In this exploratory epigenome-wide association study (EWAS), we sought to investigate associations of pre-treatment blood-derived DNA methylation with survival and response outcomes among patients with recurrent/metastatic (R/M) head and neck squamous cell carcinomas (HNSCCs) who received immune checkpoint inhibitor (ICI) therapy. Patients with R/M HNSCC were recruited from the Dana-Farber Cancer Institute, Dartmouth Cancer Center, and Rhode Island Hospital. Peripheral blood was collected prior to the start of ICI, and DNA methylation was measured using the Illumina EPIC V1 and V2 BeadArrays. After strict quality control, the top 10% most variable CpG sites (n = 67,712) were included in an EWAS of methylation with overall survival (OS) and progression-free survival (PFS). Models were adjusted for key covariates, including the DNA methylation-derived immune cell-type proportions. Among the 116 patients enrolled, 64 (55.2%) died during follow-up, and 22 (19.0%) experienced cancer progression. Of the 30 patients (25.9%) who remained progression-free, 13 showed a complete response to the ICI therapy. We identified two CpG sites (cg04079758 and cg10654272) that were associated with improved PFS (false discovery rate [FDR] < 0.05). The CpG sites cg04079758 and cg10654272 are located in the South Shelf region on chromosome 19 and in an OpenSea region on chromosome 6, respectively. Additionally, one of the CpG sites, cg04079758, was located in Kallikrein 9 (KLK9), a gene involved in tissue remodeling and inflammation within the tumor microenvironment. No CpG site was statistically significantly associated with OS. Distinct blood DNA methylation signatures may be associated with ICI therapy-related outcomes in patients with R/M HNSCC.

Abstract Study Objectives Sleep during adolescence may influence biological aging, yet few studies have examined this association using objective sleep measures. We examined the associations between accelerometry-based sleep characteristics and epigenetic age (EA), epigenetic age acceleration (EAA), and the pace of biological aging. Methods For this preliminary analysis, we used data from the HOME Study (Cincinnati, OH). At age 12 years (2016-2019), we used accelerometers to assess total sleep time, sleep efficiency, number of awakenings, and sleep fragmentation. Using DNA methylation data at the same age, we estimated EA and EAA using skinbloodHorvath, Hannum, and Wu clocks, and the pace of biological aging using DunedinPACE. We used multivariable linear regression to estimate covariate-adjusted associations between sleep and epigenetic aging. We also evaluated sex modification by including product interaction terms and examined sex-stratified models. Results Among 138 adolescents (59% female; mean age 12.29 ± 0.64 years), general trends suggested that poorer sleep quality was associated with higher EA and EAA. However, all effect estimates were imprecise, and confidence intervals included the null. Notably, we found that sex modified these relationships: higher sleep fragmentation was associated with elevated EAA among males (Hannum β= 0.885, 95%CI: 0.132, 1.638; interaction p= 0.005), but not females (β= -0.325, 95%CI: -0.866, 0.217). These associations were consistent across first-generation clocks. Conclusions Overall, we did not find consistent evidence of associations between sleep characteristics and epigenetic aging in this preliminary analysis. However, sex-stratified analyses suggested that poorer sleep quality may be associated with greater epigenetic age acceleration among males.

Supplementary Figure S3: Semi-Supervised Recursively Partitioned Mixture Model (SS-RPMM) for 10-year overall survival (OS) in NMIBC patients [For Hannum age acceleration]

Despite advances with anti-programmed death (PD)-1-based immunotherapy for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC), identifying who will benefit from these therapies remains challenging. In a prospective, multicenter study, serum levels of 92 cytokines were measured in pretreatment sera collected on 38 (discovery cohort) and 40 (validation cohort) patients with R/M HNSCC undergoing anti-PD-1-based immunotherapy. A prognostic cytokine score comprised of four cytokines (CXCL1, MCP-4, IL6, CD40) was developed using Cox elastic net modeling within the discovery cohort and showed concordance indices of 0.71 and 0.79 for overall survival (OS) in the discovery and validation cohorts, respectively. High cytokine scores were linked to worse OS (p < 0.01, adjusted HR = 2.07, 95

