Cg05575921 Methylation Research Articles

Non-cigarette tobacco products, aryl-hydrocarbon receptor repressor gene methylation and smoking-related health outcomes

IntroductionCigarette smoking leads to altered DNA methylation at the aryl-hydrocarbon receptor repressor (AHRR) gene. However, it remains unknown whether pipe or cigar smoking is associated with AHRR methylation. We evaluated...

Aromatic Hydrocarbon Receptor Repressor (AHRR) is a biomarker of ambient air pollution exposure and Coronary Artery Disease (CAD)

Two hundred and twenty subjects were recruited while undergoing cardiac catheterization. AHRR cg05575921 methylation was shown to be significantly decreased in ever smokers compared to never smokers (Mean± SD = 64.2 ± 17.2 vs 80.1 ± 11.1 respectively; P < 0.0001). In addition, higher urinary levels of 2-OHNAP and 2-OHFLU were significantly associated with more AHRR cg05575921 hypomethylation, even after correcting for smoking (β[95%CI]= −4.161[−7.553, −0.769]; P = 0.016 and −5.190[−9.761, −0.618]; P = 0.026, respectively) but not 1-OHPYR (β[95%CI]= −3.545 [−10.935, 3.845]; P = 0.345). Additionally, hypomethylation of AHRR ROI was significantly associated with obstructive coronary artery disease (CAD) after adjusting for smoking, age, sex, diabetes and dyslipidemia (OR [95%CI] = 1.024[1.000 - 1.048]; P = 0.046). Results of this study necessitate further validation to potentially consider clinical incorporation of AHRR methylation status as an early predictive biomarker for the potential association between ambient air pollution and CAD.

Association of DNA methylation signatures with cognitive performance among smokers and ex-smokers.

Alterations in DNA methylation profiles have been associated with cancer, and can be influenced by environmental factors such as smoking. A small but growing literature indicates there are reproducible and robust differences in methylation levels among smokers, never smokers, and ex-smokers. Here, we compared differences in salivary DNA methylation levels among current and ex-smokers (at least 2 years abstinent). Smokers (n=26) and ex-smokers (n=30) provided detailed smoking histories, completed the Paced Auditory Serial Addition Test (PASAT), and submitted a saliva sample. Whole-genome DNA methylation from saliva was performed, and ANCOVA models and a receiver operating characteristic (ROC) curve were used for the differences between groups and the performance of significant CpG sites. After controlling for race, age, and gender, smokers had significantly lower methylation levels than ex-smokers in two CpG sites: cg05575921 (AHRR) and cg21566642 (ALPPL2). Based on the ROC analyses, both CpGs had strong classification potentials (cg05575921 AUC=0.97 and cg21566642 AUC=0.93) in differentiating smoking status. Across all subjects, the percent methylation of cg05575921 (AHRR) and cg21566642 (ALPPL2) positively correlated with the length of the last quit attempt (r=0.65 and 0.64, respectively, p<0.001) and PASAT accuracy (r=0.29 and 0.30, respectively, p<0.05). In spite of the small sample size and preliminary research, our results replicate previously reported differences in AHRR hypomethylation among smokers. Furthermore, we show that the duration of smoking abstinence is associated with a recovery of methylation in ex-smokers, which may be linked to a reduced risk of smoking-associated diseases. The association with cognitive performance suggests that the hypomethylation of AHRR in saliva may reflect systemic exposure to cigarette-related toxicants that negatively affect cognitive performance, and should be validated in larger studies.

Abstract 93: Utility of the Cytosponge for Aetiological Research in Esophageal Squamous Cell Carcinoma: Proof-of-Concept Pilot Study Using Tobacco-Specific Methylation Markers

