Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies

The evolutionary pattern of mutations in glioblastoma reveals therapy-mediated selection

Glioblastoma presents as a heterogeneous disease with poor prognosis despite the use of multimodal therapy. Analysis of genomic DNA changes between initial diagnosis and recurrence in response to standard treatment protocols would enhance understanding of disease progression and better inform new treatment strategies. A cohort of 21 patients with primary glioblastoma were examined between diagnosis and first recurrence. This study presented a rare opportunity to characterize molecular alterations in tumors observed in three patients who received no therapeutic intervention, other than surgery, offering a unique control. We focused this study by comparing the dynamic mutation profiles between the primary tumors and their matched recurrent counterparts. Molecular profiling of tumors was performed using multiplexed targeted deep sequencing of 409 well characterized cancer-associated genes, achieving a mean read depth of 1272 x. Three levels of evidence suggested an evolutionary pattern consistent with a response to therapy-mediated selection pressures exists in treated patients: 1) variant burden was reduced in recurrent tumors, 2) neutral evolutionary dynamics apparent in untreated tumors shifted toward a non-neutral mode of evolution in treated patients at recurrence, and 3) the recurrent tumor of one patient displayed an increased mutation rate attributable to a temozolomide-associated hypermutator phenotype. Our observations suggest that current treatment modalities are likely to fail in achieving long term remission with the majority of relapse samples containing distinct mutations when compared to primary diagnostic samples. Oncotarget 2018 9(8):7844-7858

Case-control study on uveal melanoma (RIFA): rational and design

BACKGROUND: Although a rare disease, uveal melanoma is the most common primary intraocular malignancy in adults, with an incidence rate of up to 1.0 per 100,000 persons per year in Europe. Only a few consistent risk factors have been identified for this disease. We present the study design of an ongoing incident case-control study on uveal melanoma (acronym: RIFA study) that focuses on radiofrequency radiation as transmitted by radio sets and wireless telephones, occupational risk factors, phenotypical characteristics, and UV radiation. METHODS/DESIGN: We conduct a case-control study to identify the role of different exposures in the development of uveal melanoma. The cases of uveal melanoma were identified at the Division of Ophthalmology, University of Essen, a referral centre for tumours of the eye. We recruit three control groups: population controls, controls sampled from those ophthalmologists who referred cases to the Division of Ophthalmology, University of Duisburg-Essen, and sibling controls. For each case the controls are matched on sex and age (five year groups), except for sibling controls. The data are collected from the study participants by short self-administered questionnaire and by telephone interview. During and at the end of the field phase, the data are quality-checked. To estimate the effect of exposures on uveal melanoma risk, we will use conditional logistic regression that accounts for the matching factors and allows to control for potential confounding

Tobacco smoking and gastric cancer: meta-analyses of published data versus pooled analyses of individual participant data (StoP Project).

Tobacco smoking is one of the main risk factors for gastric cancer, but the magnitude of the association estimated by conventional systematic reviews and meta-analyses might be inaccurate, due to heterogeneous reporting of data and publication bias. We aimed to quantify the combined impact of publication-related biases, and heterogeneity in data analysis or presentation, in the summary estimates obtained from conventional meta-analyses. We compared results from individual participant data pooled-analyses, including the studies in the Stomach Cancer Pooling (StoP) Project, with conventional meta-analyses carried out using only data available in previously published reports from the same studies. From the 23 studies in the StoP Project, 20 had published reports with information on smoking and gastric cancer, but only six had specific data for gastric cardia cancer and seven had data on the daily number of cigarettes smoked. Compared to the results obtained with the StoP database, conventional meta-analyses overvalued the relation between ever smoking (summary odds ratios ranging from 7% higher for all studies to 22% higher for the risk of gastric cardia cancer) and yielded less precise summary estimates (SE ≤2.4 times higher). Additionally, funnel plot asymmetry and corresponding hypotheses tests were suggestive of publication bias. Conventional meta-analyses and individual participant data pooled-analyses reached similar conclusions on the direction of the association between smoking and gastric cancer. However, published data tended to overestimate the magnitude of the effects, possibly due to publication biases and limited the analyses by different levels of exposure or cancer subtypes

