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247 Distinct immune cell composition for lung squamous cell carcinoma (SCCA) and adenocarcinoma (ADCA)
S42 viability assays as well as flow cytometry analysis of cell cycle distribution were performed in the presence and absence of different cell death inhibitors and iHDACs to define cell death pathways involved in iHDACs induced cell death. Results: We have found that the actual trigger for iHDACs-induced apoptosis are serine proteases and AIF, but not p53, p21 and caspases, as previously suggested. Our studies show that endoplasmic reticulum stress and proteosome inhibition are induced by iHDACs and participate in iHDACs-induced cell death. In addition we have found that XBP1, RPS6KA5, NANOS, DUSP10 and other genes are regulated by iHDACS and participate in iHDACs-induced cell death as demonstrated by siRNAs experiments. Conclusion: Our studies show putative new targets for developing antineoplasic agents suitable for chemorresistant tumors based in the iHDACs-induced cell death pathway. No conflict of interest. 246 POSTER Blood vessel endothelial activation, egfl7 expression, and inflammatory infiltrate in breast cancer D. Pannier1 , G. Lauridant1 , M.C. Baranzelli1 , E. Bogart1 , J. Bonneterre1 , F. Soncin2 . 1 Oscar Lambret Center, Nord, Lille Cedex, France; 2 Biology Institute of Lille, Nord, Lille cedex, France Background: Egfl7 is produced by blood vessel endothelial cells and we previously showed in preclinical models that it promotes tumor escape from immunity by repressing endothelial cell activation. Here, we evaluated the activation of peritumoral vessels in breast cancers in correlation with egfl7 expression in endothelial cells, and immune infiltration. Material and Methods: Breast cancers (203 invasive [158 ductular, 33 lobular], and 37 in situ) were obtained from 236 patients treated in 2005. Ratio of activated (membrane ICAM1+ immunostaining) and egfl7+ /CD34+ vessels (by in situ hybridation and immunostaining) were estimated in peritumoral aeras using a semi-quantitative scale. The inflammatory infiltrate was scored by CD3e staining of peritumoral areas excluding tumor tissues per se. Slides were read twice by two different observers. Statistics used Chi2 and Fisher’s exact tests for categorical data and Kruskall-Wallis test for continuous variables. Results: Invasive tumors were 77% ductular, 16% lobular and 21.2% (n = 43) were Scarff-Bloom Richardson III. HER2 was overexpressed in 12.6% (n = 26) of samples. 6.7% were triple negative (ER 0, PR 0, HER2 0, n = 14). There was a strong and significant inverse correlation between ICAM1+ /CD34+ and egfl7+ /CD34+ ratios (p = 0.015). When the ICAM1+ /CD34+ ratio increased, the egfl7+ /CD34+ ratio decreased significantly (p = 0.004), and vice-versa (test for trend across ordered groups). CD3e expression in peritumoral areas was low in 99% of slides and showed no correlation with ICAM1+ /CD34+ or egfl7+ /CD34+ ratios. Tumor escape from immunity is regulated in part by endothelium activation and Egfl7 promotes this mechanism. In accordance, we show here in a large cohort of breast cancers that egfl7 expression is correlated with a less activated endothelium in peritumoral vessels. On the other hand, no correlation between egfl7 expression and infiltration of T-lymphocytes in peritumoral areas was found, mostly because the infiltration in these areas was very low. Conclusions: We confirm in human tumors that Egfl7 plays a regulatory role in endothelial activation in relation to immune infiltration. Egfl7 is thus an interesting therapeutic target to improve immunotherapies. No conflict of interest. 247 POSTER Distinct immune cell composition for lung squamous cell carcinoma (SCCA) and adenocarcinoma (ADCA) J. Kargl1 , S. Busch1 , H. Metz1 , K.H. Kim1 , A.M. Houghton1 . 1 Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, USA Background: Lung cancer is the leading cause of cancer deaths worldwide, with only about 15% of lung cancer patients surviving for five years. Recent approaches in cancer therapy target immunosuppressive factors in the tumor microenvironment (TME) capable of derailing an effective immune response, using checkpoint inhibitors (e.g. anti-PD1/PDL-1). Unfortunately, only ~20% of patients benefit from these treatments and appropriate studies are of importance to determine the underlying molecular mechanisms. Lung cancer is a heterogeneous disease classified by multiple histologic subtypes, with ADCA and SCCA representing the majority of non-small cell lung cancer (NSCLC). Material and Methods: To evaluate the complexity of the immune cell composition of the TME of human NSCLC we examined tumor and nonadjacent lung tissue from the same patients. Tissue specimen (n = 51)
