Prenatal Particulate Matter Exposure Is Associated with Saliva DNA Methylation at Age 15: Applying Cumulative DNA Methylation Scores as an Exposure Biomarker

Background: Exposure in utero to particulate matter (PM2.5 and PM10) is associated with maladaptive health outcomes. Although exposure to prenatal PM2.5 and PM10 has cord blood DNA methylation signatures at birth, signature persistence into childhood and saliva cross-tissue applicability has not been tested. Methods: In the Fragile Families and Child Wellbeing Study, a United States 20-city birth cohort, average residential PM2.5 and PM10 during the three months prior to birth was estimated using air quality monitors with inverse distance weighting. Saliva DNA methylation at ages 9 (n = 749) and 15 (n = 793) was measured using the Illumina HumanMethylation 450 k BeadArray. Cumulative DNA methylation scores for particulate matter were estimated by weighting participant DNA methylation at each site by independent meta-analysis effect estimates and standardizing the sums. Using a mixed-effects regression analysis, we tested the associations between cumulative DNA methylation scores at ages 9 and 15 and PM exposure during pregnancy, adjusted for child sex, age, race/ethnicity, maternal income-to-needs ratio, nonmartial birth status, and saliva cell-type proportions. Results: Our study sample was 50.5% male, 56.3% non-Hispanic Black, and 19.8% Hispanic, with a median income-to-needs ratio of 1.4. Mean exposure levels for PM2.5 were 27.9 μg/m3/day (standard deviation: 7.0; 23.7% of observations exceeded safety standards) and for PM10 were 15.0 μg/m3/day (standard deviation: 3.1). An interquartile range increase in PM2.5 exposure (10.73 μg/m3/day) was associated with a −0.0287 standard deviation lower cumulative DNA methylation score for PM2.5 (95% CI: −0.0732, 0.0158, p = 0.20) across all participants. An interquartile range increase in PM10 exposure (3.20 μg/m3/day) was associated with a −0.1472 standard deviation lower cumulative DNA methylation score for PM10 (95% CI: −0.3038, 0.0095, p = 0.06) across all participants. The PM10 findings were driven by the age 15 subset where an interquartile range increase in PM10 exposure was associated with a −0.024 standard deviation lower cumulative DNA methylation score for PM10 (95% CI: −0.043, −0.005, p = 0.012). Findings were robust to adjustment for PM exposure at ages 1 and 3. Conclusion: In utero PM10-associated DNA methylation differences were identified at age 15 in saliva. Benchmarking the timing and cell-type generalizability is critical for epigenetic exposure biomarker assessment.

Investigating the DNA methylation profile of e-cigarette use

Background Little evidence exists on the health effects of e-cigarette use. DNA methylation may serve as a biomarker for exposure and could be predictive of future health risk. We aimed to investigate the DNA methylation profile of e-cigarette use. Results Among 117 smokers, 117 non-smokers and 116 non-smoking vapers, we evaluated associations between e-cigarette use and epigenome-wide methylation from saliva. DNA methylation at 7 cytosine-phosphate-guanine sites (CpGs) was associated with e-cigarette use at p < 1 × 10–5 and none at p < 5.91 × 10–8. 13 CpGs were associated with smoking at p < 1 × 10–5 and one at p < 5.91 × 10–8. CpGs associated with e-cigarette use were largely distinct from those associated with smoking. There was strong enrichment of known smoking-related CpGs in the smokers but not the vapers. We also tested associations between e-cigarette use and methylation scores known to predict smoking and biological ageing. Methylation scores for smoking and biological ageing were similar between vapers and non-smokers. Higher levels of all smoking scores and a biological ageing score (GrimAge) were observed in smokers. A methylation score for e-cigarette use showed poor prediction internally (AUC 0.55, 0.41–0.69) and externally (AUC 0.57, 0.36–0.74) compared with a smoking score (AUCs 0.80) and was less able to discriminate lung squamous cell carcinoma from adjacent normal tissue (AUC 0.64, 0.52–0.76 versus AUC 0.73, 0.61–0.85). Conclusions The DNA methylation profile for e-cigarette use is largely distinct from that of cigarette smoking, did not replicate in independent samples, and was unable to discriminate lung cancer from normal tissue. The extent to which methylation related to long-term e-cigarette use translates into chronic effects requires further investigation.

