Daniel Y. Weng

Racial differences in genome-wide methylation profiling and gene expression in breast tissues from healthy women

Breast cancer is more common in European Americans (EAs) than in African Americans (AAs) but mortality from breast cancer is higher among AAs. While there are racial differences in DNA methylation and gene expression in breast tumors, little is known whether such racial differences exist in breast tissues of healthy women. Genome-wide DNA methylation and gene expression profiling was performed in histologically normal breast tissues of healthy women. Linear regression models were used to identify differentially-methylated CpG sites (CpGs) between EAs (n = 61) and AAs (n = 22). Correlations for methylation and expression were assessed. Biological functions of the differentially-methylated genes were assigned using the Ingenuity Pathway Analysis. Among 485 differentially-methylated CpGs by race, 203 were hypermethylated in EAs, and 282 were hypermethylated in AAs. Promoter-related differentially-methylated CpGs were more frequently hypermethylated in EAs (52%) than AAs (27%) while gene body and intergenic CpGs were more frequently hypermethylated in AAs. The differentially-methylated CpGs were enriched for cancer-associated genes with roles in cell death and survival, cellular development, and cell-to-cell signaling. In a separate analysis for correlation in EAs and AAs, different patterns of correlation were found between EAs and AAs. The correlated genes showed different biological networks between EAs and AAs; networks were connected by Ubiquitin C. To our knowledge, this is the first comprehensive genome-wide study to identify differences in methylation and gene expression between EAs and AAs in breast tissues from healthy women. These findings may provide further insights regarding the contribution of epigenetic differences to racial disparities in breast cancer.

Patients with colorectal cancer associated with Lynch syndrome and MLH1 promoter hypermethylation have similar prognoses

Purpose:Mismatch repair–deficient (dMMR) colorectal cancer (CRC) is caused by Lynch syndrome (LS) in 3% and sporadic inactivation of MLH1 by hypermethylation (MLH1-hm) in 12% of cases. It is not clear whether outcomes between LS-associated and MLH1-hm CRC differ. The objective of this study was to explore differences in clinical factors and outcomes in these two groups.Methods:Patients with dMMR CRC identified by immunohistochemistry staining and treated at a single institution from 1998 to 2012 were included. MLH1-hm was established with BRAF mutational analysis or hypermethylation testing. Patients’ charts were accessed for information on pathology, germ-line MMR mutation testing, and clinical course.Results:A total of 143 patients had CRC associated with LS (37 patients, 26%) or MLH1-hm (106 patients, 74%). Patients with LS were younger, more often male, presented more often with stage III disease, and had more metachronous disease than patients with MLH1-hm tumors. There was no difference in cancer-specific survival (CSS) between the groups; overall survival was longer in patients with LS, but this difference was minimal after adjusting for age and stage at diagnosis.Conclusion:CSS did not differ in LS-associated CRC compared with MLH1-hm CRC, suggesting that they carry a similar prognosis.Genet Med 18 9, 863–868.

Discovery and replication of microRNAs for breast cancer risk using genome-wide profiling

Background Genome-wide miRNA expression may be useful for predicting breast cancer risk and/or for the early detection of breast cancer. Results A 41-miRNA model distinguished breast cancer risk in the discovery study (accuracy of 83.3%), which was replicated in the independent study (accuracy = 63.4%, P=0.09). Among the 41 miRNA, 20 miRNAs were detectable in serum, and predicted breast cancer occurrence within 18 months of blood draw (accuracy 53%, P=0.06). These risk-related miRNAs were enriched for HER-2 and estrogen-dependent breast cancer signaling. Materials and Methods MiRNAs were assessed in two cross-sectional studies of women without breast cancer and a nested case-control study of breast cancer. Using breast tissues, a multivariate analysis was used to model women with high and low breast cancer risk (based upon Gail risk model) in a discovery study of women without breast cancer (n=90), and applied to an independent replication study (n=71). The model was then assessed using serum samples from the nested case-control study (n=410). Conclusions Studying breast tissues of women without breast cancer revealed miRNAs correlated with breast cancer risk, which were then found to be altered in the serum of women who later developed breast cancer. These results serve as proof-of-principle that miRNAs in women without breast cancer may be useful for predicting breast cancer risk and/or as an adjunct for breast cancer early detection. The miRNAs identified herein may be involved in breast carcinogenic pathways because they were first identified in the breast tissues of healthy women.

