Data from Gene Methylation and Cytological Atypia in Random Fine-Needle Aspirates for Assessment of Breast Cancer Risk
Vered StearnsMary Jo FacklerSidra HafeezZoila A. Lopez‐BujandaRobert T. ChattertonLisa K. JacobsNagi F. KhouriDavid IvancicKara KenneyChristina ShehataStacie C. JeterJudith A. WolfmanCarola M. ZallesPeng HuangSeema A. KhanSaraswati Sukumar
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<div>Abstract<p>Methods to determine individualized breast cancer risk lack sufficient sensitivity to select women most likely to benefit from preventive strategies. Alterations in DNA methylation occur early in breast cancer. We hypothesized that cancer-specific methylation markers could enhance breast cancer risk assessment. We evaluated 380 women without a history of breast cancer. We determined their menopausal status or menstrual cycle phase, risk of developing breast cancer (Gail model), and breast density and obtained random fine-needle aspiration (rFNA) samples for assessment of cytopathology and cumulative methylation index (CMI). Eight methylated gene markers were identified through whole-genome methylation analysis and included novel and previously established breast cancer detection genes. We performed correlative and multivariate linear regression analyses to evaluate DNA methylation of a gene panel as a function of clinical factors associated with breast cancer risk. CMI and individual gene methylation were independent of age, menopausal status or menstrual phase, lifetime Gail risk score, and breast density. CMI and individual gene methylation for the eight genes increased significantly (<i>P</i> < 0.001) with increasing cytological atypia. The findings were verified with multivariate analyses correcting for age, log (Gail), log (percent density), rFNA cell number, and body mass index. Our results demonstrate a significant association between cytological atypia and high CMI, which does not vary with menstrual phase or menopause and is independent of Gail risk and mammographic density. Thus, CMI is an excellent candidate breast cancer risk biomarker, warranting larger prospective studies to establish its utility for cancer risk assessment. <i>Cancer Prev Res; 9(8); 673–82. ©2016 AACR</i>.</p></div>Keywords:
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A new noninvasive screening tool for colorectal neoplasia detects epigenetic alterations exhibited by gastrointestinal tumor cells shed into stool. There is insufficient existing data to determine temporal associations between colorectal cancer (CRC) progression and aberrant DNA methylation. To evaluate the feasibility of using fecal DNA methylation status to determine CRC progression, we collected stool samples from 14 male SD rats aged six weeks, and administered subcutaneous injections of either 1,2-dimethylhydrazine or saline weekly. p16 DNA methylation statuses in tumorous and normal colon tissue, and from stool samples were determined using methylation-specific PCR. Additionally, p16 methylation was detected in stool DNA from 85.7% of the CRC rats. The earliest change in p16 methylation status in the DMH-treated group stool samples occurred during week nine; repeatabilities were 57.1% in week 19 (p = 0.070) and 85.7% in week 34 (p = 0.005). A temporal correlation was evidenced between progression of CRC and p16 methylation status, as evidenced by DMH-induced rat feces. Using fecal DNA methylation status to determine colorectal tissue methylation status can reveal CRC progression. Our data suggests that p16 promoter methylation is a feasible epigenetic marker for the detection and may be useful for CRC screening.
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DNA methylation measured in white blood cell DNA is increasingly being used as in studies of cancer susceptibility. However, little is known about the correlation between different assays to measure global methylation and whether the source of DNA matters when examining methylation profiles in different blood cell types. Using information from 620 women, 217 and 403 women with DNA available from granulocytes (Gran), and total white blood cells (WBC), respectively, and 48 women with DNA available from four different sources (WBC, Gran, mononuclear (MN), and lymphoblastoid cell lines (LCL)), we compared DNA methylation for three repetitive elements (LINE1, Sat2, Alu) by MethyLight, luminometric methylation assay (LUMA), and [3H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LBC, MN, or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [3H]-methyl acceptance, LINE1, and Alu assays. Methylation in MN was correlated with methylation in WBC for the [3H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant positive correlations ranging from 0.3-0.7 for each cell type with one exception (Sat2 and Alu in MN). . Among the 620 women stratified by DNA source, correlations among assays were highest for the three repetitive elements (range 0.39-0.64). Results from the LUMA assay were modestly correlated with LINE1 (0.18-0.20). These results suggest that both assay and source of DNA are critical components in the interpretation of global DNA methylation patterns from WBC.
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Age is a key risk factor for breast cancer and epigenetic alterations may contribute to age-related increases in breast cancer risk, though the relation of age-related methylation in normal breast tissues with altered methylation in breast tumors is unclear. We investigated the relation of age with DNA methylation in normal breast tissues genome-wide using two data sets from the Gene Expression Omnibus (GEO) database (GSE32393 and GSE31979). We validated our observations in an independent set of normal breast tissues, examined age-related methylation in normal breast for enrichment of genomic features, and compared age-related methylation in normal tissue with methylation alterations in breast tumors. Between the two array-based methylation data sets, there were 204 CpG loci with significant (P < 0.05) and consistent age-related methylation, 97% of which were increases in methylation. Our validation sets confirmed the direction of age-related DNA methylation changes in all measured regions. Among the 204 age-related CpG loci, we observed a significant enrichment for CpG islands (P = 8.7E-6) and polycomb group protein target genes (P = 0.03). In addition, 24 of the 204 CpGs with age-related methylation in normal breast were significantly differentially methylated between normal and breast tumor tissues. We identified consistent age-related methylation changes in normal breast tissue that are further altered in breast tumors and may represent early events contributing to breast carcinogenesis. This work identifies age-related methylation in normal breast tissue and begins to deconstruct the contribution of aging to epigenetic alterations present in breast tumors.
