We used gene expression profiling to identify biological differences in the microdissected tumor epithelium and tumor stroma that may exist between African-American and European-American breast cancer patients from the greater Baltimore area. African-Americans from this area, and for most of the U.S., have ancestral links to specific regions in West Africa and experience an increased frequency of aggressive breast cancer similar to West African women 
Our study focused on the discovery of differentially regulated biological processes comparing African-American and European-American breast cancer patients as opposed to the discovery of specific genes. This approach was used as it may reveal differences in oncogenic pathways and the tumor microenvironment, and therefore may provide insight into therapeutic opportunities 
. Using this design, we found that biological processes related to chemotaxis, angiogenesis, endoplasmic reticulum function, and cell cycle control were most significantly enriched for genes that are differentially expressed by race/ethnicity. Of those processes, only the cell cycle control association appeared to be significantly confounded by differences in other tumor markers such as the race/ethnic difference in the tumor ER status.
One of our most interesting observations was the presence of an interferon signature in tumors of the African-American patients. The rationale of examining our datasets for an interferon signature was based on our previous observation in prostate cancer showing that interferon γ-responsive genes are up-regulated in African-American tumors 
. Interferons are commonly induced by pathogens and is a key pro-inflammatory cytokine in inflammation and autoimmune disease. In tumor biology, interferon γ is the master regulator of the Th1 response and enhances tumor immunogenicity and abrogated tumor development in mouse models 
. However, an interferon γ signature can be merely an indicator of a chronic inflammation and has been observed as a component of breast cancer progression in HER2-transgenic mice 
. Currently, we are uncertain why breast tumors from African-American patients have an interferon signature, or why interferon-regulated genes were more differentially expressed in the ER-positive than ER-negative tumors, although this may reflect estrogenic regulation of host immunity 
. The signature in the tumor epithelium could partly be caused by infiltrating immune cells, which cannot be completely separated from the tumor epithelial cells by LCM. Several of the interferon-related genes in our tumor signature have previously been reported as key genes in the cellular defense against bacterial and viral pathogens 
and in the promotion of all steps of breast tumorigenesis including tumor development, growth, survival, and metastasis 
. We hypothesize that etiologic agents may induce the signature in breast and prostate tumors of African-Americans. Alternatively, chronic inflammation and/or specific genetic variations in immune-related genes could be more prevalent among these African-American cancer patients and cause the heightened interferon activity in their tumors.
There have been two recent reports that investigated gene expression variations between individuals with European ancestry and individuals with African ancestry (Nigeria) using lymphoblastoid cell lines 
. Analogous to the present study, the authors assessed the enrichment of biological processes and pathways by genes that are differentially expressed by race/ethnicity. Notably, processes related to antimicrobial humoral response, inflammation mediated by chemokines and cytokines, histamine H1 receptor-mediated signaling pathway, toll-receptor signaling pathway, and the VEGF signaling pathway were identified. The results from these two studies suggest that differences in the genetic background between healthy volunteers of European ancestry and those from Nigeria cause gene expression differences affecting host immune response, inflammation and chemotaxis, and angiogenesis. These findings are consistent with our findings in breast tumors, and while preliminary, raise the possibility that differences in common genetic variations among African-American and European-American breast cancer patients may lead to group-specific alterations in cancer-related pathways that control host response, inflammation, and tumor angiogenesis.
Few others have studied race/ethnic differences in the expression of tumor markers in breast cancer and observed that p16, p53, and cyclin E were more commonly expressed in tumors from African-American patients than European-American patients 
. p16 is encoded by CDKN2A
. This gene was also up-regulated in the African-American tumors of this study, as indicated by the microarray data. Nuclear accumulation of p53 protein in tumor cells is a surrogate for a functional impairment of the p53 pathway while the overexpression of cyclin E is most frequently caused by a post-transcriptional mechanism that leads to the accumulation of hyperactive low molecular weight cyclin E isoforms 
. Consistent with the previous reports, p53 and cyclin E protein accumulation was also more common in African-American tumors than European-American tumors in the present study.