Supplementary Figure S5: Kaplan-Meier analysis of 10-year overall survival based on the grouping results from both partDSA and SS-RPMM in all NMIBC patients [For Hannum age acceleration]

Supplementary Table S5: The information of optimal CpGs selected by SS-RPMM using the model controlling for Pheno and Hannum age acceleration respectively

Tumor tissue DNA methylation has been proposed as a potential biomarker for immune checkpoint inhibitor (ICI) response. However, less is known about blood DNA methylation as a potential biomarker for ICI treatment related outcomes in recurrent/metastatic (R/M) head and neck squamous cell carcinomas (HNSCC). Investigate associations of pre-treatment blood-derived DNA methylation profiles with survival and response outcomes among patients with R/M HNSCC who receive ICI therapy. Patients with R/M HNSCC were recruited from the Dana-Farber Cancer Institute, Dartmouth Cancer Center, and Rhode Island Hospital in an ongoing prospective study in the U.S. (data cutoff: September 2024). Peripheral blood was collected from each patient prior to start of ICI based therapy. DNA methylation was measured using the Illumina EPIC array V1 and V2. After strict quality control, the top 10% most variable CpG sites (n=67,713) were included in the analysis. Epigenome-wide association study analysis was conducted for overall survival (OS), progression-free survival (PFS), and treatment response (durable benefit, non-durable benefit, and non-benefit) in association with normalized CpG beta-values. Models were adjusted for age, sex, smoking status, and DNA methylation-derived estimated blood cell type proportions. Among the 113 subjects enrolled so far, 59 (52.2%) died during follow-up and 18 (15.9%) experienced cancer progression. Of 36 patients (31.9%) who have remained progression-free survivors, 11 patients showed a complete response to the ICI therapy. The median OS and PFS were 428 and 279 days, respectively. Approximately half of the patients were categorized in the durable benefit group (n=57, 50.4 %), followed by the non-benefit group (n=30, 26.5 %) and the non-durable benefit group (n=26, 23.0 %). We identified two CpG sites (cg04997743 and cg25104727) that were associated with improved OS; and three (cg03090869, cg19041076, and cg22088248) that were associated with worse OS (false discovery rate [FDR] < 0.05). Similarly, six CpG sites (cg04079758, cg10654272, cg14132225, cg14812628, cg20071413, and cg25104727) were associated with improved PFS while a single CpG (cg24138433) was correlated with worse PFS (FDR < 0.05). The CpG sites associated with OS or PFS were annotated to CpG islands chr11:69924338-69925197, chr18:72916107-72917233, and chr19:51505914-51506363. Additionally, these CpG sites were located in genes that are broadly characterized by metabolism-related (ZADH2 and PPT1) or inflammation-related (KLK9 and ANO1) pathways. No CpG site was associated with the treatment response categories (FDR > 0.85). Distinct blood DNA methylation signatures may be associated with ICI therapy-related outcomes in patients with R/M HNSCC. Gyeyoon Yim, Lucas Salas, Kartik Sehgal, Keisuke Shirai, Rondi Butler, Julianna Miller, Hannah Stolrow, Md Saiful Islam Saif, Devin Koestler, John Wiencke, Robert Haddad, Karl Kelsey, Brock Christensen. Blood DNA methylation in association with immune checkpoint inhibitor related outcomes among patients with head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6410.

