Gochanour, Benjamin R.

BACKGROUND: Although diabetes is a known driver of heart failure with preserved ejection fraction, whether antecedent hyperinsulinemia drives preclinical diastolic dysfunction (DD) progression before overt hyperglycemia remains unknown. Therefore, we investigated whether elevated endogenous insulin and insulin resistance independently predict longitudinal DD progression and adverse cardiovascular outcomes. METHODS: In a community-based cohort study, we evaluated 1191 participants from the Olmsted County Heart Function Study (median age, 58.7 [interquartile range, 52.4–66.6 years]) who underwent echocardiography at baseline and after 4 years. Fasting insulin and homeostatic model assessment of insulin resistance were assessed. The primary end point was DD progression. Secondary outcomes (mortality, heart failure) were assessed in 2030 participants followed for a median of 10.97 (95% CI, 10.90–11.05) years for heart failure and a median of 18.1 (95% CI, 18.0–18.2) years for mortality. RESULTS: DD progression occurred in 7.5% of participants. Baseline fasting insulin and homeostatic model assessment of insulin resistance strongly predicted progression ( P <0.001). In multivariable analysis, every 1-SD increase in log-transformed insulin was associated with 65% increased odds of progression (odds ratio, 1.65 [95% CI, 1.33–2.07]; P <0.001), adjusting for age, creatinine, and NT-proBNP (N-terminal pro-B-type natriuretic peptide). Similar results were observed for homeostatic model assessment of insulin resistance (odds ratio, 1.66 [95% CI, 1.34–2.08]; P <0.001). In secondary analyses, elevated insulin independently predicted all-cause (hazard ratio, 1.11 per 1-SD [95% CI, 1.02–1.21]) and cardiovascular mortality (hazard ratio, 1.16 [95% CI, 1.02–1.33]). Homeostatic model assessment of insulin resistance was similarly predictive of all-cause (hazard ratio, 1.11 [95% CI, 1.03–1.20]) and cardiovascular mortality (hazard ratio, 1.16 [95% CI, 1.02–1.32]). CONCLUSIONS: Elevated endogenous insulin and insulin resistance are predictors of progressive DD and long-term mortality, even after accounting for body mass index. Metabolic dysregulation may drive myocardial stiffening before diabetes onset, identifying a potential early therapeutic window for heart failure with preserved ejection fraction prevention.

Abstract Rationale Pulmonary hypertension (PH) is associated with significant morbidity and mortality. Ground-glass opacities (GGOs) are common in Group 1 PH, but their clinical significance is unclear. Objectives We sought to characterize the clinical features and outcomes of patients with Group 1 PH with and without GGOs in the PVDOMICS study, a prospective multicenter cohort study aimed at deep phenotyping PH. Methods Patients with incident and prevalent PH were enrolled across seven U.S. centers. We included patients with Group 1 PH and excluded those with parenchymal lung disease or without chest imaging, resulting in a cohort of 242 patients. Results GGOs were common among patients with Group 1 PH (43% prevalence), associated with female sex, younger age, prostanoid use, and longer disease duration. GGOs were more common among patients with familial pulmonary arterial hypertension and pulmonary veno-occlusive disease. GGOs were associated with established markers of disease severity, including echocardiographic (right ventricular systolic pressure and tricuspid annular plane systolic excursion), biomarkers (N-terminal pro B-type natriuretic peptide), and worse hemodynamics (higher mean pulmonary artery pressure, pulmonary vascular resistance, and pulmonary artery wedge pressure). GGOs were associated with worse transplant-free survival (hazard ratio, 2.49; 95% confidence interval = 1.43–4.32; P = 0.001) and had independent prognostic value for predicting transplant-free survival after adjusting for European Society of Cardiology and European Respiratory Society risk stratification (hazard ratio, 2.19; 95% confidence interval = 1.20–3.99; P = 0.01). Conclusions Overall, GGOs were associated with specific clinical characteristics and disease phenotypes, as well as worse hemodynamics, longer disease duration, prostanoid use, and worse survival. Future studies evaluating the pathophysiology and “omic” correlates of GGOs are warranted. Clinical trial registered with www.clinicaltrials.gov (NCT02980887).