Abstract Purpose: Esophageal cancer caused 544,076 deaths in 2020. It has two major subtypes: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). A geographically intriguing African Esophageal Cancer Corridor has very high incidence and mortality rates for ESCC. We propose that ESCC research in this corridor might benefit from leveraging advances in tools used for the early detection of EAC, namely using the esophageal sponge cytology. The Cytosponge™ was developed by the University of Cambridge Fitzgerald-laboratory. This pill-on-a-string is an endoscopy-alternative and may form an attractive appropriate health technology in Africa. However, use of the Cytosponge™ for ESCC research in Africa is limited to the CytoSCCAPE study which demonstrated high acceptability and safety of administering the device among 102 asymptomatic adult community volunteers in Kilimanjaro, Tanzania. Methods: In the present proof-of-concept pilot study, we investigated whether FFPE cytology blocks obtained in the CytoSCCAPE study could be used for exposure-specific methylation studies, via two objectives: (i) examine DNA yield from the cytology blocks and (ii) evaluating if the isolated DNA can be used for investigating DNA methylation-based exposure analysis. Here we performed pyrosequencing to compare DNA methylation status of established tobacco-specific methylation markers (CpG cg05575921 &amp; a nearby CpG locus) in ever compared to never tobacco users. Results: Among 102 participants (mean age 50 (SD 12) years, 52% female), 58% (N=58) had good DNA yield (&amp;gt;100 ng), 19 had poor DNA concentration (range 3-54.9ng) and 25 had low DNA concentration (range 60-104ng). In total, 49 (77%) samples passed the quantification stage out of 64 samples that were pyrosequenced. Among these 49 participants, median (IQR) cg05575921 methylation was low overall: 33% (27-44) and did not differ between the 8 ever and 41 never-tobacco users (p=0.85). Hypomethylation in tobacco consumers was not observed, in contrast to work published using blood DNA. Conclusion: We discuss possible reasons for this inconsistency, which include low cellularity and deparaffinization of FFPE sections and provide recommendations for future Cytosponge™ studies for ESCC etiological research. Citation Format: Hannah Simba, Fazlur Rahman Taludkar, Daniel Middleton, Blandina Mmbaga, Alex Mremi, Valerie McCormack. Utility of the Cytosponge for Aetiological Research in Esophageal Squamous Cell Carcinoma: Proof-of-Concept Pilot Study Using Tobacco-Specific Methylation Markers [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 93.

Development and validation of a simple general population lung cancer risk model including AHRR-methylation

IntroductionScreening reduces lung cancer mortality of high-risk populations. Currently proposed screening eligibility criteria only identify half of those individuals, who later develop lung cancer. This study aimed to develop and validate a sensitive and simple model for predicting 10-year lung cancer risk. MethodsUsing the 1991–94 examination of The Copenhagen City Heart Study in Denmark, 6,820 former or current smokers from the general population were followed for lung cancer within 10 years after examination. Logistic regression of baseline variables (age, sex, education, chronic obstructive pulmonary disease, family history of lung cancer, smoking status and cumulative smoking, secondhand smoking, occupational exposures to dust and fume, body mass index, lung function, plasma C-reactive protein, and AHRR(cg05575921) methylation) identified the best predictive model. The model was validated among 3,740 former or current smokers from the 2001-03 examination, also followed for 10 years. A simple risk chart was developed with Poisson regression. ResultsAge, sex, education, smoking status, cumulative smoking, and AHRR(cg05575921) methylation identified 65 of 88 individuals who developed lung cancer in the validation cohort. The highest risk group, consisting of less educated men aged >65 with current smoking status and cumulative smoking >20 pack-years, had absolute 10-year risks varying from 4% to 16% by AHRR(cg05575921) methylation. ConclusionA simple risk chart including age, sex, education, smoking status, cumulative smoking, and AHRR(cg05575921) methylation, identifies individuals with 10-year lung cancer risk from below 1% to 16%. Including AHRR(cg05575921) methylation in the eligibility criteria for screening identifies smokers who would benefit the most from screening.

Predictors of Smoking in Older Adults and an Epigenetic Validation of Self-Report

There are several established predictors of smoking, but it is unknown if these predictors operate similarly for young and old smokers. We examined clinical data from the National Lung Screening Trial (NLST) to determine the predictive ability of gender, body mass index (BMI), marital status, and race on smoking behavior, with emphasis on gender interactions. In addition, we validated the self-report of smoking behaviors for a subgroup that had available epigenetic data in the form of cg05575921 methylation. Participants were N=9572 current or former smokers from the NLST biofluids database, age 55-74, minimum of 30 pack years, and mostly White. A subgroup of N=3084 who had DNA were used for the self-report validation analysis. The predictor analysis was based on the larger group and used penalized logistic regression to predict the self-report of being a former or current smoker at baseline. Cg05575921 methylation showed a moderate ability to discriminate among former and current smokers, AUC = 0.85 (95% confidence interval = [0.83, 0.86]). The final selected variables for the prediction model were BMI, gender, BMI by gender, age, divorced (vs. married), education, and race. The gender by BMI interaction was such that males had a higher probability of current smoking for lower BMI, but this switched to females having higher current smoking for overweight to obese. There is evidence that the self-reported smoking behavior in NLST is moderately accurate. The results of the primary analysis are consistent with the general smoking literature, and our results provide additional specificity regarding the gender by BMI interaction. Body weight issues might play a role in smoking cessation for older established smokers in a similar manner as younger smokers. It could be that women have less success with cessation when their BMI increases.

