Search

Despite increased sophistication in DNA methylation (DNAm) measurement and methods, conducting studies in specific populations such as the hematopoietic stem cell transplant (HCT) population, presents unique challenges and study design considerations. In this article, we explain the motivation for investigating DNAm in the HCT population, highlighting important study design features and key findings in a longitudinal prospective pilot study of DNAm in 32 patients undergoing autologous HCT in Central Virginia, USA. We also discuss limitations and challenges to generating robust results. We observed that HCT does not prevent high-quality DNA from being extracted from whole blood for DNAm research and that longitudinal prospective studies that span pre- and 2-months post-HCT are feasible. Critically, we did not observe significant impacts of cancer diagnosis, time since transplant, age, or chromosomal sex on overall DNAm data dimensionality. These observations demonstrate that while extreme care is required to ensure generalizable, accurate, and interpretable results, researchers should not avoid HCT-DNAm research simply for fear that the transplant procedure or presence of a cancer diagnosis will prevent meaningful conclusions from being drawn. DNAm is an attractive biomarker that is understudied in patients undergoing HCT and needs to expand to improve precise prediction of HCT outcomes.

Supplementary Figures S1-3, Tables S1-4. Supplemental Figure S1. Racial differences in breast tumor methylation at candidate CpG probes when race is self-reported or defined by AIMs markers. Supplemental Figure S2. Methylation detected at KCNK4 and DSC2 by the GoldenGate Cancer Panel I array or quantitative methylation-specific PCR. Supplemental Figure S3. Kaplan-Meier plots showing disease-specific survival in AAs and non-AAs according to tumor methylation level for the top probes varying by race. Cases were divided into three methylation groups (high, intermediate, low) based on beta values in the combined dataset, and Kaplan-Meier plots were generated within AAs and non-AAs. Log-rank p-values are given for differences according to methylation level within each racial group. Supplemental Table S1. Reported genomic variants overlapping CpG probes and their frequencies by ancestry Supplemental Table S2. CpGs significantly differentially methylated by race in HR+ or HR- breast tumors Supplemental Table S3. Correlation between 450K CpG methylation and gene expression in TCGA breast tumors for genes showing differential methylation by race in CBCS Supplemental Table S4. Comparison of methylation in lymphoblastoid cell lines from African Americans and Caucasian Americans

Background: African American (AA) women are diagnosed with more advanced breast cancers and have worse survival than white women, but a comprehensive understanding of the basis for this disparity remains unclear. Analysis of DNA methylation, an epigenetic mechanism that can regulate gene expression, could help to explain racial differences in breast tumor clinical biology and outcomes.Methods: DNA methylation was evaluated at 1,287 CpGs in the promoters of cancer-related genes in 517 breast tumors of AA (n = 216) or non-AA (n = 301) cases in the Carolina Breast Cancer Study (CBCS).Results: Multivariable linear regression analysis of all tumors, controlling for age, menopausal status, stage, intrinsic subtype, and multiple comparisons [false discovery rate (FDR)], identified seven CpG probes that showed significant (adjusted P < 0.05) differential methylation between AAs and non-AAs. Stratified analyses detected an additional four CpG probes differing by race within hormone receptor–negative (HR−) tumors. Genes differentially methylated by race included DSC2, KCNK4, GSTM1, AXL, DNAJC15, HBII-52, TUSC3, and TES; the methylation state of several of these genes may be associated with worse survival in AAs. TCGA breast tumor data confirmed the differential methylation by race and negative correlations with expression for most of these genes. Several loci also showed racial differences in methylation in peripheral blood leukocytes (PBL) from CBCS cases, indicating that these variations were not necessarily tumor-specific.Conclusions: Racial differences in the methylation of cancer-related genes are detectable in both tumors and PBLs from breast cancer cases.Impact: Epigenetic variation could contribute to differences in breast tumor development and outcomes between AAs and non-AAs. Cancer Epidemiol Biomarkers Prev; 24(6); 921–30. ©2015 AACR.

Background: Although obesity is associated with breast cancer incidence and prognosis, the underlying mechanisms are poorly understood. Identification of obesity-associated epigenetic changes in breast tissue may advance mechanistic understanding of breast cancer initiation and progression. The goal of this study, therefore, was to investigate associations between obesity and gene methylation in breast tumors.Methods: Using the Illumina GoldenGate Cancer I Panel, we estimated the association between body mass index (BMI) and gene methylation in 345 breast tumor samples from phase I of the Carolina Breast Cancer Study, a population-based case–control study. Multivariable linear regression was used to identify sites that were differentially methylated by BMI. Stratification by tumor estrogen receptor (ER) status was also conducted.Results: In the majority of the 935 probes analyzed (87%), the average beta value increased with obesity (BMI ≥ 30). Obesity was significantly associated with differential methylation (FDR q < 0.05) in just two gene loci in breast tumor tissue overall and in 21 loci among ER-positive tumors. Obesity was associated with methylation of genes that function in immune response, cell growth, and DNA repair.Conclusions: Obesity is associated with altered methylation overall, and with hypermethylation among ER-positive tumors in particular, suggesting that obesity may influence the methylation of genes with known relevance to cancer. Some of these differences in methylation by obese status may influence levels of gene expression within breast cells.Impact: If our results are validated, obesity-associated methylation sites could serve as targets for prevention and treatment research. Cancer Epidemiol Biomarkers Prev; 24(3); 580–6. ©2015 AACR.