DNA methylation and breast tumor clinicopathological features: The Western New York Exposures and Breast Cancer (WEB) study

ABSTRACT We evaluated the association between methylation of 9 genes, SCGB3A1, GSTP1, RARB, SYK, FHIT, CDKN2A, CCND2, BRCA1, and SFN in tumor samples from 720 breast cancer cases with clinicopathological features of the tumors and survival. Logistic regression was used to estimate odds ratios (OR) of methylation and Cox proportional hazards models to estimate hazard ratios (HR) between methylation and breast cancer related mortality. Estrogen receptor (ER) and progesterone receptor (PR) positivity were associated with increased SCGB3A1 methylation among pre- and post-menopausal cases. Among premenopausal women, compared with Stage 0 cases, cases of invasive cancer were more likely to have increased methylation of RARB (Stage I OR = 4.7, 95% CI: 1.1–19.0; Stage IIA/IIB OR = 9.7, 95% CI: 2.4–39.9; Stage III/IV OR = 5.6, 95% CI: 1.1–29.4) and lower methylation of FHIT (Stage I OR = 0.2, 95% CI: 0.1–0.9; Stage IIA/IIB OR = 0.2, 95% CI: 0.1–0.8; Stage III/IV OR = 0.6, 95% CI: 0.1–3.4). Among postmenopausal women, methylation of SYK was associated with increased tumor size (OR = 1.7, 95% CI: 1.0–2.7) and higher nuclear grade (OR = 2.0, 95% CI 1.2–3.6). Associations between methylation and breast cancer related mortality were observed among pre- but not post-menopausal women. Methylation of SCGB3A1 was associated with reduced risk of death from breast cancer (HR = 0.41, 95% CI: 0.17–0.99) as was BRCA1 (HR = 0.41, 95% CI: 0.16–0.97). CCND2 methylation was associated with increased risk of breast cancer mortality (HR = 3.4, 95% CI: 1.1–10.5). We observed differences in methylation associated with tumor characteristics; methylation of these genes was also associated with breast cancer survival among premenopausal cases. Understanding of the associations of DNA methylation with other clinicopathological features may have implications for prevention and treatment.

Persistent alterations of gene expression profiling of human peripheral blood mononuclear cells from smokers

The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco‐related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross‐sectional study. We profiled global transcriptional changes in cultured smokers’ peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well‐characterized smokers’ blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5‐fold‐change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5‐fold‐change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty‐one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non‐small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect.

Landscape of genome-wide age-related DNA methylation in breast tissue

Despite known age-related DNA methylation (aDNAm) changes in breast tumors, little is known about aDNAm in normal breast tissues. Breast tissues from a cross-sectional study of 121 cancer-free women, were assayed for genome-wide DNA methylation. mRNA expression was assayed by microarray technology. Analysis of covariance was used to identify aDNAm’s. Altered methylation was correlated with expression of the corresponding gene and with DNA methyltransferase protein DNMT3A, assayed by immunohistochemistry. Publically-available TCGA-BRCA data were used for replication. 1,214 aDNAm’s were identified; 97% with increased methylation, and all on autosomes. Sites with increased methylation were predominantly in CpG lslands and non-enhancers. aDNAm’s with decreased methylation were generally located in intergenic regions, non-CpG Islands, and enhancers. Of the aDNAm’s identified, 650 are known to be involved in cancer, including ESR1 and beta-estradiol responsive genes. Expression of DNMT3A was positively associated with age. Two aDNAm’s showed borderline significant associations with DNMT3A expression; KRR1 (OR 6.57, 95% CI: 2.51–17.23) and DHRS12 (OR 6.08, 95% CI: 2.33–15.86). A subset of aDNAm’s co-localized within vulnerable regions for somatic mutations in cancers including breast cancer. Expression of C19orf48 was inversely and significantly correlated with its methylation level. In the TCGA dataset, 84% and 64% of the previously identified aDNAm’s were correlated with age in both normal-adjacent and tumor breast tissues, with differential associations by histological subtype. Given the similarity of findings in the breast tissues of healthy women and breast tumors, aDNAm’s may be one pathway for increased breast cancer risk with age.

Abstract 246: Assessing microbial dysbiosis of electronic cigarettes and cigarette smokers using oral and lung microbiome

The link between smoking tobacco and changes in the oral microbiome in response to tobacco smoking are well established. It is not known if there are changes in response to electronic cigarettes (e-cig). These changes in the microbiome are associated with increased numbers of disease causing pathogens. Currently there are no published studies that have investigated the relationship of smoking tobacco on both the oral and lung microbiome. There is insufficient evidence showing whether changes in oral cavity and lung microbiome are also seen in e-cig users. We will study the oral cavity and lung of non-smokers, smokers and e-cig users to examine concordance between oral cavity and the lungs as well as comparing the three groups, examining the microbiomes and expression of inflammatory markers. We hypothesize that microbial dysbiosis and expression of inflammatory cytokines will differ for smokers and non-smokers; and that e-cig users will have microbial dysbiosis similar to cigarette smokers. A cross-sectional study is being conducted on three groups, 1) never-smokers, 2) cigarette smokers, and 3) e-cig users. For each study participant, saliva and bronchoalveolar lavage (BAL) are being collected to measure microbiome. RNA is extracted from saliva and BAL samples for total transcriptome analysis using RNA-seq. This analysis will detect human and bacterial reads thereby allowing observations of bacterial communities as well as human inflammatory cytokine response to bacterial presence. 85% to 98% of BAL sample reads aligned to the human genome compared to less than 50% from saliva samples. The alignment results allow us to deduce that the majority of reads from BAL samples are human and that the majority of the reads in saliva samples are bacterial. Preliminary results show detection of human RNA expression and of bacterial reads are present in both saliva and BAL samples. More samples are being processed and the comparison of BAL and saliva samples between the three groups will be discussed. Citation Format: Kevin Ying, Min-Ae Song, Daniel Y. Weng, Quentin Nickerson, David Frankhouser, Pearlly S. Yan, Ralf Bundschuh, Mark D. Wewers, Ewy Mathe, Jo L. Freudenheim, Peter G. Shields. Assessing microbial dysbiosis of electronic cigarettes and cigarette smokers using oral and lung microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 246. doi:10.1158/1538-7445.AM2017-246