DNA Methylation Regulator-Meditated Modification Patterns Define the Distinct Tumor Microenvironment in Lung Adenocarcinoma

BackgroundEpigenetic changes of lung adenocarcinoma (LUAD) have been reported to be a relevant factor in tumorigenesis and cancer progression. However, the molecular mechanisms responsible for DNA methylation patterns in the tumor immune-infiltrating microenvironment and in cancer immunotherapy remain unclear.MethodsWe conducted a global analysis of the DNA methylation modification pattern (DMP) and immune cell-infiltrating characteristics of LUAD patients based on 21 DNA methylation regulators. A DNA methylation score (DMS) system was constructed to quantify the DMP model in each patient and estimate their potential benefit from immunotherapy.ResultsTwo DNA methylation modification patterns able to distinctly characterize the immune microenvironment characterization were identified among 513 LUAD samples. A lower DMS, characterized by increased CTLA-4/PD-1/L1 gene expression, greater methylation modifications, and tumor mutation burden, characterized a noninflamed phenotype with worse survival. A higher DMS, characterized by decreased methylation modification, a greater stromal-relevant response, and immune hyperactivation, characterized an inflamed phenotype with better prognosis. Moreover, a lower DMS indicated an increased mutation load and exhibited a poor immunotherapeutic response in the anti-CTLA-4/PD-1/PD-L1 cohort.ConclusionEvaluating the DNA methylation modification pattern of LUAD patients could enhance our understanding of the features of tumor microenvironment characterization and may promote more favorable immunotherapy strategies.

DNA methylation patterns at and beyond the histological margin of early-stage invasive lung adenocarcinoma radiologically manifested as pure ground-glass opacity

Background Early-stage lung cancers radiologically manifested as ground-glass opacities (GGOs) have been increasingly identified, among which pure GGO (pGGO) has a good prognosis after local resection. However, the optimal surgical margin is still under debate. Precancerous lesions exist in tumor-adjacent tissues beyond the histological margin. However, potential precancerous epigenetic variation patterns beyond the histological margin of pGGO are yet to be discovered and described. Results A genome-wide high-resolution DNA methylation analysis was performed on samples collected from 15 pGGO at tumor core (TC), tumor edge (TE), para-tumor tissues at the 5 mm, 10 mm, 15 mm, 20 mm beyond the tumor, and peripheral normal (PN) tissue. TC and TE were tested with the same genetic alterations, which were also observed in histologically normal tissue at 5 mm in two patients with lower mutation allele frequency. According to the difference of methylation profiles between PN samples, 2284 methylation haplotype blocks (MHBs), 1657 differentially methylated CpG sites (DMCs), and 713 differentially methylated regions (DMRs) were identified using reduced representation bisulfite sequencing (RRBS). Two different patterns of methylation markers were observed: Steep (S) markers sharply changed at 5 mm beyond the histological margin, and Gradual (G) markers changed gradually from TC to PN. S markers composed 86.2% of the tumor-related methylation markers, and G markers composed the other 13.8%. S-marker-associated genes enriched in GO terms that were related to the hallmarks of cancer, and G-markers-associated genes enriched in pathways of stem cell pluripotency and transcriptional misregulation in cancer. Significant difference in DNA methylation score was observed between peripheral normal tissue and tumor-adjacent tissues 5 mm further from the histological margin (p < 0.001 in MHB markers). DNA methylation score at and beyond 10 mm from histological margin is not significantly different from peripheral normal tissues (p > 0.05 in all markers). Conclusions According to the methylation pattern observed in our study, it was implied that methylation alterations were not significantly different between tissues at or beyond P10 and distal normal tissues. This finding explained for the excellent prognosis from radical resections with surgical margins of more than 15 mm. The inclusion of epigenetic characteristics into surgical margin analysis may yield a more sensitive and accurate assessment of remnant cancerous and precancerous cells in the surgical margins.