Supplemental Table 1. Correlation between BMI-associated methylation sites (overall and among ER-positive tumors) and gene expression in breast tumors from The Cancer Genome Atlas (TCGA)

Background Blood cancers may be potentially cured with hematopoietic stem cell transplantation (HCT); however, standard pre-assessments for transplant eligibility do not capture all contributing factors for transplant outcomes. Epigenetic biomarkers predict outcomes in various diseases. This pilot study aims to explore epigenetic changes (epigenetic age and differentially methylated genes) in patients before and after autologous HCT, that can serve as potential biomarkers to better predict HCT outcomes. Methods This study used a prospective longitudinal study design to compare genome wide DNA methylation changes in 36 autologous HCT eligible patients recruited from the Cellular Immunotherapies and Transplant clinic at a designated National Cancer Center. Results Genome-wide DNA methylation, measured by the Illumina Infinium Human Methylation 850K BeadChip, showed a significant difference in DNA methylation patterns post-HCT compared to pre-HCT. Compared to baseline levels of DNA methylation pre-HCT, 3358 CpG sites were hypo-methylated and 3687 were hyper-methylated. Identified differentially methylated positions overlapped with genes involved in hematopoiesis, blood cancers, inflammation and immune responses. Enrichment analyses showed significant alterations in biological processes such as immune response and cell structure organization, however no significant pathways were noted. Though participants had an advanced epigenetic age compared to chronologic age before and after HCT, both epigenetic age and accelerated age decreased post-HCT. Conclusion Epigenetic changes, both in epigenetic age and differentially methylated genes were observed in autologous HCT recipients, and should be explored as biomarkers to predict transplant outcomes after autologous HCT in larger, longitudinal studies.

Problem identification Although frailty is an important parameter in treatment planning and in predicting prognosis and overall survival among patients with hematologic malignancies and recipients of hematopoietic stem cell transplantation, frailty assessment tools are not standardized in clinical care settings. The purpose of this article is to provide an overview of the literature on frailty assessment tools in these patient populations. Literature search A systematic search of CINAHL®, Embase®, MEDLINE®, PubMed®, and Web of Science was performed using keywords and controlled vocabulary for the concepts "bone marrow transplants," "hematologic neoplasms," and "frailty." Data evaluation Extracted data included study type, diagnosis, transplantation status, frailty tools used, and outcome measures. Synthesis A systematic search resulted in 24 studies that met the inclusion criteria. There were significant differences in how various groups define and assess frailty. Implications for practice Addressing the lack of standardized frailty assessments will assist healthcare providers to routinely integrate frailty measures in clinical assessments to identify those at risk for poor outcomes, improving personalized care.

Fibromyalgia (FM) is a chronic noncommunicable disorder characterized by a constellation of symptoms that include fatigue, depression and chronic pain. FM affects 2%–8% of the U.S. population, 2% of the global population, with 61%–90% of FM diagnoses attributed to women. Key causal factors leading to the development and severity of FM-related symptoms have not yet been identified. The purpose of this article is to report relationships among identified metabolites and levels of fatigue, depression, pain severity, and pain interference in a sample of 20 women with FM. In this secondary analysis, we conducted global metabolomic analysis and examined the data for relationships of metabolite levels with self-reported symptoms of fatigue, depression, pain severity, and pain interference. Results revealed six metabolites (6-deoxy-hexose; pantothenic acid; ergothioneine; l-carnitine; n-acetylserotonin; butyrobetaine) and their associated metabolic pathways such as carnitine synthesis, lipid oxidation, tryptophan metabolism, beta-alanine metabolism and pantothenic and Coenzyme-A biosynthesis that were either positively or inversely related to pain severity, pain interference, or both. The preliminary data presented suggest that metabolites representing energy, amino acid, or lipid classification may be associated with pain symptom severity and interference in women with FM. Future work will confirm these findings in a large, comparative cohort, targeting metabolites and metabolite pathways to better understand the relationships of metabolites and symptomology.

Tenofovir disoproxil fumarate (TDF) is a common component of antiretroviral therapy in hepatitis B virus (HBV)-HIV co-infected adults but few studies have evaluated worsening renal function and bone turnover, known effects of TDF.Adults from eight North American sites were enrolled in this cohort study. Research assessments were conducted at entry and every 24 weeks for ≤192 weeks. Bone markers were tested at baseline, week 96 and week 192 from stored serum. We evaluated changes in markers of renal function and bone turnover over time and potential contributing factors.A total of 115 patients were prospectively followed; median age 49 years, 91% male and 52% non-Hispanic Black. Duration of HIV was 20.5 years. TDF use ranged from 80% to 92% throughout follow-up. Estimated glomerular filtration rate (eGFR) (ml/min/1.73mIn this HBV-HIV cohort with high prevalence of TDF use, several biomarkers of renal function and bone turnover indicated worsening status over approximately 4 years, highlighting the importance of clinical awareness in co-infected adults.

