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Lung diseases, such as cystic fibrosis and COPD, are characterized by mucus obstruction and chronic airway inflammation, but their mechanistic link remains poorly understood. Here, we focus on the function of the mucostatic airway microenvironment on epigenetic reprogramming of airway macrophages (AM) and resulting transcriptomic and phenotypical changes. Using a mouse model of muco-obstructive lung disease (Scnn1b-transgenic), we identify epigenetically controlled, differentially regulated pathways and transcription factors involved in inflammatory responses and macrophage polarization. Functionally, AMs from Scnn1b-transgenic mice have reduced efferocytosis and phagocytosis, and excessive inflammatory responses upon lipopolysaccharide challenge, mediated through enhanced Irf1 function and expression. Ex vivo stimulation of wild-type AMs with native mucus impairs efferocytosis and phagocytosis capacities. In addition, mucus induces gene expression changes, comparable with those observed in AMs from Scnn1b-transgenic mice. Our data show that mucostasis induces epigenetic reprogramming of AMs, leading to changes favoring tissue damage and disease progression. Targeting these altered AMs may support therapeutic approaches in patients with muco-obstructive lung diseases., Muco-obstructive lung diseases are characterised by airway macrophage (AM) populations which may have epigenetic changes. Here using a mouse model the authors show epigenetic alteration of AMs with changes in LPS response, phagocytosis and efferocytosis similar to culture with mucus in vitro.

The high plasticity of lung epithelial cells, has for many years, confounded the correct identification of the cell-of-origin of lung adenocarcinoma (LUAD), one of the deadliest malignancies worldwide. Here, we employ lineage-tracing mouse models to investigate the cell of origin of Eml4-Alk LUAD, and show that Club and Alveolar type 2 (AT2) cells give rise to tumours. We focus on Club cell originated tumours and find that Club cells experience an epigenetic switch by which they lose their lineage fidelity and gain an AT2-like phenotype after oncogenic transformation. Single-cell transcriptomic analyses identified two trajectories of Club cell evolution which are similar to the ones used during lung regeneration, suggesting that lung epithelial cells leverage on their plasticity and intrinsic regeneration mechanisms to give rise to a tumour. Together, this study highlights the role of Club cells in LUAD initiation, identifies the mechanism of Club cell lineage infidelity, confirms the presence of these features in human tumours, and unveils key mechanisms conferring LUAD heterogeneity.

Context Adipose tissue inflammation and dysregulated energy homeostasis are key mechanisms linking obesity and cancer. Distinct adipose tissue depots strongly differ in their metabolic profiles; however, comprehensive studies of depot-specific perturbations among patients with cancer are lacking. Objective We compared transcriptome profiles of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) from patients with colorectal cancer and assessed the associations of different anthropometric measures with depot-specific gene expression. Design Whole transcriptomes of VAT and SAT were measured in 233 patients from the ColoCare Study, and visceral and subcutaneous fat area were quantified via CT. Results VAT compared with SAT showed elevated gene expression of cytokines, cell adhesion molecules, and key regulators of metabolic homeostasis. Increased fat area was associated with downregulated lipid and small molecule metabolism and upregulated inflammatory pathways in both compartments. Comparing these patterns between depots proved specific and more pronounced gene expression alterations in SAT and identified unique associations of integrins and lipid metabolism–related enzymes. VAT gene expression patterns that were associated with visceral fat area poorly overlapped with patterns associated with self-reported body mass index (BMI). However, subcutaneous fat area and BMI showed similar associations with SAT gene expression. Conclusions This large-scale human study demonstrates pronounced disparities between distinct adipose tissue depots and reveals that BMI poorly correlates with fat mass–associated changes in VAT. Taken together, these results provide crucial evidence for the necessity to differentiate between distinct adipose tissue depots for a correct characterization of gene expression profiles that may affect metabolic health of patients with colorectal cancer.

International audience; Adult "T cell" acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that is associated with poor outcomes, requiring additional therapeutic options. The DNA methylation landscapes of adult T-ALL remain undercharacterized. Here, we systematically analyzed the DNA methylation profiles of normal thymic-sorted T cell subpopulations and 143 primary adult T-ALLs as part of the French GRAALL 2003–2005 trial. Our results indicated that T-ALL is epigenetically heterogeneous consisting of five subtypes (C1-C5), which were either associated with co-occurring DNA methyltransferase 3 alpha (DNMT3A)/isocitrate dehydrogenase [NADP(+)] 2 (IDH2) mutations (C1), TAL bHLH transcription factor 1, erythroid differentiation factor (TAL1) deregulation (C2), T cell leukemia homeobox 3 (TLX3) (C3), TLX1/in cis-homeobox A9 (HOXA9) (C4), or in trans-HOXA9 overexpression (C5). Integrative analysis of DNA methylation and gene expression identified potential cluster-specific oncogenes and tumor suppressor genes. In addition to an aggressive hypomethylated subgroup (C1), our data identified an unexpected subset of hypermethylated T-ALL (C5) associated with poor outcome and primary therapeutic response. Using mouse xenografts, we demonstrated that hypermethylated T-ALL samples exhibited therapeutic responses to the DNA hypomethylating agent 5-azacytidine, which significantly (survival probability; P = 0.001 for C3, 0.01 for C4, and 0.0253 for C5) delayed tumor progression. These findings suggest that epigenetic-based therapies may provide an alternative treatment option in hypermethylated T-ALL.

