Researching COVID to enhance recovery (RECOVER) pediatric study protocol: Rationale, objectives and design.

IMPORTANCE: The prevalence, pathophysiology, and long-term outcomes of COVID-19 (post-acute sequelae of SARS-CoV-2 [PASC] or "Long COVID") in children and young adults remain unknown. Studies must address the urgent need to define PASC, its mechanisms, and potential treatment targets in children and young adults. OBSERVATIONS: We describe the protocol for the Pediatric Observational Cohort Study of the NIH's REsearching COVID to Enhance Recovery (RECOVER) Initiative. RECOVER-Pediatrics is an observational meta-cohort study of caregiver-child pairs (birth through 17 years) and young adults (18 through 25 years), recruited from more than 100 sites across the US. This report focuses on two of four cohorts that comprise RECOVER-Pediatrics: 1) a de novo RECOVER prospective cohort of children and young adults with and without previous or current infection; and 2) an extant cohort derived from the Adolescent Brain Cognitive Development (ABCD) study (n = 10,000). The de novo cohort incorporates three tiers of data collection: 1) remote baseline assessments (Tier 1, n = 6000); 2) longitudinal follow-up for up to 4 years (Tier 2, n = 6000); and 3) a subset of participants, primarily the most severely affected by PASC, who will undergo deep phenotyping to explore PASC pathophysiology (Tier 3, n = 600). Youth enrolled in the ABCD study participate in Tier 1. The pediatric protocol was developed as a collaborative partnership of investigators, patients, researchers, clinicians, community partners, and federal partners, intentionally promoting inclusivity and diversity. The protocol is adaptive to facilitate responses to emerging science. CONCLUSIONS AND RELEVANCE: RECOVER-Pediatrics seeks to characterize the clinical course, underlying mechanisms, and long-term effects of PASC from birth through 25 years old. RECOVER-Pediatrics is designed to elucidate the epidemiology, four-year clinical course, and sociodemographic correlates of pediatric PASC. The data and biosamples will allow examination of mechanistic hypotheses and biomarkers, thus providing insights into potential therapeutic interventions. CLINICAL TRIALS.GOV IDENTIFIER: Clinical Trial Registration: http://www.clinicaltrials.gov. Unique identifier: NCT05172011.

Researching COVID to Enhance Recovery (RECOVER) adult study protocol: Rationale, objectives, and design.

IMPORTANCE: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or other health effects after the acute phase of infection; termed post-acute sequelae of SARS-CoV-2 infection (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are ill-defined. The objectives of the Researching COVID to Enhance Recovery (RECOVER) Multi-site Observational Study of PASC in Adults (RECOVER-Adult) are to: (1) characterize PASC prevalence; (2) characterize the symptoms, organ dysfunction, natural history, and distinct phenotypes of PASC; (3) identify demographic, social and clinical risk factors for PASC onset and recovery; and (4) define the biological mechanisms underlying PASC pathogenesis. METHODS: RECOVER-Adult is a combined prospective/retrospective cohort currently planned to enroll 14,880 adults aged ≥18 years. Eligible participants either must meet WHO criteria for suspected, probable, or confirmed infection; or must have evidence of no prior infection. Recruitment occurs at 86 sites in 33 U.S. states, Washington, DC and Puerto Rico, via facility- and community-based outreach. Participants complete quarterly questionnaires about symptoms, social determinants, vaccination status, and interim SARS-CoV-2 infections. In addition, participants contribute biospecimens and undergo physical and laboratory examinations at approximately 0, 90 and 180 days from infection or negative test date, and yearly thereafter. Some participants undergo additional testing based on specific criteria or random sampling. Patient representatives provide input on all study processes. The primary study outcome is onset of PASC, measured by signs and symptoms. A paradigm for identifying PASC cases will be defined and updated using supervised and unsupervised learning approaches with cross-validation. Logistic regression and proportional hazards regression will be conducted to investigate associations between risk factors, onset, and resolution of PASC symptoms. DISCUSSION: RECOVER-Adult is the first national, prospective, longitudinal cohort of PASC among US adults. Results of this study are intended to inform public health, spur clinical trials, and expand treatment options. REGISTRATION: NCT05172024.