Abstracts with corresponding clinicopathologic information were analyzed by flow cytometry and immunohistochemistry. Next generation sequencing for T-cell receptors (TCRs) was performed to assess the TCR repertoire. Results: Surprisingly we found that immune cells comprise about 70% of the tumor mass in NSCLC. Many immune cell types are significantly increased in tumor tissue, when compared to normal lung, such as CD3+ and CD4+ T-cells, as well as B-cells. Notably, CD4+ cell subtypes including Th17 and regulatory T cells (Treg), and IL-17-expressing gdT cells are increased. In contrast, decreased presence of Th1 cells was observed in tumor tissue. Remarkably, immune cell compositions are unique for lung ADCA and SCCA. In SCCA we observed a Treg predominant signature with significant decreases in Th1 and Th17 cells when compared to ADCA. SCCA displays immune suppressive cell content indicated by low levels of CD8 effector memory RA cells (CD8+ CCR7− CD45RA+ ), high expression of exhaustion markers on CD8 cells (PD-1 and TIM3) as well as significantly more EpCAM+ cells expressing PDL-1. Further, a more clonal TCR b-chain repertoire and higher frequency of the top 10 clones were observed in SCCA. The presence of tumor-associated neutrophils (TANs) at the sites of tumorigenesis inversely correlates with CD8+ cell content. In contrast, ADCA represents a Th17 signature, revealing elevated levels of IL17-expressing CD4+ T-cells and gd T cells. While SCCA immune cell content clusters well, immune cell composition in ADCA is very heterogeneous. Preliminary data suggest that driver-mutations in ADCA such as EGFR predict distinct immune cell composition. Conclusions: These data provide evidence that the immune cell composition present within different NSCLC subtypes displays unique phenotypes and identifying the immunosuppressive factors in different subsets will be important for successful immune-based therapy. No conflict of interest. 248 POSTER Fibroblast Growth Factor Receptor (FGFR)3 sustains acquired resistance to trastuzumab in gastric cancer patients G. Piro1 , C. Carbone1 , I. Cataldo2 , F. Di Nicolantonio3 , S. Giacopuzzi4 , F. Boschi5 , M. Zanotto1 , V. Merz1 , M.M. Mina1 , A. Sbarbati5 , G. De Manzoni4 , A. Scarpa2 , G. Tortora1 , D. Melisi1 . 1 University of Verona, Medicine, Verona, Italy; 2 University of Verona, Pathology and Diagnostics, Verona, Italy; 3 University of Turin, Oncology, Turin, Italy; 4 University of Verona, Surgery, Verona, Italy; 5 University of Verona, Neurological Sciences, Verona, Italy Background: Trastuzumab has been recently demonstrated as valuable treatment in HER2+ gastric cancer (GC). However the majority of patients who achieve an initial response to trastuzumab-based regimens develop resistance within 1 year of treatment. This study was aimed at identifying the molecular mechanisms responsible for this resistance. Material and Methods: A GFP+/luciferase+, HER-2 positive, trastuzumab sensitive NCI-N87 GC orthotopic nude mouse model was used to select resistant models to this agent. Tumor growth was measured by using an IVIS Spectrum Imaging System. Differentially expressed transcripts between trastuzumab-resistant and sensitive GC cell lines were measured by Illumina whole-genome microarray, and tested for network and functional interrelatedness by IPA software. Expression of FGFR3, HER2, total AKT, phosphorylated (p)AKT, and ZEB1 was measured by immunohistochemical staining in pre- vs. post-treatment biopsies from GC patients progressing under trastuzumab-based treatment. Results: NCI-N87 orthotopic tumor bearing mice were kept under treatment until the tumors suddenly recurred while on continuous therapy with trastuzumab. Four NCI-N87 trastuzumab resistant (N87-TR) cell lines were established from different excised tumors by repeated GFP flow cytometry sorting and their effective resistance was verified in vitro and in vivo. Microarray analysis showed the dowregulation of HER2, the induction of epithelial-to-mesenchymal transition, and indicated FGFR3 as one of the top 10 upregulated genes in N87-TR cell lines. We found a significant and consistent association of N87-TR gene expression profiles with the activation of the mTOR signaling. Accordingly, N87-TR cell lines showed significantly lower expression of all HER family members, E-cadherin and pERK1/2, and higher levels of FGFR3, vimentin, ZEB1, and pAKT than did sensitive control. In vitro, N87-TR cell lines demonstrated a higher sensitivity to the FGFR3 inhibitor dovitinib than did trastuzumab sensitive control. Treatment with dovitinib reduced the expression of pAKT, ZEB1, and migration in N87-TR cell lines. Oral dovitinib significantly reduced tumor burden and prolonged mice survival duration in N87-TR mouse models, whereas it was ineffective on trastuzumab-sensitive GC tumors. A significantly higher expression of FGFR3 and a lower expression of HER2 was observed in biopsies from GC patients progressing under trastuzumab-based therapies if compared with respective pre-treatment biopsies.