The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes., Helmholtz Association, Korean National Cancer Center, Korean National Research Fund, Oxford NIHR BRC, Arthritis Research UK, Projekt DEAL

An individual’s inherited genetic variation may contribute to the ‘angiogenic switch’, which is essential for blood supply and tumor growth of microscopic and macroscopic tumors. Polymorphisms in angiogenesis-related genes potentially predispose to colorectal cancer (CRC) or affect the survival of CRC patients. We investigated the association of 392 single nucleotide polymorphisms (SNPs) in 33 angiogenesis-related genes with CRC risk and survival of CRC patients in 1754 CRC cases and 1781 healthy controls within DACHS (Darmkrebs: Chancen der Verhütung durch Screening), a German population-based case-control study. Odds ratios and 95% confidence intervals (CI) were estimated from unconditional logistic regression to test for genetic associations with CRC risk. The Cox proportional hazard model was used to estimate hazard ratios (HR) and 95% CIs for survival. Multiple testing was adjusted for by a false discovery rate. No variant was associated with CRC risk. Variants in EFNB2, MMP2 and JAG1 were significantly associated with overall survival. The association of the EFNB2 tagging SNP rs9520090 (p < 0.0001) was confirmed in two validation datasets (p-values: 0.01 and 0.05). The associations of the tagging SNPs rs6040062 in JAG1 (p-value 0.0003) and rs2241145 in MMP2 (p-value 0.0005) showed the same direction of association with overall survival in the first and second validation sets, respectively, although they did not reach significance (p-values: 0.09 and 0.25, respectively). EFNB2, MMP2 and JAG1 are known for their functional role in angiogenesis and the present study points to novel evidence for the impact of angiogenesis-related genetic variants on the CRC outcome.

Background In cancer, normal epigenetic patterns are disturbed and contribute to gene expression changes, disease onset, and progression. The cancer epigenome is composed of the epigenetic patterns present in the tumor-initiating cell at the time of transformation, and the tumor-specific epigenetic alterations that are acquired during tumor initiation and progression. The precise dissection of these two components of the tumor epigenome will facilitate a better understanding of the biological mechanisms underlying malignant transformation. Chronic lymphocytic leukemia (CLL) originates from differentiating B cells, which undergo extensive epigenetic programming. This poses the challenge to precisely determine the epigenomic ground state of the cell-of-origin in order to identify CLL-specific epigenetic aberrations. Methods We developed a linear regression model, methylome-based cell-of-origin modeling (Methyl-COOM), to map the cell-of-origin for individual CLL patients based on the continuum of epigenomic changes during normal B cell differentiation. Results Methyl-COOM accurately maps the cell-of-origin of CLL and identifies CLL-specific aberrant DNA methylation events that are not confounded by physiologic epigenetic B cell programming. Furthermore, Methyl-COOM unmasks abnormal action of transcription factors, altered super-enhancer activities, and aberrant transcript expression in CLL. Among the aberrantly regulated transcripts were many genes that have previously been implicated in T cell biology. Flow cytometry analysis of these markers confirmed their aberrant expression on malignant B cells at the protein level. Conclusions Methyl-COOM analysis of CLL identified disease-specific aberrant gene regulation. The aberrantly expressed genes identified in this study might play a role in immune-evasion in CLL and might serve as novel targets for immunotherapy approaches. In summary, we propose a novel framework for in silico modeling of reference DNA methylomes and for the identification of cancer-specific epigenetic changes, a concept that can be broadly applied to other human malignancies.

DNA methylation is recognized as one of several epigenetic regulators of gene expression and as potential driver of carcinogenesis through gene-silencing of tumor suppressors and activation of oncogenes. However, abnormal methylation, even of promoter regions, does not necessarily alter gene expression levels, especially if the gene is already silenced, leaving the exact mechanisms of methylation unanswered. Using a large cohort of matching DNA methylation and gene expression samples of colorectal cancer (CRC; n = 77) and normal adjacent mucosa tissues (n = 108), we investigated the regulatory role of methylation on gene expression. We show that on a subset of genes enriched in common cancer pathways, methylation is significantly associated with gene regulation through gene-specific mechanisms. We built two classification models to infer gene regulation in CRC from methylation differences of tumor and normal tissues, taking into account both gene-silencing and gene-activation effects through hyper- and hypo-methylation of CpGs. The classification models result in high prediction performances in both training and independent CRC testing cohorts (0.92

