Abstract Background: CTCs play a major role in tumor dissemination and progression, and represent one of the key components of the metastatic cascade. The aim of this study was to identify signaling pathways associated with presence of CTCs in primary breast cancer (PBC) patients using a comprehensive genomics approach. Methods: This translational study included 78 patients with PBC. CTCs were detected before surgery by quantitative RT-PCR assay for expression of epithelial (EP; CK19) or epithelial-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1). Total DNA and RNA were extracted, in parallel, from fresh frozen primary tumor and the microRNA and mRNA expression profiles were obtained using Human microRNA Microarray v21.0 and SurePrint G3 Human Gene Expression v3 (Agilent Technologies). Next generation sequencing (NGS) was performed by Illumina Multiplex Sequencing using MiSeq Sequencing Reagent Kit V3. Results:Mutations in BRCA1/2 genes in tumor tissue were more common in patients with epithelial CTCs (CTC_EP) compared to patients without epithelial CTCs in peripheral blood (23.5% vs. 0%, p = 0.02), while there were no mutations in specific genes associated with CTC with EMT phenotype (CTC_EMT).Further, we identified 90 genes and 7 miRs that were expressed at significantly different levels in tumors with presence of CTC_EP and 199 genes and 13 miRs specifically associated with CTC_EMT, compared to tumors with non-detectable CTCs. We also identified 39 overlapping genes and 7 miRs, that were expressed at significantly different levels in tumors with CTC_EP and/or CTC_EMT compared to tumors with non-detectable CTCs. Overlapping genes and miRs with highest different levels in expression were ATAD3A, TMEM201, DCPS, DOCK9-AS2, TRAF2 and miR-5195-3p, miR-188-5p, miR-6780a-5p, miR-6757-5p. Signalling pathways associated with these genomic alterations belong to several critically functional groups, such as immune response, signal transduction, cell proliferation, cell cycle progression, or apoptosis were significantly differentially based on CTCs status. Conclusions: We identified for the first time various genomic alterations in primary tumor tissue of PBC associated with different CTCs subpopulations in peripheral blood. We hypothesize that these genomic alterations could play a role in tumor dissemination and progression and might lead to identification of new therapeutic targets. Citation Format: Mego M, Tokar T, Minarik G, Hajduk M, Karaba M, Benca J, Sedlackova T, Repiska G, Krasnicanova L, Macuch J, Sieberova G, Pindak D, Cristofanilli M, Reuben JM, Jurisica I, Mardiak J. Comprehensive analysis of genomic alterations in tumor tissue associated with presence of various subpopulations of circulating tumor cells (CTCs) in primary breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-05.