Lung and breast cancer are the two most common causes of malignant pleural effusion (MPE). MPE diagnosis plays a crucial role in determining staging and therapeutic interventions in these cancers. However, our understanding of the pathogenesis and progression of MPE at the molecular level is limited. Extracellular Vesicles (EVs) and their contents, including microRNAs (miRNAs), can be isolated from all bodily fluids, including pleural fluid. This study aims to compare EV-miRNA patterns of expression in MPE caused by breast (BA-MPE) and lung (LA-MPE) adenocarcinomas compared to the control group of heart-failure-induced effusions (HF-PE). We conducted an analysis of 24 pleural fluid samples (8 LA-MPE, 8 BA-MPE, and 8 HF-PE). Using NanoString technology, we profiled miRNAs within EVs isolated from 12 cases. Bioinformatic analysis demonstrated differential expression of miR-1246 in the MPE group vs. HF-PE group and miR-150-5p and miR-1246 in the BA-MPE vs. LA-MPE group, respectively. This difference was demonstrated and validated in an independent cohort using real-time PCR (RT-PCR). miRNA-1246 demonstrated 4-fold increased expression (OR: 3.87, 95% CI: 0.43, 35) in the MPE vs. HF-PE group, resulting in an area under the curve of 0.80 (95% CI: 0.60, 0.99). The highest accuracy for differentiating MPE vs. HF-PE was seen with a combination of miRNAs compared to each miRNA alone. Consistent with prior studies, this study demonstrates dysregulation of specific EV-based miRNAs in breast and lung cancer; pleural fluid provides direct access for the analysis of these EV-miRNAs as biomarkers and potential targets and may provide insight into the underlying pathogenesis of tumor progression. These findings should be explored in large prospective studies.

Background Hematopoietic stem cell transplant (HSCT) is a potentially curative treatment for hematologic malignancies, with 22 000 HSCTs performed annually in the United States. However, decreased quality of life (QoL) is a frequent and concerning state reported by HSCT recipients. Objectives We sought to determine if measurements of frailty and cognitive impairment were associated with fatigue and QoL in adult HSCT recipients after autologous HSCT. Methods Using a longitudinal study design, 32 participants 18 years or older receiving autologous HSCT were recruited from a bone marrow transplant clinic. Each participant completed 2 visits: pre-HSCT and post-HSCT. At each visit, participants completed assessment tools to measure frailty, cognitive impairment, fatigue, and QoL (assesses physical, social/family, emotional, functional, and transplant-related well-being). Results Participants with increased fatigue scores reported decreased QoL pre- and post-HSCT. Participants with increased frailty showed decreased functional well-being before HSCT and showed correlations with decreased physical, social, and transplant-related well-being post-HSCT. As expected, fatigued participants also showed increased frailty post-HSCT. Participants showed significant changes in physical well-being and fatigue between pre-HSCT and post-HSCT visits. Conclusion Data analyses from this pilot study show significant correlations between subsets of QoL with fatigue and frailty in autologous HSCT participants pre- and post-HSCT. Implications for practice Understanding the impact of frailty on fatigue and QoL in HSCT recipients is critical to assist nurses in initiating educational and behavioral interventions to help mitigate the effects of HSCT.

Purpose: In recent years, researchers have noted an “obesity paradox,” where individuals with obesity survive sepsis at higher rates than their nonobese counterparts. This systematic review summarizes the literature on studies examining the association between obesity and 1-year mortality among patients admitted with sepsis, severe sepsis, or septic shock. Materials and Methods: Using a comprehensive search strategy, a systematic review was conducted to identify studies examining the association of obesity and sepsis mortality. PubMed, Cumulative Index of Nursing and Allied Health Literature, and Elton B. Stephens Company host databases were searched for the terms sepsis, obesity, mortality, and adult. Results: The initial search identified 189 studies, 9 of which met inclusion criteria. Of these, four provided evidence that obese or very obese patients with sepsis have lower mortality than nonobese patients. Methodologic differences in the remaining five studies, which reported conflicting results, limit generalizability. Conclusion: This systematic review on the association of obesity and sepsis mortality found three studies that demonstrated lower sepsis mortality among obese patients in the first 30 days and one showing that this protective effect extends up to 1 year. Given the increased number of patients surviving sepsis, it is important to consider long-term mortality and further describe the variables associated with increased survival.

A nontargeted plasma metabolomic analysis was conducted to compare differentially expressed metabolites in women with and without fibromyalgia (FM) using data and samples collected from two parent studies in women with FM (n = 20) and comparative data collected from newly recruited age-matched women (n = 20). Blood plasma samples were analyzed for metabolite content using liquid chromatography mass spectrometry. Consolidation of positive and negative ion mode metabolomics data with fold change (>2 or

Sepsis is a life-threatening syndrome that occurs in response to a severe infection. In recent years, the understanding of the pathobiology of sepsis has been refined, with research describing an altered host response as the underlying cause. Survivors of sepsis often have long hospital stays and suffer from subsequent frailty and long-term health consequences. Predicting attributes of sepsis survivors remains challenging; however, an obesity paradox exists, wherein obese individuals survive sepsis at higher rates than their normal-weight counterparts. We present a model that describes the relationships between sepsis and obesity, focusing on inflammation as a shared pathway for dysregulation in obese and healthy-weight adults. Understanding the interaction of these complex variables is an important first step toward developing interventions and treatments to augment sepsis survival.

