Background Reproductive factors, particularly parity, have differential effects on breast cancer risk according to estrogen receptor (ER) status, especially among African American (AA) women. One mechanism could be through DNA methylation, leading to altered expression levels of genes important in cell fate decisions. Methods Using the Illumina 450K BeadChip, we compared DNA methylation levels in paraffin-archived tumor samples from 383 AA and 350 European American (EA) women in the Women’s Circle of Health Study (WCHS). We combined 450K profiles with RNA-seq data and prioritized genes based on differential methylation by race, correlation between methylation and gene expression, and biological function. We measured tumor protein expression and assessed its relationship to DNA methylation. We evaluated associations between reproductive characteristics and DNA methylation using linear regression. Results 410 loci were differentially methylated by race, with the majority unique to ER− tumors. FOXA1 was hypermethylated in tumors from AA versus EA women with ER− cancer, and increased DNA methylation correlated with reduced RNA and protein expression. Importantly, parity was positively associated with FOXA1 methylation among AA women with ER− tumors (P = 0.022), as was number of births (P = 0.026), particularly among those who did not breastfeed (P = 0.008). These same relationships were not observed among EA women, although statistical power was more limited. Conclusions Methylation and expression of FOXA1 is likely impacted by parity and breastfeeding. Because FOXA1 regulates a luminal gene expression signature in progenitor cells and represses the basal phenotype, this could be a mechanism that links these reproductive exposures with ER− breast cancer.

BackgroundDNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples.ResultsFor 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER– tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types.Materials and MethodsRestored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER−) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort.ConclusionsFFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.

African American (AA) women are more likely than European American (EA) women to be diagnosed with aggressive, estrogen receptor (ER) negative breast cancer. Differences in microRNA (miRNA) expression patterns have not been well studied as potential mechanisms underlying this racial disparity. In this study, we performed a whole-genome miRNA expression profiling in 58 (29 AA and 29 EA) fresh-frozen breast tumors, with clinical characteristics (e.g., ER status, histological grade, stage) comparable between AA and EA samples, and in 10 (5 AA and 5 EA) normal breast tissues obtained from women undergoing reduction mammoplasty, with pathology determined free from any abnormalities. Unsupervised hierarchical clustering showed that miRNA expression patterns clearly distinguish breast cancer from normal breast tissue. In tumors, a number of miRNAs are significantly differentially expressed between tumor subtypes and are associated with other clinicopathological factors, such as tumor grade and lymph node status. We identified 64 differentially expressed (mean fold change>2 and FDR<0.05) miRNAs between ER negative and ER positive tumors. Interestingly, we observed that there were 13 miRNAs differentially expressed between ER negative and ER positive breast tumors regardless of race, 28 miRNAs differentially expressed only in EA tumors and 23 miRNAs were differentially expressed in AA samples only. Specifically, the top most differentially expressed miRNAs from EA women include: several members of miR-17-92 cluster (miR-17, miR-18a, miR-19a, miR-20a), miR-508, miR509, and miR514a that are up-regulated, and miR-1, miR-133a, miR133b and miR206, which are down-regulated, in ER negative compared to ER positive tumors. In AA women, however, up-regulated miRNAs include miR-105, miR-106b, miR-135b, miR-520b; down-regulated ones include miR-216a, miR217, miR342, miR375, and miR378a. Further, several of these miRNAs, such as miR-17, miR-18a, miR-133a, miR-206, have been shown to regulate various target genes involved in apoptosis, cell cycle, invasion, or angiogenesis. In summary, in this genome-wide miRNA expression profiling analysis by next generation sequencing, our results suggest that miRNA expression patterns may differ by tumor subtypes between AA and EA breast samples. These initial results will provide the basis for the functional analysis of the identified miRNAs, and findings could contribute to a better understanding of the biology of breast cancer disparities and more targeted preventative and therapeutic strategies. Citation Format: Zhihong Gong, Dan Wang, Allyson Espinal, Qiang Hu, Lara Sucheston-Campbell, Li Tang, Jo Freudenheim, Peter Shields, Carl Morrison, Steven Belinsky, Song Liu, Kitaw Demissie, Michael Higgins, Christine Ambrosone. Genome-wide microRNA profiling analysis of breast cancer among African American and European American women. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A31.

