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PDF file, 437KB, Supplementary Table 1: Characteristics of the leukocyte subtype study population.Supplementary Table 2: Characteristics of the study population in the HNSCC data set. Supplementary Table 3: Characteristics of the study population in the Bladder cancer data set. Supplementary Table 4: The top 50 differentially methylated regions (DMRs) among the leukocyte subtypes (false discovery rate q-values < 0.001 for all). Supplementary Figure 1: Diagram illustrating Semi-Supervised Recursively Partitioned Mixture Models (SS-RPMM). Supplementary Figure 2: Diagram representing the analytic workflow the HNSCC data set. Supplementary Figure 3: Diagrams representing the analytic workflow the ovarian cancer data set. Supplementary Figure 4: Diagrams representing the analytic workflow the bladder cancer data set. Supplementary Figure 5: Results from the SS-RPMM analysis of the Ovarian cancer data set.

Supplemental Figure 2. Deduplication and alignment statistics. a. Bismark v0.10.1 was used for alignment and deduplication. Duplicates were discarded using deduplicate_bismark perl script. With total number of alignments analyzed (red), total number of duplicated alignments removed (green) and total count of de-duplicated sequences (blue). b. Total sequence pairs analyzed (yellow), number of paired-end alignments with a unique hit (green), sequence pairs with no alignment (light blue), sequence pairs that did not map (blue), sequence pairs that were discarded due to no genomic sequence extracted (pink)

J non-overlapping L-DMRs derived from the Illumina HM450 reference leukocyte methylation data.

Expression of ABC transporter genes in 2D and in 3D cultures.

Mitochondria contribute to tumor growth through multiple metabolic pathways, regulation of extracellular pH, calcium signaling, and apoptosis. Using the Mitochondrial Nuclear Exchange (MNX) mouse models, which pair nuclear genomes with different mitochondrial genomes, we previously showed that mitochondrial SNPs regulate mammary carcinoma tumorigenicity and metastatic potential in genetic crosses. Here, we tested the hypothesis that polymorphisms in stroma significantly affect tumorigenicity and experimental lung metastasis. Using syngeneic cancer cells (EO771 mammary carcinoma and B16-F10 melanoma cells) injected into wild-type and MNX mice (i.e., same nuclear DNA but different mitochondrial DNA), we showed mt-SNP–dependent increases (C3H/HeN) or decreases (C57BL/6J) in experimental metastasis. Superoxide scavenging reduced experimental metastasis. In addition, expression of lung nuclear-encoded genes changed specifically with mt-SNP. Thus, mitochondrial–nuclear cross-talk alters nuclear-encoded signaling pathways that mediate metastasis via both intrinsic and extrinsic mechanisms.Significance:Stromal mitochondrial polymorphisms affect metastatic colonization through reactive oxygen species and mitochondrial–nuclear cross-talk.

Supplemental Figure 3. Integrative analysis of methylation and gene expression. The methylation level for the five most differentially expressed genes (listed at the top of each page), for which also methylation data was available, is shown in Panels A-E for each gene. Chromosomal position of the corresponding gene is shown on the x-axis, and the methylation in percentage is shown on the y-axis. Each star represents one CpG. CpG''s for BB are shown in green, while CpG''s for FB are shown in orange. For both types a LOWESS (Locally Weighted Scatterplot Smoothing) smoother was used to create a "regression line". The boxplot in panel F for each gene presents the normalized gene expression for both samples (BB and FB) for the five previously picked genes. Again, green indicates a BB-sample and orange indicates a FB-sample. Each box visualizes two normalized gene expressions (two replicates).

Supplementary Figure S1: Genotyping of MNX strains. Representative gels showing genotyping of MNX and matching wild-type strains. Total DNA was isolated from tail clips of weanling mice. DNA was amplified using primers that span A: 9461 Câ†'T polymorphism in C57BL/6J mitochondria which does not allow the incorporation of a Bcl1 restriction site, which is present in FVB/NJ or B: 9348 Aâ†'G polymorphism present in BALB/cJ but not FVB/NJ which does not allow the restriction site for Pflf1 to incorporate, this DNA was then exposed to restriction enzymes, A: Bcl1 and B: Pflf1 and resolved on an agarose gel.

Supplementary Table S1: Primer sequences for RFLP sites and Her2 transgene

Supplemental Figure 7. Apoptosis Analysis. Annexin V-FITC/propidium iodide (PI) staining assay were used to measure apoptosis in MEFs harvested from the following strains: FF, FC, BB, FB, CC, CH, HH, HC (as described in Table 1). Apoptotic cells were detected by flow cytometry and analyzed using FACS Diva.

Drug development for first-line treatment of epithelial ovarian cancer (EOC) has been stagnant for almost three decades. Traditional cell culture methods for primary drug screening do not always accurately reflect clinical disease. To overcome this barrier, we grew a panel of EOC cell lines in three-dimensional (3D) cell cultures to form multicellular tumor spheroids (MCTS). We characterized these MCTS for molecular and cellular features of EOC and performed a comparative screen with cells grown using two-dimensional (2D) cell culture to identify previously unappreciated anticancer drugs. MCTS exhibited greater resistance to chemotherapeutic agents, showed signs of senescence and hypoxia, and expressed a number of stem cell–associated transcripts including ALDH1A and CD133, also known as PROM1. Using a library of clinically repurposed drugs, we identified candidates with preferential activity in MCTS over 2D cultured cells. One of the lead compounds, the dual COX/LOX inhibitor licofelone, reversed the stem-like properties of ovarian MCTS. Licofelone also synergized with paclitaxel in ovarian MCTS models and in a patient-derived tumor xenograft model. Importantly, the combination of licofelone with paclitaxel prolonged the median survival of mice (>141 days) relative to paclitaxel (115 days), licofelone (37 days), or vehicle (30 days). Increased efficacy was confirmed by Mantel–Haenszel HR compared with vehicle (HR = 0.037) and paclitaxel (HR = 0.017). These results identify for the first time an unappreciated, anti-inflammatory drug that can reverse chemotherapeutic resistance in ovarian cancer, highlighting the need to clinically evaluate licofelone in combination with first-line chemotherapy in primary and chemotherapy-refractory EOC.Significance: This study highlights the use of an in vitro spheroid 3D drug screening model to identify new therapeutic approaches to reverse chemotherapy resistance in ovarian cancer. Cancer Res; 78(15); 4370–85. ©2018 AACR.

Supplemental Figure 1. Validation of Methyl-Seq and RNA-Seq. Several hypo-/hyper-methylated and down-/up-regulated genes were selected for validation using methylation specific primers (64) or quantitative PCR, respectively. Graphs depict fold-change between MNX strains and corresponding wild-type mice are shown to the left. All gene expression data were normalized to beta actin. Each bar combines pools A and B and the experiments were performed in triplicate. Error bars represent standard error. Representative gels showing unmethylated (U) and methylated (M) DNA are shown. The table summarizes specific genes tested and whether the RNA-or Methyl-Seq data were and were not validated.

Results from a Cox-regression model fit to the Bladder cancer data set.

Using a novel mouse model, a mitochondrial-nuclear exchange model termed MNX, we tested the hypothesis that inherited mitochondrial haplotypes alter primary tumor latency and metastatic efficiency. Male FVB/N-Tg(MMTVneu)202Mul/J (Her2) transgenic mice were bred to female MNX mice having FVB/NJ nuclear DNA with either FVB/NJ, C57BL/6J, or BALB/cJ mtDNA. Pups receiving the C57BL/6J or BALB/cJ mitochondrial genome (i.e., females crossed with Her2 males) showed significantly (P < 0.001) longer tumor latency (262 vs. 293 vs. 225 days), fewer pulmonary metastases (5 vs. 7 vs. 15), and differences in size of lung metastases (1.2 vs. 1.4 vs. 1.0 mm diameter) compared with FVB/NJ mtDNA. Although polyoma virus middle T–driven tumors showed altered primary and metastatic profiles in previous studies, depending upon nuclear and mtDNA haplotype, the magnitude and direction of changes were not the same in the HER2-driven mammary carcinomas. Collectively, these results establish mitochondrial polymorphisms as quantitative trait loci in mammary carcinogenesis, and they implicate distinct interactions between tumor drivers and mitochondria as critical modifiers of tumorigenicity and metastasis. Cancer Res; 77(24); 6941–9. ©2017 AACR.