Researching COVID to enhance recovery (RECOVER) pediatric study protocol: Rationale, objectives and design.

Importance: The prevalence, pathophysiology, and long-term outcomes of COVID-19 (post-acute sequelae of SARS-CoV-2 [PASC] or "Long COVID") in children and young adults remain unknown. Studies must address the urgent need to define PASC, its mechanisms, and potential treatment targets in children and young adults. Observations: We describe the protocol for the Pediatric Observational Cohort Study of the NIH's RE searching COV ID to E nhance R ecovery (RECOVER) Initiative. RECOVER-Pediatrics is an observational meta-cohort study of caregiver-child pairs (birth through 17 years) and young adults (18 through 25 years), recruited from more than 100 sites across the US. This report focuses on two of five cohorts that comprise RECOVER-Pediatrics: 1) a de novo RECOVER prospective cohort of children and young adults with and without previous or current infection; and 2) an extant cohort derived from the Adolescent Brain Cognitive Development (ABCD) study ( n =10,000). The de novo cohort incorporates three tiers of data collection: 1) remote baseline assessments (Tier 1, n=6000); 2) longitudinal follow-up for up to 4 years (Tier 2, n=6000); and 3) a subset of participants, primarily the most severely affected by PASC, who will undergo deep phenotyping to explore PASC pathophysiology (Tier 3, n=600). Youth enrolled in the ABCD study participate in Tier 1. The pediatric protocol was developed as a collaborative partnership of investigators, patients, researchers, clinicians, community partners, and federal partners, intentionally promoting inclusivity and diversity. The protocol is adaptive to facilitate responses to emerging science. Conclusions and Relevance: RECOVER-Pediatrics seeks to characterize the clinical course, underlying mechanisms, and long-term effects of PASC from birth through 25 years old. RECOVER-Pediatrics is designed to elucidate the epidemiology, four-year clinical course, and sociodemographic correlates of pediatric PASC. The data and biosamples will allow examination of mechanistic hypotheses and biomarkers, thus providing insights into potential therapeutic interventions. Clinical Trialsgov Identifier: Clinical Trial Registration: http://www.clinicaltrials.gov . Unique identifier: NCT05172011.

Pragmatic evaluation of events and benefits of lipid lowering in older adults (PREVENTABLE): Trial design and rationale.

Whether initiation of statins could increase survival free of dementia and disability in adults aged ≥75 years is unknown. PREVENTABLE, a double-blind, placebo-controlled randomized pragmatic clinical trial, will compare high-intensity statin therapy (atorvastatin 40 mg) with placebo in 20,000 community-dwelling adults aged ≥75 years without cardiovascular disease, disability, or dementia at baseline. Exclusion criteria include statin use in the prior year or for >5 years and inability to take a statin. Potential participants are identified using computable phenotypes derived from the electronic health record and local referrals from the community. Participants will undergo baseline cognitive testing, with physical testing and a blinded lipid panel if feasible. Cognitive testing and disability screening will be conducted annually. Multiple data sources will be queried for cardiovascular events, dementia, and disability; survival is site-reported and supplemented by a National Death Index search. The primary outcome is survival free of new dementia or persisting disability. Co-secondary outcomes are a composite of cardiovascular death, hospitalization for unstable angina or myocardial infarction, heart failure, stroke, or coronary revascularization; and a composite of mild cognitive impairment or dementia. Ancillary studies will offer mechanistic insights into the effects of statins on key outcomes. Biorepository samples are obtained and stored for future study. These results will inform the benefit of statins for increasing survival free of dementia and disability among older adults. This is a pioneering pragmatic study testing important questions with low participant burden to align with the needs of the growing population of older adults.

Antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in All of Us Research Program Participants, 2 January to 18 March 2020.