6057 Background: The incidence rate of human papillomavirus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) has persistently increased over the past decade and is anticipated to rise further over the next decade. Cost effective, accurate, and less invasive screening options for HPV(+)OPSCC have remained stagnant leading to the interrogation of circulating tumor DNA in liquid biopsies such as plasma. We hypothesized that candidate methylated DNA markers (MDMs), previously described by our group, would be present in the plasma of patients with HPV(+)OPSCC and absent in controls. Methods: HPV(+)OPSCC cases were enrolled from a high-volume referral practice. Cancer-free control patients were identified from the surrounding 7-county catchment area and frequency matched to cases by age and alcohol use. cfDNA was extracted from 4 mL of plasma and bisulfite converted prior to Target Enrichment Long-probe Quantitative Amplified Signal assays for 15 MDMs. Strand counts for individual MDMs were normalized to a methylated genomic reference marker. An MDM score was derived via random forest modeling of the 15 MDMs. Area under the receiver operator characteristic curve (AUC) for discriminating cases from controls was evaluated for individual markers and for the MDM score. The sensitivity of the MDM score at 95% specificity was also evaluated. Results: The study consisted of 96 HPV(+)OPSCC and 100 cancer-free controls. The study population was predominately white (95%) with a median age of 61 yrs (IQR: 52-69 yrs). Eighty-nine percent of the cases were male with cancers affecting either the tonsil in 50 (52%) or base of the tongue in 46 (48%) patients; by design 50% of controls were male. Stage I:II:III:IV disease was present in 66%:22%:9%:3% of the cases. The median (IQR) AUC across the 15 MDMs was 0.83 (0.82-0.85) overall: 0.78 (0.77-0.83) for stage I disease and 0.92 (0.91-0.93) for stage II-IV disease. The MDM score for the combined panel of 15 MDMs achieved a cross-validated AUC of 0.93 (CI: 0.89-0.97) overall: 0.90 (CI: 0.84-0.96) for stage I disease and 0.98 (CI: 0.97-1.00) for stage II-IV disease. At 95% specificity, the cross-validated sensitivity of the MDM score for HPV(+)OPSCC was 80% (CI: 71-87%) overall: 73% (CI: 60-83%) for stage I disease and 94% (CI: 78-99%) for stage II-IV disease. Conclusions: We validated a set of previously discovered MDM markers in HPV(+)OPSCC in a robust case control cohort using plasma. The MDMs were detected in both early and late-stage disease. Additional prospective studies in larger intended use cohorts are needed to validate our results for clinical use.

Background Right ventricular (RV) maladaptation to elevated pulmonary afterload is the primary determinant of outcomes in pulmonary artery (PA) hypertension; however, the pathobiological mechanisms underlying RV decompensation remain poorly understood. Methods We performed global untargeted metabolomics on plasma from 55 patients who underwent gold‐standard RV‐PA coupling measurements using multibeat pressure volume loop assessment in a single‐center cohort and from 1027 patients with coupling surrogate measurements in a larger multicenter cohort, the PVDOMICS (Pulmonary Vascular Disease Phenomics) study. Age and sex‐adjusted linear regression was performed to identify associations between metabolites and coupling metrics. Additionally, we performed a metabolic flux analysis using gene expression data from RV tissue in an independent cohort of 32 patients. Partial least squares–discriminant analysis was used to identify metabolites and reactions characteristic of the decompensated RV. Results RV‐PA coupling was negatively associated with tricarboxylic acid (TCA) cycle intermediate levels. Specifically, plasma α‐ketoglutarate and fumarate were significantly associated with all coupling metrics in both cohorts. Metabolic flux analysis indicated that decompensated RVs exhibited aberrant TCA cycle activity, including reduced acetyl coenzyme A entry and increased lactate elimination, suggesting a shift from the TCA cycle toward glycolysis at the RV tissue level. Conclusions We identify an association between circulating TCA cycle intermediate levels and RV‐PA uncoupling in 2 independent cohorts, and dysregulated TCA cycle metabolism in decompensated PA hypertension RVs, suggesting that aberrant TCA cycle metabolism could represent a hallmark of RV maladaptation in PA hypertension. Further study of this pathway is warranted to develop novel biomarkers of RV function or RV‐targeted therapies.