AimOur goal was to investigate individual susceptibility to periodontitis by conducting an epigenome-wide association study using peripheral blood.Materials and MethodsFor this analysis, we included 1077 African American and 457 European American participants of the Atherosclerosis Risk in Communities (ARIC) study who had completed a dental examination or reported being edentulous at visit 4 and had available data on DNA methylation. DNA methylation levels were compared by periodontal disease severity and edentulism to identify differentially methylated regions (DMRs) and evaluate the CpGs belonging to those DMRs using multinominal logistic regression.ResultsWe identified a region in geneZFP57(6p22.1) that was significantly hypomethylated in severe periodontal disease compared to no/mild periodontal disease in European American participants. A separate region in an unknown gene (located in Chr10: 743,992-744,958) demonstrated significant positive association with edentulism compared to no/mild periodontal disease in African American participants. Four CpGs in a region located withinHOXA4were significantly hypermethylated in severe periodontal disease compared to no/mild periodontal disease in African American participants.ConclusionsOur study highlights epigenetic variations inZPF57andHOXA4that were significantly and reproducibly associated with periodontitis. Future studies should evaluate gene regulatory mechanisms in the candidate regions of these loci.CLINICAL RELEVANCEScientific Rationale for StudyWithout altering the DNA sequence, epigenetic effects (e.g., DNA methylation changes) can alter gene activity and influence host response to periodontal infections. Our well-powered study investigates individual susceptibility to periodontitis by conducting a thorough assessment of periodontitis-related DNA methylation levels in blood.Principal FindingsWe identified two gene regions,ZPF57andHOXA4, that are differentially methylated in individuals with compared to those without periodontitis.Practical implicationsStudying differential leukocyte DNA methylation patterns may point to candidate regions and underlying gene regulatory mechanisms that play a key role in the progression and/or susceptibility to periodontitis.

Selenium is an important micronutrient for foetal development. MicroRNAs play an important role in the function of the placenta, in communication between the placenta and maternal systems, and their expression can be altered through environmental and nutritional cues. To investigate the associations between placental selenium concentration and microRNA expression in the placenta, our observational study included 393 mother-child pairs from the New Hampshire Birth Cohort Study (NHBCS) and the Rhode Island Child Health Study (RICHS). Placental selenium concentrations were quantified using inductively coupled plasma mass spectrometry, and microRNA transcripts were measured using RNA-seq. We fit negative binomial additive models for assessing the association between selenium and microRNAs. We used the microRNA Data Integration Portal (mirDIP) to predict the target mRNAs of the differentially expressed microRNAs and verified the relationships between miRNA and mRNA targets in a subset of samples using existing whole transcriptome data (N = 199). We identified a non-monotonic association between selenium concentration and the expression of miR-216a-5p/miR-217-5p cluster (effective degrees of freedom, EDF = 2.44 and 2.08; FDR = 3.08 × 10−5) in placenta. Thirty putative target mRNAs of miR-216a-5p and/or miR-217-5p were identified computationally and empirically and were enriched in selenium metabolic pathways (driven by selenoprotein coding genes, TXNRD2 and SELENON). Our findings suggest that selenium influences placental microRNA expression. Further, miR-216a-5p and its putative target mRNAs could be the potential mechanistic targets of the health effect of selenium.

Background There is considerable evidence that epigenetic mechanisms and DNA methylation are critical drivers of immune cell lineage differentiation and activation. However, there has been limited coordinated investigation of common epigenetic pathways among cell lineages. Further, it remains unclear if long-lived memory cell subtypes differentiate distinctly by cell lineages. Results We used the Illumina EPIC array to investigate the consistency of DNA methylation in B cell, CD4 T, and CD8 T naïve and memory cells states. In the process of naïve to memory activation across the three lineages, we identify considerable shared epigenetic regulation at the DNA level for immune memory generation. Further, in central to effector memory differentiation, our analyses revealed specific CpG dinucleotides and genes in CD4 T and CD8 T cells with DNA methylation changes. Finally, we identified unique DNA methylation patterns in terminally differentiated effector memory (TEMRA) CD8 T cells compared to other CD8 T memory cell subtypes. Conclusions Our data suggest that epigenetic alterations are widespread and essential in generating human lymphocyte memory. Unique profiles are involved in methylation changes that accompany memory genesis in the three subtypes of lymphocytes.

Background Tumor-based classification of human glioma portends patient prognosis, but considerable unexplained survival variability remains. Host factors (eg, age) also strongly influence survival times, partly reflecting a compromised immune system. How blood epigenetic measures of immune characteristics and age augment molecular classifications in glioma survival has not been investigated. We assess the prognostic impact of immune cell fractions and epigenetic age in archived blood across glioma molecular subtypes for the first time. Methods We evaluated immune cell fractions and epigenetic age in archived blood from the University of California San Francisco Adult Glioma Study, which included a training set of 197 patients with IDH-wild type, 1p19q intact, TERT wild type (IDH/1p19q/TERT-WT) glioma, an evaluation set of 350 patients with other subtypes of glioma, and 454 patients without glioma. Results IDH/1p19q/TERT-WT patients had lower lymphocyte fractions (CD4+ T, CD8+ T, natural killer, and B cells) and higher neutrophil fractions than people without glioma. Recursive partitioning analysis delineated 4 statistically significantly different survival groups for patients with IDH/1p19q/TERT-WT based on an interaction between chronological age and 2 blood immune factors, CD4+ T cells, and neutrophils. Median overall survival ranged from 0.76 years (95% confidence interval = 0.55-0.99) for the worst survival group (n = 28) to 9.72 years (95% confidence interval = 6.18 to not available) for the best (n = 33). The recursive partitioning analysis also statistically significantly delineated 4 risk groups in patients with other glioma subtypes. Conclusions The delineation of different survival groups in the training and evaluation sets based on an interaction between chronological age and blood immune characteristics suggests that common host immune factors among different glioma types may affect survival. The ability of DNA methylation-based markers of immune status to capture diverse, clinically relevant information may facilitate noninvasive, personalized patient evaluation in the neuro-oncology clinic.

Batch effect Reduction of mIcroarray data with Dependent samples usinG Empirical Bayes (BRIDGE) is a recently developed statistical method to address the issue of batch effect correction in batch-confounded microarray studies with dependent samples. The key component of the BRIDGE methodology is the use of samples run as technical replicates in two or more batches, “bridging samples”, to inform batch effect correction/attenuation. While previously published results indicate a relationship between the number of bridging samples, M, and the statistical power of downstream statistical testing on the batch-corrected data, there is of yet no formal statistical framework or user-friendly software, for estimating M to achieve a specific statistical power for hypothesis tests conducted on the batch-corrected data. To fill this gap, we developed pwrBRIDGE, a simulation-based approach to estimate the bridging sample size, M, in batch-confounded longitudinal microarray studies. To illustrate the use of pwrBRIDGE, we consider a hypothetical, longitudinal batch-confounded study whose goal is to identify Alzheimer’s disease (AD) progression-associated genes from amnestic mild cognitive impairment (aMCI) to AD in human blood after a 5-year follow-up. pwrBRIDGE helps researchers design and plan batch-confounded microarray studies with dependent samples to avoid over- or under-powered studies.