Background Catheter ablation is an effective treatment for atrial fibrillation ( AF ), but high levels of post‐procedure inflammation predict adverse clinical events. Ascorbic acid ( AA ) has shown promise in reducing inflammation but is untested in this population. We sought to test the feasibility, safety, and preliminary effects on inflammatory biomarkers in the CITRIS‐AF (Vitamin C Intravenous Treatment In the Setting of Atrial Fibrillation Ablation) pilot study. Methods and Results Patients scheduled to undergo AF ablation (N=20) were randomized 1:1 to double‐blinded treatment with AA (200 mg/kg divided over 24 hours) or placebo. C‐reactive protein and interleukin‐6 levels were obtained before the first infusion and repeated at 24 hours and 30 days. Pain levels within 24 hours and early recurrence of AF within 90 days were recorded. Median and interquartile range were aged 63 (56–70) years, 13 (65%) men, and 18 (90%) white. Baseline data were similar between the 2 groups except ejection fraction. Baseline C‐reactive protein levels were 2.56 (1.47–5.87) mg/L and similar between groups ( P =0.48). Change in C‐reactive protein from baseline to 24 hours was +10.79 (+6.56–23.19) mg/L in the placebo group and +3.01 (+0.40–5.43) mg/L in the AA group ( P =0.02). Conversely, change in interleukin‐6 was numerically higher in the AA group, though not statistically significant ( P =0.32). One patient in each arm developed pericarditis; no adverse events related to the infusions were seen. There were no significant differences between aggregated post‐procedure pain levels within 24 hours or early recurrence of AF (both P >0.05). Conclusions High‐dose AA is safe and well tolerated at the time of AF ablation and may be associated with a blunted rise in C‐reactive protein, although consistent findings were not seen in interleukin‐6 levels. Further studies are needed to validate these findings and explore the potential benefit in improving clinically relevant outcomes. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 03148236.

// Benjamin Garcia-Bloj 1, 2, 4 , Colette Moses 1, 2 , Agustin Sgro 1 , Janice Plani-Lam 1 , Mahira Arooj 1, 5 , Ciara Duffy 1, 2 , Shreyas Thiruvengadam 1 , Anabel Sorolla 1 , Rabab Rashwan 1 , Ricardo L. Mancera 5 , Andrea Leisewitz 4 , Theresa Swift-Scanlan 6 , Alejandro H. Corvalan 3, 4 , Pilar Blancafort 1, 2 1 Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia 2 School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia 3 Advanced Center for Chronic Diseases (ACCDiS), Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 4 Faculty of Medicine, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 5 School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth WA 6845, Australia 6 School of Nursing, Virginia Commonwealth University, Richmond, Virginia, 23298, USA Correspondence to: Pilar Blancafort, email: pilar.blancafort@uwa.edu.au Keywords: CRISPR/dCas9, ZF, TALE, tumor suppressor genes, gene reactivation Received: June 01, 2016 Accepted: July 19, 2016 Published: August 09, 2016 ABSTRACT The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO , in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

Introduction Children with severe asthma may benefit from non-pharmacological approaches to symptom management, yet little is known regarding the impact of mind-body approaches such as mindful yoga in this population. This pilot study examined the tolerability, feasibility, acceptability and immediate effects of a mindful yoga intervention in children with severe asthma. Method Fifteen children ages 7–11 with severe asthma participated in a single mindful yoga session, followed by semi-structured interviews with the child and caregiver. Vital signs and pulmonary function tests were collected pre- and post-intervention. Results All children reported positive experiences and caregivers were generally supportive of their participation. One child experienced self-limited bronchoconstriction with asthma symptoms. There was no significant change in lung function or vital signs after the mindful yoga session. Discussion Our results warrant additional larger studies to evaluate the safety and benefits of mindful yoga in children with severe asthma.

Background: Although obesity is associated with breast cancer incidence and prognosis, the underlying mechanisms are poorly understood. Identification of obesity-associated epigenetic changes in breast tissue may advance mechanistic understanding of breast cancer initiation and progression. The goal of this study, therefore, was to investigate associations between obesity and gene methylation in breast tumors. Methods: Using the Illumina GoldenGate Cancer I Panel, we estimated the association between body mass index (BMI) and gene methylation in 345 breast tumor samples from phase I of the Carolina Breast Cancer Study, a population-based case–control study. Multivariable linear regression was used to identify sites that were differentially methylated by BMI. Stratification by tumor estrogen receptor (ER) status was also conducted. Results: In the majority of the 935 probes analyzed (87%), the average beta value increased with obesity (BMI ≥ 30). Obesity was significantly associated with differential methylation (FDR q < 0.05) in just two gene loci in breast tumor tissue overall and in 21 loci among ER-positive tumors. Obesity was associated with methylation of genes that function in immune response, cell growth, and DNA repair. Conclusions: Obesity is associated with altered methylation overall, and with hypermethylation among ER-positive tumors in particular, suggesting that obesity may influence the methylation of genes with known relevance to cancer. Some of these differences in methylation by obese status may influence levels of gene expression within breast cells. Impact: If our results are validated, obesity-associated methylation sites could serve as targets for prevention and treatment research. Cancer Epidemiol Biomarkers Prev; 24(3); 580–6. ©2015 AACR.

Tobacco smoking is a risk factor in several cancers, yet its roles as a putative etiologic exposure or poor prognostic factor in breast cancer are less clear. Altered DNA methylation contributes to breast cancer development and may provide a mechanistic link between smoking and gene expression changes leading to cancer development or progression. Using a cancer-focused array, we examined methylation at 933 CpGs in 517 invasive breast tumors in the Carolina Breast Cancer Study to determine whether methylation patterns differ by exposure to tobacco smoke. Multivariable generalized linear regression models were used to compare tumor methylation profiles between smokers and never smokers, overall, or stratified on hormone receptor (HR) status. Modest differences in CpG methylation were detected at p