Using Cg05575921 methylation to predict lung cancer risk: a potentially bias-free precision epigenetics approach

ABSTRACT The decision to engage in lung cancer screening (LCS) necessitates weighing benefits versus harms. Previously, clinicians in the United States have used the PLCOM2012 algorithm to guide LCS decision-making. However, that formula contains race and gender-based variables. Previously, using data from a European study, Bojesen and colleagues have suggested that cg05575921 methylation could guide decision-making. To test this hypothesis in a more diverse American population, we examined DNA and clinical data from 3081 subjects from the National Lung Screening Trial (NLST) study. Using survival analysis, we found a simple linear predictor consisting of age, pack-year consumption and cg05575921, to have the best predictive power among several alternatives (AUC = 0.66). Results showed that the highest quartile of risk was more than 2-fold more likely to develop lung cancer than those in the lowest quartile. Race, ethnicity, and gender had no effect on prediction with both cg05575921 and pack years contributing equally (both p < 0.003) to risk prediction. Current smokers had considerably lower methylation than former smokers (46% vs 67%; p < 0.001) with the average methylation of those who quit approaching 80% after 25 years of cessation. Finally, current male smokers had lower mean cg05575921 percentage than female smokers (46% vs 49%; p < 0.001). We conclude that cg05575921 (along with age and pack years) can be used to guide LCS decision-making, and additional studies might focus on how best to use methylation to inform decision-making.

Clinical Implication of Smoking-Related Aryl-Hydrocarbon Receptor Repressor (AHRR) Hypomethylation in Japanese Adults.

Tobacco smoking is a leading preventable cause of morbidity and mortality worldwide; still, the success rate of smoking cessation is low in general. From the viewpoint of public health and clinical care, an objective biomarker of long-term smoking behavior is sought. This study assessed DNA methylation as a biomarker of smoking in a hospital setting through a combination of molecular approaches including genetic, DNA methylation and mRNA expression analyses. First, in an epigenome-wide association study involving Japanese individuals with chronic cardiovascular disease (n=94), genome-wide significant smoking association was identified at 2 CpG sites on chromosome 5, with the strongest signal at cg05575921 located in intron 3 of the aryl-hydrocarbon receptor repressor (AHRR) gene. Highly significant (P<1×10-27) smoking-cg05575921 association was validated in 2 additional panels (n=339 and n=300). For the relationship of cg05575921 methylation extent with time after smoking cessation and cumulative cigarette consumption among former smokers, smoking-related hypomethylation was found to remain for ≥20 years after smoking cessation and to be affected by multiple factors, such as cis-interaction of genetic variation. There was a significant inverse correlation (P=0.0005) between cg05575921 methylation extent and AHRR mRNA expression. The present study results support that reversion of AHRR hypomethylation can be a quantifiable biomarker for progress in and observance of smoking cessation, although some methodological points need to be considered.

AHRR (cg05575921) Methylation Safely Improves Specificity of Lung Cancer Screening Eligibility Criteria: A Cohort Study.

Screening reduces lung cancer mortality, but specificities of eligibility criteria are low. We tested if leukocyte AHRR(cg05575921) methylation improves specificity of lung cancer screening eligibility criteria. A total of 9,206 and 5,370 individuals of the 1991 to 1994 and 2001 to 2003 examinations of the Copenhagen City Heart Study, Denmark, were followed for lung cancer within 5 years after examination and mortality. Screening eligibility criteria (DANTE, DLCST, ITALUNG, LUSI, NELSON, NLST, and PLCOM2012) were evaluated, and AHRR (cg05575921) methylation extent at different methylation cut points was added. The model with the lowest number of eligible individuals per 5-year lung cancer was validated within the 2001 to 2003 examination. Eligibility criteria identified risk-groups ranging from 3,182 (DANTE) to 1,641 (ITALUNG) individuals. The positive predictive value was highest for PLCOM2012 (3.2%), while DANTE showed the highest negative predictive value (99.7%). Adding AHRR (cg05575921) methylation led to higher specificities for all criteria. Number of eligible individuals per 5-year lung cancer varied from 38 (NELSON) to 27 (NLST) with AHRR (cg05575921) methylation <55%. This last model led to a 21.9% lower screening burden and increased (P < 0.05) specificity of 84.0%. Findings were reproduced among the 5,334 individuals of the 2001 to 2003 examination. Adding AHRR (cg05575921) methylation on top of current eligibility criteria for lung cancer screening improves specificity by excluding those individuals with the lowest risk. The results point toward a potential clinical use of AHRR (cg05575921) methylation, which is a cost-effective measurement compared with lung CT scanning, to provide additional predictive risk information to identify eligible smokers for lung cancer screening. See related commentary by Hung, p. 698.

