Abstract P1-05-11: Biological characterization of tumor-associated leukocytes in positive and negative lymph node breast cancer patients
Authors: ElGhonaimy EA , El-Shinawi M , Abd-El-Tawab R , El Mamlouk T , Sloane BF , Mohamed MM 
Published: August 2014

Abstract
Background: Leukocytes plays crucial role in breast cancer progression and lymph node metastasis. Veins draining the breast through the axillary tributaries contain tumor-associated leukocytes migrating away from tumor microenvironment. Collecting tumor-associated leukocytes and characterizing their immunophenotype and biological properties in positive and negative lymph node patients may elucidate their role in lymph node metastasis.
Aims: Using an innovative surgical approach previously described, wecollectedtumor-associated leukocytes from venous drainage of the breast during breast cancer surgery of positive and negative lymph node (LN)metastasis patients. Isolated leukocytes were subjected to: 1) immunophenotyping; 2) cytokine profiling; and 3) assessment of the expression level of total and phospho NF-kβ.
Material and Methods: We enrolled 40 patients diagnosed with breast cancer from Ain Shams University Hospitals. Patients were sub-grouped into negative (zero) and positive (4) LN metastasis patients. All enrolled patients had not received neo-adjuvant chemotherapy. During modified radical mastectomy or conservative breast surgery, 15–20 ml of blood were collected from axillary vein tributaries for the isolation of tumor-associated leukocytes by Ficoll-Hypaque density gradient centrifugation. The immunophenotype of the isolated mononuclear cells was assessed using flow cytometry to identify the percentage of T lymphocytes (CD3+) and their subpopulations (CD4+ and CD8+), NK cells (CD56+) and B-lymphocytes (CD19+). Tumor-associated leukocytes isolated from positive and negative LN patients were seeded overnight in appropriate culture media at 37°C in a humidified CO2 incubator. Conditioned media containing secreted cytokines/chemokines were profiled using RayBio human cytokine antibody arrayIIIand then analyzed and quantified using ImageJ software. In addition, using immunoblottingwe assessed the level of expression of total and phospho NF-kβ in leukocyte lysates from positive and negative LN patients.
Result: Flow cytometric analysis revealed a significant increase in the percentage of T lymphocytes and T helper(CD3+/CD4+) cells collected from axillary tributaries of positive LN patients as compared to negative LN patients. Leukocytesisolated from positive LN patients were characterized by more than five-foldincreases in IL-12, Thrombopoietin, IL-13, INF-ϒ, TARC, IGF-1, IL-3, TNF-α, IL-4, GCSF, IL-5 and IL-1α. Moreover, we detected an increase in cellular expression of total and phospho NF-kβ in leukocytes isolated from positive LN as compared to negative LN breast cancer patients.
Conclusion: Our results demonstrate for the first time that tumor-associated leukocytes of positive LN patients possess different cytokine patterns than those ofnegative LN patients. Moreover, we identified major cytokines that may contribute to LN metastasis and could be therapeutically targeted.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-11.