Purpose The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation that has been reported to be associated with survival after chronic disease diagnoses, including lung cancer. We hypothesized that the inflammatory profile reflected by pre-diagnosis NLR, rather than the well-studied pre-treatment NLR at diagnosis, may be associated with increased mortality after lung cancer is diagnosed in high-risk heavy smokers. Methods We examined associations between pre-diagnosis methylation-derived NLR (mdNLR) and lung cancer-specific and all-cause mortality in 279 non-small lung cancer (NSCLC) and 81 small cell lung cancer (SCLC) cases from the β-Carotene and Retinol Efficacy Trial (CARET). Cox proportional hazards models were adjusted for age, sex, smoking status, pack years, and time between blood draw and diagnosis, and stratified by stage of disease. Models were run separately by histotype. Results Among SCLC cases, those with pre-diagnosis mdNLR in the highest quartile had 2.5-fold increased mortality compared to those in the lowest quartile. For each unit increase in pre-diagnosis mdNLR, we observed 22–23% increased mortality (SCLC-specific hazard ratio [HR] = 1.23, 95% confidence interval [CI]: 1.02, 1.48; all-cause HR = 1.22, 95% CI 1.01, 1.46). SCLC associations were strongest for current smokers at blood draw (Interaction Ps = 0.03). Increasing mdNLR was not associated with mortality among NSCLC overall, nor within adenocarcinoma (N = 148) or squamous cell carcinoma (N = 115) case groups. Conclusion Our findings suggest that increased mdNLR, representing a systemic inflammatory profile on average 4.5 years before a SCLC diagnosis, may be associated with mortality in heavy smokers who go on to develop SCLC but not NSCLC.

BackgroundTo reduce lung cancer burden in the US, a better understanding of biological mechanisms in early disease development could provide new opportunities for risk stratification.MethodsIn a nested case-control study, we measured blood leukocyte DNA methylation levels in pre-diagnostic samples collected from 430 men and women in the 1989 CLUE II cohort. Median time from blood drawn to diagnosis was 14 years for all participants. We compared DNA methylation levels by case/control status to identify novel genomic regions, both single CpG sites and differentially methylated regions (DMRs), while controlling for known DNA methylation changes associated with smoking using a previously described pack-years based smoking methylation score. Stratification analyses were conducted by time from blood draw to diagnosis, histology, and smoking status.ResultsWe identified sixteen single CpG sites and forty DMRs significantly associated with lung cancer risk (q < 0.05). The identified genomic regions were associated with genes including H19, HOXA4, RUNX3, BRICD5, PLXNB2, and RP13. For the single CpG sites, the strongest association was noted for cg09736286 in the DIABLO gene (OR [for 1 SD] = 2.99, 95% CI: 1.95-4.59, P-value = 4.81 × 10−7). For the DMRs, we found that CpG sites in the HOXA4 region were hypermethylated in cases compared to controls.ConclusionThe single CpG sites and DMRs that we identified represented significant measurable differences in lung cancer risk, providing new insights into the biological processes of early lung cancer development and potential biomarkers for lung cancer risk stratification.

In the United States, cardiovascular disease is the leading cause of death and the rate of maternal mortality remains among the highest of any industrialized nation. Maternal cardiometabolic health throughout gestation and postpartum is representative of placental health and physiology. Both proper placental functionality and placental microRNA expression are essential to successful pregnancy outcomes, and both are highly sensitive to genetic and environmental sources of variation. Placental pathologies, such as preeclampsia, are associated with maternal cardiovascular health but may also contribute to the developmental programming of chronic disease in offspring. However, the role of more subtle alterations to placental function and microRNA expression in this developmental programming remains poorly understood. We performed small RNA sequencing to investigate microRNA in placentae from the Rhode Island Child Health Study (n = 230). MicroRNA counts were modeled on maternal family history of cardiovascular disease using negative binomial generalized linear models. MicroRNAs were considered to be differentially expressed at a false discovery rate (FDR) less than 0.10. Parallel mRNA sequencing data and bioinformatic target prediction software were then used to identify potential mRNA targets of differentially expressed microRNAs. Nine differentially expressed microRNAs were identified (FDR < 0.1). Bioinformatic target prediction revealed 66 potential mRNA targets of these microRNAs, many of which are implicated in TGFβ signaling pathway but also in pathways involving cellular metabolism and immunomodulation. A robust association exists between familial cardiovascular disease and placental microRNA expression which may be implicated in both placental insufficiencies and the developmental programming of chronic disease.

Background Identifying blood-based DNA methylation patterns is a minimally invasive way to detect biomarkers in predicting age, characteristics of certain diseases and conditions, as well as responses to immunotherapies. As microarray platforms continue to evolve and increase the scope of CpGs measured, new discoveries based on the most recent platform version and how they compare to available data from the previous versions of the platform are unknown. The neutrophil dexamethasone methylation index (NDMI 850) is a blood-based DNA methylation biomarker built on the Illumina MethylationEPIC (850K) array that measures epigenetic responses to dexamethasone (DEX), a synthetic glucocorticoid often administered for inflammation. Here, we compare the NDMI 850 to one we built using data from the Illumina Methylation 450K (NDMI 450). Results The NDMI 450 consisted of 22 loci, 15 of which were present on the NDMI 850. In adult whole blood samples, the linear composite scores from NDMI 450 and NDMI 850 were highly correlated and had equivalent predictive accuracy for detecting DEX exposure among adult glioma patients and non-glioma adult controls. However, the NDMI 450 scores of newborn cord blood were significantly lower than NDMI 850 in samples measured with both assays. Conclusions We developed an algorithm that reproduces the DNA methylation glucocorticoid response score using 450K data, increasing the accessibility for researchers to assess this biomarker in archived or publicly available datasets that use the 450K version of the Illumina BeadChip array. However, the NDMI850 and NDMI450 do not give similar results in cord blood, and due to data availability limitations, results from sample types of newborn cord blood should be interpreted with care.

The mammalian kidney has extensive repair capacity; however, identifying adult renal stem cells has proven elusive. We applied an epigenetic marker of foetal cell origin (FCO) in diverse human tissues as a probe for developmental cell persistence, finding a 5.4-fold greater FCO proportion in kidney. Normal kidney FCO proportions averaged 49% with extensive interindividual variation. FCO proportions were significantly negatively correlated with immune-related gene expression and positively correlated with genes expressed in the renal medulla, including those involved in renal organogenesis (e.g., FGF2, PAX8, and HOXB7). FCO associated genes also mapped to medullary nephron segments in mouse and rat, suggesting evolutionary conservation of this cellular compartment. Renal cancer patients whose tumours contained non-zero FCO scores survived longer. The kidney appears unique in possessing substantial foetal epigenetic features. Further study of FCO-related gene methylation may elucidate regenerative regulatory programmes in tissues without apparent discrete stem cell compartments.