Abstract 13530: Epi+Gen CHD: A More Sensitive and Cost-Effective Tool to Assess Risk and Monitor Treatment Response for Coronary Heart Disease

Coronary heart disease (CHD) is preventable, but current lipid-based risk assessment tools such as The Framingham Risk Score (FRS) and ASCVD Pooled Cohort Equation (PCE) have limitations. Using DNA-based genetic and epigenetic biomarkers, we have developed a more sensitive and cost-effective incident CHD risk assessment tool, Epi+Gen CHD, that can provide actionable insights and monitor treatment response. This test can be administered remotely via telemedicine using at-home sampling, or in a clinical setting. Epi+Gen CHD was developed and validated using machine learning techniques and datasets from the Framingham Heart Study (FHS) and Intermountain Healthcare (IM). It incorporates five SNPs and three methylation markers for predicting 3-year incident CHD. It was more sensitive than FRS and PCE and had high generalizability across cohorts with sensitivity/specificity of 79/75% in the FHS test set and 75/72% in the IM set. In contrast, the sensitivity/specificity was 15/93% in FHS and 31/89% in IM for FRS, and sensitivity/specificity was 41/74% in FHS and 69/55% in IM for the PCE. To assess cost effectiveness, we conducted cost-utility analyses of Epi+Gen CHD. As compared to the PCE, we found that the use of Epi+Gen CHD was associated with both better survival and highly competitive negative incremental cost-effective ratios (cost savings) ranging from -$42,000 to -$-8,000 per quality-adjusted life year with and without accounting for a secondary test. Finally, to understand if the epigenetic biomarkers in Epi+Gen CHD could be leveraged to monitor treatment response, we performed a proof-of-concept study that assessed the three Epi+Gen CHD methylation sites in 39 subjects before and after 3 months of biochemically verified smoking cessation, then analyzed the relationship between change in methylation at each of the sites to the change in smoking intensity (cg05575921 methylation). We showed that in those who quit smoking, methylation change at one Epi+Gen CHD site (cg00300879) was significantly associated with change in smoking intensity (p &lt; 0.04). We conclude that Epi+Gen CHD can identify patients at risk for CHD, save more lives, reduce more healthcare spending and guide prevention interventions better than current methods.

The relationship of smoking to cg05575921 methylation in blood and saliva DNA samples from several studies

Numerous studies have shown that cg05575921 methylation decreases in response to smoking. However, secondary to methodological issues, the magnitude and dose dependency of that response is as of yet unclear. This lack of certainty is a barrier to the use of DNA methylation clinically to assess and monitor smoking status. To better define this relationship, we conducted a joint analysis of methylation sensitive PCR digital (MSdPCR) assessments of cg05575921 methylation in whole blood and/or saliva DNA to smoking using samples from 421 smokers and 423 biochemically confirmed non-smokers from 4 previously published studies. We found that cg05575921 methylation manifested a curvilinear dose dependent decrease in response to increasing cigarette consumption. In whole blood DNA, the Receiver Operating Characteristic (ROC) Area Under the Curve (AUC) of cg05575921 methylation for predicting daily smoking status was 0.98. In saliva DNA, the gross AUC was 0.91 with correction for cellular heterogeneity improving the AUC to 0.94. Methylation status was significantly associated with the Fagerstrom Test for Nicotine Dependence score, but with significant sampling heterogeneity. We conclude that MSdPCR assessments of cg05575921 methylation are a potentially powerful, clinically implementable tool for the assessment and management of smoking.

An Examination of Risk Factors for Tobacco and Cannabis Smoke Exposure in Adolescents Using an Epigenetic Biomarker.

