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A. Ramirez-Tirado

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    MINI 02 - Immunotherapy (ID 92)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI02.03 - Overexpression of CD47, Decrease of Apoptosis and Phagocytosis of Neutrophils in Advanced Non-Small Cell Lung Cancer Patients (ID 2265)

      10:45 - 12:15  |  Author(s): A. Ramirez-Tirado

      • Abstract
      • Presentation
      • Slides

      Lung cancer is the leading cause of cancer death worldwide and most of the patients are diagnosed with advanced disease. Inflammatory components play a key role in tumor progression and survival. Neutrophils are increased in blood of patients with lung cancer and they are associated with poor clinical outcomes. CD47 is a protein which control cell communication, apoptosis, adhesion and proliferation and it has been found increased in cancer and related with phagocytosis evasion mechanism.The aim of this study was to evaluate CD47 expression levels on peripheral neutrophils, also assess the phenotype, apoptosis, activation state, reactive oxygen species production of neutrophils between patients with Non-Small Cell Lung Cancer (NSCLC) and healthy subjects.

      Fifty NSCLC patients (stage IIIB and IV) naive to treatment and 25 healthy subjects were analized for: CD47 peripheral blood expression, neutrophils phenotype and activation state, evaluation of apoptosis and phagocytosis by flow cytometry. Reactive oxygen species (ROS) production by circulating neutrophils upon stimulation with PMA was assessed by flow cytometry. For the phagocytosis assay, PMNC were labeled with CMFDA and were cultured in RPMI for 24 hrs. To obtain apoptotic target cells, 24h PMNC were labeled with Annexin-V. For the evaluation of phagocytosis, the neutrophils from NSCLC patients were co-cultured with THP-1 cells. The percentage of phagocytosis was assessed by flow cytometry.

      Our results showed a lower percentage of total CD47 in peripheral blood cells in NSCLC patients compared to controls [P=0.042]. Mean Fluorescence Intensity (MFI) of CD47 was higher in patients [P<0.001]. The percentage of CD66b+ cells characterized as neutrophils was higher in patients as well as their MFI of CD47 [P< 0.001]. MFI of CD66b was higher in patients [P< 0.0178]. This would be related with a more activated state. We found that a higher disease stage (IIIB vs. IV) associated with a higher MFI of CD47 [P=0.020]. Plasma pro-inflammatory cytokines, was increased in patients compare to controls IL-6 (P<0.002), IL-8 (P<0.001), IL-12p70 (P<0.008), TNF (0.010) and IFN-g (P<0.001). MFI of CD47 >1635.5 was associated with a higher median Overall survival (P= 0.007). We found a decrease of AnnexinV+/7AAD+ in neutrophils of patients [P=0.0317]. Caspases 3 and 7 were found decreased in neutrophils of patients [P= 0.049]. Oxygen species (ROS) production of neutrophils upon PMA stimulation was increased in patients [P=0.029], suggesting it might play a role in immune effector function. Phagocytosis of apoptotic neutrophils by differentiated THP-1 cells was decrease in cancer patients cells (P=0.0445). Mean fluorescence Intensity of CD47 was increased after 24 hrs in patients [P=0.0408]. This result suggests that neutrophils from patients avoid being engulfed and this may be associated with overexpression of CD47.

      Taken together, these findings suggest that these are altered mechanisms by which neutrophils evade anti-tumor immune response and their increased expression of CD47 is a potential therapeutic target for NSCLC.

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