The aim of this work is to present the changes in classical illicit and licit drug, as well as stimulant designer drug (SDD) consumption of suspected drug users in South-East Hungary between 2008 and 2015. Urine and/or blood samples of 2976 subjects were analyzed for these groups of substances of which 1777 (59.7%) were tested positive. THC was the most frequent (32.2%) substance, followed by classical stimulants (amphetamine, metamphetamine, MDMA, cocain) (21.4%), SDDs (17.0%), benzodiazepines (15.5%), medical opiates (codeine without morphine, methadone, tramadol) (4.03%), and morphine with or without 6-acethyl-morphine (1.98%). The annual rate of cannabis consumption continuously decreased after 2010. The use of classical stimulants was constant, except for a significant increase in 2015. Benzodiazepine incidence increased and remained steady after 2011. Medical opiate and morphine frequency was variable. SDDs were found in the highest number in 2012-13, exceeding the frequency of classical stimulants. The most prevalent SDDs were as follows: 2010 - mephedrone, 2011 - 4-MEC, methylone, MDPV, 4-FMC, and 4-FA, and 2012-2015 - pentedrone. Beside pentedrone, 3-MMC, αPVP, αPHP, and 4-CMC were detected with a notable number in this period. Multi-drug use was found in 30-43% of suspects tested positive between 2008 and 2014, which elevated to 52% in 2015. The frequency of substances in the biological samples corresponded to their seizure rate. When SDDs were included on the NPS list, their frequency in biological samples and in seized materials slightly decreased or did not change. However, a marked decrease was observed following classification as illicit drugs.

Background: Epigenetic disturbances are crucial in cancer initiation, potentially with pleiotropic effects, and may be influenced by the genetic background. Methods: In a subsets (ASSET) meta-analytic approach, we investigated associations of genetic variants related to epigenetic mechanisms with risks of breast, lung, colorectal, ovarian and prostate carcinomas using 51,724 cases and 52,001 controls. False discovery rate–corrected P values (q values < 0.05) were considered statistically significant. Results: Among 162,887 imputed or genotyped variants in 555 candidate genes, SNPs in eight genes were associated with risk of more than one cancer type. For example, variants in BABAM1 were confirmed as a susceptibility locus for squamous cell lung, overall breast, estrogen receptor (ER)–negative breast, and overall prostate, and overall serous ovarian cancer; the most significant variant was rs4808076 [OR = 1.14; 95% confidence interval (CI) = 1.10–1.19; q = 6.87 × 10−5]. DPF1 rs12611084 was inversely associated with ER-negative breast, endometrioid ovarian, and overall and aggressive prostate cancer risk (OR = 0.93; 95% CI = 0.91–0.96; q = 0.005). Variants in L3MBTL3 were associated with colorectal, overall breast, ER-negative breast, clear cell ovarian, and overall and aggressive prostate cancer risk (e.g., rs9388766: OR = 1.06; 95% CI = 1.03–1.08; q = 0.02). Variants in TET2 were significantly associated with overall breast, overall prostate, overall ovarian, and endometrioid ovarian cancer risk, with rs62331150 showing bidirectional effects. Analyses of subpathways did not reveal gene subsets that contributed disproportionately to susceptibility. Conclusions: Functional and correlative studies are now needed to elucidate the potential links between germline genotype, epigenetic function, and cancer etiology. Impact: This approach provides novel insight into possible pleiotropic effects of genes involved in epigenetic processes. Cancer Epidemiol Biomarkers Prev; 26(6); 816–25. ©2017 AACR.

DNA methylation profiling offers unique insights into human development and diseases. Often the analysis of complex tissues and cell mixtures is the only feasible option to study methylation changes across large patient cohorts. Since DNA methylomes are highly cell type specific, deconvolution methods can be used to recover cell type-specific information in the form of latent methylation components (LMCs) from such 'bulk' samples. Reference-free deconvolution methods retrieve these components without the need for DNA methylation profiles of purified cell types. Currently no integrated and guided procedure is available for data preparation and subsequent interpretation of deconvolution results. Here, we describe a three-stage protocol for reference-free deconvolution of DNA methylation data comprising: (i) data preprocessing, confounder adjustment using independent component analysis (ICA) and feature selection using DecompPipeline, (ii) deconvolution with multiple parameters using MeDeCom, RefFreeCellMix or EDec and (iii) guided biological inference and validation of deconvolution results with the R/Shiny graphical user interface FactorViz. Our protocol simplifies the analysis and guides the initial interpretation of DNA methylation data derived from complex samples. The harmonized approach is particularly useful to dissect and evaluate cell heterogeneity in complex systems such as tumors. We apply the protocol to lung cancer methylomes from The Cancer Genome Atlas (TCGA) and show that our approach identifies the proportions of stromal cells and tumor-infiltrating immune cells, as well as associations of the detected components with clinical parameters. The protocol takes slightly3 d to complete and requires basic R skills.

