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Introduction: The presence of Disseminated (DTCs) and Circulating Tumor Cells (CTCs) in the bone marrow (BM) and the blood of breast cancer (BC) patients are poor prognostic factors. We have recently shown that CTCs derived from metastatic BC patients, overexpressed CXCR4 and JUNB and this expression was related to clinical outcome. In this study we applied the same methodology to BM of non-metastatic BC patients at primary diagnosis, before the onset of therapy. Methods: Bilateral BM (10 ml) aspirations were assessed for the detection of DTCs by a Ficoll density gradient centrifugation and subsequent centrifugation of the BM cells onto glass slides [1x106 mononuclear cells (MNCs) per slide]. Triple staining experiments with panytokeratin/CXCR4/JUNB antibodies were performed. An expression pattern of the examined proteins was created, using three different BC cell lines (SKBR3, MDA-MB231, and MCF7). Consequently, 10 BM aspirations from BC patients were analyzed. Mean intensity for each examined molecule was calculated automatically with the ARIOL system. Results: Quantification of CXCR4 among BC cell lines revealed that CXCR4 expression followed the subsequent hierarchy SKBR3>MCF7>MDA-MB231 with higher expression in SKBR3 and lower in MDA-MB 231 cells, respectively. Accordingly, the expression pattern of JUNB in cell lines was SKBR3>MCF7>MDA-MB231. Consequently, we analyzed 10 BM samples from primary BC patients. The (CXCR4+JUNB+CK+) phenotype was observed in 90% (9/10) of the samples. In addition, the (CXCR4-JUNB+CK+) phenotype was detected in 20% (2/10) of the patients. Interestingly, the (CXCR4+JUNB-CK+) phenotype was not observed in any of the patient's samples. Quantification of CXCR4 expression revealed that the mean intensity in DTCs (19.1±2) was statistically higher than in MCF7 (13±0.51; p=0.006) and MDA-MB231 (10.22±0.7; p= 0.0002) cell lines. It was also higher compared to normal donors' PBMCs (10.6±1.01; p=0.0004). In addition, the mean intensity of JUNB was statistically increased in DTCs (17.1±1.66) compared to MCF7 (8.9±0.56; p=0.00001) and MDA-MB231 (6.6±0.15; p= 0.00001) cells, respectively. Furthermore, it was increased compared to normal donors' PBMCs (5.2±0.15; p=0.0002). In two patients, we had available samples after treatment. In one patient, DTCs were eliminated after neoadjuvant therapy, for the other patient, DTCs were reduced from 10 to two DTCs. However, these cells were double positive for CXCR4 and JUNB. Conclusion: CXCR4 and JUNB were overexpressed in DTCs derived from primary BC patients, in agreement to previous findings in CTCs, implying an upregulation of the CXCR4 pathway in these cells. Quantification of this expression potentially defines a subgroup of patients with high expression of CXCR4 and JUNB that could benefit from targeted therapies. Citation Format: Galatea Kallergi, Oliver Hoffmann, Nefeli Zacharopoulou, Christos Stournaras, Vassilis Georgoulias, Sabine Kasmir-Bauer. Overexpression of CXCR4 and JUNB in disseminated tumor cells (DTCs) isolated from breast cancer patients before neoadjuvant treatment [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 5186.