Clinical and epigenetic features of colorectal cancer patients with somatic POLE proofreading mutations

Background Mutations in the POLE gene result in an ultra-hypermutated phenotype in colorectal cancer (CRC); however, the molecular characterisation of epigenetic alterations remains unclear. We examined the genetic and epigenetic profiles of POLE-mutant CRC to elucidate the clinicopathological features of the associated genetic and epigenetic alterations. Results Tumour tissues (1,013) obtained from a cohort of patients with CRC were analysed to determine associations between the proofreading domain mutations of POLE with various clinicopathological variables, microsatellite instability (MSI) status, BRAF and KRAS mutations, and the methylation status of key regions of MLH1, MGMT, and SFRP2 promoters by calculating the methylation scores (range 0–6). Only four cases (0.4%) exhibited pathogenic POLE hotspot mutations (two p.P286R [c.857C > G], one p.V411L [c.1231G > C], and p.S459F [c.1376C > T] each), which were mutually exclusive to BRAF and KRAS mutations and MSI. CRC patients were divided into four subgroups: patients with POLE mutations (POLE, 0.4%, n = 4), patients with both MSI and extensive methylation in MLH1 (MSI-M, 2.9%, n = 29), patients with MSI but no extensive methylation in MLH1 (MSI-U, 3.6%, n = 36), and patients without MSI (non-MSI, 93.2%, n = 944). The POLE group was younger at diagnosis (median 52 years, P < 0.0001), with frequent right-sided tumour localisation (frequency of tumours located in the right colon was 100%, 93.1%, 36.1%, and 29.9% in POLE, MSI-M, MSI-U, and non-MSI, respectively; P < 0.0001), and was diagnosed at an earlier stage (frequency of stages I–II was 100%, 72.4%, 77.8%, and 46.6% in POLE, MSI-M, MSI-U, and non-MSI, respectively, P < 0.0001). The mean methylation score in POLE was not different from that in MSI-U and non-MSI, but the methylation signature was distinct from that of the other subgroups. Additionally, although the examined number of POLE-mutant tumours was small, the number of CD8-positive cells increased in tumours with partial methylation in the MLH1 gene. Conclusions CRC patients with POLE proofreading mutations are rare. Such mutations are observed in younger individuals, and tumours are primarily located in the right colon. Diagnosis occurs at an earlier stage, and distinct epigenetic alterations may be associated with CD8 cell infiltration.

Investigating the DNA methylation profile of e-cigarette use

Rationale and objectivesLittle evidence exists on the health effects of e-cigarette use. DNA methylation may serve as a biomarker for exposure and could be predictive of future health risk. We aimed to investigate the DNA methylation profile of e-cigarette use.MethodsAmong 117 smokers, 117 non-smokers and 116 non-smoking vapers, we evaluated associations between e-cigarette use and epigenome-wide methylation from saliva. We tested associations between e-cigarette use and methylation scores known to predict smoking and smoking-related disease. We assessed the ability of a methylation score for predicting e-cigarette use and for discriminating lung cancer.Measurements and Main Results7 CpGs were identified in relation to e-cigarette use at p<1×10−5 and none at p<5.91×10−8. 13 CpGs were associated with smoking at p<1×10−5 and one at p<5.91×10−8. CpGs associated with e-cigarette use were largely distinct from those associated with smoking. There was strong enrichment of known smoking-related CpGs in the smokers but not the vapers. A methylation score for e-cigarette use showed poor prediction internally (AUC 0.55, 0.41-0.69) and externally (AUC 0.57, 0.36-0.74) compared with a smoking score (AUCs 0.80) and was less able to discriminate lung squamous cell carcinoma from adjacent normal tissue (AUC 0.64, 0.52-0.76 versus AUC 0.73, 0.61-0.85).ConclusionsThe DNA methylation profile for e-cigarette use is largely distinct from that of cigarette smoking, did not replicate in independent samples, and was unable to discriminate lung cancer from normal tissue. The extent to which methylation related to long-term e-cigarette use translates into chronic effects requires further investigation.Key MessagesWhat is the key question?Is there a DNA methylation signature of e-cigarette use and is it distinct from that of smoking?What is the bottom line?Smoke exposure is known to lead to widespread changes in DNA methylation which have been identified in different populations and samples, persist for many years after smoking cessation, and may act as a biomarker for smoking-related disease risk and mortality. Whether a similar methylation profile exists in relation to e-cigarette use has not been widely investigated.Why read on?We obtained saliva samples from 116 e-cigarette users and compared their DNA methylation profile with 117 smokers and 117 non-smokers. The e-cigarette users in this study had a minimal smoking history, and so we were able to distinguish the effects of e-cigarette use from those of smoke exposure. Overall, we found that the methylation profile associated with e-cigarette use is less pronounced and distinct from that associated with cigarette smoking.