BACKGROUND: With limited severe acute respiratory syndrome coronavirus (SARS-CoV-2) testing capacity in the United States at the start of the epidemic (January-March 2020), testing was focused on symptomatic patients with a travel history throughout February, obscuring the picture of SARS-CoV-2 seeding and community transmission. We sought to identify individuals with SARS-CoV-2 antibodies in the early weeks of the US epidemic. METHODS: All of Us study participants in all 50 US states provided blood specimens during study visits from 2 January to 18 March 2020. Participants were considered seropositive if they tested positive for SARS-CoV-2 immunoglobulin G (IgG) antibodies with the Abbott Architect SARS-CoV-2 IgG enzyme-linked immunosorbent assay (ELISA) and the EUROIMMUN SARS-CoV-2 ELISA in a sequential testing algorithm. The sensitivity and specificity of these ELISAs and the net sensitivity and specificity of the sequential testing algorithm were estimated, along with 95% confidence intervals (CIs). RESULTS: The estimated sensitivities of the Abbott and EUROIMMUN assays were 100% (107 of 107 [95% CI: 96.6%-100%]) and 90.7% (97 of 107 [83.5%-95.4%]), respectively, and the estimated specificities were 99.5% (995 of 1000 [98.8%-99.8%]) and 99.7% (997 of 1000 [99.1%-99.9%]), respectively. The net sensitivity and specificity of our sequential testing algorithm were 90.7% (97 of 107 [95% CI: 83.5%-95.4%]) and 100.0% (1000 of 1000 [99.6%-100%]), respectively. Of the 24 079 study participants with blood specimens from 2 January to 18 March 2020, 9 were seropositive, 7 before the first confirmed case in the states of Illinois, Massachusetts, Wisconsin, Pennsylvania, and Mississippi. CONCLUSIONS: Our findings identified SARS-CoV-2 infections weeks before the first recognized cases in 5 US states.

Mini-Review of Laboratory Operations in Biobanking: Building Biobanking Resources for Translational Research.

Biobanks have become integral to improving population health. We are in a new era in medicine as patients, health professionals, and researchers increasingly collaborate to gain new knowledge and explore new paradigms for diagnosing and treating disease. Many large-scale biobanking efforts are underway worldwide at the institutional, national, and even international level. When linked with subject data from questionnaires and medical records, biobanks serve as valuable resources in translational research. A biobank must have high quality samples that meet researcher's needs. Biobank laboratory operations require an enormous amount of support-from lab and storage space, information technology expertise, and a laboratory management information system to logistics for sample movement, quality management systems, and appropriate facilities. A paramount metric of success for a biobank is the concept of every biospecimen coming to the repository belongs to a participant who has something to contribute to research for a healthier future. This article will discuss the importance of biorepository operations, specific to the collection and storage of participants materials. Specific focus will be given to maintaining the quality of samples, along with the various levels of support biorepositories need to fulfill their purpose and ensure the integrity of each specimen is maintained.

Impact of Diverse Data Sources on Computational Phenotyping.

Electronic health records (EHRs) are widely adopted with a great potential to serve as a rich, integrated source of phenotype information. Computational phenotyping, which extracts phenotypes from EHR data automatically, can accelerate the adoption and utilization of phenotype-driven efforts to advance scientific discovery and improve healthcare delivery. A list of computational phenotyping algorithms has been published but data fragmentation, i.e., incomplete data within one single data source, has been raised as an inherent limitation of computational phenotyping. In this study, we investigated the impact of diverse data sources on two published computational phenotyping algorithms, rheumatoid arthritis (RA) and type 2 diabetes mellitus (T2DM), using Mayo EHRs and Rochester Epidemiology Project (REP) which links medical records from multiple health care systems. Results showed that both RA (less prevalent) and T2DM (more prevalent) case selections were markedly impacted by data fragmentation, with positive predictive value (PPV) of 91.4 and 92.4%, false-negative rate (FNR) of 26.6 and 14% in Mayo data, respectively, PPV of 97.2 and 98.3%, FNR of 5.2 and 3.3% in REP. T2DM controls also contain biases, with PPV of 91.2% and FNR of 1.2% for Mayo. We further elaborated underlying reasons impacting the performance.