Background The clinical course of prostate cancer (PCa) is highly variable, demanding an individualized approach to therapy. Overtreatment of indolent PCa cases, which likely do not progress to aggressive stages, may be associated with severe side effects and considerable costs. These could be avoided by utilizing robust prognostic markers to guide treatment decisions. Results We present a random forest-based classification model to predict aggressive behaviour of prostate cancer. DNA methylation changes between PCa cases with good or poor prognosis (discovery cohort with n = 70) were used as input. DNA was extracted from formalin-fixed tumour tissue, and genome-wide DNA methylation differences between both groups were assessed using Illumina HumanMethylation450 arrays. For the random forest-based modelling, the discovery cohort was randomly split into a training (80%) and a test set (20%). Our methylation-based classifier demonstrated excellent performance in discriminating prognosis subgroups in the test set (Kaplan-Meier survival analyses with log-rank p value n = 222) and the TCGA PRAD cohort (n = 477) for external validation, AUCs for sensitivity analyses were 77.1% and 68.7%, respectively. Cancer progression-related DNA hypomethylation was frequently located in ‘partially methylated domains’ (PMDs)—large-scale genomic areas with progressive loss of DNA methylation linked to mitotic cell division. We selected several candidate genes with differential methylation in gene promoter regions for additional validation at the protein expression level by immunohistochemistry in > 12,000 tissue micro-arrayed PCa cases. Loss of ZIC2 protein expression was associated with poor prognosis and correlated with significantly shorter time to biochemical recurrence. The prognostic value of ZIC2 proved to be independent from established clinicopathological variables including Gleason grade, tumour stage, nodal stage and prostate-specific-antigen. Conclusions Our results highlight the prognostic relevance of methylation loss in PMD regions, as well as of several candidate genes not previously associated with PCa progression. Our robust and externally validated PCa classification model either directly or via protein expression analyses of the identified top-ranked candidate genes will support the clinical management of prostate cancer.

Aims Harmful alcohol drinking habits, even among Roma children and adolescents, are more common than in the majority population. The aim of the study was to evaluate the genetic susceptibility of Roma to hazardous alcohol consumption compared to the Hungarian general population. Methods A total of 1273 samples from the population of segregated Hungarian Roma colonies and 2967 samples from the Hungarian general population were genotyped for 25 polymorphisms. Differences in genotype and allele distributions were investigated. Genetic risk scores (GRS) were generated to estimate the joint effect of individual single-nucleotide polymorphisms (SNPs). After unweighted and weighted GRS were calculated the distribution of scores in study populations was compared. Results The allele frequencies differed significantly between the study populations for 17 SNPs ( P

Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer. It is defined by cholangiocytic differentiation and has poor prognosis. Recently, epigenetic processes have been shown to play an important role in cholangiocarcinogenesis. We performed an integrative analysis on 52 iCCAs using both genetic and epigenetic data with a specific focus on DNA methylation components. We found recurrent isocitrate dehydrogenase 1 (IDH1) and IDH2 (28%) gene mutations, recurrent arm-length copy number alterations (CNAs), and focal alterations such as deletion of 3p21 or amplification of 12q15, which affect BRCA1 Associated Protein 1, polybromo 1, and mouse double minute 2 homolog. DNA methylome analysis revealed excessive hypermethylation of iCCA, affecting primarily the bivalent genomic regions marked with both active and repressive histone modifications. Integrative clustering of genetic and epigenetic data identified four iCCA subgroups with prognostic relevance further designated as IDH, high (H), medium (M), and low (L) alteration groups. The IDH group consisted of all samples with IDH1 or IDH2 mutations and showed, together with the H group, a highly disrupted genome, characterized by frequent deletions of chromosome arms 3p and 6q. Both groups showed excessive hypermethylation with distinct patterns. The M group showed intermediate characteristics regarding both genetic and epigenetic marks, whereas the L group exhibited few methylation changes and mutations and a lack of CNAs. Methylation-based latent component analysis of cell-type composition identified differences among these four groups. Prognosis of the H and M groups was significantly worse than that of the L group. Conclusion: Using an integrative genomic and epigenomic analysis approach, we identified four major iCCA subgroups with widespread genomic and epigenomic differences and prognostic implications. Furthermore, our data suggest differences in the cell-of-origin of the iCCA subtypes.

Background: Metabolic and transcriptomic differences between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) compartments, particularly in the context of obesity, may play a role in colorectal carcinogenesis. We investigated the differential functions of their metabolic compositions. Objectives: Biochemical differences between adipose tissues (VAT compared with SAT) in patients with colorectal carcinoma (CRC) were investigated by using mass spectrometry metabolomics and gene expression profiling. Metabolite compositions were compared between VAT, SAT, and serum metabolites. The relation between patients’ tumor stage and metabolic profiles was assessed. Design: Presurgery blood and paired VAT and SAT samples during tumor surgery were obtained from 59 CRC patients (tumor stages I–IV) of the ColoCare cohort. Gas chromatography time-of-flight mass spectrometry and liquid chromatography quadrupole time-of-flight mass spectrometry were used to measure 1065 metabolites in adipose tissue (333 identified compounds) and 1810 metabolites in serum (467 identified compounds). Adipose tissue gene expression was measured by using Illumina’s HumanHT-12 Expression BeadChips. Results: Compared with SAT, VAT displayed elevated markers of inflammatory lipid metabolism, free arachidonic acid, phospholipases (PLA2G10), and prostaglandin synthesis–related enzymes (PTGD/PTGS2S). Plasmalogen concentrations were lower in VAT than in SAT, which was supported by lower gene expression of FAR1, the rate-limiting enzyme for ether-lipid synthesis in VAT. Serum sphingomyelin concentrations were inversely correlated (P = 0.0001) with SAT adipose triglycerides. Logistic regression identified lipids in patients’ adipose tissues, which were associated with CRC tumor stage. Conclusions: As one of the first studies, we comprehensively assessed differences in metabolic, lipidomic, and transcriptomic profiles between paired human VAT and SAT and their association with CRC tumor stage. We identified markers of inflammation in VAT, which supports prior evidence regarding the role of visceral adiposity and cancer. This trial was registered at clinicaltrials.gov as {"type":"clinical-trial","attrs":{"text":"NCT02328677","term_id":"NCT02328677"}}NCT02328677.