Replication of Lung Cancer Susceptibility Loci at Chromosomes 15q25, 5p15, and 6p21: A Pooled Analysis From the International Lung Cancer Consortium

Background Genome-wide association studies have identified three chromosomal regions at 15q25, 5p15, and 6p21 as being associated with the risk of lung cancer. To confirm these associations in independent studies and investigate heterogeneity of these associations within specific subgroups, we conducted a coordinated genotyping study within the International Lung Cancer Consortium based on independent studies that were not included in previous genome-wide association studies. Methods Genotype data for single-nucleotide polymorphisms at chromosomes 15q25 (rs16969968, rs8034191), 5p15 (rs2736100, rs402710), and 6p21 (rs2256543, rs4324798) from 21 case-control studies for 11 645 lung cancer case patients and 14 954 control subjects, of whom 85% were white and 15% were Asian, were pooled. Associations between the variants and the risk of lung cancer were estimated by logistic regression models. All statistical tests were two-sided. Results Associations between 15q25 and the risk of lung cancer were replicated in white ever-smokers (rs16969968: odds ratio [OR] = 1.26, 95% confidence interval [CI] = 1.21 to 1.32, Ptrend = 2 × 10−26), and this association was stronger for those diagnosed at younger ages. There was no association in never-smokers or in Asians between either of the 15q25 variants and the risk of lung cancer. For the chromosome 5p15 region, we confirmed statistically significant associations in whites for both rs2736100 (OR = 1.15, 95% CI = 1.10 to 1.20, Ptrend = 1 × 10−10) and rs402710 (OR = 1.14, 95% CI = 1.09 to 1.19, Ptrend = 5 × 10−8) and identified similar associations in Asians (rs2736100: OR = 1.23, 95% CI = 1.12 to 1.35, Ptrend = 2 × 10−5; rs402710: OR = 1.15, 95% CI = 1.04 to 1.27, Ptrend = .007). The associations between the 5p15 variants and lung cancer differed by histology; odds ratios for rs2736100 were highest in adenocarcinoma and for rs402710 were highest in adenocarcinoma and squamous cell carcinomas. This pattern was observed in both ethnic groups. Neither of the two variants on chromosome 6p21 was associated with the risk of lung cancer. Conclusions In this international genetic association study of lung cancer, previous associations found in white populations were replicated and new associations were identified in Asian populations. Future genetic studies of lung cancer should include detailed stratification by histolog

Imprinted CDKN1C Is a Tumor Suppressor in Rhabdoid Tumor and Activated by Restoration of SMARCB1 and Histone Deacetylase Inhibitors

SMARCB1 is deleted in rhabdoid tumor, an aggressive paediatric malignancy affecting the kidney and CNS. We hypothesized that the oncogenic pathway in rhabdoid tumors involved epigenetic silencing of key cell cycle regulators as a consequence of altered chromatin-remodelling, attributable to loss of SMARCB1, and that this hypothesis if proven could provide a biological rationale for testing epigenetic therapies in this disease. We used an inducible expression system to show that the imprinted cell cycle inhibitor CDKN1C is a downstream target for SMARCB1 and is transcriptionally activated by increased histone H3 and H4 acetylation at the promoter. We also show that CDKN1C expression induces cell cycle arrest, CDKN1C knockdown with siRNA is associated with increased proliferation, and is able to compete against the anti-proliferative effect of restored SMARCB1 expression. The histone deacetylase inhibitor (HDACi), Romidepsin, specifically restored CDKN1C expression in rhabdoid tumor cells through promoter histone H3 and H4 acetylation, recapitulating the effect of SMARCB1 on CDKNIC allelic expression, and induced cell cycle arrest in G401 and STM91-01 rhabdoid tumor cell lines. CDKN1C expression was also shown to be generally absent in clinical specimens of rhabdoid tumor, however CDKN1A and CDKN1B expression persisted. Our observations suggest that maintenance of CDKN1C expression plays a critical role in preventing rhabdoid tumor growth. Significantly, we report for the first time, parallels between the molecular pathways of SMARCB1 restoration and Romidepsin treatment, and demonstrate a biological basis for the further exploration of histone deacetylase inhibitors as relevant therapeutic reagents in the treatment of rhabdoid tumor

Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome

Abstract Background Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with a population frequency of approximately 1 in 10,000. The most common epigenetic defect in BWS is a loss of methylation (LOM) at the 11p15.5 imprinting centre, KCNQ1OT1 TSS-DMR, and affects 50% of cases. We hypothesised that genetic factors linked to folate metabolism may play a role in BWS predisposition via effects on methylation maintenance at KCNQ1OT1 TSS-DMR. Results Single nucleotide variants (SNVs) in the folate pathway affecting methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), 5-methyltetrahydrofolate-homocysteine S-methyltransferase (MTR), cystathionine beta-synthase (CBS) and methionine adenosyltransferase (MAT1A) were examined in 55 BWS patients with KCNQ1OT1 TSS-DMR LOM and in 100 unaffected cases. MTHFR rs1801133: C>T was more prevalent in BWS with KCNQ1OT1 TSS-DMR LOM (p T, rs150331990: A>G and rs757460628: G>A encoding NP_001124295 p.Arg136Cys, p.His1118Arg and p.Arg1223His, respectively. These variants have population frequencies of less than 1 in 1000 and were absent from 100 control cases. Functional characterization using a hemimethylated DNA trapping assay revealed a reduced methyltransferase activity relative to wild-type DNMT1 for each variant ranging from 40 to 70% reduction in activity. Conclusions This study is the first to examine folate pathway genetics in BWS and to identify rare DNMT1 missense variants in affected individuals. Our data suggests that reduced DNMT1 activity could affect maintenance of methylation at KCNQ1OT1 TSS-DMR in some cases of BWS, possibly via a maternal effect in the early embryo. Larger cohort studies are warranted to further interrogate the relationship between impaired MTHFR enzymatic activity attributable to MTHFR rs1801133: C>T, dietary folate intake and BWS

Glioma Through the Looking GLASS: Molecular Evolution of Diffuse Gliomas and the Glioma Longitudinal AnalySiS Consortium

Adult diffuse gliomas are a diverse group of brain neoplasms that inflict a high emotional toll on patients and their families. The Cancer Genome Atlas (TCGA) and similar projects have provided a comprehensive understanding of the somatic alterations and molecular subtypes of glioma at diagnosis. However, gliomas undergo significant cellular and molecular evolution during disease progression. We review the current knowledge on the genomic and epigenetic abnormalities in primary tumors and after disease recurrence, highlight the gaps in the literature, and elaborate on the need for a new multi-institutional effort to bridge these knowledge gaps and how the Glioma Longitudinal AnalySiS Consortium (GLASS) aims to systemically catalog the longitudinal changes in gliomas. The GLASS initiative will provide essential insights into the evolution of glioma toward a lethal phenotype, with the potential to reveal targetable vulnerabilities, and ultimately, improved outcomes for a patient population in need

Measuring performance on the Healthcare Access and Quality Index for 195 countries and territories and selected subnational locations: A systematic analysis from the Global Burden of Disease Study 2016