Abstracts with corresponding clinicopathologic information were analyzed by flow cytometry and immunohistochemistry. Next generation sequencing for T-cell receptors (TCRs) was performed to assess the TCR repertoire. Results: Surprisingly we found that immune cells comprise about 70% of the tumor mass in NSCLC. Many immune cell types are significantly increased in tumor tissue, when compared to normal lung, such as CD3+ and CD4+ T-cells, as well as B-cells. Notably, CD4+ cell subtypes including Th17 and regulatory T cells (Treg), and IL-17-expressing gdT cells are increased. In contrast, decreased presence of Th1 cells was observed in tumor tissue. Remarkably, immune cell compositions are unique for lung ADCA and SCCA. In SCCA we observed a Treg predominant signature with significant decreases in Th1 and Th17 cells when compared to ADCA. SCCA displays immune suppressive cell content indicated by low levels of CD8 effector memory RA cells (CD8+ CCR7− CD45RA+ ), high expression of exhaustion markers on CD8 cells (PD-1 and TIM3) as well as significantly more EpCAM+ cells expressing PDL-1. Further, a more clonal TCR b-chain repertoire and higher frequency of the top 10 clones were observed in SCCA. The presence of tumor-associated neutrophils (TANs) at the sites of tumorigenesis inversely correlates with CD8+ cell content. In contrast, ADCA represents a Th17 signature, revealing elevated levels of IL17-expressing CD4+ T-cells and gd T cells. While SCCA immune cell content clusters well, immune cell composition in ADCA is very heterogeneous. Preliminary data suggest that driver-mutations in ADCA such as EGFR predict distinct immune cell composition. Conclusions: These data provide evidence that the immune cell composition present within different NSCLC subtypes displays unique phenotypes and identifying the immunosuppressive factors in different subsets will be important for successful immune-based therapy. No conflict of interest. 248 POSTER Fibroblast Growth Factor Receptor (FGFR)3 sustains acquired resistance to trastuzumab in gastric cancer patients G. Piro1 , C. Carbone1 , I. Cataldo2 , F. Di Nicolantonio3 , S. Giacopuzzi4 , F. Boschi5 , M. Zanotto1 , V. Merz1 , M.M. Mina1 , A. Sbarbati5 , G. De Manzoni4 , A. Scarpa2 , G. Tortora1 , D. Melisi1 . 1 University of Verona, Medicine, Verona, Italy; 2 University of Verona, Pathology and Diagnostics, Verona, Italy; 3 University of Turin, Oncology, Turin, Italy; 4 University of Verona, Surgery, Verona, Italy; 5 University of Verona, Neurological Sciences, Verona, Italy Background: Trastuzumab has been recently demonstrated as valuable treatment in HER2+ gastric cancer (GC). However the majority of patients who achieve an initial response to trastuzumab-based regimens develop resistance within 1 year of treatment. This study was aimed at identifying the molecular mechanisms responsible for this resistance. Material and Methods: A GFP+/luciferase+, HER-2 positive, trastuzumab sensitive NCI-N87 GC orthotopic nude mouse model was used to select resistant models to this agent. Tumor growth was measured by using an IVIS Spectrum Imaging System. Differentially expressed transcripts between trastuzumab-resistant and sensitive GC cell lines were measured by Illumina whole-genome microarray, and tested for network and functional interrelatedness by IPA software. Expression of FGFR3, HER2, total AKT, phosphorylated (p)AKT, and ZEB1 was measured by immunohistochemical staining in pre- vs. post-treatment biopsies from GC patients progressing under trastuzumab-based treatment. Results: NCI-N87 orthotopic tumor bearing mice were kept under treatment until the tumors suddenly recurred while on continuous therapy with trastuzumab. Four NCI-N87 trastuzumab resistant (N87-TR) cell lines were established from different excised tumors by repeated GFP flow cytometry sorting and their effective resistance was verified in vitro and in vivo. Microarray analysis showed the dowregulation of HER2, the induction of epithelial-to-mesenchymal transition, and indicated FGFR3 as one of the top 10 upregulated genes in N87-TR cell lines. We found a significant and consistent association of N87-TR gene expression profiles with the activation of the mTOR signaling. Accordingly, N87-TR cell lines showed significantly lower expression of all HER family members, E-cadherin and pERK1/2, and higher levels of FGFR3, vimentin, ZEB1, and pAKT than did sensitive control. In vitro, N87-TR cell lines demonstrated a higher sensitivity to the FGFR3 inhibitor dovitinib than did trastuzumab sensitive control. Treatment with dovitinib reduced the expression of pAKT, ZEB1, and migration in N87-TR cell lines. Oral dovitinib significantly reduced tumor burden and prolonged mice survival duration in N87-TR mouse models, whereas it was ineffective on trastuzumab-sensitive GC tumors. A significantly higher expression of FGFR3 and a lower expression of HER2 was observed in biopsies from GC patients progressing under trastuzumab-based therapies if compared with respective pre-treatment biopsies.