Quality of life (QoL) is an important clinical outcome in cancer patients. We investigated associations between dietary patterns and QoL changes in colorectal cancer (CRC) patients. The study included 192 CRC patients with available EORTC QLQ-C30 data before and 12 months post-surgery and food frequency questionnaire data at 12 months post-surgery. Principal component analysis was used to identify dietary patterns. Multivariate regression models assessed associations between dietary patterns and QoL changes over time. We identified four major dietary patterns: “Western” dietary pattern characterized by high consumption of potatoes, red and processed meat, poultry, and cakes, “fruit&vegetable” pattern: high intake of vegetables, fruits, vegetable oils, and soy products, “bread&butter” pattern: high intake of bread, butter and margarine, and “high-carb” pattern: high consumption of pasta, grains, nonalcoholic beverages, sauces and condiments. Patients following a “Western” diet had lower chances to improve in physical functioning (OR = 0.45 [0.21–0.99]), constipation (OR = 0.30 [0.13–0.72]) and diarrhea (OR: 0.44 [0.20–0.98]) over time. Patients following a “fruit&vegetable” diet showed improving diarrhea scores (OR: 2.52 [1.21–5.34]. A “Western” dietary pattern after surgery is inversely associated with QoL in CRC patients, whereas a diet rich in fruits and vegetables may be beneficial for patients’ QoL over time.

Pancreatic acinar cell carcinoma (ACC) is an aggressive exocrine tumor with largely unknown biology. Here, to identify potential targets for personalized treatment, we perform integrative genome-wide and epigenome-wide analyses. The results show frequently aberrant DNA methylation, abundant chromosomal amplifications and deletions, and mutational signatures suggesting defective DNA repair. In contrast to pancreatic ductal adenocarcinoma, no recurrent point mutations are detected. The tumor suppressors ID3, ARID1A, APC, and CDKN2A are frequently impaired also on the protein level and thus potentially affect ACC tumorigenesis. Consequently, this work identifies promising therapeutic targets in ACC for drugs recently approved for precision cancer therapy., Pancreatic acinar cell carcinoma (ACC) is an aggressive exocrine tumor with largely unknown biology. Here, the authors perform genome- and epigenome-wide analyses from normal and ACC pancreatic tissue that identify aberrations in genome stability and cell cycle control.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by mutations activating RAS signaling. Established clinical and genetic markers fail to fully recapitulate the clinical and biological heterogeneity of this disease. Here we report DNA methylome analysis and mutation profiling of 167 JMML samples. We identify three JMML subgroups with unique molecular and clinical characteristics. The high methylation group (HM) is characterized by somatic PTPN11 mutations and poor clinical outcome. The low methylation group is enriched for somatic NRAS and CBL mutations, as well as for Noonan patients, and has a good prognosis. The intermediate methylation group (IM) shows enrichment for monosomy 7 and somatic KRAS mutations. Hypermethylation is associated with repressed chromatin, genes regulated by RAS signaling, frequent co-occurrence of RAS pathway mutations and upregulation of DNMT1 and DNMT3B, suggesting a link between activation of the DNA methylation machinery and mutational patterns in JMML., Juvenile myelomonocytic leukemia (JMML) is an aggressive disease with limited options for treatment. Here, the authors analyse the DNA methylome and mutational profile of JMML to define three subgroups with unique molecular and clinical characteristics.

The majority of long noncoding RNAs (lncRNAs) is still poorly characterized with respect to function, interactions with protein-coding genes, and mechanisms that regulate their expression. As for protein-coding RNAs, epigenetic deregulation of lncRNA expression by alterations in DNA methylation might contribute to carcinogenesis. To provide genome-wide information on lncRNAs aberrantly methylated in breast cancer we profiled tumors of the C3(1) SV40TAg mouse model by MCIp-seq (Methylated CpG Immunoprecipitation followed by sequencing). This approach detected 69 lncRNAs differentially methylated between tumor tissue and normal mammary glands, with 26 located in antisense orientation of a protein-coding gene. One of the hypomethylated lncRNAs, 1810019D21Rik (now called Esrp2-antisense (as)) was identified in proximity to the epithelial splicing regulatory protein 2 (Esrp2) that is significantly elevated in C3(1) tumors. ESRPs were shown previously to have a dual role in carcinogenesis. Both gain and loss have been associated with poor prognosis in human cancers, but the mechanisms regulating expression are not known. In-depth analyses indicate that coordinate overexpression of Esrp2 and Esrp2-as inversely correlates with DNA methylation. Luciferase reporter gene assays support co-expression of Esrp2 and the major short Esrp2-as variant from a bidirectional promoter, and transcriptional regulation by methylation of a proximal enhancer. Ultimately, this enhancer-based regulatory mechanism provides a novel explanation for tissue-specific expression differences and upregulation of Esrp2 during carcinogenesis. Knockdown of Esrp2-as reduced Esrp2 protein levels without affecting mRNA expression and resulted in an altered transcriptional profile associated with extracellular matrix (ECM), cell motility and reduced proliferation, whereas overexpression enhanced proliferation. Our findings not only hold true for the murine tumor model, but led to the identification of an unannotated human homolog of Esrp2-as which is significantly upregulated in human breast cancer and associated with poor prognosis.