Characteristics Associated With Recruitment and Re-contact in Mayo Clinic Biobank.

Objective: To better understand the characteristics associated with a participant's willingness to consent to the Mayo Clinic Biobank (MCB) and examine factors associated with willingness to participate in follow-up studies embedded within MCB that require re-contact and participant approval. Participants and Methods: Consent rates were compared across patient demographics to the MCB. Rates of participation to follow-up studies were also compared across demographics and request types. Results: Among 272,102 Mayo Clinic patients invited to the MCB, 48,314 (19%) consented across the three recruitment sites within 90 days of initial invitation. A significant age by gender interaction was identified, showing young males consent at a lower rate than young females and older males consent at a higher rate than older females. Over the recruitment time frame of 2009-2015, there was a significant decrease in consent rates (decline of 2.5%/year). Of the 57,041 consented MCB participants, 33,487 participants (59%) have been invited to participate in follow-up studies via re-contact. Follow-up studies of the MCB may require participants to provide additional samples, complete questionnaires, and/or release their identity to a research team. MCB participants have been invited to enroll in a median of two studies (IQR: 1-3). Seventy-one percent of participants consented to at least one follow-up study, with individual follow-up study consent rates ranging from 14 to 87% depending on study type, with a median consent rate of 61% (IQR: 47-70%). Studies requesting return of a questionnaire had the highest participation rates. White participants, older participants, and participants with some college or a degree were significantly more likely to participate to follow-up studies, while there was no association with gender. Conclusion: Consent rates among younger and non-white patients were lower than in older, white patients. However, we also found that participation rates among those already enrolled in the biobank were much higher than those seen in new recruitment efforts, external to an existing biobank. We thus demonstrate an important way that biobanks can advance precision medicine goals: through provision of populations from which studies can draw participants for future studies.

The impact of sample processing on inflammatory markers in serum: Lessons learned.

Objectives: To investigate the effect of sample handling on inflammatory cytokines in serum and highlight challenges with using samples pre-collected from biobanks for biomarker research.Methods: Cytokine concentrations (IL-1ß, IL-2, IL-6, IL-8, IL-10, TNFα, and IFNγ) were measured in serum samples of 205 patients with bipoldar disorder (BD) from the Mayo Clinic Bipolar Disorder Biobank and 205 non-psychiatric controls from the Mayo Clinic Biobank. As cytokine concentrations varied by recruitment site, post-hoc models were used to test the effect of clinical variables and pre-processing time on cytokines. To evaluate the effect of pre-processing time experimentally, cytokines were assayed in serum and plasma from 6 healthy volunteers processed at different time points.Results: Cytokine levels were significantly higher in the BD group. However, both cytokine levels and pre-processing times differed by recruitment site, and post-hoc analyses revealed that pre-processing time was significantly associated with several cytokines. An experiment using samples from healthy volunteers confirmed that concentrations for most cytokines increased with longer pre-processing times.Conclusions: Delays in processing influence cytokine concentrations in blood samples. Given the increasing use of biobanks in research, this study highlights the need to carefully evaluate sample collection and handling methods when designing biomarker studies.

Association of mitochondrial DNA copy number with self-rated health status.