Background: Although European American (EA) women have higher incidence of breast cancer than African American (AA) women, AA women are more often diagnosed with aggressive estrogen receptor negative (ER-) tumors, the risk of which increases with parity, lack of breastfeeding, and an early age at menarche. DNA methylation could be affected by reproductive factors and may be a potential molecular mechanism driving differences in tumor etiology. In a small study using fresh frozen breast tissue (n=58 AA, 80 EA), we previously found more differentially methylated loci (DMLs) in ER- tumors from AA and EA women than in ER+ tumors. Methods: To follow up on these preliminary findings, we used the Illumina 450K platform to determine genome-wide DNA methylation profiles in formalin fixed paraffin embedded (FFPE) breast tumors from 383 AA and 350 EA women who participated in the Women's Circle of Health Study, a case control study conducted in NY and NJ. We identified DMLs by race and ER status, as well as differentially methylated regions (DMRs). DMLs in eight genes were validated using the Sequenom EpiTYPER platform. Recursively partitioned mixture modeling (RPMM) package in R was used to examine relationships between methylation clusters and reproductive factors, information available from in-person interviews. In addition, using fresh frozen tumor tissue from 50 patients treated at Roswell Park Cancer Institute, we performed RNA sequencing on samples with methylation data available from a prior study, and used Spearmen's correlation to compare methylation and gene expression of DMLs and DMRs. Results: In assessing average methylation by location relative to CpG-islands (CGIs), we found that CGIs and shores in ER- tumors were significantly hypomethylated compared to CGI and shores in ER+ tumors from AA women, but not in tumors from EA women. We also identified 410 DMLs (Δβ>0.10 & FDR<0.05) between AA and EA breast tumors (race-associated DMLs, raDMLs), the majority of which were unique to ER- tumors (n = 260) and hypomethylated in AAs. These loci were enriched for immune response genes and several cell adhesion and inflammatory pathways. RPMM showed that parity, but not breastfeeding or age at menarche, was significantly associated with methylation class of ER- breast tumors. Of the genes that had ER- specific raDMLs, FOXA1 and THSD4 DNA methylation was highly correlated with gene expression (rho=-0.80, rho=+0.87, respectively; pvalue< 2.2x10-16). Summary: ER- tumors from AA women, but not from EA women, exhibited global hypomethylation at CGI and shores compared to ER+ tumors and contained the majority of raDMLs, many of which were associated with genes involved in immune and inflammatory response. The FOXA1 gene, which encodes a pioneer factor previously implicated in suppressing the molecular phenotype of basal breast cancer cells, was found to be methylated and silenced in the majority of ER- tumors analyzed for expression, consistent with aggressiveness of these tumors. These results, and the novel finding of associations between parity and DNA methylation signature, bring us a step forward in understanding the heterogeneous population of ER- tumors and provide insight into mechanisms of racial disparities in breast cancer. Citation Format: Allyson C. Espinal, Dan Wang, Lara Sucheston-Campbell, Song Liu, Qiang Hu, Li Tang, Gary Zirpoli, Thaer Khoury, Song Yao, Kitaw Demissie, Elisa V. Bandera, Christine B. Ambrosone, Michael J. Higgins. Methylation differences in breast tumor DNA from African American and European women are predominant in estrogen receptor (ER) negative breast cancer and are associated with childbearing. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B47.

African American (AA) women are more likely than European American (EA) women to be diagnosed with aggressive, estrogen receptor (ER) negative breast cancer. Differences in microRNA (miRNA) expression patterns have not been well studied as potential mechanisms underlying this racial disparity. In this study, we performed a whole-genome miRNA expression profiling in 58 (29 AA and 29 EA) fresh-frozen breast tumors, with clinical characteristics (e.g., ER status, histological grade, stage) comparable between AA and EA samples, and in 10 (5 AA and 5 EA) normal breast tissues obtained from women undergoing reduction mammoplasty, with pathology determined free from any abnormalities. Unsupervised hierarchical clustering showed that miRNA expression patterns clearly distinguish breast cancer from normal breast tissue. In tumors, a number of miRNAs are significantly differentially expressed, i.e., DEmiRs defined as >2-fold change in expression and FDR<0.05, between tumor subtypes and by race. We identified 61 DEmiRs between ER negative and ER positive breast tumors; of these, 14 miRNAs were differentially expressed regardless of race; 29 miRNAs were specific in EA tumors only; and 18 were in AA samples only. Specifically, the top most differentially expressed miRNAs from EA women include: several members of miR-17-92 cluster (miR-17,-18a,-19a, -20a), miR-508, -509,and -514a that are up-regulated, and miR-1, -133a, -133b, and -206, which are down-regulated, in ER negative compared to ER positive tumors. In AA women, however, up-regulated miRNAs include miR-105, -106b, -135b, and -520b; down-regulated ones include miR-216a, -217, -1303, and -378a. We also identified 14 DEmiRs between AA and EA tumor samples, with 5 miRNAs specific in ER- tumors, such as miR-9, -106b, and 9 miRNAs in ER+ tumors, such as miR-1, -133a, -133b, and -206. Further, several of these miRNAs, such as miR-17, miR-18a, miR-133a, miR-206, miR-9, have been shown to regulate various target genes involved in apoptosis, cell cycle, invasion, or angiogenesis. We also found that several miRNAs, such as miR-1, -133a, -216a, are associated with improved or reduced recurrence-free or overall survival. In summary, in this genome-wide miRNA expression profiling analysis by next generation sequencing, our results suggest that miRNA expression patterns may differ by tumor subtypes between AA and EA breast samples. These initial results will provide the basis for the functional analysis of the identified miRNAs, and findings could contribute to a better understanding of the biology of breast cancer disparities and more targeted preventative and therapeutic strategies. Citation Format: Zhihong Gong, Dan Wang, Allyson Espinal, Qiang Hu, Xuan Peng, Lara Sucheston-Campbell, Li Tang, Jo Freudenheim, Peter Shields, Carl Morrison, Steven Belinsky, Song Liu, Kitaw Demissie, Michael Higgins, Christine Ambrosone. Identification of differentially expressed miRNAs of breast cancer among African American and European American 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 1960.