Summary Identifying the earliest somatic changes in prostate cancer can give important insights into tumor evolution and aids in stratifying high- from low-risk disease. We integrated whole genome, transcriptome and methylome analysis of early-onset prostate cancers (diagnosis ???55 years). Characterization across 292 prostate cancer genomes revealed age-related genomic alterations and a clock-like enzymatic-driven mutational process contributing to the earliest mutations in prostate cancer patients. Our integrative analysis identified four molecular subgroups, including a particularly aggressive subgroup with recurrent duplications associated with increased expression of ESRP1, which we validate in 12,000 tissue microarray tumors. Finally, we combined the patterns of molecular co-occurrence and risk-based subgroup information to deconvolve the molecular and clinical trajectories of prostate cancer from single patient samples.

The prevalence of smoking in Romani of both genders is significantly higher than in the general population. Our aim was to determine whether a genetic susceptibility contributes to the high prevalence of smoking among Roma in a study based on data collected from cross-sectional surveys.Twenty single nucleotide polymorphisms known to be closely related to smoking behavior were investigated in DNA samples of Hungarian Roma (N = 1273) and general (N = 2388) populations. Differences in genotype and allele distribution were investigated. Genetic risk scores (GRSs) were generated to estimate the joint effect of single nucleotide polymorphisms in genes COMT, CHRNA3/4/5, CYP2A6, CTNNA3, DRD2, MAOA, KCNJ6, AGPHD1, ANKK1, TRPC7, GABRA4, and NRXN1. The distribution of scores in study populations was compared. Age, gender, and body mass index were considered as confounding factors.Difference in allele frequencies between the study populations remained significant for 16 polymorphisms after multiple test correction (p.003). Unexpectedly, the susceptible alleles were more common in the general population, although the protective alleles were more prevalent among Roma. The distribution of unweighted GRS in Roma population was left shifted compared to general population (p.001). Furthermore, the median weighted GRS was lower among the subjects of Roma population compared to the subjects of general population (p.001) even after adjustment for confounding factors.The harmful smoking behavior of the Roma population could not be accounted for by genetic susceptibility; therefore, interventions aimed at smoking prevention and cessation should focus on cultural and environmental factors.This is the first study designed to determine whether genetic background exists behind the harmful behavior of the smoking of the Roma population. Although the frequencies of susceptible and protective alleles strongly differ between the Hungarian Roma and general populations, it is shown that calculated GRSs being significantly higher in the general population, which do not support the hypothesis on the genetic susceptibility of the Roma population. Interventions aimed at smoking cessation in the Roma population should preferentially target cultural and environmental factors.

Identification of abuse and frequency patterns of stimulant designer drugs (SDDs) provides important information for their risk assessment and legislative control. In the present study urine and/or blood samples of suspected drug users in criminal cases were analysed by GC-MS for 38 SDDs, and for the most frequent illicit and psychoactive licit drugs in Hungary. Between July 2012 and June 2013, 2744 suspected drug users were sampled in Budapest and during 2012 and 2013, 774 persons were sampled in South-East Hungary (Csongrad County - neighbour the Romanian and Serbian borders). In Budapest 71.4% of cases, and in South-East Hungary 61% of cases were positive for at least one substance. Pentedrone was the most frequent SDD in both regions; however, the frequency distribution of the remaining drugs was highly diverse. SDDs were frequently present in combination with other drugs - generally with amphetamine or other stimulants, cannabis and/or benzodiazepines. The quarterly distribution of positive samples indicated remarkable seasonal changes in the frequency and pattern of consumption. Substances placed on the list of illicit drugs (mephedrone, 4-fluoro-amphetamine, MDPV, methylone, 4-MEC) showed a subsequent drop in frequency and were replaced by other SDDs (pentedrone, 3-MMC, methiopropamine, etc.). Newly identified compounds from seized materials were added to the list of new psychoactive substances ("Schedule C"). While the risk assessment of substances listed in Schedule C has to be performed within 2 years after scheduling, continuous monitoring of their presence and frequency among drug users is essential. In summary, our results suggest which substances should be dropped from the list of SDDs measured in biological samples; while the appearance of new substances from seized materials indicate the need for developing adequate standard analytical methods.

BackgroundDespite the extensive research approaches applied to characterise malignant melanoma, no specific molecular markers are available that are clearly related to the progression of this disease. In this study, our aims were to define a gene expression signature associated with the clinical outcome of melanoma patients and to provide an integrative interpretation of the gene expression -, copy number alterations -, and promoter methylation patterns that contribute to clinically relevant molecular functional alterations.MethodsGene expression profiles were determined using the Affymetrix U133 Plus2.0 array. The NimbleGen Human CGH Whole-Genome Tiling array was used to define CNAs, and the Illumina GoldenGate Methylation platform was applied to characterise the methylation patterns of overlapping genes.ResultsWE IDENTIFIED TWO SUBCLASSES OF PRIMARY MELANOMA: one representing patients with better prognoses and the other being characteristic of patients with unfavourable outcomes. We assigned 1,080 genes as being significantly correlated with ulceration, 987 genes were downregulated and significantly enriched in the p53, Nf-kappaB, and WNT/beta-catenin pathways. Through integrated genome analysis, we defined 150 downregulated genes whose expression correlated with copy number losses in ulcerated samples. These genes were significantly enriched on chromosome 6q and 10q, which contained a total of 36 genes. Ten of these genes were downregulated and involved in cell-cell and cell-matrix adhesion or apoptosis. The expression and methylation patterns of additional genes exhibited an inverse correlation, suggesting that transcriptional silencing of these genes is driven by epigenetic events.ConclusionUsing an integrative genomic approach, we were able to identify functionally relevant molecular hotspots characterised by copy number losses and promoter hypermethylation in distinct molecular subtypes of melanoma that contribute to specific transcriptomic silencing and might indicate a poor clinical outcome of melanoma.

