IntroductionOur efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. In order to provide the specimens that will facilitate such an understanding, The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is, to our knowledge, the only biorepository in the world prospectively established to collect normal, healthy breast tissue from volunteer donors. As a first initiative toward a molecular understanding of the biology and developmental genetics of the normal mammary gland, the effect of the menstrual cycle and hormonal contraceptives on DNA expression in the normal breast epithelium was examined.MethodsUsing normal breast tissue from 20 premenopausal donors to KTB, the changes in the mRNA of the normal breast epithelium as a function of phase of the menstrual cycle and hormonal contraception were assayed using next-generation whole transcriptome sequencing (RNA-Seq).ResultsIn total, 255 genes representing 1.4% of all genes were deemed to have statistically significant differential expression between the two phases of the menstrual cycle. The overwhelming majority (221; 87%) of the genes have higher expression during the luteal phase. These data provide important insights into the processes occurring during each phase of the menstrual cycle. There was only a single gene significantly differentially expressed when comparing the epithelium of women using hormonal contraception to those in the luteal phase.ConclusionsWe have taken advantage of a unique research resource, the KTB, to complete the first-ever next-generation transcriptome sequencing of the epithelial compartment of 20 normal human breast specimens. This work has produced a comprehensive catalog of the differences in the expression of protein-coding genes as a function of the phase of the menstrual cycle. These data constitute the beginning of a reference data set of the normal mammary gland, which can be consulted for comparison with data developed from malignant specimens, or to mine the effects of the hormonal flux that occurs during the menstrual cycle.

Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER−,PR−,HER2−). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90% of TNBCs revealing an over-expressed central network. In conclusion, Use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos.

Background: Gene expression data from pancreatic cancer, histologically normal tissue adjacent to the cancer and normal pancreas reveals that adjacent normal has already acquired a number of transcriptional alterations and is not, therefore, an appropriate baseline for comparison with cancers. (Gadaleta et al., 2011) The purpose of this study was to determine if this is also the case for breast cancer and, if so, to identify the differences in gene expression between adjacent normal and normal breast. Methods: RNA-Seq data from breast cancer and adjacent normal was downloaded from the TCGA (The Cancer Genome Atlas) data portal. The epithelia from 20 frozen tissue cores from healthy premenopausal donors to the Susan G. Komen for the the Cure® Tissue Bank at the IU Simon Cancer Center were microdissected and the RNA isolated. RNA-seqeuncing was carried out using the Life Technologies SOLiD Platform. RPKM gene expression values from TCGA and sequencing of the Komen normal tissues were merged, quantile normalized, and batch effect corrected. Normalization and differential gene expression was performed using Partek Genomics Suite. Results: Principal component analysis (PCA) reveals complete separation between adjacent normal and healthy normal breast tissue. Setting a maximum FDR (false discover rate) of 5%, 2239 genes are differentially expressed between adjacent normal and healthy normal. Ingenuity pathway analysis reveals that the Fos, Jun and TGFbeta pathways are active in the adjacent normal. Conclusions: Tissue adjacent to a primary breast cancer is not normal when using healthy breast tissue as a comparator. As RNA-Seq data is digital, it is possible to quantify the changes in gene expression starting from healthy normal to normal adjacent to tumor to tumor. Increasing and decreasing gene expression values provide clues to the fundamental molecular changes occurring in histologically normal appearing adjacent tissue. The differences in gene expression we have identified are some of the earliest changes in breast carcinogenesis and provide insight into the etiology of this disease and, potentially, its prevention. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-03-02.