Objective: Evolving patterns of nicotine and cannabis use by adolescents require new tools to understand the changing epidemiology of these substances. Here we describe the use of a novel epigenetic biomarker sensitive to both tobacco and cannabis smoke in a longitudinal sample of high-risk adolescents. We examine risk factors for positivity for this epigenetic biomarker in comparison to positivity for conventional serum biomarkers of nicotine and cannabis use.Method: Eastern Iowa 10th graders who had a friend or family member who smoked were eligible to participate in a longitudinal study over 10–12th grades. Subjects provided self-report data on nicotine, tobacco, and cannabis use patterns as well as blood samples that were used for serum cotinine and THC assays. DNA was prepared for analysis of methylation at the CpG cg05575921, a sensitive indicator of smoke exposure. Relationships between positivity for each these biomarkers and a variety of risk factors, including demographics, family and peer relationships, psychopathology, willingness to smoke, and perceptions of typical cigarette and cannabis users, were examined at the 10th (n = 442), 11th (n = 376), and 12th (n = 366) grade timepoints.Results: A increasing proportion of subjects were positive for cotinine (5–16%), THC (3–10%), and cg05575921 methylation (5–7%) across timepoints, with some overlap. Self-reported combusted tobacco and cannabis use was strongly correlated with all biomarkers, whereas cg05575921 methylation was not correlated with reported e-cigarette use. Dual users, defined as those positive for nicotine and THC in the 12th grade showed the greatest cumulative smoke exposure, indicated by cg05575921 methylation. Subjects reported more positive attitudes toward cannabis users than cigarette smokers, and willingness to smoke and positive perceptions of tobacco and cannabis smokers were significant risk factors for biomarker positivity across timepoints.Conclusion: We conclude that measurement of cg05575921 methylation in adolescents is a useful tool in detecting tobacco smoking in adolescents, and may be a novel tool for the detection of cannabis smoking and cannabis and tobacco co-use, though non-combusted forms of nicotine use do not appear to be detectable by this method.

The Reversion of DNA Methylation at Coronary Heart Disease Risk Loci in Response to Prevention Therapy

Coronary heart disease (CHD) is preventable, but the methods for assessing risk and monitoring response rely on imprecise lipid-based assessments. Recently, we have shown that an integrated genetic–epigenetic test that includes three methylation-sensitive digital PCR assays predicts 3-year risk for incident CHD better than lipid-based methods. However, whether methylation sites change in response to therapies that alter CHD risk is not known. Therefore, we assessed methylation at these three incident CHD-related sites in DNA from 39 subjects before and after three months of biochemically verified smoking cessation, then analyzed the relationship between change in methylation at each of the sites to the change in smoking intensity as assessed by cg05575921 methylation. We found that, in those who quit smoking, methylation change at one CHD risk marker (cg00300879) was significantly associated with change in cg05575921 methylation (p &lt; 0.04). We conclude that changes in incident CHD-related methylation occur within three months of cessation of smoking, a major risk factor for CHD. This suggests that the effectiveness of treatment of other CHD risk factors, such as high cholesterol, may be similarly quantifiable using epigenetic approaches. Further studies to determine the relationship of changes of methylation status in response to treatment of other CHD risk factors are indicated.

The Reversion of cg05575921 Methylation in Smoking Cessation: A Potential Tool for Incentivizing Healthy Aging.

Smoking is the largest preventable cause of mortality and the largest environmental driver of epigenetic aging. Contingency management-based strategies can be used to treat smoking but require objective methods of verifying quitting status. Prior studies have suggested that cg05575921 methylation reverts as a function of smoking cessation, but that it can be used to verify the success of smoking cessation has not been unequivocally demonstrated. To test whether methylation can be used to verify cessation, we determined monthly cg05575921 levels in a group of 67 self-reported smokers undergoing biochemically monitored contingency management-based smoking cessation therapy, as part of a lung imaging protocol. A total of 20 subjects in this protocol completed three months of cotinine verified smoking cessation. In these 20 quitters, the reversion of cg05575921 methylation was dependent on their initial smoking intensity, with methylation levels in the heaviest smokers reverting to an average of 0.12% per day over the 3-month treatment period. In addition, we found suggestive evidence that some individuals may have embellished their smoking history to gain entry to the study. Given the prominent effect of smoking on longevity, we conclude that DNA methylation may be a useful tool for guiding and incentivizing contingency management-based approaches for smoking cessation.

