Combined exome and transcriptome sequencing of non-muscle-invasive bladder cancer: associations between genomic changes, expression subtypes, and clinical outcomes.

BACKGROUND: Three-quarters of bladder cancer patients present with early-stage disease (non-muscle-invasive bladder cancer, NMIBC, UICC TNM stages Ta, T1 and Tis); however, most next-generation sequencing studies to date have concentrated on later-stage disease (muscle-invasive BC, stages T2+). We used exome and transcriptome sequencing to comprehensively characterise NMIBCs of all grades and stages to identify prognostic genes and pathways that could facilitate treatment decisions. Tumour grading is based upon microscopy and cellular appearances (grade 1 BCs are less aggressive, and grade 3 BCs are most aggressive), and we chose to also focus on the most clinically complex NMIBC subgroup, those patients with grade 3 pathological stage T1 (G3 pT1) disease. METHODS: Whole-exome and RNA sequencing were performed in total on 96 primary NMIBCs including 22 G1 pTa, 14 G3 pTa and 53 G3 pT1s, with both exome and RNA sequencing data generated from 75 of these individual samples. Associations between genomic alterations, expression profiles and progression-free survival (PFS) were investigated. RESULTS: NMIBCs clustered into 3 expression subtypes with different somatic alteration characteristics. Amplifications of ARNT and ERBB2 were significant indicators of worse PFS across all NMIBCs. High APOBEC mutagenesis and high tumour mutation burden were both potential indicators of better PFS in G3pT1 NMIBCs. The expression of individual genes was not prognostic in BCG-treated G3pT1 NMIBCs; however, downregulated interferon-alpha and gamma response pathways were significantly associated with worse PFS (adjusted p-value < 0.005). CONCLUSIONS: Multi-omic data may facilitate better prognostication and selection of therapeutic interventions in patients with G3pT1 NMIBC. These findings demonstrate the potential for improving the management of high-risk NMIBC patients and warrant further prospective validation

Defining the frequency of human papillomavirus and polyomavirus infection in urothelial bladder tumours

Given the contradictory nature of the literature regarding the role of human papillomaviruses and polyomaviruses in the pathogenesis of urothelial bladder cancer (UBC), we sought to investigate the frequency of their involvement in a large cohort of primary UBCs. DNA was extracted from 689 fresh-frozen UBC tissues and screened for the presence of high-risk human papillomavirus (HPV) types 16 and 18 and BKV/JCV genomic DNA by qPCR. In positive cases, viral identity was confirmed by Sanger sequencing and viral gene expression was analysed by RT-PCR or immunohistochemistry. All 689 UBCs were negative for HPV18. One UBC from a female patient with areas of squamous differentiation was positive for HPV16. The qPCR data indicated variable levels of polyomavirus in 49 UBCs. In the UBCs with low Cts we were able to confirm that 23 were BKV and 6 were JCV by Sanger sequencing. Polyomavirus large T antigen expression was low but detectable in 70% of the sequencing-confirmed polyomavirus positive samples. Thus, in United Kingdom patients, the presence of HPV DNA sequences is extremely rare in UBC (<1% of cases). Polyomavirus DNA (predominantly BKV) is more common in UBC, but still only detectable in 7% of cases and in many of these cases at low copy number. We have performed the largest virus screening to date in UBC, finding that HPV16, HPV18 and HPyV are unlikely to be common causative agents in UBC

Low-fidelity DNA synthesis by human DNA polymerase theta

Human DNA polymerase theta (pol θ or POLQ) is a proofreading-deficient family A enzyme implicated in translesion synthesis (TLS) and perhaps in somatic hypermutation (SHM) of immunoglobulin genes. These proposed functions and kinetic studies imply that pol θ may synthesize DNA with low fidelity. Here, we show that when copying undamaged DNA, pol θ generates single base errors at rates 10- to more than 100-fold higher than for other family A members. Pol θ adds single nucleotides to homopolymeric runs at particularly high rates, exceeding 1% in certain sequence contexts, and generates single base substitutions at an average rate of 2.4 × 10−3, comparable to inaccurate family Y human pol κ (5.8 × 10−3) also implicated in TLS. Like pol κ, pol θ is processive, implying that it may be tightly regulated to avoid deleterious mutagenesis. Pol θ also generates certain base substitutions at high rates within sequence contexts similar to those inferred to be copied by pol θ during SHM of immunoglobulin genes in mice. Thus, pol θ is an exception among family A polymerases, and its low fidelity is consistent with its proposed roles in TLS and SHM

Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

AimsModulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy.MethodsAn industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses.ResultsSeveral specific APE1 inhibitors were isolated by this approach. The IC(50) for APE1 inhibition ranged between 30 nM and 50 μM. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines.ConclusionsOur study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma

The Base Excision Repair Pathway Is Required for Efficient Lentivirus Integration

