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The "delayed infection hypothesis" states that a paucity of infections in early childhood may lead to higher risks of childhood leukemia (CL), especially acute lymphoblastic leukemia (ALL). Using prospectively collected data from six population-based birth cohorts we studied the association between birth order (a proxy for pathogen exposure) and CL. We explored whether other birth or parental characteristics modify this association. With 2.2 × 10 6 person-years of follow-up, 185 CL and 136 ALL cases were ascertained. In Cox proportional hazards models, increasing birth order (continuous) was inversely associated with CL and ALL; hazard ratios (HR) = 0.88, 95% confidence interval (CI): (0.77-0.99) and 0.85: (0.73-0.99), respectively. Being later-born was associated with similarly reduced hazards of CL and ALL compared to being first-born; HRs = 0.78: 95% CI: 0.58-1.05 and 0.73: 0.52-1.03, respectively. Successive birth orders were associated with decreased CL and ALL risks (P for trend 0.047 and 0.055, respectively). Multivariable adjustment somewhat attenuated the associations. We found statistically significant and borderline interactions between birth weight (p = 0.024) and paternal age (p = 0.067), respectively, in associations between being later-born and CL, with the lowest risk observed for children born at <3 kg with fathers aged 35+ (HR = 0.18, 95% CI: 0.06-0.50). Our study strengthens the theory that increasing birth order confers protection against CL and ALL risks, but suggests that this association may be modified among subsets of children with different characteristics, notably advanced paternal age and lower birth weight. It is unclear whether these findings can be explained solely by infectious exposures., (© 2018 UICC.)

The large geographic variations in the incidence of gastric cancer (GC) are likely due to differential environmental exposures, in particular to Helicobacter pylori (H. pylori) infection. We aimed to investigate the impact of H. pylori on the epigenome in normal gastric mucosa and methylation changes associated with cancer risk independent of H. pylori. A discovery set of normal gastric mucosa from GC cases (n = 42) and controls (n = 42), nested in a large case-control study and stratified by H. pylori status, were subjected to genome-wide methylation profiling. Single-nucleotide polymorphism arrays from peripheral blood leukocytes were used to conduct methylation quantitative trait loci (mQTL) analysis. A validation set of gastric mucosa samples (n = 180) was used in the replication phase. We found 1,924 differentially methylated positions (DMPs) and 438 differentially methylated regions (DMRs) associated with H. pylori infection, most of which were hypermethylated. Significant methylation alterations identified in the initial set were successfully replicated. Furthermore, the H. pylori-associated DMP/Rs showed marked stability ('epigenetic memory') after H. pylori clearance. Interestingly, we found 152 DMRs associated with cancer risk independent of the H. pylori status in normal gastric mucosa. The methylation score derived from three biomarkers was a strong predictor of GC. Finally, the mQTL analysis indicated that the H. pylori- and cancer-specific methylation signatures were minimally affected by genetic variation. The comprehensively characterized methylome changes associated with H. pylori infection and GC risk in our study might serve as potential biomarkers for early cancer progression in tumour-free gastric mucosa., (© 2018 International Agency for Research on Cancer (IARC/WHO); licensed by UICC.)

