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D. Madeddu
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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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P2.01-062 - Impact of the Tissue Distribution of Subpopulations of TILs and PD-L1 Expression on the Clinical Outcome of NSCLC (ID 5715)
14:30 - 14:30 | Author(s): D. Madeddu
- Abstract
Background:
The number and function of tumor infiltrating lymphocytes (TILs) represent an important prognostic factor in cancer. Among the multiple immune escape mechanisms triggered by cancer, the PD-1/PD-L1 checkpoint seems to play a central role. Accordingly, PD-1/PD-L1 inhibitors have shown significant clinical results in multiresistant NSCLC. However, the role of this immune checkpoint on tumor biology and clinical outcome remains to be determined. To this end, the number and distribution of subpopulations of TILs together with the quantification of PD-L1 expression were immunohistochemically assessed in NSCLC and their impact on patients survival evaluated.
Methods:
Histologic sections from 106 NSCLC (46 ADC,60 SCC) were morphometrically analysed after immunohistochemical assessment of the incidence of CD3+, CD8+ and PD-1+ TILs and their proximal or distal location with respect to neoplastic cells. A comparative evaluation between immuneperoxidase and immunofluorescence (IF) on control tissues and on serial sections from the same cases was undertaken using three different anti-PD-L1 antibodies (clones:28-8,SP142 and M4420). Following suitability criteria, PD-L1 was measured by confocal quantitative IF. Neoplastic and stromal expression of PD-L1 was ascertained by the simultaneous IF detection of Cytokeratin (CK). Morphometric data and clinical records were subjected to Kaplan Meier estimation.
Results:
The gradient of lymphocyte subsets according to their hierarchical phenotype was maintained in both NSCLC, however, the number of CD3[+] TILs was 1.8-fold higher in ADC vs SCC in the presence of similar density of CD8[+] and PD-1[+ ]cells. EGFR and K-RAS mutations conditioned the ADC immune microenvironment by altering CD8[+] and PD-1[+] distribution. High intra- and inter-patients variability in PD-L1 levels was expectedly observed although the average value in SCC samples was higher compared to ADC . K-RAS and to a less extent EGFR mutations were associated with a lower PD-L1 expression. Significant PD-L1 labelling of stromal cells was present in 10% of cases. Interestingly, a lower expression of CK in cells with high PD-L1 signal and the occasional presence of neoplastic plugs overexpressing PD-L1 and lacking CK were documented. Although the number of TILs and PD-L1 levels tended to positively correlate with OS in the entire population of NSCLC, in the individulal cohort of ADC and SCC patients only low number of intratumor PD-1[+] lymphocytes was statistically associated with a significant increased (>10months) OS.
Conclusion:
High levels of PD-L1 and reduction of its cellular target are associated with improved clinical outcome in NSCLC suggesting that adoption by TILs of local escape from PD-L1 pressure delays tumor progression.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-050 - Isolation and Charcterization of Lymphatic Endothelial Cells from Neoplastic and Normal Human Lung (ID 6054)
14:30 - 14:30 | Author(s): D. Madeddu
- Abstract
Background:
Cell culture models may be crucial to study lung microvascular endothelium and its role in cancer progression and treatment. In addition, a high heterogeneity among endothelial cells from various districts has been demonstrated, with greater difference on the lymphatic circulatory system. Organ-specific endothelial cells are essential to elucidate signalling pathways involved in the pathogenetic mechanisms of neoplastic lung diseases and to provide novel approaches to reach the goal of a true personalized therapy. The aim of the present study was to isolate and characterize lymphatic endothelial cells from neoplastic and healthy human lung.
Methods:
A simple and unexpensive method, requiring minimal equipment and accessories was utilized to harvest, isolate and expand lymphatic endothelial cells from human lung (Lu-LECs). Specifically, samples from lung cancer (T) and spared distal lung (D) of 46 patients undergoing lobectomy or pneumonectomy for NSCLC, were processed. To obtain a pure population of Lu-LEC, a two-step purification tool based on sorting with monoclonal antibody to CD31 and podoplanin coated paramagnetic beads was employed. Immunohistochemical analysis on harvested pulmonary tissues was performed to assess the presence or absence of neoplastic cells and to identify blood and lymphatic vasculature.
Results:
The purity of cultured endothelial cells was ascertained by morphologic criteria including TEM analysis, immunocytochemistry, flow cytometry and functional assays. T and D Lu-LECs were positive for CD31. Moreover, to define the lymphatic phenotype, we examined the specific markers podoplanin, LYVE-1 and Prox-1. No significant immunophenotypic differences between D and T Lu-LEC were detected by FACS. Although T Lu-LEC were larger than D Lu-LEC, morphologic features as cytoplasmic microvescicles, Weibel-Palade Bodies and aggregate of parallel intermediate filaments were equally observed. Cells were characterized in vitro for the ability to express several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation and in the development and progression of cancer. Cultured lymphatic endothelial cells variably expressed VEGFR-2, VEGFR-3, PDGFR-beta, c-met, and IGF-1R according to D or T origin. Moreover, FGFR-1 and EGFR-1 were present in a large fraction of T and D Lu-LEC. Matrigel assay documented that T Lu-LEC more efficiently organized in tubular structures at early time point when compared to D counterpart. Conversely, wound healing assay revealed that D Lu-LEC had a superior migratory ability.
Conclusion:
Primary lines of LECs from the human lung have been consistently obtained and may represent an important tool to study NSLCL microenvironment, lymphoangiogenesis and anti-cancer therapy.