Genome-wide association studies (GWAS) have identified many genetic susceptibility loci for colorectal cancer (CRC). However, variants in these loci explain only a small proportion of familial aggregation, and there are likely additional variants that are associated with CRC susceptibility. Genome-wide studies of gene-environment interactions may identify variants that are not detected in GWAS of marginal gene effects. To study this, we conducted a genome-wide analysis for interaction between genetic variants and alcohol consumption and cigarette smoking using data from the Colon Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). Interactions were tested using logistic regression. We identified interaction between CRC risk and alcohol consumption and variants in the 9q22.32/HIATL1 (Pinteraction = 1.76×10−8; permuted p-value 3.51x10-8) region. Compared to non-/occasional drinking light to moderate alcohol consumption was associated with a lower risk of colorectal cancer among individuals with rs9409565 CT genotype (OR, 0.82 [95% CI, 0.74–0.91]; P = 2.1×10−4) and TT genotypes (OR,0.62 [95% CI, 0.51–0.75]; P = 1.3×10−6) but not associated among those with the CC genotype (p = 0.059). No genome-wide statistically significant interactions were observed for smoking. If replicated our suggestive finding of a genome-wide significant interaction between genetic variants and alcohol consumption might contribute to understanding colorectal cancer etiology and identifying subpopulations with differential susceptibility to the effect of alcohol on CRC risk., Author Summary Alcohol consumption and smoking are associated with CRC risk. We performed a genome-wide analysis for interaction between genetic variants and alcohol consumption and cigarette smoking to identify potential new genetic regions associated with CRC. About 8,000 CRC cases and 8,800 controls were included in alcohol-related analysis and over 11,000 cases and 11,000 controls were involved in smoking-related analysis. We identified interaction between variants at 9q22.32/HIATL1 and alcohol consumption in relation to CRC risk (Pinteraction = 1.76×10−8). If replicated our suggested finding of the interaction between genetic variants and alcohol consumption might contribute to understanding colorectal cancer etiology and identifying subpopulations with differential susceptible to the effect of alcohol on CRC risk.

The 7q31 locus contains several genes affected in cancer progression. Although evidences exist regarding its impact on tumorigenesis, the role of genetic alterations and the expressions of locus-related genes are still controversial. Our study aimed to define the 7q31 copy number alterations in primary melanomas, primary-metastatic tumor pairs and cell lines. Data were correlated with clinical-pathological parameters. Genetic data show that 7q31 copy number distribution was heterogeneous in both primary and metastatic tumors. Extra copies were highly accompanied by chromosome 7 polisomy, and significantly increased in primary lesions with poor prognosis. Additionally, we determined the mRNA and protein levels of the locus-related CAV1 and TES genes. TES mRNA level was associated with metastatic location. CAV1 mRNA and protein levels were significantly higher in thicker tumors, however, lack of protein was also observed in a subpopulation of thin lesions. Expressions of CAV1 and TES were not associated with 7q31 alterations. In conclusion, 7q31 amplification can predict unfavorable outcome. Alterations of TES mRNA level may predict the location of metastasis. CAV1 possibly affect the cancer cell invasion.

The aim of our study was to evaluate whether any association exists between metabolic syndrome (MS) and ACE I/D and AGT M235T gene polymorphisms in Hungarians as an example of European Caucasian population. Study subjects of our cross-sectional study were recruited from the Hungarian General Practitioners’ Morbidity Sentinel Stations Program. The study population ( n = 1762) approximates very well the age and sex distribution of the general Hungarian population. MS was defined according to the latest diagnostic criteria proposed by the International Diabetes Federation. The frequency of DD genotype (31.36% vs. 25.42%, p = 0.006) and the frequency of D allele (0.56 vs. 0.51, p = 0.006) were significantly higher in the metabolic group than in the non-metabolic group. The distribution of the AGT M235T polymorphism was similar in each group investigated. Association was shown in the case of patients in whom central obesity was combined with elevated TG and low HDL cholesterol level ( p = 0.024 and p = 0.022). It suggests that ACE I/D polymorphism is likely to be involved in lipid metabolism.