Air pollution is associated with a broad range of adverse health effects, including mortality and morbidity due to cardio- and cerebrovascular diseases (CCVD), but the molecular mechanisms involved are not entirely understood. This study aims to investigate the involvement of oxidative stress and inflammation in the causal chain, and to identify intermediate biomarkers that are associated retrospectively with the exposure and prospectively with the disease. We designed a case-control study on CCVD nested in a cohort of 18,982 individuals from the EPIC-Italy study. We measured air pollution, inflammatory biomarkers, and whole-genome DNA methylation in blood collected up to 17 years before the diagnosis. The study sample includes all the incident CCVD cases among former- and never-smokers, with available stored blood sample, that arose in the cohort during the follow-up. We identified enrichment of altered DNA methylation in "ROS/Glutathione/Cytotoxic granules" and "Cytokine signaling" pathways related genes, associated with both air pollution (multiple comparisons adjusted p for enrichment ranging from 0.01 to 0.03 depending on pollutant) and with CCVD risk (P = 0.04 and P = 0.03, respectively). Also, Interleukin-17 was associated with higher exposure to NO 2 (P = 0.0004), NO x (P = 0.0005), and CCVD risk (OR = 1.79; CI 1.04-3.11; P = 0.04 comparing extreme tertiles). Our findings indicate that chronic exposure to air pollution can lead to oxidative stress, which in turn activates a cascade of inflammatory responses mainly involving the "Cytokine signaling" pathway, leading to increased risk of CCVD. Inflammatory proteins and DNA methylation alterations can be detected several years before CCVD diagnosis in blood samples, being promising preclinical biomarkers. Environ. Mol. Mutagen. 59:234-246, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The interaction between the (epi)genetic makeup of an individual and his/her environmental exposure record (exposome) is accepted as a determinant factor for a significant proportion of human malignancies. Recent evidence has highlighted the key role of epigenetic mechanisms in mediating gene-environment interactions and translating exposures into tumorigenesis. There is also growing evidence that epigenetic changes may be risk factor-specific ("fingerprints") that should prove instrumental in the discovery of new biomarkers in cancer. Here, we review the state of the science of epigenetics associated with environmental stimuli and cancer risk, highlighting key developments in the field. Critical knowledge gaps and research needs are discussed and advances in epigenomics that may help in understanding the functional relevance of epigenetic alterations. Key elements required for causality inferences linking epigenetic changes to exposure and cancer are discussed and how these alterations can be incorporated in carcinogen evaluation and in understanding mechanisms underlying epigenome deregulation by the environment., (© 2017 International Agency for Research on Cancer (IARC/WHO); licensed by UICC.)

Previous studies have revealed a robust association between exposure to asbestos and human lung cancer. Accumulating evidence has highlighted the role of epigenome deregulation in the mechanism of carcinogen-induced malignancies. We examined the impact of asbestos on DNA methylation. Our genome-wide studies (using Illumina HumanMethylation450K BeadChip) of lung cancer tissue and paired normal lung from 28 asbestos-exposed or non-exposed patients, mostly smokers, revealed distinctive DNA methylation changes. We identified a number of differentially methylated regions (DMR) and differentially variable, differentially methylated CpGs (DVMC), with individual CpGs further validated by pyrosequencing in an independent series of 91 non-small cell lung cancer and paired normal lung. We discovered and validated BEND4, ZSCAN31 and GPR135 as significantly hypermethylated in lung cancer. DMRs in genes such as RARB (FDR 1.1 × 10 -19 , mean change in beta [Δ] -0.09), GPR135 (FDR 1.87 × 10 -8 , mean Δ -0.09) and TPO (FDR 8.58 × 10 -5 , mean Δ -0.11), and DVMCs in NPTN, NRG2, GLT25D2 and TRPC3 (all with p <0.05, t-test) were significantly associated with asbestos exposure status in exposed versus non-exposed lung tumors. Hypomethylation was characteristic to DVMCs in lung cancer tissue from asbestos-exposed subjects. When DVMCs related to asbestos or smoking were analyzed, 96% of the elements were unique to either of the exposures, consistent with the concept that the methylation changes in tumors may be specific for risk factors. In conclusion, we identified novel DNA methylation changes associated with lung tumors and asbestos exposure, suggesting that changes may be present in causal pathway from asbestos exposure to lung cancer., (© 2017 UICC.)

Advances in laboratory sciences offer much in the challenge to unravel the complex etiology of cancer and to therefore provide an evidence-base for prevention. One area where improved measurements are particularly important to epidemiology is exposure assessment; this requirement has been highlighted through the concept of the exposome. In addition, the ability to observe genetic and epigenetic alterations in individuals exposed to putative risk factors also affords an opportunity to elucidate underlying mechanisms of carcinogenesis, which in turn may allow earlier detection and more refined molecular classification of disease. In this context the application of omics technologies to large population-based studies and their associated biobanks raise exciting new avenues of research. This review considers the areas of genomics, transcriptomics, epigenomics and metabolomics and the evidence to date that people exposed to well-defined factors (for example, tobacco, diet, occupational exposures, environmental pollutants) have specific omics profiles. Although in their early stages of development these approaches show promising evidence of distinct exposure-derived biological effects and indicate molecular pathways that may be particularly relevant to the carcinogenic process subsequent to environmental and lifestyle exposures. Such an interdisciplinary approach is vital if the full benefits of advances in laboratory sciences and investments in large-scale prospective cohort studies are to be realized in relation to cancer prevention., (Copyright © 2013 Wiley Periodicals, Inc.)