Abstract 2777: One-carbon metabolism genetic variant and genome-wide DNA methylation in breast tissues from healthy women

Altered DNA methylation is an early event in carcinogenesis. Little is known about the mechanism of altered methylation in breast tissue; possible factors include diet such as alcohol and folate intake, and genetic variation for enzymes in one carbon metabolism. Examination of the association of these factors with methylation in breast tissues from healthy women provides insight into these changes. Blood and glandular breast tissues from 81 women with no history of cancer and who underwent reduction mammoplasty were assayed. The 96-plex Illumina BeadXpress® or TaqMan® SNP Genotyping assays assessed SNPs, genome-wide DNA methylation profiling was performed using the Illumina Infinium HumanMethylation450 BeadChip.The Affymetrix GeneChip Human Trascriptome Array 2.0 was used to compare gene expression level with methylation change in fresh frozen breast tissues. Biological networks of differentially-methylated (DM) genes were assigned using the Ingenuity Pathway Analysis (IPA). Fifty-seven CpG sites were DM in comparisons of genotype for eight SNPs in FTHFD, MTHFD1, MTHFR, MTR, MTRR, and TYMS (P Citation Format: Min-Ae Song, Theodore M. Brasky, Catalin Marian, Daniel Y. Weng, Cenny Taslim, Adana A. Llanos, Ramona G. Dumitrescu, Zhenhua Liu, Joel B. Mason, Bhaskar V. Kallakury, Jo L. Freudenheim, Peter G. Shields. One-carbon metabolism genetic variant and genome-wide DNA methylation in breast tissues from healthy women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2777.

Abstract 3639: DNA methylation in SYK, GSTP1, & FHIT genes: associations with parity and time since birth.

Introduction: Breast cancer risk is transiently increased following pregnancy; these tumors, referred to as pregnancy associated breast cancer (PABC) have poorer prognosis. As more women delay pregnancy until older age this increase in risk following pregnancy is projected to mean increased numbers of PABCs. Epigenetic events are important in the development and progression of cancer. Methylation status of specific genes may potentially be useful as screening targets in clinical practice. We examined DNA methylation for three genes of interest, Spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase expressed in hematopoietic and non-hematopoietic breast epithelial cells, exhibiting tumor suppressor qualities, Fragile histadine triad (FHIT), encoding a protein involved in cell differentiation and apoptosis, and Glutathione S-transferase P1 (GSTP1), a phase II detoxification enzyme in most cell types acting on carcinogens, environmental pollutants, and drugs. We compared DNA methylation in these genes in breast tissues from healthy premenopausal women by parity status and time since last birth (nulliparous, 10 years) to understand changes in methylation associated with recent pregnancy. Methods: DNA samples were obtained from fresh frozen tissues of 81 premenopausal women undergoing reduction mammoplasty with no prior history of cancer except non-melanoma skin cancer. Genomic DNA was modified using EZ DNA Methylation Gold Kit and sequenced using Pyro Q-CpG TM Software. Two-sample t-tests and 1-way Analysis of Variance were used to examine differences in mean methylation in the three genes by parity status and time since last birth. Generalized linear regression models were used to compare mean methylation levels adjusted for age, race, menopausal status, and family history of breast cancer. Results: Parous healthy premenopausal women had a higher adjusted mean methylation in all three genes than their nulliparous counterparts. Mean percent methylation of SYK (0.98 vs. 0.87; P=0.56) and FHIT (2.08 vs. 1.85, P=0.50) were 12% higher in parous versus nulliparous women. Methylation of GSTP1 was 7% higher among parous versus nulliparous women (0.70 vs. 0.65; P=0.82); however, differences were not statistically significant. For GSTP1, there was a suggestion of higher mean percent methylation among women who gave birth more recently ( 10 years: 0.64) or nulliparous women (0.69; P=0.64). There were no clear differences by recency of birth for FHIT or SYK. Conclusions: Mean percent methylation of SYK, GSTP1, and FHIT may be suggestive of changes in methylation among parous women, and for GSTP1, particularly higher DNA methylation among women who gave birth more recently. Given the small sample size, these findings are preliminary, and additional studies are needed to better understand methylation in PABC risk. Citation Format: Ruth N. Kagwima, Adana A. LLanos, Theodore M. Brasky, Daniel Y. Weng, Jo L. Freudenheim, Peter G. Shields. DNA methylation in SYK, GSTP1, & FHIT genes: associations with parity and time since birth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3639. doi:10.1158/1538-7445.AM2013-3639