PURPOSE: In aging adults, mitochondrial dysfunction may be an important contributor. We evaluated the association between mitochondrial DNA (mtDNA) copy number, which is a biomarker for mitochondrial function, and self-rated health status. PATIENTS AND METHODS: We conducted a cross-sectional study of patients enrolled within the Mayo Clinic Biobank. We utilized the questionnaire and sequence data from 944 patients. We examined the association between mtDNA copy number and self-rated health status with 3 collapsed categories for the latter variable (excellent/very good, good, and fair/poor). For analysis, we used proportional odds models after log-transforming mtDNA copy number, and we adjusted for age and sex. RESULTS: We found the median age at enrollment was 61 years (25th-75th percentile: 51-71), and 64% reported excellent or very good health, 31% reported good health, and 6% reported fair/poor health. Overall, the median mtDNA copy number was 88.9 (25th-75th percentile: 77.6-101.1). Higher mtDNA copy number was found for subjects reporting better self-rated health status after adjusting for age, sex, and comorbidity burden (OR =2.3 [95% CI: 1.2-4.5] for having better self-rated health for a one-unit increase in log-transformed mtDNA copy number). CONCLUSION: We found that a higher mtDNA copy number is associated with better self-rated health status after adjustment for age, sex, and comorbidity burden. The current study implies that mtDNA copy number may serve as a biomarker for self-reported health. Further studies, potentially including cohort studies, may be required.

Germline whole exome sequencing and large-scale replication identifies FANCM as a likely high grade serous ovarian cancer susceptibility gene.

We analyzed whole exome sequencing data in germline DNA from 412 high grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas Project and identified 5,517 genes harboring a predicted deleterious germline coding mutation in at least one HGSOC case. Gene-set enrichment analysis showed enrichment for genes involved in DNA repair (p = 1.8×10-3). Twelve DNA repair genes - APEX1, APLF, ATX, EME1, FANCL, FANCM, MAD2L2, PARP2, PARP3, POLN, RAD54L and SMUG1 - were prioritized for targeted sequencing in up to 3,107 HGSOC cases, 1,491 cases of other epithelial ovarian cancer (EOC) subtypes and 3,368 unaffected controls of European origin. We estimated mutation prevalence for each gene and tested for associations with disease risk. Mutations were identified in both cases and controls in all genes except MAD2L2, where we found no evidence of mutations in controls. In FANCM we observed a higher mutation frequency in HGSOC cases compared to controls (29/3,107 cases, 0.96 percent; 13/3,368 controls, 0.38 percent; P=0.008) with little evidence for association with other subtypes (6/1,491, 0.40 percent; P=0.82). The relative risk of HGSOC associated with deleterious FANCM mutations was estimated to be 2.5 (95% CI 1.3 - 5.0; P=0.006). In summary, whole exome sequencing of EOC cases with large-scale replication in case-control studies has identified FANCM as a likely novel susceptibility gene for HGSOC, with mutations associated with a moderate increase in risk. These data may have clinical implications for risk prediction and prevention approaches for high-grade serous ovarian cancer in the future and a significant impact on reducing disease mortality.

PPM1D Mosaic Truncating Variants in Ovarian Cancer Cases May Be Treatment-Related Somatic Mutations.

Mosaic truncating mutations in the protein phosphatase, Mg(2+)/Mn(2+)-dependent, 1D (PPM1D) gene have recently been reported with a statistically significantly greater frequency in lymphocyte DNA from ovarian cancer case patients compared with unaffected control patients. Using massively parallel sequencing (MPS) we identified truncating PPM1D mutations in 12 of 3236 epithelial ovarian cancer (EOC) case patients (0.37%) but in only one of 3431 unaffected control patients (0.03%) (P = .001). All statistical tests were two-sided. A combination of Sanger sequencing, pyrosequencing, and MPS data suggested that 12 of the 13 mutations were mosaic. All mutations were identified in post-chemotherapy treatment blood samples from case patients (n = 1827) (average 1234 days post-treatment in carriers) rather than from cases collected pretreatment (less than 14 days after diagnosis, n = 1384) (P = .002). These data suggest that PPM1D variants in EOC cases are primarily somatic mosaic mutations caused by treatment and are not associated with germline predisposition to EOC.

Contribution of Germline Mutations in the RAD51B, RAD51C, and RAD51D Genes to Ovarian Cancer in the Population.