Pre-diagnosis circulating leukocyte profiles, including higher white blood cell (WBC) counts and neutrophil-to-lymphocyte ratios (NLR), have been reported to be associated with non-small cell lung cancer (NSCLC) risk. Higher neutrophil levels were recently reported to drive NSCLC-WBC count associations, implicating the innate immune response in NSCLC risk and etiology. Though not directly measurable in bio-banked blood, leukocyte subtype proportions can be estimated using established deconvolution algorithms applied to genome-wide DNA methylation data. We previously reported that NSCLC risk was elevated in heavy smokers with greater pre-diagnosis methylation-derived NLR (mdNLR). Here, we examine the influence of each pre-diagnosis mdNLR leukocyte subtype (neutrophils, and lymphocytes: B, Natural Killer, CD8+T, and CD4+T cells), separately on NSCLC risk. In our nested case-control study from the Beta Carotene and Retinol Efficacy Trial of heavy smokers (≥20 pack years), 243 NSCLC cases were 1:1 matched to controls on age (±5 years), sex, race and ethnicity, enrollment (±2 years), smoking status (ever/never), asbestos exposure, and follow-up time. Methylation was assayed on the Illumina EPIC array in whole blood collected on average 4.4 years (range 0.1-10.1) before diagnosis in NSCLC cases. We assessed conditional logistic regression models for each mdNLR-related leukocyte subtype, dichotomized at the median in controls, and further adjusted for continuous age and smoking pack years at blood draw. We evaluated NSCLC risk overall, and in subgroup strata: histotype, sex, smoking status, asbestos exposure, stage at diagnosis, age at blood draw, age at diagnosis, and time between blood draw and diagnosis. A greater than median level of neutrophils was suggestively associated with NSCLC risk (OR=1.40, 95% CI: 0.93-2.12); this association was restricted to squamous cell carcinoma (SCC n=103 pairs; 2.00, 1.03-3.86), with no association for adenocarcinoma (n=132 pairs; 0.99, 0.57-1.73). SCC-neutrophil associations were strongest among former smokers (n=26 pairs; 6.81, 1.48-31.3), cases with ≤ 4.4 years between blood draw and diagnosis (n=48 pairs; 3.00, 1.10-8.14), cases aged > 64.8 years at blood draw (n=56 pairs; 2.66, 1.05-6.73), and cases diagnosed at stage III or IV (n=61 pairs; 2.21, 0.92-5.30). Greater than median lymphocyte levels were suggestively associated with reduced NSCLC (0.71, 0.47-1.08) and SCC risk (0.62, 0.32-1.21), but less so for adenocarcinoma (0.85, 0.48-1.50). Though statistically imprecise, SCC-lymphocyte associations were strongest for CD4+T cells (0.56, 0.28-1.12). Our results indicate that greater pre-diagnosis neutrophil levels may be a biomarker for SCC, but not adenocarcinoma, in heavy smokers. Given the descriptive nature of this analysis, small sample size, and multiple testing, additional research is needed to replicate these findings. Citation Format: Laurie Grieshober, Stefan Graw, Matt J. Barnett, Gary E. Goodman, Chu Chen, Devin C. Koestler, Carmen J. Marsit, Jennifer A. Doherty. Pre-diagnostic methylation-based leukocyte profiles and non-small cell lung cancer risk in heavy smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3005.

Immune cell profiles in peripheral blood have been associated with bladder cancer outcomes, however, their association with response to immunotherapy and the tumor microenvironment is a major unresolved issue. Although tumor growth can be attenuated via the activation of tumor-infiltrating effector T cells, the relationship between tumor infiltration and immune activation remains unclear. This study explored the interaction between bladder cancer outcomes and immune profiles within peripheral blood and a tumor microenvironment (TME) based on DNA methylation profiles. Peripheral blood and the matched tumor FFPE DNA methylation profiles of 60 non-muscle-invasive bladder cancer (NMIBC) and 12 muscle-invasive bladder cancer (MIBC) patients. Cell-type deconvolution approaches were applied to estimate 12 peripheral immune cell-type proportions and 17 cell-type proportions within TME. We found a positive correlation between dendritic cell proportions in the TME with peripheral CD8T memory cell proportions (r = 0.35, P = 0.003) and a negative correlation between dendritic cell proportions in the TME with peripheral regulatory T cell proportions (r = -0.28, P = 0.021). In addition, monocyte cell proportions in TME had a positive correlation with peripheral B memory (r = 0.37, P = 0.002) and CD8T memory cell proportions (r = 0.43, P = 0.0002). To investigate associations of bladder cancer outcomes with immune cell profiles, using Cox proportional hazard models, we observed an association between the fraction of dendritic cells and the hazard of death (HR = 1.27, 95% CI = 1.06-1.53). Further, a high endothelial cell proportion was significantly associated with an increased hazard of death and tumor recurrence (HR = 1.06, 95% CI = 1.01-1.13) in TME. In addition, the peripheral neutrophil-to-lymphocyte ratio (HR = 1.49, 95% CI = 1.01-2.22), monocyte (HR = 1.17, 95% CI = 1.05-1.31), neutrophil (HR = 1.04, 95% CI = 1.01-1.07), and basophil (HR = 1.35, 95% CI = 1.01-1.81) cell proportions were associated with an increased hazard of death and tumor recurrence. Our results integrated the information on bladder cancer outcomes and cell profiles in TME and peripheral blood, providing biomarkers for estimating bladder cancer prognosis using genome-scale DNA methylation measures. Citation Format: Ji-Qing Chen, Lucas A. Salas, John K. Wiencke, Devin C. Koestler, Annette M. Molinaro, Angeline S. Andrew, John D. Seigne, Margaret R. Karagas, Karl T. Kelsey, Brock C. Christensen. Integration of associations of immune profiles in peripheral blood and tumor microenvironment with bladder cancer outcomes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6675.

Structured AbstractBackgroundPancreatic cancer is projected to become the second leading cause of cancer-related death by 2030 in the United States. DNA methylation (DNAm) age may reflect age-related variations in the biological changes and abnormalities related to cancer development.MethodWe conducted a pooled analysis using prediagnostic blood samples of pancreatic cancer cases and matched controls selected from the Nurses’ Health Study (NHS), the Physician’s Health Study (PHS), and the Health Professionals Follow-up Study (HPFS). We used three DNAm aging clocks (Hannum, Horvath, and PhenoAge) to estimate subjects’ DNAm age, epigenetic age acceleration (AA) and intrinsic epigenetic age acceleration (IEAA) metrics. We performed conditional logistic regression and multivariable Cox proportional hazard regression to examine associations between six AA and IEAA metrics and risk of pancreatic cancer and survival, respectively.ResultsA total of 393 incidence pancreatic cancer cases and 431 matched controls from the NHS, PHS, and HPFS cohorts were included in this analysis. The medians of all three epigenetic AA and three IEAA metrics were consistently above zero (indicating accelerated age) among cases, while they were below zero (indicating decelerated age) among the matched controls. Comparing participants in the highest quartile of age acceleration metrics, the pancreatic cancer risks were significantly increased by 67% to 83% for Hannum and PhenoAge AA or IEAA metrics with minimal of 7- to 9-years accelerated ages. Except for Hovarth AA and IEAA metrics, there were significant dose-response trends, such that higher age accelerations were associated with higher pancreatic cancer risk, but the relationships were nonlinear. Stratified analyses showed heterogeneous associations, varying by participants’ characteristics and by epigenetic AA or IEAA metrics. As time to diagnosis increased, the ORs of pancreatic cancer for the Hannum AA and Horvath AA or IEAA metrics trended upwards, while the ORs for the PhenoAge AA or IEAA and Hannum IEAA metrics trended downward. Overall, we observed no significant association between pancreatic cancer survival and any of the prediagnostic epigenetic AA or IEAA metrics.ConclusionOur results indicate DNAm age acceleration is associated with an increased risk of pancreatic cancer in a nonlinear, dose-response manner. Epigenetic IEAA metrics may be a useful addition to current methods for pancreatic cancer risk prediction.

BackgroundMyeloid-derived suppressor cells (MDSCs), which include monocytic (mMDSCs) and granulocytic (gMDSCs) cells, are an immunosuppressive, heterogeneous population of cells upregulated in cancer and other pathologic conditions, in addition to normal conditions of stress. The origin of MDSCs is debated, and the regulatory pattern responsible for gMDSC differentiation remains unknown. Since DNA methylation (DNAm) contributes to lineage differentiation, we have investigated whether it contributes to the acquisition of the gMDSC phenotype.ResultsUsing the Illumina EPIC array to measure DNAm of gMDSCs and neutrophils from diverse neonatal and adult blood sources, we found 189 differentially methylated CpGs between gMDSCs and neutrophils with a core of ten differentially methylated CpGs that were consistent across both sources of cells. Genes associated with these loci that are involved in immune responses includeVCL, FATS, YAP1, KREMEN2, UBTF,MCC-1, andEFCC1. In two cancer patient groups that reflected those used to develop the methylation markers (head and neck squamous cell carcinoma (HNSCC) and glioma), all of the CpG loci were differentially methylated, reaching statistical significance in glioma cases and controls, while one was significantly different in the smaller HNSCC group.ConclusionsOur findings indicate that gMDSCs have a core of distinct DNAm alterations, informing future research on gMDSC differentiation and function.