BACKGROUND: Standardized death rates from chronic liver diseases (CLDs) in Hungary are much higher than the European Union average. Carrying the alcohol dehydrogenase 1B 48His allele (rs1229984 or ADH1B*2) could decrease the risk of alcoholism, but with persistent drinking may confer a greater risk of CLDs. The aim of this study was to assess the prevalence of this polymorphism in the Hungarian population and its association with alcohol consumption and with CLDs. METHODS AND RESULTS: A total of 278 cases with diagnosed CLDs and 752 controls without any alterations in liver function, all males aged 45-64, were screened for ADH1B Arg48His polymorphism. ADH1B*2 allele frequencies in controls and cases were 8.31% and 4.50%, respectively (chi(2) = 9.2; P = 0.01). Carrying the ADH1B*2 allele was associated with significantly lower odds ratio (OR) for drinking frequency (OR = 0.63; P = 0.003), the number of positive answers on CAGE (Cut-down, Annoyed, Guilt, Eye-opener) assessment (OR = 0.58; P = 0.005) and a positive CAGE status (OR = 0.55; P = 0.007). There was a significant association between ADH1B*2 and CLDs (OR = 0.50; P = 0.003), but it disappeared after adjusting for CAGE status and scores (OR = 0.67 P = 0.134; OR = 0.67 P = 0.148, respectively) and weakened after adjusting for drinking frequency (OR = 0.61; P = 0.045). Among heavy drinkers the presence of ADH1B*2 did not increase the risk of cirrhosis but there was a significant interaction between genotype and CAGE status (P = 0.003, P = 0.042), with ADH1B*2 conferring reduced risk of CLDs in CAGE negatives. CONCLUSION: In Hungarians, the ADH1B 48His allele reduces the risk of alcoholism, but not the risk of chronic liver disease among heavy drinkers.

Amplification of the 11q13 chromosomal region is a common event in primary melanomas. Several candidate genes are localized at this sequence; however, their role in melanoma has not been clearly defined. The aim of this study was to develop an accurate method for determining the amplification pattern of six candidate genes that map to this amplicon core and to elucidate the possible relationship between BRAF, NRAS mutations and CCND1 copy number alterations, all of which are key components of the MAP kinase pathway. Characterization of gene copy numbers was performed by quantitative PCR and, as an alternative method, fluorescence in situ hybridization was used to define the CCND1 amplification pattern at the single cell level. Samples with amplified CCND1 (32%) were further analyzed for copy number alterations for the TAOS1, FGF3, FGF19, FGF4 and EMS1 genes. Co-amplification of the CCND1 and TAOS1 was present in 15% of tumors and was more frequent in ulcerated lesions (P=0.017). Furthermore, 56% of primary melanomas had either BRAF or NRAS mutations, but these two mutations were not present in any of the lesions analyzed. Of these cases, 34% also had CCND1 amplification. There was a significant relationship between NRAS activating mutations and UV exposure (P=0.005). We did not find correlations between CCND1 gene amplification status and any of the patients' clinicopathological parameters. However, CCND1 amplification simultaneously with either BRAF or NRAS activation mutations was observed mainly in primary tumors with ulcerated surfaces (P=0.028). We assume that co-amplification of these candidate genes in the 11q13 region or CCND1 gene alterations along with either BRAF or NRAS mutations might be more important for prognosis than the presence of these alterations alone.

In melanoma, the presence of promoter related hypermethylation has previously been reported, however, no methylation-based distinction has been drawn among the diverse melanoma subtypes. Here, we investigated DNA methylation changes associated with melanoma progression and links between methylation patterns and other types of somatic alterations, including the most frequent mutations and DNA copy number changes. Our results revealed that the methylome, presenting in early stage samples and associated with the BRAF(V600E) mutation, gradually decreased in the medium and late stages of the disease. An inverse relationship among the other predefined groups and promoter methylation was also revealed except for histologic subtype, whereas the more aggressive, nodular subtype melanomas exhibited hypermethylation as well. The Breslow thickness, which is a continuous variable, allowed for the most precise insight into how promoter methylation decreases from stage to stage. Integrating our methylation results with a high-throughput copy number alteration dataset, local correlations were detected in the MYB and EYA4 genes. With regard to the effects of DNA hypermethylation on melanoma patients' survival, correcting for clinical cofounders, only the KIT gene was associated with a lower overall survival rate. In this study, we demonstrate the strong influence of promoter localized DNA methylation changes on melanoma initiation and show how hypermethylation decreases in melanomas associated with less favourable clinical outcomes. Furthermore, we establish the methylation pattern as part of an integrated apparatus of somatic DNA alterations.

Patients infected with the human immunodeficiency virus exhibit a progressive decline in the CD4 T-cell number, resulting in immunodeficiency and increased susceptibility to opportunistic infections and malignancies. Although CD4 T cell production is impaired in patients infected with HIV, there is now increasing evidence that the primary basis of T cell depletion is accelerated apoptosis of CD4 and CD8 T cells. The rate of lymphocyte apoptosis in HIV infection correlates inversely with the progression of the disease: it is low in long-term progressors and in patients undergoing highly active antiretroviral therapy. Interestingly, only a minor fraction of apoptotic lymphocytes are infected by HIV, indicating that the enhanced apoptosis does not necessarily always serve to remove the HIV+ cells and results from mechanisms other than direct infection. Thus, understanding and influencing the mechanisms of HIV-associated lymphocyte apoptosis may lead to new therapies for HIV disease. In this paper the potential effects of retinoids on CD4 T cell apoptosis is discussed.