PURPOSE: The aim of this study was to estimate the contribution of deleterious mutations in the RAD51B, RAD51C, and RAD51D genes to invasive epithelial ovarian cancer (EOC) in the population and in a screening trial of individuals at high risk of ovarian cancer. PATIENTS AND METHODS: The coding sequence and splice site boundaries of the three RAD51 genes were sequenced and analyzed in germline DNA from a case-control study of 3,429 patients with invasive EOC and 2,772 controls as well as in 2,000 unaffected women who were BRCA1/BRCA2 negative from the United Kingdom Familial Ovarian Cancer Screening Study (UK_FOCSS) after quality-control analysis. RESULTS: In the case-control study, we identified predicted deleterious mutations in 28 EOC cases (0.82%) compared with three controls (0.11%; P < .001). Mutations in EOC cases were more frequent in RAD51C (14 occurrences, 0.41%) and RAD51D (12 occurrences, 0.35%) than in RAD51B (two occurrences, 0.06%). RAD51C mutations were associated with an odds ratio of 5.2 (95% CI, 1.1 to 24; P = .035), and RAD51D mutations conferred an odds ratio of 12 (95% CI, 1.5 to 90; P = .019). We identified 13 RAD51 mutations (0.65%) in unaffected UK_FOCSS participants (RAD51C, n = 7; RAD51D, n = 5; and RAD51B, n = 1), which was a significantly greater rate than in controls (P < .001); furthermore, RAD51 mutation carriers were more likely than noncarriers to have a family history of ovarian cancer (P < .001). CONCLUSION: These results confirm that RAD51C and RAD51D are moderate ovarian cancer susceptibility genes and suggest that they confer levels of risk of EOC that may warrant their use alongside BRCA1 and BRCA2 in routine clinical genetic testing.

Germline Mutations in the BRIP1, BARD1, PALB2, and NBN Genes in Women With Ovarian Cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, responsible for 13 000 deaths per year in the United States. Risk prediction based on identifying germline mutations in ovarian cancer susceptibility genes could have a clinically significant impact on reducing disease mortality. METHODS: Next generation sequencing was used to identify germline mutations in the coding regions of four candidate susceptibility genes-BRIP1, BARD1, PALB2 and NBN-in 3236 invasive EOC case patients and 3431 control patients of European origin, and in 2000 unaffected high-risk women from a clinical screening trial of ovarian cancer (UKFOCSS). For each gene, we estimated the prevalence and EOC risks and evaluated associations between germline variant status and clinical and epidemiological risk factor information. All statistical tests were two-sided. RESULTS: We found an increased frequency of deleterious mutations in BRIP1 in case patients (0.9%) and in the UKFOCSS participants (0.6%) compared with control patients (0.09%) (P = 1 x 10(-4) and 8 x 10(-4), respectively), but no differences for BARD1 (P = .39), NBN1 ( P = .61), or PALB2 (P = .08). There was also a difference in the frequency of rare missense variants in BRIP1 between case patients and control patients (P = 5.5 x 10(-4)). The relative risks associated with BRIP1 mutations were 11.22 for invasive EOC (95% confidence interval [CI] = 3.22 to 34.10, P = 1 x 10(-4)) and 14.09 for high-grade serous disease (95% CI = 4.04 to 45.02, P = 2 x 10(-5)). Segregation analysis in families estimated the average relative risks in BRIP1 mutation carriers compared with the general population to be 3.41 (95% CI = 2.12 to 5.54, P = 7×10(-7)). CONCLUSIONS: Deleterious germline mutations in BRIP1 are associated with a moderate increase in EOC risk. These data have clinical implications for risk prediction and prevention approaches for ovarian cancer and emphasize the critical need for risk estimates based on very large sample sizes before genes of moderate penetrance have clinical utility in cancer prevention.

Genome-wide investigation of regional blood-based DNA methylation adjusted for complete blood counts implicates BNC2 in ovarian cancer.

