Abstract P5-08-16: Molecular landscape of the breasts of women at high risk for breast cancer
Authors: Marino Natascia , German Rana , Nakshatri Harikrishna , Vode Ashley , Liu Jun , Huang Jie , Rush Douglas B , Cao Sha , Storniolo Anna Maria V 
Published: March 2020

Abstract
Background: Despite advances in breast cancer diagnosis and treatment, there is a critical need to identify the determinants of breast cancer susceptibility in order to define new strategies to target cancer initiation and progression. The Susan G. Komen Tissue Bank at Indiana University (KTB), the only repository worldwide of non-malignant breast samples, can address key scientific questions by examining molecular changes, including epigenetic (DNA methylation) and transcriptomic alterations that occur in breasts of cancer-free women who are considered at high risk for developing breast cancer.
Methods: Disease-free breast tissue cores donated by 80 high-risk (Tyrer-Cuzick lifetime risk ≥20%) and 80 average-risk women were processed for whole methylome (Diagenode MeCap Seq Library and NextSeq 75_sequencing) and whole transcriptome (Illumina TruSeq Stranded mRNA HT Library followed by NextSeq 75_sequencing) profiling. The two groups were matched according to age, racial background, and menopausal status. The cohort included 131 pre-menopausal and 29 post-menopausal women (mean age 38.6, SD 12.1). Frozen breast tissue cores with either abundant or moderate epithelial cellularity were selected. For the gene expression analysis, the reads were mapped to the human genome reference, GRCh38.p12 using the alignment software STAR ver. 2.5.2b and the read counts per gene were obtained using featureCounts ver. 1.6.3. For the evaluation of the epigenetically regulated genes, we focused our investigation on two types of functionally relevant epigenetic panels: the first panel includes 666 cancer-related markers (or pan-cancer) described by Shegafinia et al 2018, and the second panel includes 16 epigenetic markers identified in normal breast tissues-derived cells as connected with breast epithelial cell differentiation*.
Results: In a preliminary analysis, we searched for transcriptomic differences in the breast tissues of high-risk women (N=43) as compared with that of matched average-risk subjects (N=48).The differential expression analysis identified 126 differentially expressed genes (DEGs, FDR&lt;0.05, fold change &gt;2), including 117 upregulated and 9 downregulated in the high-risk group. Interestingly, pathway analysis (Ingenuity Pathway Analysis, IPA v06_01) showed that most of these genes are involved with cancer. The sub-category “tumorigenesis of tissues” as defined by IPA included 71 genes. Through TCGA database analysis, using the UALCAN portal, we searched among our candidate genes for those targets that showed both overexpression and hypomethylation in breast tumors as compared with the normal breast, or vice versa, downregulation and hypermethylation in breast cancer. This pipeline allowed us to identify 17 genes potentially epigenetically regulated and involved in breast cancer susceptibility and the early phases of cancer development. Among those, two genes, FAM83A and PLA2G3, belong to a family of genes previously described as epigenetically dysregulated in multiple cancers (TCGA dataset, and Shegafinia et al 2018) and transform breast epithelial cell in vitro (FAM83A), thus reaffirming robustness of our approach in identifying genes associated with breast cancer risk
Conclusion: The discovery of epigenetically regulated genes associated with breast cancer risk will open the doors to a deeper understanding of the process of cancer initiation and progression. The present study identified 17 gene alterations, two of which are epigenetically regulated in cancer. Our findings highlight an opportunity to address molecular alterations potentially linked with breast cancer susceptibility and risk, using a unique resource of normal breast samples.
*Unpublished data by Dr. H. Nakshatri
Citation Format: Natascia Marino, Rana German, Harikrishna Nakshatri, Ashley Vode, Jun Liu, Jie Huang, Douglas B Rush, Sha Cao, Anna Maria V Storniolo. Molecular landscape of the breasts of women at high risk for breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-08-16.