DNA methylation changes from primary cultures through senescence-bypass in Syrian hamster fetal cells initially exposed to benzo[a]pyrene

Current chemical testing strategies are limited in their ability to detect non-genotoxic carcinogens (NGTxC). Epigenetic anomalies develop during carcinogenesis regardless of whether the molecular initiating event is associated with genotoxic (GTxC) or NGTxC events; therefore, epigenetic markers may be harnessed to develop new approach methodologies that improve the detection of both types of carcinogens. This study used Syrian hamster fetal cells to establish the chronology of carcinogen-induced DNA methylation changes from primary cells until senescence-bypass as an essential carcinogenic step. Cells exposed to solvent control for 7 days were compared to naïve primary cultures, to cells exposed for 7 days to benzo[a]pyrene, and to cells at the subsequent transformation stages: normal colonies, morphologically transformed colonies, senescence, senescence-bypass, and sustained proliferation in vitro. DNA methylation changes identified by reduced representation bisulphite sequencing were minimal at day-7. Profound DNA methylation changes arose during cellular senescence and some of these early differentially methylated regions (DMRs) were preserved through the final sustained proliferation stage. A set of these DMRs (e.g., Pou4f1, Aifm3, B3galnt2, Bhlhe22, Gja8, Klf17, and L1l) were validated by pyrosequencing and their reproducibility was confirmed across multiple clones obtained from a different laboratory. These DNA methylation changes could serve as biomarkers to enhance objectivity and mechanistic understanding of cell transformation and could be used to predict senescence-bypass and chemical carcinogenicity

Pediatric pan-central nervous system tumor analysis of immune-cell infiltration identifies correlates of antitumor immunity

Here, using methylCIBERSORT, the authors characterize the tumour-immune microenvironment of paediatric central nervous system (CNS) tumours and its association with tumour type and prognosis. These findings suggest that immuno-methylomic profiling may inform immunotherapy approaches in paediatric patients with CNS tumour

SARS-like WIV1-CoV poised for human emergence

The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV highlights the continued risk of cross-species transmission leading to epidemic disease. This manuscript describes efforts to extend surveillance beyond sequence analysis, constructing chimeric and full-length zoonotic coronaviruses to evaluate emergence potential. Focusing on SARS-like virus sequences isolated from Chinese horseshoe bats, the results indicate a significant threat posed by WIV1-CoV. Both full-length and chimeric WIV1-CoV readily replicated efficiently in human airway cultures and in vivo, suggesting capability of direct transmission to humans. In addition, while monoclonal antibody treatments prove effective, the SARS-based vaccine approach failed to confer protection. Together, the study indicates an ongoing threat posed by WIV1-related viruses and the need for continued study and surveillance

DNA methylation-based classification of glioneuronal tumours synergises with histology and radiology to refine accurate molecular stratification

AIMS: Glioneuronal tumours (GNTs) are poorly distinguished by their histology and lack robust diagnostic indicators. Previously, we showed that common GNTs comprise two molecularly distinct groups, correlating poorly with histology. To refine diagnosis, we constructed a methylation-based model for GNT classification, subsequently evaluating standards for molecular stratification by methylation, histology and radiology. METHODS: We comprehensively analysed methylation, radiology and histology for 83 GNT samples: a training cohort of 49, previously classified into molecularly defined groups by genomic profiles, plus a validation cohort of 34. We identified histological and radiological correlates to molecular classification and constructed a methylation-based support vector machine (SVM) model for prediction. Subsequently, we contrasted methylation, radiological and histological classifications in validation GNTs. RESULTS: By methylation clustering, all training and 23/34 validation GNTs segregated into two groups, the remaining 11 clustering alongside control cortex. Histological review identified prominent astrocytic/oligodendrocyte-like components, dysplastic neurons, and a specific glioneuronal element as discriminators between groups. However, these were present in only a subset of tumours. Radiological review identified location, margin definition, enhancement, and T2 FLAIR-rim sign as discriminators. When validation GNTs were classified by SVM, 22/23 classified correctly, comparing favourably against histology and radiology which resolved 17/22 and 15/21 respectively where data were available for comparison. CONCLUSIONS: Diagnostic criteria inadequately reflect glioneuronal tumour biology, leaving a proportion unresolvable. In the largest cohort of molecularly defined glioneuronal tumours, we develop molecular, histological, and radiological approaches for biologically meaningful classification and demonstrate almost all cases are resolvable, emphasising the importance of an integrated diagnostic approach