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Abstract Breast cancer is a frequent female malignant tumor with high mortality and poor prognosis. Peroxidasin like (PXDNL) has many biological functions, including characteristic activity of hormone biosynthesis, host defense, and cell motility. In addition, PXDNL is closely connected with the progression of breast cancer. In this study, we found that PXDNL may be an independent prognostic biomarker of breast cancer. We tested the mRNA expression of PXDNL in breast cancer by detecting The Cancer Genome Atlas (TCGA) database. The chi-squared test was used to evaluate clinical correlation. The receiver operating characteristic (ROC) curves were drawn to evaluate diagnosis potential in breast cancer. Subsequently, survival analyses were performed to identify the relevance between the expression of PXDNL and the overall survival/relapse-free survival of patients with breast cancer. Univariate/multivariate Cox regression model was executed to detect risk factors affecting the prognosis of patients with breast cancer. PXDNL is highly expressed in breast cancer tissues and is related to survival status of patients. The ROC curve showed that PXDNL had beneficial diagnostic ability in breast cancer. Survival analysis indicated that patients with breast cancer with high PXDNL expression generally had decreased overall survival/relapse-free survival. Univariate/multivariate Cox model analyses further suggested an association between PXDNL expression and prognosis of patients with breast cancer. High PXDNL expression is a potential and independent prognostic biomarker in breast cancer.
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17ß-Estradiol, an epigenetic modulator, is involved in the increased prevalence of migraine in women. Together with the prophylactic efficacy of valproate, which influences DNA methylation and histone modification, this points to the involvement of epigenetic mechanisms. Epigenetic studies are often performed on leukocytes, but it is unclear to what extent methylation is similar in other tissues. Therefore, we investigated methylation of migraine-related genes that might be epigenetically regulated (CGRP-ergic pathway, estrogen receptors, endothelial NOS, as well as MTHFR) in different migraine-related tissues and compared this to methylation in rat as well as human leukocytes. Further, we studied whether 17ß-estradiol has a prominent role in methylation of these genes. Female rats (n = 35) were ovariectomized or sham-operated and treated with 17β-estradiol or placebo. DNA was isolated and methylation was assessed through bisulphite treatment and mass spectrometry. Human methylation data were obtained using the Illumina 450k genome-wide methylation array in 395 female subjects from a population-based cohort study. We showed that methylation of the Crcp, Calcrl, Esr1 and Nos3 genes is tissue-specific and that methylation in leukocytes was not correlated to that in other tissues. Interestingly, the interindividual variation in methylation differed considerably between genes and tissues. Furthermore we showed that methylation in human leukocytes was similar to that in rat leukocytes in our genes of interest, suggesting that rat may be a good model to study human DNA methylation in tissues that are difficult to obtain. In none of the genes a significant effect of estradiol treatment was observed.
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DNA promoter methylation of tumor suppressor genes and global DNA hypomethylation are common features of head and neck cancers. Our goal was to identify early DNA methylation changes in oral premalignant lesions (OPL) that may serve as predictive markers of developing oral squamous cell carcinoma (OSCC). Using high-throughput DNA methylation profiles of 24 OPLs, we found that the top 86 genes differentially methylated between patients who did or did not develop OSCC were simultaneously hypermethylated, suggesting that a CpG island methylation phenotype may occur early during OSCC development. The vast majority of the 86 genes were nonmethylated in normal tissues and hypermethylated in OSCC versus normal mucosa. We used pyrosequencing in a validation cohort of 44 patients to evaluate the degree of methylation of AGTR1, FOXI2, and PENK promoters CpG sites that were included in the top 86 genes and of LINE1 repetitive element methylation, a surrogate of global DNA methylation. A methylation index was developed by averaging the percent methylation of AGTR1, FOXI2, and PENK promoters; patients with a high methylation index had a worse oral cancer-free survival (P = 0.0030). On the other hand, patients with low levels of LINE1 methylation had a significantly worse oral cancer-free survival (P = 0.0153). In conclusion, AGTR1, FOXI2, and PENK promoter methylation and LINE1 hypomethylation may be associated with an increased risk of OSCC development in patients with OPLs.
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DNA methylation is a well-characterized epigenetic modification that plays an important role in the regulation of gene expression. There is growing evidence on the involvement of epigenetic mechanisms in disease onset, including cancer. Environmental factors seem to induce changes in DNA methylation affecting human health. However, little is known about basal methylation levels in healthy people and about the correlation between environmental factors and different methylation profiles. We investigated the effect of seasonality on basal methylation by testing methylation levels in the long interspersed nucleotide element-1 (LINE-1) and in two cancer-related genes (RASSF1A and MGMT) of 88 healthy male heavy smokers involved in an Italian randomized study; at enrolment the subjects donated a blood sample collected in different months. Methylation analyses were performed by pyrosequencing. Mean methylation percentage was higher in spring and summer for the LINE1, RASSF1A and MGMT genes (68.26%, 2.35%, and 9.52% respectively) compared with autumn and winter (67.43%, 2.17%, and 8.60% respectively). In particular, LINE-1 was significantly hypomethylated (p = 0.04 or 0.05 depending on the CpG island involved) in autumn and winter compared with spring and summer. Seasonality seems to be a modifier of methylation levels and this observation should be taken into account in future analyses.
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