Due to its potential as a biomarker for early cancer detection, blood-based DNA methylation (DNAm) is of interest in cancer research. Specifically, highly predictive mechanisms for early detection of epithelial ovarian cancer (EOC) are desired, so previous studies have compared DNAm between EOC cases and controls. However, case-control studies are confounded by the distribution of white blood cell types through an immune response induced by the cancer. Rather than determining the distribution of the cell types manually or investigating isolated cell types, an alternative approach involves the use of complete blood count (CBC), which is routinely collected. In the analysis of an EOC case-control study of DNAm, we incorporate CBC measures to adjust for this confounding and compare DNAm between 242 EOC cases and 181 age-matched controls (assayed on the Illumina Infinium HumanMethylation27 or HumanMethylation450 Beadchips), at both the individual CpG and CpG island levels. We found that adjustment for leukocyte distribution using CBC measurements dramatically reduced confounding, with 62 single CpG sites found to be associated with EOC status after adjustment (P < 5E-8). Additionally, regional DNAm was assessed by applying principal components analysis to CpG islands. The top associated CpG island (P = 7E-6) was located in the promoter/transcription start site of the human basonuclin 2 gene (BNC2), a known susceptibility gene for EOC risk identified through GWAS. Follow-up studies are necessary to establish the role of BNC2 in blood-based DNA and EOC, including prospective studies to validate this region as a potential biomarker and predictor of EOC susceptibility.

Methylation of leukocyte DNA and ovarian cancer: relationships with disease status and outcome.

BACKGROUND: Genome-wide interrogation of DNA methylation (DNAm) in blood-derived leukocytes has become feasible with the advent of CpG genotyping arrays. In epithelial ovarian cancer (EOC), one report found substantial DNAm differences between cases and controls; however, many of these disease-associated CpGs were attributed to differences in white blood cell type distributions. METHODS: We examined blood-based DNAm in 336 EOC cases and 398 controls; we included only high-quality CpG loci that did not show evidence of association with white blood cell type distributions to evaluate association with case status and overall survival. RESULTS: Of 13,816 CpGs, no significant associations were observed with survival, although eight CpGs associated with survival at p < 10-3, including methylation within a CpG island located in the promoter region of GABRE (p = 5.38 x 10-5, HR = 0.95). In contrast, 53 CpG methylation sites were significantly associated with EOC risk (p <5 x10-6). The top association was observed for the methylation probe cg04834572 located approximately 315 kb upstream of DUSP13 (p = 1.6 x10-14). Other disease-associated CpGs included those near or within HHIP (cg14580567; p =5.6x10-11), HDAC3 (cg10414058; p = 6.3x10-12), and SCR (cg05498681; p = 4.8x10-7). CONCLUSIONS: We have identified several CpGs in leukocytes that are differentially methylated by case-control status. Since a retrospective study design was used, we cannot differentiate whether DNAm was etiologic or resulting from EOC; thus, prospective studies of EOC-associated loci are the critical next step.

Tumor hypomethylation at 6p21.3 associates with longer time to recurrence of high-grade serous epithelial ovarian cancer.

To reveal biologic mechanisms underlying clinical outcome of high-grade serous (HGS) epithelial ovarian carcinomas (EOC), we evaluated the association between tumor epigenetic changes and time to recurrence (TTR). We assessed methylation at approximately 450,000 genome-wide CpGs in tumors of 337 Mayo Clinic (Rochester, MN) patients. Semi-supervised clustering of discovery (n=168) and validation (n=169) sets was used to determine clinically relevant methylation classes. Clustering identified two methylation classes based on 60 informative CpGs, which differed in TTR in the validation set [R vs. L class, P=2.9×10(-3), HR=0.52; 95% confidence interval (CI), 0.34-0.80]. Follow-up analyses considered genome-wide tumor mRNA expression (n=104) and CD8 T-cell infiltration (n=89) in patient subsets. Hypomethylation of CpGs located in 6p21.3 in the R class associated with cis upregulation of genes enriched in immune response processes (TAP1, PSMB8, PSMB9, HLA-DQB1, HLA-DQB2, HLA-DMA, and HLA-DOA), increased CD8 T-cell tumor infiltration (P=7.6×10(-5)), and trans-regulation of genes in immune-related pathways (P=1.6×10(-32)). This is the most comprehensive assessment of clinical outcomes with regard to epithelial ovarian carcinoma tumor methylation to date. Collectively, these results suggest that an epigenetically mediated immune response is a predictor of recurrence and, possibly, treatment response for HGS EOC.