The IGF2 methylation score for adrenocortical cancer: an ENSAT validation study

Adrenocortical carcinoma (ACC) is diagnosed using the histopathological Weiss score (WS), but remains clinically elusive unless it has metastasized or grows locally invasive. Previously, we proposed the objective IGF2 methylation score as diagnostic tool for ACC. This multicenter European cohort study validates these findings. Patient and tumor characteristics were obtained from adrenocortical tumor patients. DNA was isolated from frozen specimens, where after DMR2, CTCF3, and H19 were pyrosequenced. The predictive value of the methylation score for malignancy, defined by the WS or metastasis development, was assessed using receiver operating characteristic curves and logistic and Cox regression analyses. Seventy-six ACC patients and 118 patients with adrenocortical adenomas were included from seven centers. The methylation score and tumor size were independently associated with the pathological ACC diagnosis (OR 3.756 95% CI 2.224–6.343; OR 1.467 95% CI 1.202–1.792, respectively; Hosmer–Lemeshow test P = 0.903), with an area under the curve (AUC) of 0.957 (95% CI 0.930–0.984). The methylation score alone resulted in an AUC of 0.910 (95% CI 0.866–0.952). Cox regression analysis revealed that the methylation score, WS and tumor size predicted development of metastases in univariate analysis. In multivariate analysis, only the WS predicted development of metastasis (OR 1.682 95% CI 1.285–2.202; P < 0.001). In conclusion, we validated the high diagnostic accuracy of the IGF2 methylation score for diagnosing ACC in a multicenter European cohort study. Considering the known limitations of the WS, the objective IGF2 methylation score could potentially provide extra guidance on decisions on postoperative strategies in adrenocortical tumor patients.

Creating and validating a DNA methylation-based proxy for Interleukin-6

Chronic inflammation is a pervasive feature of ageing and may be linked to age-related cognitive decline. However, population studies evaluating its relationship with cognitive functioning have produced heterogeneous results. A potential reason for this is the variability of inflammatory mediators which could lead to misclassifications of individuals' persisting levels of inflammation. The epigenetic mechanism DNA methylation has shown utility in indexing environmental exposures and could potentially be leveraged to provide proxy signatures of chronic inflammation. We conducted an elastic net regression of interleukin-6 (IL-6) in a cohort of 895 older adults (mean age: 69 years) to develop a DNA methylation-based predictor. The predictor was tested in an independent cohort (n=7,028 [417 with measured IL-6], mean age: 51 years).We examined the association between the DNA methylation IL-6 score and serum IL-6, its association with age and established correlates of circulating IL-6, and with cognitive ability. A weighted score from 12 DNA methylation sites optimally predicted IL-6 (independent test set R2=5.1%). In the independent test cohort, both measured IL-6, and the DNA methylation proxy, increased as a function of age (serum IL-6: n=417, β=0.02, SE=0.004 p=1.3x10-7; DNAm IL-6 score: n=7,028, β=0.02, SE=0.0009, p<2x10-16). Serum IL-6 was not found to associate with cognitive ability (n=417, β=-0.06, SE=0.05, p=0.19); however, an inverse association was identified between the DNA methylation score and cognitive functioning (n=7,028, β=-0.14, SE=0.02, pFDR=1.5x10-14). These results suggest DNA methylation-based predictors can be used as proxies for inflammatory markers, potentially allowing for reliable insights into the relationship between chronic inflammation and pertinent health outcomes.