AHRR methylation in heavy smokers: associations with smoking, lung cancer risk, and lung cancer mortality

BackgroundA low level of methylation at cg05575921 in the aryl-hydrocarbon receptor repressor (AHRR) gene is robustly associated with smoking, and some studies have observed associations between cg05575921 methylation and increased lung cancer risk and mortality. To prospectively examine whether decreased methylation at cg05575921 may identify high risk subpopulations for lung cancer screening among heavy smokers, and mortality in cases, we evaluated associations between cg05575921 methylation and lung cancer risk and mortality, by histotype, in heavy smokers.MethodsThe β-Carotene and Retinol Efficacy Trial (CARET) included enrollees ages 45–69 with ≥ 20 pack-year smoking histories and/or occupational asbestos exposure. A subset of CARET participants had cg05575921 methylation available from HumanMethylationEPIC assays of blood collected on average 4.3 years prior to lung cancer diagnosis in cases. Cg05575921 methylation β-values were treated continuously for a 10% methylation decrease and as quintiles, where quintile 1 (Q1, referent) represents high methylation and Q5, low methylation. We used conditional logistic regression models to examine lung cancer risk overall and by histotype in a nested case-control study including 316 lung cancer cases (diagnosed through 2005) and 316 lung cancer-free controls matched on age (±5 years), sex, race/ethnicity, enrollment year, current/former smoking, asbestos exposure, and follow-up time. Mortality analyses included 372 lung cancer cases diagnosed between 1985 and 2013 with available methylation data. We used Cox proportional hazards models to examine mortality overall and by histotype.ResultsDecreased cg05575921 methylation was strongly associated with smoking, even in our population of heavy smokers. We did not observe associations between decreased pre-diagnosis cg05575921 methylation and increased lung cancer risk, overall or by histotype. We observed linear increasing trends for lung cancer-specific mortality across decreasing cg05575921 methylation quintiles for adenocarcinoma and small cell carcinoma (P-trends = 0.01 and 0.04, respectively).ConclusionsIn our study of heavy smokers, decreased cg05575921 methylation was strongly associated with smoking but not increased lung cancer risk. The observed association between cg05575921 methylation and increased mortality in adenocarcinoma and small cell histotypes requires further examination. Our results do not support using decreased cg05575921 methylation as a biomarker for lung cancer screening risk stratification.

AHRR hypomethylation as an epigenetic marker of smoking history predicts risk of myocardial infarction in former smokers

Background and aimsSmoking causes cardiovascular disease. AHRR hypomethylation at the cg05575921 site is associated with active and former smoking status at baseline, and cumulative amount of tobacco smoked. We tested the hypothesis that AHRR cg05575921 hypomethylation as an epigenetic marker of smoking history predicts the risk of myocardial infarction in former smokers. MethodsWe included 10,510 individuals with methylation extent measurements and information on smoking status from the Copenhagen City Heart Study (CCHS), a prospective, cohort study of the general population carried out from 1991 to 2003. The endpoint myocardial infarction was retrieved from the national Danish Patient Registry and the national Danish Causes of Death Registry. ResultsFor individuals in the 1st (lowest) quartile of AHRR cg05575921 methylation (≤49% methylation extent), 99% were ever smokers at baseline (active and former smokers combined) compared to 42% in the 4th (highest) quartile (>62% methylation extent). For former smokers, the cumulative incidence of myocardial infarction was higher in the lowest methylation extent (1st-50th percentile) compared to the highest methylation extent (51st-100th percentile). Compared to never smokers, the multivariable adjusted subhazard ratio for myocardial infarction was 1.09 (95%CI:0.88–1.35) for former smokers with the highest methylation degree, 1.38 (1.06–1.80) for active smokers with the highest methylation extent, 1.39 (1.08–1.78) for former smokers with the lowest methylation extent, and 1.61 (1.35–1.92) for active smokers with the lowest methylation extent. ConclusionsAHRR cg05575921 hypomethylation as an epigenetic marker of smoking history predicts risk of myocardial infarction, particularly in former smokers. Further, AHRR hypomethylation, regardless of smoking status, was associated with increased risk of myocardial infarction.

Abstract A20: AHRR hypomethylation in heavy smokers: Associations with lung cancer risk and mortality