Poor placental function is a common cause of intrauterine growth restriction, which in turn is associated with increased risks of adverse health outcomes. Our prior work suggests that birthweight and childhood obesity-associated genetic variants functionally impact placental function and that placental microRNA are associated with birthweight. To address the influence of the placenta beyond birth, we assessed the relationship between placental microRNAs and early childhood growth.Using the SITAR package, we generated two parameters that describe individual weight trajectories of children (0-5 years) in the New Hampshire Birth Cohort Study (NHBCS, n = 238). Using negative binomial generalized linear models, we identified placental microRNAs that relate to growth parameters (FDR 0.1), while accounting for sex, gestational age at birth, and maternal parity.Genes targeted by the six growth trajectory-associated microRNAs are enriched (FDR 0.05) in growth factor signaling (TGF/beta: miR-876; EGF/R: miR-155, Let-7c; FGF/R: miR-155; IGF/R: Let-7c, miR-155), calmodulin signaling (miR-216a), and NOTCH signaling (miR-629).Growth-trajectory microRNAs target pathways affecting placental proliferation, differentiation and function. Our results suggest a role for microRNAs in regulating placental cellular dynamics and supports the Developmental Origins of Health and Disease hypothesis that fetal environment can have impacts beyond birth.We found that growth trajectory associated placenta microRNAs target genes involved in signaling pathways central to the formation, maintenance and function of placenta; suggesting that placental cellular dynamics remain critical to infant growth to term and are under the control of microRNAs. Our results contribute to the existing body of research suggesting that the placenta plays a key role in programming health in the offspring. This is the first study to relate molecular patterns in placenta, specifically microRNAs, to early childhood growth trajectory.

DNA methylation microarrays can be employed to interrogate cell-type composition in complex tissues. Here, we expand reference-based deconvolution of blood DNA methylation to include 12 leukocyte subtypes (neutrophils, eosinophils, basophils, monocytes, naïve and memory B cells, naïve and memory CD4 + and CD8 + T cells, natural killer, and T regulatory cells). Including derived variables, our method provides 56 immune profile variables. The IDOL (IDentifying Optimal Libraries) algorithm was used to identify libraries for deconvolution of DNA methylation data for current and previous platforms. The accuracy of deconvolution estimates obtained using our enhanced libraries was validated using artificial mixtures and whole-blood DNA methylation with known cellular composition from flow cytometry. We applied our libraries to deconvolve cancer, aging, and autoimmune disease datasets. In conclusion, these libraries enable a detailed representation of immune-cell profiles in blood using only DNA and facilitate a standardized, thorough investigation of immune profiles in human health and disease.

Poor placental function is a common cause of intrauterine growth restriction, which in turn is associated with increased risks of perinatal morbidity, mortality and long-term adverse health outcomes. Our prior work suggests that birthweight and childhood obesity-associated genetic variants functionally impact placental function and that placental microRNA are associated with birthweight. To address the influence of the placenta beyond birth, we assessed the relationship between placental microRNAs and early childhood growth. Using the SITAR package, we generated two parameters that describe individual weight trajectories of children (0-5 years) in the New Hampshire Birth Cohort Study (NHBCS). Using negative binomial generalized linear models, we identified placental microRNAs that associate with growth parameters (FDR

MicroRNAs are non-coding RNAs that regulate gene expression post-transcriptionally. In the placenta, the master regulator of fetal growth and development, microRNAs shape the basic processes of trophoblast biology and specific microRNA have been associated with fetal growth. To comprehensively assess the role of microRNAs in placental function and fetal development, we have performed small RNA sequencing to profile placental microRNAs from two independent mother-infant cohorts: the Rhode Island Child Health Study (n=225) and the New Hampshire Birth Cohort Study (n=317). We modeled microRNA counts on infant birthweight percentile (BWP) in each cohort, while accounting for race, sex, parity and technical factors, using negative binomial generalized linear models. We identified microRNAs that were differentially expressed (DEmiRs) with BWP at false discovery rate (FDR) less than 0.05 in both cohorts. hsa-miR-532-5p (miR-532) was positively associated with BWP in both cohorts. By integrating parallel whole transcriptome and small RNA sequencing in the RICHS cohort, we identified putative targets of miR-532. These targets are enriched for pathways involved in adipogenesis, adipocytokine signaling, energy metabolism and hypoxia response, and included Leptin, which we further demonstrated to have decreasing expression with increasing BWP, particularly in male infants. Overall, we have shown a robust and reproducible association of miR-532 with BWP, which could influence BWP through regulation of adipocytokines Leptin and Adiponectin.

Objective The aim of this study was to test whether increased energy expenditure (EE), independent of physical activity, reduces acute diet-induced weight gain through tighter coupling of energy intake to energy demand and enhanced metabolic adaptations. Methods Indirect calorimetry and quantitative magnetic resonance imaging were used to assess energy metabolism and body composition during 7-day high-fat/high-sucrose (HFHS) feeding in male and female mice housed at divergent temperatures (20°C vs. 30°C). Results As previously observed, 30°C housing resulted in lower total EE and energy intake compared with 20°C mice regardless of sex. Interestingly, housing temperature did not impact HFHS-induced weight gain in females, whereas 30°C male mice gained more weight than 20°C males. Energy intake coupling to EE during HFHS feeding was greater in 20°C versus 30°C housing, with females greater at both temperatures. Fat mass gain was greater in 30°C mice compared with 20°C mice, whereas females gained less fat mass than males. Strikingly, female 20°C mice gained considerably more fat-free mass than 30°C mice. Reduced fat mass gain was associated with greater metabolic flexibility to HFHS, whereas fat-free mass gain was associated with diet-induced adaptive thermogenesis. Conclusions These data reveal that EE and sex interact to impact energy homeostasis and metabolic adaptation to acute HFHS feeding, altering weight gain and body composition change.

Purpose Ewing Sarcoma Family of Tumors (ESFT), the second most common pediatric osseous malignancy, are characterized by the pathognomonic chromosomal EWS-ETS translocation. Outside of tumor biopsy, no clinically relevant ESFT biomarkers exist. Additionally, tumor burden assessment at diagnosis, monitoring of disease responsiveness to therapy, and detection of disease recurrence are limited to radiographic imaging. To identify new, clinically relevant biomarkers we evaluated the proteome of a subset of ESFT-derived small extracellular vesicles (sEVs). Materials and methods We performed the first high quality proteomic study of ESFT-derived sEVs from 5 ESFT cell lines representing the most common EWS-ETS fusion types and identified 619 proteins composing the core ESFT sEV proteome. We compared these core proteins to databases of common plasma-based proteins and sEV-associated proteins found within healthy plasma to identify proteins unique or enriched within ESFT. Results From these analyses, two membrane bound proteins with biomarker potential were selected, CD99/MIC2 and NGFR, to develop a liquid-based assay enriching of ESFT-associated sEVs and detection of sEV mRNA cargo (i.e., EWS-ETS transcripts). We employed this immuno-enrichment approach to diagnosis of ESFT utilizing plasma (250 μl) from both localized and metastatic ESFT pediatric patients and cancer-free controls, and showed significant diagnostic power [AUC = 0.92, p = 0.001 for sEV numeration, with a PPV = 1.00, 95% CI = (0.63, 1.00) and a NPV = 0.67, 95% CI = (0.30, 0.93)]. Conclusions In this study, we demonstrate utilization of circulating ESFT-associated sEVs in pediatric patients as a source of minimally invasive diagnostic and potentially prognostic biomarkers.