Supplementary Figure S1: The distribution of chronological age, methylation age, age acceleration, and each immune cell profile

Supplementary Table S3: Cox proportional hazards models of immune cell proportions and NMIBC patient outcomes (For Hannum Age Acceleration)

Background Evaluating individual health outcomes does not capture co-morbidities children experience. Purpose We aimed to describe profiles of child neurodevelopment and anthropometry and identify their predictors. Methods Using data from 501 mother-child pairs (age 3-years) in the Maternal-Infant Research on Environmental Chemicals (MIREC) Study, a prospective cohort study, we developed phenotypic profiles by applying latent profile analysis to twelve neurodevelopmental and anthropometric traits. Using multinomial regression, we evaluated odds of phenotypic profiles based on maternal, sociodemographic, and child level characteristics. Results For neurodevelopmental outcomes, we identified three profiles characterized by Non-optimal (9%), Typical (49%), and Optimal neurodevelopment (42%). For anthropometric outcomes, we observed three profiles of Low (12%), Average (61%), and Excess Adiposity (27%). When examining joint profiles, few children had both Non-optimal neurodevelopment and Excess Adiposity (2%). Lower household income, lower birthweight, younger gestational age, decreased caregiving environment, greater maternal depressive symptoms, and male sex were associated with increased odds of being in the Non-optimal neurodevelopment profile. Higher pre-pregnancy body mass index was associated with increased odds of being in the Excess Adiposity profile. Conclusions Phenotypic profiles of child neurodevelopment and adiposity were associated with maternal, sociodemographic, and child level characteristics. Few children had both non-optimal neurodevelopment and excess adiposity.

Human cytomegalovirus (CMV) is a beta-herpes virus that may play a role in head and neck squamous cell carcinoma (HNSCC). CMV is a highly prevalent latent infection among older US adults, and viral reactivation occurs in response to immune suppression and inflammation. Previously, in a study of 184 HNSCC we reported that elevated CMV IgG titers were associated with worse patient survival. Here, we have expanded our evaluation of CMV IgG and patient survival in 983 cases from the CoHANDS study. CMV positive patients had significantly worse HNSCC survival (p<0.003); this association remained after controlling for age, sex and stage (HR=1.30, 95% CI 1.02- 1.74). The association between CMV and outcome was independent of the known association between human papilloma virus infection (HPV) and patient survival. Next, we used nCounter gene expression data to evaluate immune infiltration and inflammation in 34 tumors from HNSCC patients with and without active CMV infection; active infection was defined as having DNAemia, the presence of cell-free CMV DNA in serum. Tumors from patients with active CMV infection were more likely to contain CMV transcript (p<0.06) and have increased T-cell infiltration (p<0.05), however there were no other differences in the immune infiltrate. The Tumor Inflammation Signature (TIS), a gene expression indicator that correlates with response to immune checkpoint inhibitors, was significantly higher in CMV positive patients (p=0.007). CMV positive patients were 12 times more likely to have an elevated TIS score (adjusted OR 12.4, 95% CI 1.2 - 124.2). In summary, HNSCC patients who are CMV IgG positive have poor survival and tumors from patients with active CMV infection have both increased T-cell infiltration and elevated tumor inflammation. These findings may have potential implications for immune therapy in HNSCC. Heather H. Nelson, DeVon Hunter-Schlichting, Rondi Butler, Karl Kelsey. Human cytomegalovirus, tumor characteristics and patient survival in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6233.

Supplementary Table S4: Cox proportional hazards models of immune cell proportions and NMIBC patient outcomes (For Pheno Age Acceleration; NMIBC patients without BCG treatment; N = 512)

