive Bladder Cancer.

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.