Symptom-based stratification of patients with primary Sjögren's syndrome: multi-dimensional characterisation of international observational cohorts and reanalyses of randomised clinical trials

Background Heterogeneity is a major obstacle to developing effective treatments for patients with primary Sjögren's syndrome. We aimed to develop a robust method for stratification, exploiting heterogeneity in patient-reported symptoms, and to relate these differences to pathobiology and therapeutic response. Methods We did hierarchical cluster analysis using five common symptoms associated with primary Sjögren's syndrome (pain, fatigue, dryness, anxiety, and depression), followed by multinomial logistic regression to identify subgroups in the UK Primary Sjögren's Syndrome Registry (UKPSSR). We assessed clinical and biological differences between these subgroups, including transcriptional differences in peripheral blood. Patients from two independent validation cohorts in Norway and France were used to confirm patient stratification. Data from two phase 3 clinical trials were similarly stratified to assess the differences between subgroups in treatment response to hydroxychloroquine and rituximab. Findings In the UKPSSR cohort (n=608), we identified four subgroups: Low symptom burden (LSB), high symptom burden (HSB), dryness dominant with fatigue (DDF), and pain dominant with fatigue (PDF). Significant differences in peripheral blood lymphocyte counts, anti-SSA and anti-SSB antibody positivity, as well as serum IgG, κ-free light chain, β2-microglobulin, and CXCL13 concentrations were observed between these subgroups, along with differentially expressed transcriptomic modules in peripheral blood. Similar findings were observed in the independent validation cohorts (n=396). Reanalysis of trial data stratifying patients into these subgroups suggested a treatment effect with hydroxychloroquine in the HSB subgroup and with rituximab in the DDF subgroup compared with placebo. Interpretation Stratification on the basis of patient-reported symptoms of patients with primary Sjögren's syndrome revealed distinct pathobiological endotypes with distinct responses to immunomodulatory treatments. Our data have important implications for clinical management, trial design, and therapeutic development. Similar stratification approaches might be useful for patients with other chronic immune-mediated diseases. Funding UK Medical Research Council, British Sjogren's Syndrome Association, French Ministry of Health, Arthritis Research UK, Foundation for Research in Rheumatology

SARS-like WIV1-CoV poised for human emergence

Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations

A mechanistic evaluation of the Syrian hamster embryo cell transformation assay (pH 6.7) and molecular events leading to senescence bypass in SHE cells

The implementation of the Syrian hamster embryo cell transformation assay (SHE CTA) into test batteries and its relevance in predicting carcinogenicity has been long debated. Despite prevalidation studies to ensure reproducibility and minimise the subjective nature of the assay’s endpoint, an underlying mechanistic and molecular basis supporting morphological transformation (MT) as an indicator of carcinogenesis is still missing. We found that only 20 % of benzo(a)pyrene-induced MT clones immortalised suggesting that, alone, the MT phenotype is insufficient for senescence bypass. From a total of 12 B(a)P- immortalised MT lines, inactivating p53 mutations were identified in 30 % of clones, and the majority of these were consistent with the potent carcinogen’s mode of action. Expression of p16 was commonly silenced or markedly reduced with extensive promoter methylation observed in 45 % of MT clones, while Bmi1 was strongly upregulated in 25 % of clones. In instances where secondary events to MT appeared necessary for senescence bypass, as evidenced by a transient cellular crisis, clonal growth correlated with monoallelic deletion of the CDKN2A/B locus. The findings further implicate the importance of p16 and p53 pathways in regulating senescence while providing a molecular evaluation of SHE CTA -derived variant MT clones induced by benzo(a)pyrene