Human cytomegalovirus (CMV) is a common infection that can have a profound impact on the immune system which, in turn, has the potential to impact cancer risk. Natural Killer (NK) cells are the front-line defense against viral infection and CMV seropositive individuals develop a specialized subset of NK cells, the adaptive NK (aNK), in response to CMV reactivation. A defining feature of aNK is surface expression of the NKG2C receptor, encoded by the KLRC2 gene. There is a polymorphic 15.5 kb deletion of the KLRC2 gene and approximately 5% of the Caucasian population completely lacks the NKG2C receptor due to this polymorphism. KLRC2 gene copy number has been shown to influence steady-state levels of aNK cells in CMV seropositive individuals, and deletion of the gene is associated with both early life CMV infection and CMV viremia after transplantation. Here, we have evaluated whether the polymorphic KLRC2 gene deletion is associated with active CMV infection and risk of head and neck squamous cell carcinoma (HNSCC) using the resources of the CoHANDS epidemiology study. KLRC2 deletion was measured using PCR with gel visualization in 1, 096 cases and 1, 179 controls. Among a subset of the controls (n=207) we measured CMV DNAemia, the presence of cell free CMV DNA in serum, which is an established biomarker of active CMV infection. KLRC2 deletion was associated with the presence of CMV DNAemia: among those with one copy of KLRC2 the risk of active infection was 2.74 (95% CI 1.30 - 5.78), and among those with homozygous deletion of KLRC2 the risk was 5.17 (95% CI 1.56 - 17.16). These results from a healthy population are consistent with prior reports that the KLRC2 gene is important in the control of CMV reactivation. We then evaluated KLRC2 as a risk factor for HNSCC. Homozygous deletion of the KLRC2 gene was strongly associated with increased risk of HNSCC after adjusting for age and sex (OR 3.99; 95% CI 2.62 - 6.10). Collectively, these data support the hypothesis that active CMV infection may be a risk factor for HNSCC. Heather H. Nelson, DeVon Hunter-Schlichting, Rondi Butler, Anna Prizment, Bharat Thyagarajan, Karl Kelsey. KLRC2 gene deletion is associated with active cytomegalovirus infection and risk of head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2293.

Supplementary Table S6: Characteristics of subjects of each group based on the grouping results from both partDSA and SS-RPMM in all NMIBC patients

DNA methylation (DNAm)-predicted deconvolution uses cell-specific methylation markers of leukocytes in peripheral blood to estimate immune cell type proportions. Validity is high for predicted proportions of immune cell types. Given known age-related immune system changes and changes in predicted immune cell proportions by age, we assessed whether the correlations between predicted and measured proportions differ across age. Such information is needed for cancer studies on immunoaging in cohorts without CBC differential or stored whole blood. Lymphocyte, neutrophil, monocyte, eosinophil, and basophil proportions were previously estimated using DNAm deconvolution (Salas et al., PMID: 35140201) and measured by CBC among Black participants (N=151; 62% female) without cancer in the Atherosclerosis Risk in Communities study. Pearson correlation coefficients were calculated in age tertiles (T1: 47-53, T2: 54-60, T3: 61-67 years). To minimize confounding by health factors, we stratified by more (no diabetes, BMI <30 kg/m2, and non-smoker) and fewer (≤ 2 of the factors) positive health factors. Correlations in age T1 and T3 were compared after Fisher’s z-transformation. Correlations between predicted and measured proportion were similar across age tertiles, albeit slightly weaker in the oldest tertile (Table, except for basophils). This same pattern was present stratifying by health factors, although the correlations appeared slightly stronger for more vs. fewer positive health factors (e.g., age T1, T3 - neutrophils: more 0.81, 0.77; fewer 0.78, 0.66). However, all p-values were > 0.1 comparing correlations in the youngest and oldest age tertiles. For each common immune cell type, correlations between DNAm-predicted and measured proportion were generally similar across mid- to older adulthood, supporting use of the algorithm in immunoaging studies. Studies with participant diversity, including ages ≥ 70 yrs are needed. Zhike Lin, Dominique S. Michaud, Naisi Zhao, Vernon A. Burk, Eric Boerwinkle, Jan Bressler, Devin C. Koestler, Karl T. Kelsey, Elizabeth A. Platz. Correlation between DNA-methylation predicted and measured immune-cell type proportions by age in ARIC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2278.

