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T. Kucharczyk
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-055 - Lymphocytes' Subtypes Differentiation after Stimulation with Synthetic Antigen-Pulsed Dendritic Cells in Lung Adenocarcinoma Patients (ID 5055)
14:30 - 14:30 | Author(s): T. Kucharczyk
- Abstract
Background:
Immunotherapy in lung cancer is currently experiencing huge interest. The greatest efficacy demonstrated the antibodies against immune checkpoints receptors (PD-L1, CTLA-4 or LAG-3). However, an effective anti-tumor response also required tumor antigen presentation to lymphocytes in peripheral lymph nodes. To enhance this effect, vaccination with synthetic tumor antigen was conducted in many clinical trials using antigen presenting cells (APC). Unfortunately, this type of active immunotherapy has not achieved spectacular success (START or MAGRIT studies). The explanation could lie in distorted presentation of tumor antigens in peripheral lymph nodes or in activation of lymphocytes with strong immunosuppressive properties. In our study, we checked which pathways of T helper differentiation are favored by autologous dendritic cells (DCs) after synthetic tumor antigens stimulation.
Methods:
Peripheral blood was acquired from twenty unresectable and treatment-naïve lung adenocarcinoma patients. Using CD14 magnetic beads, two cell populations were isolated. CD14-negative cells were used for CD3-positive cells isolation, which were frozen until further stimulation. CD14-positive cells were cultured in CellDCGrow medium supplemented with IL-4 and GM-CSF. After TNF-a stimulation, mature DCs were incubated with tumour-specific antigens: MUC1, MAGE-A3, EGFR and CMV (positive stimulator) or only with medium (negative control). Flow cytometry and specific monoclonal antibodies were used to analyse immunophenotype of DCs: CD1a, CD11c, CD83, CD80, CD86, B7-H1 (PD-L1), B7-DC (PD-L2). Mature autologous DCs were cultured with fraction of CD3-positive cells and after 48h of mixed cultures, the expression of intracellular factors specific for different T helper cells subpopulation (T-bet – Th1, STAT-6 – Th2, ROR-gT – Th17, FoxP3 – Treg) and expression of extracellular interleukin receptors (IL-12R, IL-4R, IL-23R, TGF-bR, IL-2R) were analysed.
Results:
Autologous DCs were well generated and showed phenotype specific for fully-mature DCs. In culture with DCs after MUC1 stimulation, we observed in lymphocytes the highest expression of ROR-gT and IL-23R (p<0.05) compared with lymphocytes from cultures with other synthetic tumour antigens. Expression of IL-4R on lymphocytes was also significantly (0<0.05) higher in the culture stimulated with MUC1. The highest expression of intracellular FoxP3 factor, TGF-bR (p<0.05) and IL-2R (p=0.009) on Th cells were observed in mixed culture with DCs after MAGE-A3 stimulation.
Conclusion:
Dendritic cells stimulated with synthetic tumour antigens could activate different T helper cell populations. It could depend on the nature of antigens and could influence the effectiveness of stimulation different lymphocyte subtypes in peripheral lymph nodes. Therefore, our study clarifies some failure reasons of large clinical trials using active antigen-specific immunotherapy.
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P2.03b - Poster Session with Presenters Present (ID 465)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.03b-011 - Screening for ALK Abnormalities in Central Nervous System Metastases of Non-Small-Cell Lung Cancer (ID 5146)
14:30 - 14:30 | Author(s): T. Kucharczyk
- Abstract
Background:
Anaplastic lymphoma kinase (ALK) gene rearrangement was reported in 3-7% of primary non-small-cell lung cancer (NSCLC) and its presence is commonly associated with adenocarcinoma (AD) type and non-smoking history. ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, alectinib and ceritinib showed efficiency in patients with primary NSCLC harboring ALK gene rearrangement. Moreover, response to ALK TKIs was observed in central nervous system (CNS) metastatic lesions of NSCLC. Till date there is limited data ALK rearrangement incidence in CNS metastases of NSCLC, which could be considered as a regiment for targeted treatment. For this reason we undertook the present retrospective study to determine the frequency of ALK abnormalities in CNS metastases of NSCLC.
Methods:
The studied group included 145 patients (45 females, 100 males, median age 60 years ±8) with CNS metastases of NSCLC. The studied group was heterogeneous in terms of histology (80 adenocarcinoma, 29 squamous-cell carcinoma, 22 large-cell carcinoma, 14 not otherwise specific) and smoking status. ALK abnormalities were screened in sections obtained from formalin fixed paraffin embedded (FFPE) tissue samples. NSCLC using immunohistochemical (IHC) automated staining (BenchMark GX, Ventana, USA) and fluorescence in situ hybridization (FISH) technique (Abbot Molecular, USA).
Results:
ALK abnormalities were detected in 4.8% (7/145) of CNS metastases of NSCLC. ALK abnormalities were observed in AD and squamous-cell carcinoma (SqCC) patients (7.5%; 6/80 vs. 3.4%; 1/29, respectively). Analysis of clinical and demographic factors indicated that expression of abnormal ALK was significantly more frequently observed (p=0.0002; χ[2]=16.783) in former-smokers. Comparison of IHC and FISH results showed some discrepancies, which were caused by unspecific staining of macrophages and glial/nerve cells, which constitute the background of CNS tissues.
Conclusion:
In this retrospective study we evaluated the expression of ALK abnormal protein and ALK gene rearrangement in extremely unique material which are CNS metastatic lesions of NSCLC. The frequency of ALK abnormalities in this material could be higher or comparable to frequency of ALK gene rearrangement in primary NSCLC tumors. However, the comparison of IHC and FISH results showed discrepancies that arose from unspecific background, which was made by cells with nonmalignant origin. For this reason assessment of ALK gene rearrangement in CNS tissues require additional standardizations.