Copyright © 2018 The Author(s). Published by Elsevier Ltd. Background A key component of achieving universal health coverage is ensuring that all populations have access to quality health care. Examining where gains have occurred or progress has faltered across and within countries is crucial to guiding decisions and strategies for future improvement. We used the Global Burden of Diseases, Injuries, and Risk Factors Study 2016 (GBD 2016) to assess personal health-care access and quality with the Healthcare Access and Quality (HAQ) Index for 195 countries and territories, as well as subnational locations in seven countries, from 1990 to 2016. Methods Drawing from established methods and updated estimates from GBD 2016, we used 32 causes from which death should not occur in the presence of effective care to approximate personal health-care access and quality by location and over time. To better isolate potential effects of personal health-care access and quality from underlying risk factor patterns, we risk-standardised cause-specific deaths due to non-cancers by location-year, replacing the local joint exposure of environmental and behavioural risks with the global level of exposure. Supported by the expansion of cancer registry data in GBD 2016, we used mortality-to-incidence ratios for cancers instead of risk-standardised death rates to provide a stronger signal of the effects of personal health care and access on cancer survival. We transformed each cause to a scale of 0-100, with 0 as the first percentile (worst) observed between 1990 and 2016, and 100 as the 99th percentile (best); we set these thresholds at the country level, and then applied them to subnational locations. We applied a principal components analysis to construct the HAQ Index using all scaled cause values, providing an overall score of 0-100 of personal health-care access and quality by location over time. We then compared HAQ Index levels and trends by quintiles on the Socio-demographic Index (SDI), a summary measure of overall development. As derived from the broader GBD study and other data sources, we examined relationships between national HAQ Index scores and potential correlates of performance, such as total health spending per capita. Findings In 2016, HAQ Index performance spanned from a high of 97·1 (95% UI 95·8-98·1) in Iceland, followed by 96·6 (94·9-97·9) in Norway and 96·1 (94·5-97·3) in the Netherlands, to values as low as 18·6 (13·1-24·4) in the Central African Republic, 19·0 (14·3-23·7) in Somalia, and 23·4 (20·2-26·8) in Guinea-Bissau. The pace of progress achieved between 1990 and 2016 varied, with markedly faster improvements occurring between 2000 and 2016 for many countries in sub-Saharan Africa and southeast Asia, whereas several countries in Latin America and elsewhere saw progress stagnate after experiencing considerable advances in the HAQ Index between 1990 and 2000. Striking subnational disparities emerged in personal health-care access and quality, with China and India having particularly large gaps between locations with the highest and lowest scores in 2016. In China, performance ranged from 91·5 (89·1-93·6) in Beijing to 48·0 (43·4-53·2) in Tibet (a 43·5-point difference), while India saw a 30·8-point disparity, from 64·8 (59·6-68·8) in Goa to 34·0 (30·3-38·1) in Assam. Japan recorded the smallest range in subnational HAQ performance in 2016 (a 4·8-point difference), whereas differences between subnational locations with the highest and lowest HAQ Index values were more than two times as high for the USA and three times as high for England. State-level gaps in the HAQ Index in Mexico somewhat narrowed from 1990 to 2016 (from a 20·9-point to 17·0-point difference), whereas in Brazil, disparities slightly increased across states during this time (a 17·2-point to 20·4-point difference). Performance on the HAQ Index showed strong linkages to overall development, with high and high-middle SDI countries generally having higher scores and faster gains for non-communicable diseases. Nonetheless, countries across the development spectrum saw substantial gains in some key health service areas from 2000 to 2016, most notably vaccine-preventable diseases. Overall, national performance on the HAQ Index was positively associated with higher levels of total health spending per capita, as well as health systems inputs, but these relationships were quite heterogeneous, particularly among low-to-middle SDI countries. Interpretation GBD 2016 provides a more detailed understanding of past success and current challenges in improving personal health-care access and quality worldwide. Despite substantial gains since 2000, many low-SDI and middle- SDI countries face considerable challenges unless heightened policy action and investments focus on advancing access to and quality of health care across key health services, especially non-communicable diseases. Stagnating or minimal improvements experienced by several low-middle to high-middle SDI countries could reflect the complexities of re-orienting both primary and secondary health-care services beyond the more limited foci of the Millennium Development Goals. Alongside initiatives to strengthen public health programmes, the pursuit of universal health coverage hinges upon improving both access and quality worldwide, and thus requires adopting a more comprehensive view - and subsequent provision - of quality health care for all populations