Brain metastasis affects 10-30% of breast cancer patients, but it is unclear how breast cancer cells can colonize the unique and mostly cellular brain environment. Exosomes, endosomal-derived extracellular vesicles, have been reported to support tumorigenesis, mediate tumor-stromal interactions, and drive pre-metastatic niche formation, ultimately creating sites conducive to cancer development. Still, their role in brain metastasis remains to be fully understood. Glial cells, namely astrocytes, are important contributors to brain metastasis and secrete exosomes that support metastatic outgrowth in the brain. Thus, our study aims to investigate if breast cancer-derived exosomes can transform astrocytes towards a pro-metastatic phenotype. To do this, we isolated exosomes from 4T1 metastatic and 67NR non-metastatic mouse mammary carcinoma cell conditioned media using ultracentrifugation. Exosomes were characterized using Western immunoblot, dynamic light scatter analysis, and transmission electron microscopy. Using confocal microscopy, we found that mouse astrocytes can internalize 4T1 exosomes in vitro. Preliminary studies using qPCR analysis revealed that mouse astrocytes treated with 4T1 exosomes compared to those treated with 67NR exosomes exhibit differential gene expression, suggesting that there is a unique response profile associated with metastatic versus non-metastatic breast cancer-derived exosomes. To further assess this, we are investigating changes in the astrocyte secretome in response to 4T1 and 67NR exosome treatment using protein mass spectrometry. Taking a global approach, we have also been assessing differences between 4T1 and 67NR exosomal miRNA, and protein that could promote a shift in astrocytes towards a pro-metastatic phenotype. For these studies, we have implemented the Nanostring nCounter Analysis System and protein mass spectrometry, respectively. Proteomics assessment revealed distinct differences between the 4T1 metastatic and 67NR non-metastatic exosomes, whereby we identified 259 proteins that were enriched in 4T1 exosomes and 71 proteins that were decreased in 4T1 exosomes compared to 67NR’s. Proteins enriched in 4T1 exosomes were found to be associated with RNA binding and translation machinery, as well as abnormal cell adhesion, and BRCA1 interactions. Proteins identified to be decreased in 4T1 exosomes were found to play a role in IL-6 and IL-12 signaling events, the estrogen signaling pathway, and syndecan-2 signaling. These findings indicate that 4T1 and 67NR exosomes differ in protein cargo and these differences may be important in facilitating brain metastasis. Altogether, these studies will provide a more comprehensive assessment of the differences between metastatic versus non-metastatic cancer exosomes, and ultimately, provide novel insight into understanding breast cancer metastasis to the brain. Citation Format: Megan Sayyad, Madhavi Puchalapalli, Chris Canal, Theresa Swift-Scanlan, Andrew Ottens, Jennifer E. Koblinski. Metastatic v non-metastatic cancer exosomes in brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1988.

Catheter ablation is an effective treatment for atrial fibrillation (AF), but high levels of post-procedure inflammation predict adverse clinical events. Ascorbic acid (AA) has shown promise in reducing sterile inflammation but is untested in this population. Patients scheduled to undergo AF

Purpose: The challenges of cytology for accurate diagnosis of breast cancer are well recognized. We previously showed that normal and tumor tissue can be distinguished using a technique called quantitative multiplex methylation-specific PCR (QM-MSP). We hypothesized that quantitative analysis of methylated genes will provide enhanced detection of cancer cells present in cytologic specimens.Experimental Design: QM-MSP was done on ductal lavage cells from a set of 37 ductal lavage samples from women undergoing mastectomy (27 with cancer and 3 without). Duct histology information was available for each lavaged duct. QM-MSP data was assessed by measuring cumulative methylation index and by receiver operating characteristic threshold analysis. To determine the baseline level of methylation for each gene in this population, cells from 60 ducts of women at high risk of developing breast cancer were analyzed.Results: QM-MSP findings on a panel of nine genes were correlated to duct histology and ductal lavage cytology. Cytology detected cancer in 33% (7 of 21 ducts) with a specificity of 99% (92 of 93). QM-MSP detected cancer as calculated by cumulative methylation index with a sensitivity of 62% (13 of 21) and specificity of 82% (62 of 76) and by receiver operating characteristic threshold analysis with a sensitivity of 71% (15 of 21) and specificity of 83% (63 of 76).Conclusions: Compared with cytology, QM-MSP doubled the sensitivity of detection of cancer. This study provides proof of principle by showing the advantages of using methylation analyses to query cytologic specimens and indicates its potential use in diagnosis and in stratifying risk.

In recent years, several methylation-specific PCR-based techniques have been developed to identify and characterize hypermethylation of CpG dinucleotides with the primary goal of elucidating a better understanding of the role of DNA methylation in important biological processes, such as chromosome X inactivation and carcinogenesis. The specificity of methylation-specific PCR (MSP) techniques relies on amplifying sodium bisulfite-treated DNA with primers specific to predicted sequences of unmethylated and methylated DNA within the gene of interest. In the past, unmethylated and methylated reactions were singleplex and performed in separate wells. In this paper, we report a modification of the real-time quantitative multiplex MSP (QM-MSP) technique of Fackler and colleagues that can be applied to any real-time MSP experiment. Although co-amplification with multiple fluorophores is common in standard reverse transcription PCR (RT-PCR), MSP presents unique challenges both mechanistically and operationally that must be overcome in order to successfully co-amplify two methylation-specific targets. In this two-color modification, unmethylated and methylated primer/probe sets are successfully co-amplified in the same reaction using FAM™- and VIC®-labeled probes. Our modification decreases the cost and time of each real-time experiment by allowing increased throughput of clinical samples and by doubling either the number of genes or the number of samples that can be analyzed per real-time plate.