Background: Pancreatic cancer is projected to become the second most common cause of cancer-related death over the next 5 years. Because inflammation is thought to be a common trajectory for disease initiation, we sought to prospectively characterize immune profiles using DNA methylation markers and examine DNA methylation levels previously linked to inflammation biomarkers to evaluate whether these immune markers play a key role in pancreatic cancer. Methods: In a nested case–control study pooling three U.S. prospective cohort studies, DNA methylation was measured in prediagnostic leukocytes of incident pancreatic cancer cases and matched controls using the Illumina MethylationEPIC array. Differentially methylated regions were used to predict immune cell types, and CpGs previously associated with inflammatory biomarkers were selected for the analysis. DNA methylation data from a retrospective case–control study conducted in Spain (PanGenEU) was used for independent replication. Results: Immune cell proportions and ratio of cell proportions were not associated with pancreatic cancer risk in the nested case–control study. Methylation extent of CpGs residing in or near gene MNDA was significantly associated with pancreatic cancer risk in the nested case–control study and replicated in PanGenEU. Methylation level of a promoter CpG of gene PIM-1 was associated with survival in both studies. Conclusions: Using a targeted approach, we identified several CpGs that may play a role in pancreatic carcinogenesis in two large, independent studies with distinct study designs. Impact: These findings could provide insight into critical pathways that may help identify new markers of early disease and survival.

Diagnosis and treatment of Ménière's disease remains a significant challenge because of our inability to understand what is occurring on a molecular level. MicroRNA (miRNA) perilymph profiling is a safe methodology and may serve as a "liquid biopsy" equivalent. We used machine learning (ML) to evaluate miRNA expression profiles of various inner ear pathologies to predict diagnosis of Ménière's disease.Prospective cohort study.Tertiary academic hospital.Perilymph was collected during labyrinthectomy (Ménière's disease, n = 5), stapedotomy (otosclerosis, n = 5), and cochlear implantation (sensorineural hearing loss [SNHL], n = 9). miRNA was isolated and analyzed with the Affymetrix miRNA 4.0 array. Various ML classification models were evaluated with an 80/20 train/test split and cross-validation. Permutation feature importance was performed to understand miRNAs that were critical to the classification models.In terms of miRNA profiles for conductive hearing loss versus Ménière's, 4 models were able to differentiate and identify the 2 disease classes with 100% accuracy. The top-performing models used the same miRNAs in their decision classification model but with different weighted values. All candidate models for SNHL versus Ménière's performed significantly worse, with the best models achieving 66% accuracy. Ménière's models showed unique features distinct from SNHL.We can use ML to build Ménière's-specific prediction models using miRNA profile alone. However, ML models were less accurate in predicting SNHL from Ménière's, likely from overlap of miRNA biomarkers. The power of this technique is that it identifies biomarkers without knowledge of the pathophysiology, potentially leading to identification of novel biomarkers and diagnostic tests.

We conducted a multiinstitutional, placebo-controlled phase IIB trial of the lignan secoisolariciresinol diglucoside (SDG) found in flaxseed. Benign breast tissue was acquired by random periareolar fine needle aspiration (RPFNA) from premenopausal women at increased risk for breast cancer. Those with hyperplasia and ≥2% Ki-67 positive cells were eligible for randomization 2:1 to 50 mg SDG/day (Brevail) versus placebo for 12 months with repeat bio-specimen acquisition. The primary endpoint was difference in change in Ki-67 between randomization groups. A total of 180 women were randomized, with 152 ultimately evaluable for the primary endpoint. Median baseline Ki-67 was 4.1% with no difference between arms. Median Ki-67 change was −1.8% in the SDG arm (P = 0.001) and −1.2% for placebo (P = 0.034); with no significant difference between arms. As menstrual cycle phase affects proliferation, secondary analysis was performed for 117 women who by progesterone levels were in the same phase of the menstrual cycle at baseline and off-study tissue sampling. The significant Ki-67 decrease persisted for SDG (median = −2.2%; P = 0.002) but not placebo (median = −1.0%). qRT-PCR was performed on 77 pairs of tissue specimens. Twenty-two had significant ERα gene expression changes (2.0) with 7 of 10 increases in placebo and 10 of 12 decreases for SDG (P = 0.028), and a difference between arms (P = 0.017). Adverse event incidence was similar in both groups, with no evidence that 50 mg/day SDG is harmful. Although the proliferation biomarker analysis showed no difference between the treatment group and the placebo, the trial demonstrated use of SDG is tolerable and safe.

Both lipid oversupply and poor mitochondrial function (low respiration and elevated H(2)O(2) emission) have been implicated in the development of hepatic steatosis and liver injury. Mitophagy, the targeted degradation of low-functioning mitochondria, is critical for maintaining mitochondrial quality control. Here, we used intralipid injection combined with acute (4 day) and chronic (4–7wk) high-fat diets (HFD) to examine whether hepatic mitochondrial respiration would decrease and H(2)O(2) emission would increase with lipid overload. We tested these effects in male and female wild type (WT) mice and mice null for a critical mediator of mitophagy, BCL-2/adenovirus EIB 19-kDa interacting protein knockout (BNIP3 KO) housed at thermoneutral temperatures. Intralipid injection was successful in elevating serum triglycerides and nonesterified fatty acids but had no impact on hepatic mitochondrial respiratory function or H(2)O(2) emission. However, female mice had greater mitochondrial respiration on the acute HFD and lower H(2)O(2) emission across both HFD durations and were protected against hepatic steatosis. Unexpectedly, BNIP3 KO animals had greater hepatic mitochondrial respiration, better coupled respiration, and increased electron chain protein content after the 4-day HFD, compared with WT animals. Altogether, these data suggest that acute lipid overload delivered by a single intralipid bolus does not alter hepatic mitochondrial outcomes, but rather sex and genotype profoundly impact hepatic mitochondrial respiration and H(2)O(2) emission. NEW & NOTEWORTHY This is the first study focusing on hepatic mitochondrial respiratory outcomes in response to lipid overload via a high-fat diet (HFD) combined with intralipid injection. Novel findings include no effect of intralipid injection on mitochondrial outcomes of interest despite increased circulating lipid concentrations. However, we report pronounced differences in hepatic mitochondrial respiration, complex protein expression, and H(2)O(2) production by sex and BCL-2/adenovirus EIB 19-kDa interacting protein (BNIP3) genotype. Specifically, female mice had lower H(2)O(2) emission globally and on an acute HFD, females had greater hepatic mitochondrial respiration than males while BNIP3 knockout (KO) animals had greater mitochondrial coupling and complex protein expression than wild-type (WT) animals.

Mitochondria contribute to tumor growth through multiple metabolic pathways, regulation of extracellular pH, calcium signaling, and apoptosis. Using the Mitochondrial Nuclear Exchange (MNX) mouse models, which pair nuclear genomes with different mitochondrial genomes, we previously showed that mitochondrial SNPs regulate mammary carcinoma tumorigenicity and metastatic potential in genetic crosses. Here, we tested the hypothesis that polymorphisms in stroma significantly affect tumorigenicity and experimental lung metastasis. Using syngeneic cancer cells (EO771 mammary carcinoma and B16-F10 melanoma cells) injected into wild-type and MNX mice (i.e., same nuclear DNA but different mitochondrial DNA), we showed mt-SNP–dependent increases (C3H/HeN) or decreases (C57BL/6J) in experimental metastasis. Superoxide scavenging reduced experimental metastasis. In addition, expression of lung nuclear-encoded genes changed specifically with mt-SNP. Thus, mitochondrial–nuclear cross-talk alters nuclear-encoded signaling pathways that mediate metastasis via both intrinsic and extrinsic mechanisms.Significance:Stromal mitochondrial polymorphisms affect metastatic colonization through reactive oxygen species and mitochondrial–nuclear cross-talk.