Abstract Background/Introduction Close monitoring of aortic stenosis (AS) by follow-up echocardiography is recommended by all Cardiology and Imaging Societies’ guidelines. Whether these guidelines are strictly followed is unknown. Purpose We aimed to describe our institution’s practice patterns for echocardiographic monitoring in patients with aortic sclerosis (AScl) and AS of any severity. Methods Adult patients residing in our institution’s county with an echocardiographic diagnosis of AScl or AS between 01/2005-01/2022 were identified and classified into 4 groups: AScl, mild AS, moderate AS, or severe AS, at each echocardiogram during the study timeframe. Kaplan Meier analyses were used to evaluate the composite outcome, the occurrence of follow-up echocardiogram or death. Patients were censored at the time of aortic valve replacement/repair, heart transplant, or last clinical follow-up. Guideline follow-up was considered appropriate when the primary outcome occurred by 5 years (AScl/mild AS), 2 years (moderate AS), and 1 year (severe AS). Results A total of 23,726 echocardiograms from 9,578 unique patients were included. At index echocardiogram, median patient age was 74 years (IQR 65-82) and 4,806 (50.2%) were women. AScl was observed in 7,358 (76.8%) cases, mild AS in 1,442 (15.1%) cases, moderate AS in 479 (5%), and severe AS in 301 (3.1%) cases. Rates of composite outcome achievement by the end of the guideline-recommended interval were 81%, 90%, 80%, and 72% in AScl, mild, moderate, and severe AS, respectively. Rates for men were 83%, 92%, 82%, and 75% in these groups, while rates for women were 78%, 88%, 78%, and 68%, respectively (Figure 1). Follow-up rates were similar before and after 2012 (when transcatheter aortic valve replacement was approved for treatment of severe AS in high-risk surgical patients in the USA). Follow-up rates were higher in patients managed by cardiovascular providers compared to non-cardiovascular providers (Figure 2). Conclusion Depending on disease severity, between one fourth and one tenth of patients remained alive and unfollowed at the end of their guideline-recommended surveillance interval. There is significant variability in echocardiogram monitoring based on sex and referring provider specialty. Figure 1 Figure 2

Introduction: Non-invasive assays are needed to better discriminate patients with prostate cancer (PCa) to avoid over-treatment of indolent disease. We analyzed 14 methylated DNA markers (MDMs) from urine samples of patients with biopsy-proven PCa relative to healthy controls and further studied discrimination of clinically significant PCa (csPCa) from healthy controls and Gleason 6 cancers. Methods: To evaluate the panel, urine from 24 healthy male volunteers with no clinical suspicion for PCa and 24 men with biopsy-confirmed disease across all Gleason scores was collected. Blinded to clinical status, DNA from the supernatant was analyzed for methylation signal within specific DNA sequences across 14 genes (HES5, ZNF655, ITPRIPL1, MAX.chr3.6187, SLCO3A1, CHST11, SERPINB9, WNT3A, KCNB2, GAS6, AKR1B1, MAX.chr3.8028, GRASP, ST6GALNAC2) by target enrichment long-probe quantitative-amplified signal assays. Results: Utilizing an overall specificity cut-off of 100% for discriminating normal controls from PCa cases across the MDM panel resulted in 71% sensitivity (95% CI: 49–87%) for PCa detection (4/7 Gleason 6, 8/12 Gleason 7, 5/5 Gleason 8+) and 76% (50–92%) for csPCa (Gleason ≥ 7). At 100% specificity for controls and Gleason 6 patients combined, MDM panel sensitivity was 59% (33–81%) for csPCa (5/12 Gleason 7, 5/5 Gleason 8+). Conclusions: MDMs assayed in urine offer high sensitivity and specificity for detection of clinically significant prostate cancer. Prospective evaluation is necessary to estimate discrimination of patients as first-line screening and as an adjunct to prostate-specific antigen (PSA) testing.