A strong genetic association between the NOTCH4 locus on chromosome 6 and schizophrenia was recently reported. Based on the data suggesting overlapping susceptibility for schizophrenia and bipolar disorder, we genotyped the polymorphic (CTG)n encoding polyleucine repeat in exon 1 of NOTCH4 in 65 pedigrees ascertained for a genetic linkage study of bipolar disorder. In addition, we analyzed a subset of our pedigrees with psychotic features at this locus. We failed to find any association between the (CTG)n NOTCH4 polymorphism and either the bipolar or the psychotic bipolar phenotype in our 65 pedigrees.

Purpose/objectives To compare and contrast the molecular and environmental factors contributing to basal-like breast cancer and highlight the clinical implications for women with this phenotype. Data sources CINAHL® and PubMed databases, journals, and citation indices were searched using the key word basal-like in combination with breast cancer, epigenetic, treatment, subtype, risk factor, and BRCA1 to synthesize the literature on the multiple underpinnings of basal-like breast cancer. Data synthesis Research findings related to the molecular foundation of basal-like breast cancer were integrated with knowledge of nongenetic contributing risk factors. Approved therapies and those under development were summarized with the goal of improving understanding for research and practice. Conclusions Of the five subtypes of breast cancer, the basal-like subtype has the shortest survival and poorest prognosis. The development of gene expression assays with epigenetic studies has enabled reliable identification of the basal-like subtype and has shed light on novel therapeutic possibilities. Clinical trials for basal-like breast cancer are underway, and the potential for individualized treatments for women with this subtype show promise. Implications for nursing The main difficulties with basal-like breast cancer are its aggressive course, treatment refractory nature, and complex biology, all of which pose real challenges for clinical management and patient education. Oncology nurses play a pivotal role in providing holistic care and patient support. Therefore, nurses must understand the complexity of the clinical presentation and the underlying biology of this cancer subtype.

Background: African American (AA) women are diagnosed with more advanced breast cancers and have worse survival than white women, but a comprehensive understanding of the basis for this disparity remains unclear. Analysis of DNA methylation, an epigenetic mechanism that can regulate gene expression, could help to explain racial differences in breast tumor clinical biology and outcomes. Methods: DNA methylation was evaluated at 1,287 CpGs in the promoters of cancer-related genes in 517 breast tumors of AA (n = 216) or non-AA (n = 301) cases in the Carolina Breast Cancer Study (CBCS). Results: Multivariable linear regression analysis of all tumors, controlling for age, menopausal status, stage, intrinsic subtype, and multiple comparisons [false discovery rate (FDR)], identified seven CpG probes that showed significant (adjusted P < 0.05) differential methylation between AAs and non-AAs. Stratified analyses detected an additional four CpG probes differing by race within hormone receptor–negative (HR−) tumors. Genes differentially methylated by race included DSC2, KCNK4, GSTM1, AXL, DNAJC15, HBII-52, TUSC3, and TES; the methylation state of several of these genes may be associated with worse survival in AAs. TCGA breast tumor data confirmed the differential methylation by race and negative correlations with expression for most of these genes. Several loci also showed racial differences in methylation in peripheral blood leukocytes (PBL) from CBCS cases, indicating that these variations were not necessarily tumor-specific. Conclusions: Racial differences in the methylation of cancer-related genes are detectable in both tumors and PBLs from breast cancer cases. Impact: Epigenetic variation could contribute to differences in breast tumor development and outcomes between AAs and non-AAs. Cancer Epidemiol Biomarkers Prev; 24(6); 921–30. ©2015 AACR.

With the recent comprehensive mapping of cancer genomes, there is now a need for functional approaches to edit the aberrant epigenetic state of key cancer drivers to reprogram the epi-pathology of the disease. In this study we utilized a programmable DNA-binding methyltransferase to induce targeted incorporation of DNA methylation (DNAme) in the SOX2 oncogene in breast cancer through a six zinc finger (ZF) protein linked to DNA methyltransferase 3A (ZF-DNMT3A). We demonstrated long-lasting oncogenic repression, which was maintained even after suppression of ZF-DNMT3A expression in tumor cells. The de novo DNAme was faithfully propagated and maintained through cell generations even after the suppression of the expression of the chimeric methyltransferase in the tumor cells. Xenograft studies in NUDE mice demonstrated stable SOX2 repression and long-term breast tumor growth inhibition, which lasted for >100 days post implantation of the tumor cells in mice. This was accompanied with a faithful maintenance of DNAme in the breast cancer implants. In contrast, downregulation of SOX2 by ZF domains engineered with the Krueppel-associated box repressor domain resulted in a transient and reversible suppression of oncogenic gene expression. Our results indicated that targeted de novo DNAme of the SOX2 oncogenic promoter was sufficient to induce long-lasting epigenetic silencing, which was not only maintained during cell division but also significantly delayed the tumorigenic phenotype of cancer cells in vivo, even in the absence of treatment. Here, we outline a genome-based targeting approach to long-lasting tumor growth inhibition with potential applicability to many other oncogenic drivers that are currently refractory to drug design.