Abstract Hypomethylation of cg05575921 in the aryl-hydrocarbon receptor repressor (AHRR) gene is consistently and strongly associated with higher exposure to smoking (e.g., current smoking, greater number of pack years, and less time since quitting). Although cg05575921 hypomethylation has been reported to be associated with lung cancer risk and mortality, this has not been explored within heavy smokers or by histotype. We assessed cg05575921 methylation for associations with smoking behavior, prospective lung cancer risk, and mortality in 313 cases and controls from the Beta Carotene and Retinol Efficacy Trial (CARET) with ≥20 pack years of smoking and matched on age (±5 years), sex, race/ethnicity, enrollment year, smoking status, occupational asbestos exposure, and follow-up time. Methylation of cg05575921 in blood collected on average 4.3 years prior to diagnosis in cases was assayed using the 850K Illumina EPIC array. Quintiles of cg05575921 methylation were defined based on the control distribution, with the lowest quintile (Q1, referent) representing hypermethylation and the highest (Q5), hypomethylation. Among controls, increasing quintiles of cg05575921 hypomethylation were associated with current smoking status (Cochran-Armitage trend p=3.0 × 10-21), and with decreasing years since last smoked, increasing cigarettes per day, and decreasing body mass index (BMI) (ordinal ANOVA p=3.1 × 10-21, 1.3 × 10-17, and 0.002, respectively). We observed similar patterns in cases. We evaluated associations between cg05575921 methylation and lung cancer risk using logistic regression models conditioned on matching factors and adjusted for age, years since quit, and BMI. We evaluated cg05575921 methylation and lung cancer-specific mortality using Cox proportional hazards models adjusted for stage, age, sex, race, smoking status, intervention arm, asbestos exposure, and pack years smoked. We did not observe clear patterns of associations between cg05575921 methylation and lung cancer risk overall or by histotype. However, we found a linear relationship (p=0.03) between increasing cg05575921 hypomethylation and overall lung cancer mortality, with Q5 vs Q1 Hazard Ratio (HR)=1.60 (95% Confidence Interval (95% CI): 1.00-2.57) for mortality from lung cancer overall, and HR=2.09, 95% CI: 0.96-4.54 for adenocarcinoma (N=121). Our findings provide preliminary support that even among heavy smokers, cg05575921 hypomethylation is associated with more recent and extensive exposure to cigarette smoking, and that prediagnosis cg05575921 hypomethylation in lung cancer cases is associated with an increased hazard of death. However, since the CARET population represents a group who would largely qualify for lung cancer screening with annual low-dose computed tomography per the United States Preventive Task Force guidelines, our results do not provide support for the use of cg05575921 hypomethylation as an indicator for screening-based lung cancer risk stratification. Citation Format: Stefan Graw, Matt J. Barnett, Mark D. Thornquist, Gary E. Goodman, Chu Chen, Devin C. Koestler, Carmen J. Marsit, Jennifer A. Doherty, Laurie Grieshober. AHRR hypomethylation in heavy smokers: Associations with lung cancer risk and mortality [abstract]. In: Proceedings of the AACR Special Conference on Modernizing Population Sciences in the Digital Age; 2019 Feb 19-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(9 Suppl):Abstract nr A20.

AHRR cg05575921 methylation in relation to smoking and PM2.5 exposure among Taiwanese men and women

BackgroundPolycyclic aromatic hydrocarbon (PAH)-rich substances like cigarette smoke and PM2.5 induce aryl hydrocarbon receptor (AHR)-mediated aryl hydrocarbon receptor repressor (AHRR) methylation. AHRR cg05575921 and coagulation factor II (thrombin) receptor-like 3 (F2RL3) cg03636183 methylation patterns are well-established biomarkers for smoking. Even though AHRR cg05575921 methylation has recently been associated with PM2.5, the interaction between smoking and PM2.5 on AHRR methylation is yet to be fully explored. We evaluated AHRR and F2RL3 CpG sites to identify potential significant markers in relation to PM2.5 and smoking in Taiwanese adults.MethodsDNA methylation and smoking data of 948 participants aged 30–70 years were obtained from the Taiwan Biobank Database (2008–2015), while PM2.5 data were obtained from the Air Quality Monitoring Database (2006–2011).ResultsSmoking and PM2.5 were independently associated with hypomethylation (lower levels) of AHRR cg05575921, AHRR cg23576855, F2RL3 cg03636183, and F2LR3 cg21911711 after multiple-comparison correction (Bonferroni P < 0.00028409). Cg05575921 was the most hypomethylated AHRR CpG site, while cg03636183 was the most hypomethylated F2RL3 CpG site. Overall, cg05575921 was the most hypomethylated CpG site: β = − 0.03909, P < 0.0001; − 0.17536, P < 0.0001 for former and current smoking, respectively (P-trendsmoking < 0.0001) and − 0.00141, P < 0.0001 for PM2.5. After adjusting for F2RL3 cg03636183, smoking and PM2.5 remained significantly associated with cg05575921 hypomethylation: β − 0.02221, P < 0.0001; − 0.11578, P < 0.0001 for former and current smoking, respectively (P-trendsmoking < 0.0001) and − 0.0070, P = 0.0120 for PM2.5. After stratification by sex, smoking and PM2.5 remained associated (P < 0.05) with cg05575921 hypomethylation in both men (β = − 0.04274, − 0.17700, and − 0.00163 for former smoking, current smoking, and PM2.5, respectively) and women (β = − 0.01937, − 0.17255, and − 0.00105 for former smoking, current smoking, and PM2.5, respectively). After stratification by residential area, former and current smoking remained associated (P < 0.05) with cg05575921 hypomethylation: β = − 0.03918 and − 0.17536, respectively (P-trendsmoking < 0.0001). Living in the central and southern areas was also associated (P < 0.05) with cg05575921 hypomethylation: β = − 0.01356 and − 0.01970, respectively (P-trendarea < 0.0001).ConclusionSmoking and PM2.5 were independently associated with hypomethylation of cg05575921, cg23576855, cg03636183, and cg21911711. The most hypomethylated CpG site was cg05575921 and its association with smoking and PM2.5 was dose-dependent.