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy., (Copyright © 2011 UICC.)

Constitutional epimutation is one of the causes for MLH1 gene inactivation associated with hereditary non-polyposis colon cancer (HNPCC) syndrome. Here we investigate MLH1 promoter hypermethylation in 110 sporadic early-onset colorectal cancer patients. Variable levels of hypermethylation were detected in 55 patients (50%). Importantly a reduced MLH1 gene expression was found in patients with high-level methylation, with the association of microsatellite instability (MSI) in their tumor cells. Such high-level methylation accounts for 7.4% of all patients included in this study. Furthermore, we found that in one case constitutional methylation affected both alleles, indicating a post-zygotic methylation dysregulation. Our findings suggest that constitutional epimutation is a mechanism underlying early-onset colorectal cancer, although it is involved in only a small proportion of patients, who require appropriate surveillance. Our findings provide further insight into the role of aberrant constitutional methylation in colon carcinogenesis and raise the question of whether prevalent low-level methylation constitutes a potential risk factor for cancer development., (2010 Wiley-Liss, Inc.)

Recently we found a clearly reduced basal level of wt p53 protein in PARP-deficient cells. Interestingly, PARP deficiency affected only regularly spliced (RS) wt p53. No significant difference of the p53 transcription rate was observed between wt and PARP-lacking cells. To clarify whether the reduction of RS p53 protein is due to a lower translation rate or rather to its instability in the absence of functional PARP, we investigated the effect of the inhibition of proteasome activity and nuclear export on the p53 level. The p53 half-life was approximately eight-fold decreased in PARP-lacking cells. Surprisingly, treatment with three proteasome inhibitors increased RS p53 in normal but not in PARP-deficient cells. However, the inhibition of nuclear export resulted in a considerable accumulation of RS p53 in the latter. Therefore, we decided to increase concentrations of the inhibitors. Their higher concentrations strongly affected viability of normal, but not of PARP-deficient cells, about 70% of MEFs died. Interestingly, higher concentrations of proteasome inhibitors resulted in the appearance of RS p53 in PARP-lacking fibroblasts. Reconstitution of PARP-deficient cells with PARP restored the normal susceptibility to proteasome inhibitors thereby unequivocally demonstrating that the enhanced cytotoxicity of proteasome inhibitors and their action on p53 level depends on the presence of functional PARP., (Copyright 2000 Wiley-Liss, Inc.)

It has proved difficult to develop suitable models to study radiation-induced carcinogenesis by using human epithelial cells. However, immortalised human epithelial cell lines have proved useful. Unirradiated cells from the human keratinocyte cell line (HPV-G) and the human embryonic lung cell line (L132) were found to be tumourigenic in T-cell-deficient mice; thus, they are not suitable for transformation studies. Human urothelial cell lines (SV-HUC-1, NT11, BC16) and the human thyroid epithelial cell line (HTori-3) were nontumourigenic. The urothelial cell lines were refractory to radiation-induced carcinogenesis, and only one small tumour was observed in 57 mice that received irradiated cells. Whereas tumours were not produced following irradiation of these urothelial cells, changes in anchorage-independent growth were observed after a single dose of 8 Gy gamma-irradiation but not after 2 or 4 Gy. Irradiation of the human thyroid epithelial cell line (HTori-3) in vitro resulted in tumour formation. Passaging of the cells in vitro before injection did not seem to be critical. Some of the cell lines derived from the primary thyroid tumours exhibited p53 mutations in exons 5, 6, 7, and 8, as detected by single-stranded conformational polymorphism (SSCP) analysis. Thus, the human thyroid epithelial cell line (HTori-3) looks promising as a model for investigating the molecular events in radiation-induced carcinogenesis.