Introduction Breast cancer is a heterogeneous disease, with several intrinsic subtypes differing by hormone receptor (HR) status, molecular profiles, and prognosis. However, the role of DNA methylation in breast cancer development and progression and its relationship with the intrinsic tumor subtypes are not fully understood. Methods A microarray targeting promoters of cancer-related genes was used to evaluate DNA methylation at 935 CpG sites in 517 breast tumors from the Carolina Breast Cancer Study, a population-based study of invasive breast cancer. Results Consensus clustering using methylation (β) values for the 167 most variant CpG loci defined four clusters differing most distinctly in HR status, intrinsic subtype (luminal versus basal-like), and p53 mutation status. Supervised analyses for HR status, subtype, and p53 status identified 266 differentially methylated CpG loci with considerable overlap. Genes relatively hypermethylated in HR+, luminal A, or p53 wild-type breast cancers included FABP3, FGF2, FZD9, GAS7, HDAC9, HOXA11, MME, PAX6, POMC, PTGS2, RASSF1, RBP1, and SCGB3A1, whereas those more highly methylated in HR-, basal-like, or p53 mutant tumors included BCR, C4B, DAB2IP, MEST, RARA, SEPT5, TFF1, THY1, and SERPINA5. Clustering also defined a hypermethylated luminal-enriched tumor cluster 3 that gene ontology analysis revealed to be enriched for homeobox and other developmental genes (ASCL2, DLK1, EYA4, GAS7, HOXA5, HOXA9, HOXB13, IHH, IPF1, ISL1, PAX6, TBX1, SOX1, and SOX17). Although basal-enriched cluster 2 showed worse short-term survival, the luminal-enriched cluster 3 showed worse long-term survival but was not independently prognostic in multivariate Cox proportional hazard analysis, likely due to the mostly early stage cases in this dataset. Conclusions This study demonstrates that epigenetic patterns are strongly associated with HR status, subtype, and p53 mutation status and may show heterogeneity within tumor subclass. Among HR+ breast tumors, a subset exhibiting a gene signature characterized by hypermethylation of developmental genes and poorer clinicopathologic features may have prognostic value and requires further study. Genes differentially methylated between clinically important tumor subsets have roles in differentiation, development, and tumor growth and may be critical to establishing and maintaining tumor phenotypes and clinical outcomes. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0450-6) contains supplementary material, which is available to authorized users.

A genome scan of approximately 12-cM initial resolution was done on 50 of a set of 51 carefully ascertained unilineal multiplex families segregating the bipolar affective disorder phenotype. In addition to standard multipoint linkage analysis methods, a simultaneous-search algorithm was applied in an attempt to surmount the problem of genetic heterogeneity. The results revealed no linkage across the genome. The results exclude monogenic models and make it unlikely that two genes account for the disease in this sample. These results support the conclusion that at least several hundred kindreds will be required in order to establish linkage of susceptibility loci to bipolar disorder in heterogeneous populations.

The Vimentin gene plays a pivotal role in epithelial-to-mesenchymal transition and is known to be overexpressed in the prognostically poor basal-like breast cancer subtype. Recent studies have reported Vimentin DNA methylation in association with poor clinical outcomes in other solid tumors, but not in breast cancer. We therefore quantified Vimentin DNA methylation using MALDI-TOF mass spectrometry in breast tumors and matched normal pairs in association with gene expression and survival in a hospital-based study of breast cancer patients. Gene expression data via qRT-PCR in cell lines and oligomicroarray data from breast tissues were correlated with percent methylation in the Vimentin promoter. A threshold of 20 percent average methylation compared with matched normal pairs was set for bivariate and multivariate tests of association between methylation and tumor subtype, tumor histopathology, and survival. Vimentin was differentially methylated in luminal breast cancer cell lines, and in luminal A, luminal B, and HER2-enriched breast tumor subtypes, but was rare in basal-like cell lines and tumors. Increased methylation was strongly correlated with decreased mRNA expression in cell lines, and had a moderate inverse correlation in breast tumors. Vimentin methylation predicted poor overall survival independent of race, subtype, stage, nodal status, or metastatic disease and holds promise as a new prognostic biomarker for breast cancer patients.

Anticipation has been frequently found in bipolar families ascertained for linkage studies. An association of polymorphic triplet repeats with the bipolar phenotype in some pedigrees has been proposed. We have previously found linkage to chromosome 18 in a set of families with evidence of anticipation. As part of a search for CAG/CTG motifs on chromosome 18, we screened a genomic chromosome 18 cosmid library and identified 65 loci with trinucleotide repeats. Eleven of 33 genotyped loci were polymorphic, though none of these showed any evidence of instability. We performed genetic analysis of six loci in the Hopkins/Dana bipolar pedigrees ascertained for a genetic linkage study of bipolar disorder and found that the CAG repeat within the AD4D2 clone on 18q21.1 showed nominally significant over-transmission of the rare CAG23 allele (P=0.034). We have characterized all 65 trinucleotide repeats and flanking sequences with GENSCAN analysis and find that 29 were predicted to be in coding regions. These 29 trinucleotide-repeat-containing genes may be involved in functional modulation of their respective proteins, and may be candidates for other diseases or disease mechanisms that map to this region.

The purpose of this study was to assess 65 pedigrees ascertained through a Bipolar I (BPI) proband for evidence of linkage, using nonparametric methods in a genome-wide scan and for possible parent of origin effect using several analytical methods. We identified 15 loci with nominally significant evidence for increased allele sharing among affected relative pairs. Eight of these regions, at 8q24, 18q22, 4q32, 13q12, 4q35, 10q26, 2p12, and 12q24, directly overlap with previously reported evidence of linkage to bipolar disorder. Five regions at 20p13, 2p22, 14q23, 9p13, and 1q41 are within several Mb of previously reported regions. We report our findings in rank order and the top five markers had an NPL>2.5. The peak finding in these regions were D8S256 at 8q24, NPL 3.13; D18S878 at 18q22, NPL 2.90; D4S1629 at 4q32, NPL 2.80; D2S99 at 2p12, NPL 2.54; and D13S1493 at 13q12, NPL 2.53. No locus produced statistically significant evidence for linkage at the genome-wide level. The parent of origin effect was studied and consistent with our previous findings, evidence for a locus on 18q22 was predominantly from families wherein the father or paternal lineage was affected. There was evidence consistent with paternal imprinting at the loci on 13q12 and 1q41.