The gene expression profiles of breast tumors indicated that pathways related to tumor angiogenesis and chemotaxis could be functionally different between African-American and European-American patients. For further corroboration of these findings, we demonstrated higher levels of microvessel density and TAMs in African-American tumors than European-American tumors. Increased microvessel density and the infiltration of tumors by macrophages have been shown to be poor prognosis markers 
. The two markers are interrelated as TAMs are a major source of chemokines and cytokines which induce tumor angiogenesis 
. From the present study, we do not know why TAM infiltration is increased in tumors from African-American women. Possibly, tumors from African-American patients release more chemotactic cytokines to attract the infiltration of TAM than those from European-American patients. Among the most important of the chemotactic signals that attract TAM are MCP-1, CSF-1, and VEGF 
. Of those, VEGF was found to be higher expressed in the African-American tumors. We also assessed the infiltration of tumors by FoxP3-positive T-regulatory cells. The number of these cells in breast tumors has been associated with an increased risk of relapse in breast cancer 
. Although the number of these immune suppressive cells was not significantly different between African-American and European-American breast tumors, elevated numbers of these cells were found in basal-like tumors. Future studies should investigate whether these T-regulatory cells are related to poor outcome among young African-American breast cancer patients who frequently present with the basal-like subtype 
If gene expression differences exist between African-American and European-American breast cancer patients, those differences could potentially be exploited for tailored therapy. In an exploratory approach, we used the Connectivity Map database to identify small molecules that may antagonize the gene expression signature in the tumor epithelium of breast tumors that differentiated African-American patients from European-American patients. An antagonist could potentially improve the therapeutic outcome of high risk breast cancer patients, e.g., African-American patients with ER-negative tumors. Significant negative connectivity scores, indicating an antagonistic effect of the small molecule drug on the query signature, were obtained for the PI3-kinase inhibitor, LY-294002, and Rho signaling inhibitor, Y-27632, in ER-positive breast tumors and for sirolimus, resveratrol, and chlorpromazine in ER-negative tumors. Although preliminary, these findings are novel and may have implications for cancer therapy. The toxicity profile of LY-294002 excludes it from clinical trials, but its antagonist effect suggests that ER-positive African-American and European-American breast cancer patients could respond differentially to therapeutic PI3-kinase inhibitors. Sirolimus is rapamycin, which is a mTOR inhibitor and an immunosuppressant. Sirolimus and other mTOR inhibitors are currently in clinical trials, and their anti-tumor efficacy in breast cancer in preclinical models has been demonstrated 
. Resveratrol is a chemopreventive agent that has a plethora of effects in breast cancer including anti-inflammatory activities, apoptosis induction, angiogenesis inhibition, and effects on PI3-kinase signaling, among others 
. Chlorpromazine is a psychotropic agent and not an anti-cancer drug. However, recent data have shown that chlorpromazine has anti-proliferative and pro-apoptotic effects in cancer cells 
, and future studies should evaluate the activity of chlorpromazine in breast cancer.
Our study has strengths and limitations. We conducted a microarray analysis of microdissected tumor epithelium and tumor stroma, thus providing comprehensive databases of gene expression for these two cellular compartments in African-American and European-American breast tumors. The use of LCM also largely ruled out that varying amounts of tumor and stroma between African-American and European-American tumors is a confounder of the datasets. Despite the advantages of more rigorous histological sampling enabled by LCM, the approach has limitations. LCM is labor intensive and cannot be applied for the analyses of large sample sets, and further stratification of the breast tumors in the many subtypes (basal-like, ERBB+, normal-like, luminal A & B & C) was not possible in this sample set because of sample size limitations.
In conclusion, the gene expression profiles of breast tumors correspond to differences in tumor biology between African-American and European-American patients. Particularly, pathways related to chemotaxis, tumor angiogenesis, and immunobiology could be functionally different in the two patient groups. The therapeutic implications of those differences should be evaluated in future studies.