The contribution of deleterious germline mutations in BRCA1, BRCA2 and the mismatch repair genes to ovarian cancer in the population.

The aim of this study was to estimate the contribution of deleterious mutations in BRCA1, BRCA2, MLH1, MSH2, MSH6 and PMS2 to invasive epithelial ovarian cancer (EOC) in the population. The coding sequence and splice site boundaries of all six genes were amplified in germline DNA from 2240 invasive EOC cases and 1535 controls. Barcoded fragment libraries were sequenced using the Illumina GAII or HiSeq and sequence data for each subject de-multiplexed prior to interpretation. GATK and Annovar were used for variant detection and annotation. After quality control 2222 cases (99.2%) and 1528 controls (99.5%) were included in the final analysis. We identified 193 EOC cases (8.7%) carrying a deleterious mutation in at least one gene compared with 10 controls (0.65%). Mutations were most frequent in BRCA1 and BRCA2, with 84 EOC cases (3.8%) carrying a BRCA1 mutation and 94 EOC cases (4.2%) carrying a BRCA2 mutation. The combined BRCA1 and BRCA2 mutation prevalence was 11% in high-grade serous disease. Seventeen EOC cases carried a mutation in a mismatch repair gene, including 10 MSH6 mutation carriers (0.45%) and 4 MSH2 mutation carriers (0.18%). At least 1 in 10 women with high-grade serous EOC has a BRCA1 or BRCA2 mutation. The development of next generation sequencing technologies enables rapid mutation screening for multiple susceptibility genes at once, suggesting that routine clinical testing of all incidence cases should be considered.

Integrative genomic analysis identifies epigenetic marks that mediate genetic risk for epithelial ovarian cancer.

BACKGROUND: Both genetic and epigenetic factors influence the development and progression of epithelial ovarian cancer (EOC). However, there is an incomplete understanding of the interrelationship between these factors and the extent to which they interact to impact disease risk. In the present study, we aimed to gain insight into this relationship by identifying DNA methylation marks that are candidate mediators of ovarian cancer genetic risk. METHODS: We used 214 cases and 214 age-matched controls from the Mayo Clinic Ovarian Cancer Study. Pretreatment, blood-derived DNA was profiled for genome-wide methylation (Illumina Infinium HumanMethylation27 BeadArray) and single nucleotide polymorphisms (SNPs, Illumina Infinium HD Human610-Quad BeadArray). The Causal Inference Test (CIT) was implemented to distinguish CpG sites that mediate genetic risk, from those that are consequential or independently acted on by genotype. RESULTS: Controlling for the estimated distribution of immune cells and other key covariates, our initial epigenome-wide association analysis revealed 1,993 significantly differentially methylated CpGs that between cases and controls (FDR, q < 0.05). The relationship between methylation and case-control status for these 1,993 CpGs was found to be highly consistent with the results of previously published, independent study that consisted of peripheral blood DNA methylation signatures in 131 pretreatment cases and 274 controls. Implementation of the CIT test revealed 17 CpG/SNP pairs, comprising 13 unique CpGs and 17 unique SNPs, which represent potential methylation-mediated relationships between genotype and EOC risk. Of these 13 CpGs, several are associated with immune related genes and genes that have been previously shown to exhibit altered expression in the context of cancer. CONCLUSIONS: These findings provide additional insight into EOC etiology and may serve as novel biomarkers for EOC susceptibility.

Survival prediction based on inherited gene variation analysis.

There is a significant variation of outcome among ovarian cases. Clinical features such as age, stage, comorbidities, or degree of debulking are known prognostic factors for the disease. However, additional variation remains unexplained, some of which may be due to inherited factors. Here, we describe identification of survival-associated inherited variants in ovarian cancer that can enhance our current prognostic capabilities.