Agents which both reduce risk for development of breast cancer and relieve vasomotor symptoms are likely to have good uptake and adherence. We conducted a pilot study with 6 months of the tissue selective estrogen complex bazedoxifene (20 mg) and conjugated estrogen (0.45 mg) (Duavee®) to assess feasibility and effects on biomarkers. Risk biomarkers for postmenopausal breast cancer included fully automated mammographic volumetric density (Volpara®), benign breast tissue Ki-67, and serum levels of progesterone, IGF-1 and IGFBP3, bioavailable estradiol and testosterone. Exploratory biomarkers included selected estrogen and progesterone responsive gene expression in benign breast tissue. 28 peri- and post-menopausal women at increased risk for breast cancer were enrolled; 13 in Cohort A with baseline Ki-67 < 1% and 15 in Cohort B with baseline Ki-67 of 1-4%. All completed the study with > 85% drug adherence. An improvement in median hot flash score from 15 at baseline to 0 at 6 months, and menopause specific quality of life total, vasomotor and sexual domain scores were also observed (p< 0.001). Significant changes in risk biomarkers, uncorrected for multiple comparisons, were a decrease in mammographic fibroglandular volume (p=0.043); decreases in serum progesterone, bioavailable testosterone, and IGF-1 (p This study was supported in part by grants from the Breast Cancer Research Foundation (BCRF-16-049, BCRF-17-049, BCRF-18-049); and an NIH Clinical and Translational Science Award grant (UL1 TR000001, formerly UL1RR033179) awarded to the University of Kansas Medical Center, and an internal clinical pilot grant program of the KUMC Research Institute. Citation Format: Carol J Fabian, Lauren Nye, Teresa A Phillips, Onalisa Winblad, Carola M Zalles, Christy R Hagan, Merit L Goodman, Byron J Gajewski, Devin C Koestler, Prabhakar Chalise, Bruce F Kimler. Biomarker modulation by bazedoxifene and conjugated estrogen (Duavee®) in women at high risk for development of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD3-06.

PURPOSE The University of Kansas Cancer Center (KU Cancer Center) recently developed a data warehouse to Organize and Prioritize Trends to Inform KU Cancer Center (OPTIK). The OPTIK database aggregates and standardizes data collected across the bistate catchment area served by the KU Cancer Center. To improve the usability of the OPTIK database, we developed shinyOPTIK, a user-friendly, interactive web application for visualizing cancer risk factor and mortality rate data across the KU Cancer Center Catchment area. METHODS Data in the OPTIK database were first consolidated at the county level across the KU Cancer Center catchment area. Next, the shinyOPTIK development team met with the KU Cancer Center leadership to discuss the needs and priorities of the shinyOPTIK web application. shinyOPTIK was developed under the R Shiny framework and consists of a user interface (ui.R) and a web server (server.R). At present, s hinyOPTIK can be used to generate county-level geographical heatmaps; bar plots of demographic, screening, and risk factors; and line plots to visualize temporal trends at different Rural-Urban Continuum Codes (RUCCs), rural-urban status, metropolitan, or county levels across the KU Cancer Center catchment area. RESULTS Two examples, adult obesity prevalence and lung cancer mortality, are presented to illustrate how researchers can use shinyOPTIK. Each example is accompanied by post hoc visualizations to help explain key observations in terms of rural-urban disparities. CONCLUSION Although shinyOPTIK was developed to improve understanding of spatial and temporal trends across the population served by the KU Cancer Center, our hope is that the description of the steps involved in the creation of this tool along with open-source code for our application provided herein will serve as a guide for other research centers in the development of similar tools.

Background Non-muscle-invasive bladder cancer (NMIBC) patients receive frequent monitoring because ≥ 70% will have recurrent disease. However, screening is invasive, expensive, and associated with significant morbidity making bladder cancer the most expensive cancer to treat per capita. There is an urgent need to expand the understanding of markers related to recurrence and survival outcomes of NMIBC. Methods and results We used the Illumina HumanMethylationEPIC array to measure peripheral blood DNA methylation profiles of NMIBC patients (N = 603) enrolled in a population-based cohort study in New Hampshire and applied cell type deconvolution to estimate immune cell-type proportions. Using Cox proportional hazard models, we identified that increasing CD4T and CD8T cell proportions were associated with a statistically significant decreased hazard of tumor recurrence or death (CD4T: HR = 0.98, 95% CI = 0.97–1.00; CD8T: HR = 0.97, 95% CI = 0.95–1.00), whereas increasing monocyte proportion and methylation-derived neutrophil-to-lymphocyte ratio (mdNLR) were associated with the increased hazard of tumor recurrence or death (monocyte: HR = 1.04, 95% CI = 1.00–1.07; mdNLR: HR = 1.12, 95% CI = 1.04–1.20). Then, using an epigenome-wide association study (EWAS) approach adjusting for age, sex, smoking status, BCG treatment status, and immune cell profiles, we identified 2528 CpGs associated with the hazard of tumor recurrence or death (P Conclusions Our results expand on the knowledge of immune profiles and methylation alteration associated with NMIBC outcomes and represent a first step toward the development of DNA methylation-based biomarkers of tumor recurrence.

Prenatal cadmium (Cd) exposure has been implicated in both placental toxicity and adverse neurobehavioral outcomes. Placental microRNAs (miRNAs) may function to developmentally program adverse pregnancy and newborn health outcomes in response to gestational Cd exposure.In a subset of the Rhode Island Child Health Study (RICHS, n = 115) and the New Hampshire Birth Cohort Study (NHBCS, = 281), we used small RNA sequencing and trace metal analysis to identify Cd-associated expression of placental miRNAs using negative binomial generalized linear models. We predicted mRNAs targeted by Cd-associated miRNAs and relate them to neurobehavioral outcomes at birth through the integration of transcriptomic data and summary scores from the NICU Network Neurobehavioral Scale (NNNS).Placental Cd concentrations are significantly associated with the expression level of five placental miRNAs in NHBCS, with similar effect sizes in RICHS. These miRNA target genes overrepresented in nervous system development, and their expression is correlated with NNNS metrics suggestive of atypical neurobehavioral outcomes at birth.Gestational Cd exposure is associated with the expression of placental miRNAs. Predicted targets of these miRNAs are involved in nervous system development and may also regulate placental physiology, allowing their dysregulation to modify developmental programming of early life health outcomes.This research aims to address the poor understanding of the molecular mechanisms governing adverse pregnancy and newborn health outcomes in response to Gestational cadmium (Cd) exposure. Our results outline a robust relationship between Cd-associated placental microRNA expression and NICU Network Neurobehavioral Scales (NNNS) at birth indicative of atypical neurobehavior. This study utilized healthy mother-infant cohorts to describe the role of Cd-associated dysregulation of placental microRNAs as a potential mechanism by which adverse neurobehavioral outcomes are developmentally programmed.

The neutrophil dexamethasone methylation index (NDMI) is an algorithm-based biomarker to assess individuals’ exposures to dexamethasone, a synthetic glucocorticoid commonly administered for inflammation. Cortisol is the main endogenous glucocorticoid that controls vital processes including the immune response and lipid and carbohydrate metabolism. Variations in the NDMI score reflect individuals’ sensitivities of exposures to both exogenous and endogenous glucocorticoids, and this biomarker was trained using elastic net regression on Illumina’s most recent DNA methylation beadarray, the EPIC array, which contains 850,000 cytosine-guanine (CpG) sites. While technology for microarray research continues to advance over time, researchers are capable of conducting more comprehensive epigenome-wide association studies (EWAS). However, many studies are still run and archived using Illumina’s historical 450K platform with approximately 450,000 CpGs, and there are fewer published databases using the 850K EPIC array. To evaluate the cross-platform bioinformatic comparability, we performed elastic net regression modeling using predictors available in the 450K to train the NDMI. Among the 135 pre-surgery glioma cases from the UCSF Immune Profiles Study (IPS), NDMI scores between the 450K and 850K model were strongly correlated (r = 0.99, p < 0.0001). In the 311 controls from the UCSF Adult Glioma Study (AGS), similar correlations were observed (r = 0.96, p < 0.0001). We observe that NDMI remains a robust tool using historical 450K data and conclude that this algorithmic tool is capable of detecting the variations in individuals’ responses to dexamethasone.

Background Historical concerns over bone resorption and malunion of the osteocutaneous radial forearm free flap (OCRFFF) limited its widespread adoption for head and neck reconstruction, despite lack of outcomes data evaluating this notion. Methods A retrospective cohort study was performed including patients 18 years or older who underwent reconstruction of the mandible using an OCRFFF. Linear modeling and logistic regression were used to evaluate the change in bone volume and union over time. Results One hundred and twenty-one patients were included in the study. A mixed effects linear model incorporating age, sex, treatment type, and number of bone segments did not demonstrate a significant loss of bone volume over time. A logistic regression model identified lack of adjuvant treatment and time to be significantly associated with complete union. Conclusion This study supports that the OCRFFF is a stable form of osseus reconstruction for defects of the head and neck.