Abstract Introduction: Screening for prostate cancer (PCa) with molecular markers is hindered by low tumor allele fraction (TAF) in liquid biopsy samples such as blood or urine. Others have suggested that tumor allele enrichment can be achieved by evaluating fragment-level methylation patterns generated by next generation sequencing (NGS). Here, we test this concept using target capture NGS on cfDNA from prediagnostic collected urine samples to discriminate PCa from otherwise healthy men. Methods: Buffered urines were collected from 23 patients with biopsy confirmed PCa (Gleason score 6-9) and 24 otherwise healthy age matched cancer-free controls. cfDNA was isolated and enzymatic methyl-seq libraries were prepared and enriched by hybrid capture using probes mapping to 197 differentially methylated regions (DMRs) from a previous PCa/normal tissue marker discovery study in our lab. These DMRs were subsequently vetted independently using a methylome-wide dataset from a cohort of 36 clean and disease control urines to ensure low non-cancer background signal (&lt;1%). Indexed libraries were sequenced at 560X deduplicated coverage and fragment-level CpG methylation information was extracted from individual sample .bam files. Mapped sequencing reads exhibiting concordant methylation were used to derive a quantitative α-score. Reads had to contain at minimum 5 CpGs and have an α-value (% 5mC per read) &gt; 60%. Positivity for the 197 markers required an α-score greater than 6 sigma (σ) above the mean of the control arm. Values were variance-adjusted to ensure equal weighting. Beta (β) analysis (mean % 5mC across all reads) of the data was also performed for comparison. Results: Of the 197 markers, 187 were positive for one or more PCa patients. Tumor-derived fragments were detected in 22 of 23 cases, which included 8 G-6, 9 G-7, 2 G-8, and 3 G-9 patients. 1 G-7 was missed, however the sequencing coverage for this sample was significantly lower than the others due to poor library enrichment. 2 of the 24 healthy controls were positive, one with extremely high fragment counts. Upon re-review of the full control cohort, this patient had been diagnosed with bladder cancer 2 years after urine collection. The 2nd patient had was diagnosed with lung cancer in a similar timeframe. The other 22 confirmed cancer-free controls had α-scores below the 6σ threshold. Using a co-captured reference gene (B3GALT6) to mimic genomic equivalents, the TAFs ranged from 0.015% to 8%. β analysis of the data detected only 13 PCa patients (&gt;6σ) and marker positivity was lower than the fragment-level approach. Conclusion: Target capture NGS coupled with fragment-level analysis is a rational approach for sensitive and specific detection of low tumor-derived DNA in liquid biopsy samples. For PCa assay in urine, we were able to uncover positivity in cancer patients approaching 1 in 10,000 ctDNA dilution levels. These results warrant further validation studies with this and other cancers. Citation Format: Tibor Bedekovics, William R. Taylor, Zhifu Sun, Saurabh Baheti, Xiaoming Cao, Kelli N. Burger, Karen A. Doering, Patrick H. Foote, Benjamin Gochanour, Douglas, W. Mahoney, Matthew T. Gettman, John B. Kisiel. Detection of prostate cancer in urine by fragment-level DNA methylation analysis [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 2424.