Refinement of cg05575921 demethylation response in nascent smoking

The initiation of adolescent smoking is difficult to detect using carbon monoxide or cotinine assays. Previously, we and others have shown that the methylation of cg05575921 is an accurate predictor of adult smoking status. But the dose and time dependency of the demethylation response to smoking initiation in adolescents is not yet well understood. To this end, we conducted three consecutive annual in-person interviews and biological samplings of 448 high school students (wave 1 (W1)-wave 3 (W3)). At W1 (n = 448), 62 subjects reported using tobacco and 72 subjects reported using cannabis at least once in their life-time with 38 and 20 subjects having a positive cotinine and cannabinoid levels, respectively, at W1 intake. At W3 (n = 383), 67 subjects reported using tobacco and 60 subjects reported using cannabis at least once with 75 and 60 subjects having positive cotinine and cannabinoid levels, respectively, at W3. Subjects with undetectable cotinine levels at all three-time waves had stable levels of cg05575921 methylation throughout the study (88.7% at W1 and 88.8% at W3, n = 149), while subjects with positive cotinine levels at all 3 time points manifested a steady decrease in cg05575921 methylation (81.8% at W1 and 71.3% at the W3, n = 12). In those subjects with an affirmative smoking self-report at W3 (n = 17), the amount of demethylation at cg05575921 was correlated with time and intensity of smoking. We conclude that cg05575921 methylation is a sensitive, dose-dependent indicator of early stages of smoking, and may help to identify smokers in the early stages of smoking.

Abstract P251: Monitoring the Success of Smoking Cessation Using Dna Methylation Status at Cg05575921: Implications for Incentive Based Strategies for Quantitative Reduction of Harm

Cigarette smoking accounts for about 1/5 of all deaths from heart disease in the United States. Despite this grim fact, getting patients to quit smoking is extremely difficult. Encouragingly, several studies have shown that incentive based smoking cessation interventions may be more effective than traditional strategies. However, their implementation has been stymied by the lack of clinically implementable objective mechanisms to determine smoking cessation in those using nicotine replacement. Over the past several years, we and others have shown that DNA methylation status at a CpG site in the AHRR gene referred to as cg05575921 accurately classifies smoking status and predicts smoking intensity. In addition, we have developed a rapid clinically implementable digital PCR assay for assessing methylation at this locus using DNA prepared from blood or saliva. However, whether changes in DNA methylation at this locus occur quickly enough to allow the monitoring and possible incentivization of smoking cessation is unknown. To examine these possibilities, we followed 70 regular adult smokers as they underwent smoking cessation using a financial incentive based, pharmacologically assisted paradigm that stipulated no use of nicotine replacement. Consistent with prior reports, we found that as quantified by nondetectable serum cotinine levels at 1 month, 2 months and 3 months, 30% of all subjects who attempted to quit smoking in order to receive a $400 reward succeeded in quitting smoking. Over the course of their cessation trajectory, cg05575921 methylation demonstrated a stepwise increase commiserate with initial smoking intensity. Overall, the heaviest smokers, manifested a stepwise 14% increase in cg05575921 methylation while the lightest smokers manifested a 5% increase in cg05575921 methylation over the three month follow up period. Taken together with prior data, these new data demonstrate a robust reversion of cg05575921 in response to smoking cessation whose values can be used as objective evidence of smoking cessation even in those using nicotine replacement. In addition, because this method can detect and quantify partial cessation, these findings further suggest that the use of this technology together with incentive based approaches may also be useful in encouraging quantitative reduction of harm for cancer and smoking related cardiovascular risk in those who can reduce, but cannot fully quit smoking.