Identifying relationships between genetic variations and their clinical presentations has been challenged by the heterogeneous causes of a disease. It is imperative to unveil the relationship between the high-dimensional genetic manifestations and the clinical presentations, while taking into account the possible heterogeneity of the study subjects.We proposed a novel supervised clustering algorithm using penalized mixture regression model, called component-wise sparse mixture regression (CSMR), to deal with the challenges in studying the heterogeneous relationships between high-dimensional genetic features and a phenotype. The algorithm was adapted from the classification expectation maximization algorithm, which offers a novel supervised solution to the clustering problem, with substantial improvement on both the computational efficiency and biological interpretability. Experimental evaluation on simulated benchmark datasets demonstrated that the CSMR can accurately identify the subspaces on which subset of features are explanatory to the response variables, and it outperformed the baseline methods. Application of CSMR on a drug sensitivity dataset again demonstrated the superior performance of CSMR over the others, where CSMR is powerful in recapitulating the distinct subgroups hidden in the pool of cell lines with regards to their coping mechanisms to different drugs. CSMR represents a big data analysis tool with the potential to resolve the complexity of translating the clinical representations of the disease to the real causes underpinning it. We believe that it will bring new understanding to the molecular basis of a disease and could be of special relevance in the growing field of personalized medicine.

BackgroundExamining inflammation-related DNA methylation alterations in blood could help elucidate the role of inflammation in lung cancer etiology and aid discovery of factors that are key to lung cancer development and progression. In a nested case-control study, we estimated the neutrophil-to-lymphocyte ratio using a validated index, methylation-derived NLR (mdNLR), and quantified DNA methylation levels at loci previously linked with circulating concentrations of C-reactive protein (CRP). We examined associations between these measures and lung cancer risk, and among the cases, lung cancer survival, using pre-diagnostic blood samples of cases (median of 14 years before diagnosis) and controls in the CLUE I/II cohorts. Our analyses controlled for self-reported smoking and methylation-predicted cumulative smoking in order to better focus our examinations on the DNA methylation marks that are informative of the immune response profile.ResultsUsing conditional logistic regression and further adjusting for BMI, batch effects, and a smoking-based methylation score, we observed a 47% increased risk of non-small cell lung cancer (NSCLC) for one standard deviation increase in mdNLR (n = 150 pairs; OR: 1.47 [1.08, 2.02]) and found the estimated CRP Scores to be inversely associated with risk of NSCLC risk after additionally adjusting for methylation-predicted pack-years (n = 150 pairs; Score 1 OR: 0.57 [0.40, 0.81]; Score 2 OR: 0.62 [0.45, 0.84]; Score 3 OR: 0.65 [0.44, 0.95]). Using Cox proportional-hazards models and adjusting age, sex, smoking status, methylation-predicted pack-years, BMI, batch effect, and stage, we observed a 27% increased risk of dying from lung cancer for one standard deviation increase in mdNLR (n = 145 deaths in 205 cases; HR: 1.27 [1.08, 1.50]). A 50% increased risk of dying from lung cancer for one standard deviation increase in mdNLR was observed for NSCLC cases (n = 103 deaths in 149 cases; HR: 1.50 [1.19, 1.89]).ConclusionsA better understanding of inflammation-associated methylation-based biomarkers in lung cancer development could provide insight into critical pathways that may help identify new markers of early disease and survival.

RATIONALE: The novel coronavirus disease 2019 (COVID-19) is a rapidly spreading global viral pandemic with a high-risk of mortality in selected populations (i.e. elderly, immunocompromised, cardiopulmonary diseases). A hyperinflammatory state caused by excessive inflammatory cytokine production (i.e. TNFα, IL-1β, IL6, IL-8) has been attributed to the pathobiology of COVID-19-mediated acute respiratory distress syndrome, worsened lung fibrosis and increased mortality. Inflammation and inflammatory disorders signal primarily through the MAPK pathway. Activation of p38α is important for regulating inflammation, and aberrant p38α activation is associated in the pathobiology of diseases such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and tissue fibrosis. The downstream target of p38α is the protein MAPKAPK2 (MK2) and is responsible for transcriptional production of pro-inflammatory cytokines also elevated in COVID-19 inflammation. Further evidence in other severe viral illnesses (i.e. Dengue, Influenza, CMV) show activation of p38-MK2 signaling axis for mediating inflammation. Potentially, COVID-19 mediated inflammatory cytokine production may signal through p38α-MK2 axis and MK2 pathway blockade may suppress unwanted inflammation. However, it remains unknown whether reducing the inflammatory state can improve COVID-19 outcomes particularly in those with pre-existing conditions. We hypothesized that blockading this pathway would reduce inflammatory cytokine burden and improve respiratory failure-free survival in moderate-severe COVID-19 infected patients. Methods: We designed an investigator-initiated trial (IND#:149790;ClinicalTrials.gov Identifier: NCT04481685) using an oral MK2 inhibitor (ATI-450, Aclaris Pharmaceuticals) in COVID-19. This single-center trial is a Phase IIa, doubleblinded, randomized placebo-controlled proof-of-concept study. COVID-19 positive hospitalized patients with pulmonary signs and symptoms of moderate-severe hypoxic respiratory distress were randomized to ATI-450 or placebo twice-daily for up to 14 days. Results: Study enrollment is completed (n=20 subjects;n=11 male;median age 63 years old). The primary endpoint of this trial is respiratory failure-free survival at 14 days. Secondary endpoints include: changes in WHO-Ordinal scale, additional respiratory and survival outcomes, biochemical assays of circulating cytokines, and safety endpoints. Given the incomplete knowledge of MK2 pathway blockade effects on immune cell function, this study will further explore immune cell characterization in COVID-19 patients treated with ATI-450 via immunophenotyping and 10X Genomics single-cell gene expression analysis. We surmise that myeloid cell activation following COVID-19 infection contributes to localized and systemic tissue injury and will examine the effect of MK2 pathway blockade on eliciting myeloid cell inflammatory activation-suppression. Conclusion: Analyses of the safety, efficacy, and biology of MK2 inhibition, via ATI-450, in treating moderate-severe COVID-19 will be presented.

Background Exposure to the herbicide Agent Orange during the Vietnam War was widespread and is associated with numerous adverse health outcomes. A continuing concern of veterans is the possibility that exposure to the dioxin-containing herbicide might induce adverse reproductive outcomes. We sought to assess whether exposure to Agent Orange in Vietnam was associated with changes in DNA methylation in sperm in a subset of Vietnam veterans who participated in the Air Force Health Study (AFHS). Methods We studied 37 members of the AFHS chosen to have no, low, medium or high exposure to Agent Orange, based upon serum dioxin levels obtained during a series of examinations. DNA from stored semen was extracted and DNA methylation assessed on the Illumina 450 K platform. Results Initial epigenome-wide analysis returned no loci that survived control for false discovery. However, the TEAD3 gene had four different CpG sites that showed loss of DNA methylation associated with dioxin exposure. Analysis assessing regional DNA methylation changes revealed 36 gene regions, including the region of the imprinted gene H19 to have altered DNA methylation associated with high exposure compared to the low exposure group. Additional comparison of our data with sperm DNA methylation data from Russian boys exposed to dioxin found an additional 5 loci that were altered in both studies and exhibited a consistent direction of association. Conclusions Studying a small number of sperm samples from veterans enrolled in the AFHS, we did not find evidence of significant epigenome-wide alterations associated with exposure to Agent Orange. However, additional analysis showed that the H19 gene region is altered in the sperm of Agent Orange-exposed Ranch Hand veterans. Our study also replicated several findings of a prior study of dioxin-exposed Russian boys. These results provide additional candidate loci for further investigation and may have implications for the reproductive health of dioxin-exposed individuals.