Introduction: Biomarkers can facilitate the prediction of events in primary prevention. Few studies have been conducted in the community setting with analyses that incorporate many biomarkers. Hypothesis: We tested whether a multi biomarker model would outperform clinical factors and individual biomarkers for predicting MACE and Stroke/MI in subjects without known CAD. Methods: A retrospective cohort study assessed composite outcomes MACE (MI, CABG, PCI, stroke, and death) and Stroke/MI. Cox proportional hazard models were fit for individual biomarkers, adjusted for age, gender, BMI, hypertension, CAD, and GFR. Multi biomarker models were developed based on AIC values from the single-biomarker models. Bootstrap tests compared survival C-statistics and relative Integrated Discrimination Index (IDI). Results: 1131 patients in Olmsted County, MN were studied. Median age was 63.1 years, 97.5% were Caucasian, and 52.2% were female. Median BMI was 27.8. Kaplan Meier rates of MACE and Stroke/MI were 29.2% (CI = 26.5-31.8%) and 12.3% (CI = 10.3% - 14.3%), at 10 years. Adjusted NT proBNP models showed a 31% increased risk of MACE/death (HR = 1.31; CI = 1.17-1.46) and a 31% increased risk for stroke/MI (HR = 1.31; CI = 1.08-1.58). Adjusted Ceramide score models showed a 13% increased risk of MACE/death (HR = 1.13; CI = 1.04-1.24) and a 29% increased risk for stroke/MI (HR = 1.29; CI = 1.11-1.51). NT proBNP and the Ceramide score with clinical factors was better than clinical factors alone for MACE/Death (Relative IDI = 29.7%; p = 0.003) and Stroke/MI (Relative IDI = 36.1%; p = 0.034). This combined model did not outperform the strongest single biomarker model for Stroke/MI or MACE/Death. Other biomarkers were not significant when added to the combined model. Discussion: Ceramide score and NT proBNP alone and together improve the prediction of MACE and Stroke/MI in a community primary prevention cohort.

Abstract Background: Reliable non-invasive biomarkers for human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPX) are lacking. We performed a discovery and validation study of methylated DNA markers (MDMs) for detection of OPX. In addition, we sought to evaluate the MDM profiles in saliva gargle samples from normal controls (nSAL). Methods: Formalin fixed paraffin embedded OPX and gender-balanced normal oropharyngeal tissue (NOP) were pathologically reviewed and punched for DNA extraction and sequenced using reduced representation bisulfite sequencing (RRBS). A tiling algorithm was used to define candidate MDMs. Highest ranked (e.g., area under the receiver operating characteristic curve (AUC), fold-change, p-value) candidate MDMs were validated by qMSP in independent OPX and NOP samples. nSAL DNA was used to assess MDM background signal. Discrimination between OPX tissue, NOP, and nSAL was assessed using AUC with corresponding 95% confidence intervals. HPV was confirmed with p16 staining or DNA in situ hybridization. Results: RRBS discovery assessed 18 OPX, 18 NOP, and 18 normal WBC samples. 234 MDM candidates from approximately 3 × 106 CpGs at &gt;10X coverage were identified. 36 MDMs were selected for validation in independent patient samples which included 32 OPX, 36 NOP, and 35 nSAL specimens. OPX patients were older (median 51; IQR 45-54 years) than NOP patients (median 29; IQR 24-42). 69 and 61% of OPX and NOP patients were female, respectively. The majority of OPX patients were Stage I (n=27, 84%). We observed excellent discrimination between OPX and NOP in the validation set; the median AUC across 36 MDMs was 0.90 (IQR 0.87-0.95). The 10 highest performing markers (PARP15, EPDR1, SHROOM1, EMBP1.rs, IFFO1, MAX.chr19.118, ZNF763, ITGB4, FAM19A2, and MT1IP) exhibited AUCs ranging from 0.95-0.99. Strong discrimination was also observed between OPX and nSAL (Median AUC=0.97; IQR=0.93-0.99). All 10 of the highest-performing markers in tissue exhibited AUCs of at least 0.97 for discriminating OPX versus nSAL. Median fold change between NOP and nSAL across all MDMs was 1.9 (IQR 1.4-3.8), demonstrating low background signal in nSAL. Conclusions: We found strong discrimination in a panel of 36 MDMs between OPX case and control tissue as well as between OPX case tissue and normal saliva. The low background signal observed in normal saliva makes this media a strong candidate for non-invasive detection of OPX. Further studies are in development to assess the utility of measuring these MDMs in both case and control saliva and blood for detecting OPX non-invasively. Citation Format: Benjamin R. Gochanour, David M. Routman, Kathleen R. Bartemes, William R. Taylor, Douglas W. Mahoney, Rondell P. Graham, Xiaoming Cao, Calise K. Berger, Sara S. Then, Karen A. Doering, Kelli N. Burger, Patrick H. Foote, John B. Kisiel, Kathryn M. Van Abel. Discovery and validation of methylated DNA markers of human papillomavirus associated oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-039.