We previously reported linkage between bipolar disorder and a region on human chromosome (HC) 18q21. To identify genes in this region, exon trapping was performed on cosmids isolated from an HC18-specific cosmid library (LL18NC02) using 47 sequence tagged site (STS) markers from 18q21 as hybridization probes. A total of 285 unique sequences (exons) were obtained from 850 sequenced clones. Homology searching of the databases using NCBI's BLAST algorithms revealed that 31 exons have identity to known genes and/or ESTs, seven are identical to regions of finished genomic sequences in the 18q21 region, 20 have significant similarity (>30% sequence identity) to genes from human and/or other species, 19 were repetitive sequences, and 208 sequences (72%) are novel. Seventy per cent of the trapped sequences were predicted to be derived from genes using library screening and RT-PCR analyses. This represents an initial stage in characterizing genes in a susceptibility region for further study in bipolar disorder or other diseases that map to this region.

The aim of this study was to describe breast tumor subtypes by common breast cancer risk factors and to determine correlates of subtypes using baseline data from two pooled prospective breast cancer studies within a large health maintenance organization.Tumor data on 2544 invasive breast cancer cases subtyped by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Her2) status were obtained (1868 luminal A tumors, 294 luminal B tumors, 288 triple-negative tumors and 94 Her2-overexpressing tumors). Demographic, reproductive and lifestyle information was collected either in person or by mailed questionnaires. Case-only odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression, adjusting for age at diagnosis, race/ethnicity, and study origin.Compared with luminal A cases, luminal B cases were more likely to be younger at diagnosis (P = 0.0001) and were less likely to consume alcohol (OR = 0.74, 95% CI = 0.56 to 0.98), use hormone replacement therapy (HRT) (OR = 0.66, 95% CI = 0.46 to 0.94), and oral contraceptives (OR = 0.73, 95% CI = 0.55 to 0.96). Compared with luminal A cases, triple-negative cases tended to be younger at diagnosis (Por = 0.0001) and African American (OR = 3.14, 95% CI = 2.12 to 4.16), were more likely to have not breastfed if they had parity greater than or equal to three (OR = 1.68, 95% CI = 1.00 to 2.81), and were more likely to be overweight (OR = 1.82, 95% CI = 1.03 to 3.24) or obese (OR = 1.97, 95% CI = 1.03 to 3.77) if premenopausal. Her2-overexpressing cases were more likely to be younger at diagnosis (P = 0.03) and Hispanic (OR = 2.19, 95% CI = 1.16 to 4.13) or Asian (OR = 2.02, 95% CI = 1.05 to 3.88), and less likely to use HRT (OR = 0.45, 95% CI = 0.26 to 0.79).These observations suggest that investigators should consider tumor heterogeneity in associations with traditional breast cancer risk factors. Important modifiable lifestyle factors that may be related to the development of a specific tumor subtype, but not all subtypes, include obesity, breastfeeding, and alcohol consumption. Future work that will further categorize triple-negative cases into basal and non-basal tumors may help to elucidate these associations further.

The catechol-O-methyltransferase (COMT) enzyme has been widely studied due to its multiple roles in neurological functioning, estrogen biology, and methylation metabolic pathways. Numerous studies have investigated variation in the large COMT gene, with the majority focusing on single nucleotide polymorphisms (SNPs). This body of work has linked COMT genetic variation with a vast array of conditions, including several neurobehavioral disorders, pain sensitivity, and multiple human cancers. Based on COMT’s numerous biological roles and recent studies suggesting that methylation of the COMT gene impacts COMT gene expression, we comprehensively interrogated methylation in over 200 CpG dinucleotide sequences spanning the length of the COMT gene. Using saliva-derived DNA from a non-clinical sample of human subjects, we tested for associations between COMT CpG methylation and factors reported to interact with COMT genetic effects, including demographic factors and alcohol use. Finally, we tested associations between COMT CpG methylation state and COMT gene expression in breast cancer cell lines. We interrogated >200 CpGs in 13 amplicons spanning the 5’ UTR to the last exon of the CpG dinucleotide-rich COMT gene in n = 48 subjects, n = 11 cell lines and 1 endogenous 18S rRNA control. With the exception of the CpG island in the 5’UTR and 1st exon, all other CpG islands were strongly methylated with typical dynamic ranges between 50-90%. In the saliva samples, methylation of multiple COMT loci was associated with socioeconomic status or ethnicity. We found associations between methylation at numerous loci and genotype at the functional Val 158 Met SNP (rs4680), and most of the correlations between methylation and demographic and alcohol use factors were Val 158 Met allele-specific. Methylation at several of these loci also associated with COMT gene expression in breast cancer cell lines. We report the first comprehensive interrogation of COMT methylation. We corroborate previous findings of variation in COMT methylation with gene expression and the Val 158 Met genotype, and also report novel associations with socioeconomic status (SES) and ethnicity at several methylated loci. These results point to novel mechanisms for COMT regulation, which may have broad therapeutic implications.