Abstract Incidence of estrogen receptor (ER)-negative breast cancer, a subtype with worse prognosis, is higher among African American (AA) versus European American (EA) women. The reasons for this gap remain poorly defined, but likely include differences in the prevalence and biological effects of known risk factors for ER- disease (eg. parity without breastfeeding and early menarche). These exposures may influence risk of ER- cancer via multiple mechanisms, one of which is epigenetic regulation of gene expression. Altered expression of genes controlling the proliferation and/or differentiation of luminal progenitor cells in the breast is likely to affect the propensity of such cells to give rise to ER- versus ER+ cancer. We previously conducted genome-wide DNA methylation profiling of 224 ER- tumor samples from 141 AA and 83 EA women in the Women's Circle of Health Study, and identified 374 differentially methylated loci (DML) by race. Our initial analysis focused on DMLs mapping to annotated genes, and revealed that a key pro-luminal transcription factor gene, FOXA1, was hypermethylated and repressed in AA compared to EA tumors. Moreover, parity without breastfeeding, more common among AAs, was associated with increased FOXA1 methylation. In this study, motivated by the biological importance of the non-coding genome, we focused on ~100 intergenic DML, and used paired 450K and RNA-seq data to assess the relationship between CpG methylation and RNA expression of genes located up to 50 kb away. Methylation at ten DML was significantly correlated with paired gene expression (FDR q<0.05). For seven of these loci, lower methylation tracked with higher expression of the paired gene. One such CpG, hypomethylated in AA versus EA tumors, mapped to a putative enhancer element located 19 kb upstream of, and predicted to interact with, the promoter of LEMD1, a pro-tumorigenic cancer-testis antigen. Analysis of an independent set of 207 ER- breast cancers from TCGA confirmed that LEMD1 RNA expression was higher in AA compared to EA tumors (P=0.01, log2FC=0.95). Our findings suggest that epigenetic differences between AA and EA ER- tumors are linked to altered gene expression and may hold functional significance in breast cancer pathogenesis. Further studies are needed to extend these results to normal breast tissues from cancer-free women, and explore a potential role for FOXA1/LEMD1 epigenetic regulation in luminal progenitor cell homeostasis. Citation Format: Matthew F. Buas, Jianhong Chen, Qiang Hu, Song Liu, Zhihong Gong, Michael J. Higgins, Christine B. Ambrosone. Intergenic DNA methylation differences in ER- breast tumors from African American versus European American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5328.