An siRNA screen has identified several proteins throughout the base excision repair (BER) pathway of oxidative DNA damage as important for efficient HIV infection. The proteins identified included early repair factors such as the base damage recognition glycosylases OGG1 and MYH and the late repair factor POLß, implicating the entire BER pathway. Murine cells with deletions of the genes Ogg1, Myh, Neil1 and Polß recapitulate the defect of HIV infection in the absence of BER. Defective infection in the absence of BER proteins was also seen with the lentivirus FIV, but not the gammaretrovirus MMLV. BER proteins do not affect HIV infection through its accessory genes nor the central polypurine tract. HIV reverse transcription and nuclear entry appear unaffected by the absence of BER proteins. However, HIV integration to the host chromosome is reduced in the absence of BER proteins. Pre-integration complexes from BER deficient cell lines show reduced integration activity in vitro. Integration activity is restored by addition of recombinant BER protein POLß. Lentiviral infection and integration efficiency appears to depend on the presence of BER proteins

Selenium and vitamin E for prevention of non–muscle-invasive bladder cancer recurrence and progression

Importance Selenium and vitamin E have been identified as promising agents for the chemoprevention of recurrence and progression of non–muscle-invasive bladder cancer. Objective To determine whether selenium and/or vitamin E may prevent disease recurrence in patients with newly diagnosed NMIBC. Design, Setting, and Participants This multicenter, prospective, double-blinded, placebo-controlled, 2 × 2 factorial randomized clinical trial included patients with newly diagnosed NMIBC recruited from 10 secondary or tertiary care hospitals in the UK. A total of 755 patients were screened for inclusion; 484 did not meet the inclusion criteria, and 1 declined to participate. A total of 270 patients were randomly assigned to 4 groups (selenium plus placebo, vitamin E plus placebo, selenium plus vitamin E, and placebo plus placebo) in a double-blind fashion between July 17, 2007, and October 10, 2011. Eligibility included initial diagnosis of NMIBC (stages Ta, T1, or Tis); randomization within 12 months of first transurethral resection was required. Interventions Oral selenium (200 μg/d of high-selenium yeast) and matched vitamin E placebo, vitamin E (200 IU/d of d-alfa-tocopherol) and matched selenium placebo, selenium and vitamin E, or placebo and placebo. Main Outcome and Measures Recurrence-free interval (RFI) on an intention-to-treat basis (analyses completed on November 28, 2022). Results The study randomized 270 patients (mean [SD] age, 68.9 [10.4] years; median [IQR] age, 69 [63-77] years; 202 male [75%]), with 65 receiving selenium and vitamin E placebo, 71 receiving vitamin E and selenium placebo, 69 receiving selenium and vitamin E, and 65 receiving both placebos. Median overall follow-up was 5.5 years (IQR, 5.1-6.1 years); 228 patients (84%) were followed up for more than 5 years. Median treatment duration was 1.5 years (IQR, 0.9-2.5 years). The study was halted because of slow accrual. For selenium (n = 134) vs no selenium (n = 136), there was no difference in RFI (hazard ratio, 0.92; 95% CI, 0.65-1.31; P = .65). For vitamin E (n = 140) vs no vitamin E (n = 130), there was a statistically significant detriment to RFI (hazard ratio, 1.46; 95% CI, 1.02-2.09; P = .04). No significant differences were observed for progression-free interval or overall survival time with either supplement. Results were unchanged after Cox proportional hazards regression modeling to adjust for known prognostic factors. In total, 1957 adverse events were reported; 85 were serious adverse events, and all were considered unrelated to trial treatment. Conclusions and Relevance In this randomized clinical trial of selenium and vitamin E, selenium supplementation did not reduce the risk of recurrence in patients with NMIBC, but vitamin E supplementation was associated with an increased risk of recurrence. Neither selenium nor vitamin E influenced progression or overall survival. Vitamin E supplementation may be harmful to patients with NMIBC, and elucidation of the underlying biology is required

The association between social capital and mental health and behavioural problems in children and adolescents: an integrative systematic review

Background Mental health is an important component of overall health and wellbeing and crucial for a happy and meaningful life. The prevalence of mental health problems amongst children and adolescent is high; with estimates suggesting 10-20% suffer from mental health problems at any given time. These mental health problems include internalising (e.g. depression and social anxiety) and externalising behavioural problems (e.g. aggression and anti-social behaviour). Although social capital has been shown to be associated with mental health/behavioural problems in young people, attempts to consolidate the evidence in the form of a review have been limited. This integrative systematic review identified and synthesised international research findings on the role and impact of family and community social capital on mental health/behavioural problems in children and adolescents to provide a consolidated evidence base to inform future research and policy development. Methods Nine electronic databases were searched for relevant studies and this was followed by hand searching. Identified literature was screened using review-specific inclusion/exclusion criteria, the data were extracted from the included studies and study quality was assessed. Heterogeneity in study design and outcomes precluded meta-analysis/meta-synthesis, the results are therefore presented in narrative form. Results After screening, 55 studies were retained. The majority were cross-sectional surveys and were conducted in North America (n = 33); seven were conducted in the UK. Samples ranged in size from 29 to 98,340. The synthesised results demonstrate that family and community social capital are associated with mental health/behavioural problems in children and adolescents. Positive parent–child relations, extended family support, social support networks, religiosity, neighbourhood and school quality appear to be particularly important. Conclusions To date, this is the most comprehensive review of the evidence on the relationships that exist between social capital and mental health/behavioural problems in children and adolescents. It suggests that social capital generated and mobilised at the family and community level can influence mental health/problem behaviour outcomes in young people. In addition, it highlights key gaps in knowledge where future research could further illuminate the mechanisms through which social capital works to influence health and wellbeing and thus inform policy development