Supplementary Table S2: Cox proportional hazards multivariable models for demographic and tumor characteristics of 601 NMIBC patients (For Hannum Age Acceleration)

Indoor radon is a human lung carcinogen. The objective of this research is to describe residential radon distribution in a European region with high geogenic radon potential using cross‐sectional data. This study is also aimed at describing the use of census tracts as a geographical unit in radon maps and describing radon distribution according to floor level. To do this, 6080 radon measurements were carried out by the Galician Radon Laboratory (GRL, https://www.radon.gal ) with alpha track detectors located at each dwelling for at least 3 months. The dwellings measured were the primary residences of volunteer participants. All participants filled out a questionnaire on the dwelling characteristics, and a follow‐up call assured the appropriate return of radon devices to the GRL. A descriptive analysis was performed, including radon concentration distribution per floor level measured and a Galician map of indoor radon using both municipalities and census tracts as geographic units was made. In Galicia, a median radon concentration of 132 Bq/m 3 (interquartile range: 66–239 Bq/m 3 ) and a geometric mean (GM) of 126 Bq/m 3 were observed. Eighteen percent of dwellings exceeded 300 Bq/m 3 . Radon levels vary widely across and within municipalities. The latter can be observed with census tract‐level maps. A height gradient for radon concentration was observed from a GM of 289 Bq/m 3 in cellars to a GM of 89 Bq/m 3 on the fifth floor and above. Remarkably, 14% of dwellings on the second floor exceeded 300 Bq/m 3 , thus challenging the paradigm that indoor radon is only a priority for underground and ground levels in radon priority areas. Galicia is mostly composed of radon priority areas, with high heterogeneity in indoor radon distribution. Census tracts can be used in indoor radon maps as the smallest geographic unit to increase resolution and acknowledge variations within municipalities. In radon priority areas, radon surveillance should include second‐floor levels.

Abstract Background: Prior cohort studies assessing cancer risk based on immune cell subtype profiles have predominantly focused on White populations. This limitation can obscure vital insights into how cancer risks vary across races, ethnicities, or exposures. This study was designed with the purpose of examining associations between pre-diagnostic immune cell profiles and cancer risk in Black participants. Methods: In this study, DNA methylation was measured in blood samples collected between 1993 and 1995 from a subset of 2,467 Black participants in the Atherosclerosis Risk in Communities (ARIC) prospective cohort study. Immune cell subtype proportions were estimated using deconvolution based on leukocyte DNA methylation markers. The analysis included 668 incident cancers ascertained between time of blood draw and 2015. Cox proportional hazards regression models were used to analyze the association between immune cell subtype proportions and cancer incidence (all cancers; lung, breast, and prostate cancers). Models controlled for age, sex, BMI, self-reported smoking status, self-reported and methylation-derived smoking pack-years, postmenopausal hormone use, and batch effect, along with other cancer-specific factors. Results: Increased T regulatory cell (Treg) proportions were associated with significantly higher risk of lung cancer (HR: 1.22, 95% CI = 1.06, 1.41 per percent increase; n = 84 cases) and suggestively higher risk of all cancers. Increased B memory cell proportions were associated with significantly higher risk of prostate cancer (HR: 1.17, 95% CI = 1.04, 1.33; n = 173 cases), all cancers (HR: 1.13, 95% CI = 1.05, 1.22), and suggestively higher risk of lung cancer. Increased CD8+ naïve cell proportions were associated with suggestively lower risk of lung cancer and all cancers, and significantly lower risk of all cancers in participants over the median age of 55 years (HR: 0.91, 95% CI = 0.83, 0.98). Cancer risks for other immune cell subtypes were null. Conclusions: These results in Black participants align closely with prior findings in largely White populations. Deconvolution allowed for the extension of work on cancer immunity (which has largely focused on major immune cell types) to immune cell subtypes like B memory cells and CD8+ naïve cells. The results are consistent with Tregs initiating and accelerating cancer development by impeding the usual protective immune response. A large reservoir of CD8+ naïve cells may allow for a more targeted and efficient elimination of malignant clones. Further studies should assess these relationships in other cancer types and better elucidate the interplay of B cells in theorized mechanistic processes. This study provides insight into the role of immune cell subtypes in cancer risk among Black men and women. Funding: NHLBI, NCR, NPCR Citation Format: Christopher S. Semancik, Naisi Zhao, Devin Koestler, Eric Boerwinkle, Jan Bressler, Karl Kelsey, Elizabeth A. Platz, Dominique S. Michaud. DNA methylation-derived immune cell profiles and risk of cancers in Black participants in the ARIC Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 844.