Poster Session – Immunotherapy, Wednesday 29 November 2016 Poster abstracts S91 Immunotherapy 273 Poster (Board P099) PD-L1 expression and association with patient outcome in a large pediatric cohort F. Saletta 1 , R. Vilain 2 , A. Yuksel 1 , A. Gupta 3 , S. Nagabushan 3 , R. Scolyer 4 , C. Daniel 1 , J. Byrne 1 , G. McCowage 3 . 1 Children’s Hospital at Westmead, Children’s Cancer Reserach Unit, Westmead, Australia; 2 John Hunter Hospital, Division of Anatomical Pathology, Newcastle, Australia; 3 Children’s Hospital at Westmead, Cancer Centre for Children, Westmead, Australia; 4 Royal Prince Alfred Hospital, Tissue Pathology and Diagnostic Oncology, Camperdown, Australia Background: Cancers can evade the host immune system by inducing inhibitory signals. Programmed death-1 monoclonal antibody (PD-1 mAb) based inhibitors were design to block these signals and have shown promising results in adult clinical trials for the treatment of melanoma and non-small cell lung cancer. It has been proposed that programmed death- ligand 1 (PD-L1) expression represents a potential predictive biomarker of immune checkpoint blockade response in these cancer subtypes. However, literature about the prevalence of PD-L1 expression in the paediatric setting and efﬁcacy of PD-1 mAb therapy in children is lacking. Therefore, we sought to determine the frequency of PD-L1 expression in a large cohort of clinically annotated paediatric tumours and to investigate associations with clinico-pathological features and patient outcome. Materials and Methods: PD-L1 expression was analysed using immuno- histochemistry in 496 paediatric cancer patients (including neuroblastoma, medulloblastoma, low-grade and high-grade glioma, Ewing sarcoma, osteosarcoma and rhabdomyosarcoma) using tissue microarrays. Tumors with 30% cells showing positive membrane staining were considered to have high PD-L1expression. Results: PD-L1 expression of any intensity was identiﬁed in 12.9% of cases. High PD-L1 expression was found in 3.0% of cases. Neuroblastoma (n = 254) showed PD-L1 expression more commonly than any other tumor and, in particular, high PD-L1 expression (4.3%) was signiﬁcantly associated with risk of relapse (p = 0.002). In contrast, low-level PDL-1 expression (26.4%) was signiﬁcantly associated with longer overall survival (p = 0.045). There were no signiﬁcant associations with patient risk stratiﬁcation or tumor staging. Conclusions: High PD-L1 expression level in neuroblastoma patients represents an unfavourable prognostic factor associated with risk of re- lapse, whereas low PDL-1 expression was associated with improved overall survival. This work proposes a novel method to identify neuroblastoma patients with a higher likelihood of cancer recurrence, and suggests that PDL-1 expression may be a predictor of PD-1 mAb therapy efﬁcacy in paediatric cancer patients. No conﬂict of interest. 274 Poster (Board P100) Understanding the mechanisms of immunoresistance in malignant pleural mesothelioma stem cells to ﬁnd new therapeutic tools V. Milosevic 1 , J. Kopecka 1 , I.C. Salaroglio 1 , C. Riganti 1 . 1 University of Turin, Department of Oncology, Turin, Italy Malignant pleural mesothelioma (MPM) is a huge medical problem worldwide with a poor prognosis for the poor response to multimodal therapy and the intrinsic immunoresistance. Indeed, MPM creates a strongly immunosuppressive microenvironment. Tumor-derived stem cells (SCs) are responsible for MPM dissemination and progression, but it is not known if they can also exert immunosuppressive properties. Aim of this work was to investigate if MPM-derived stem cells could be responsible for MPM immunoresistance. From biopsies and pleural effusions of MPM patients we collected and stabilized MPM cell lines. We isolated the SC component by sorting the SOX2 + Oct4 + Nanog + ALDH bright cells and checked them for their clonogenicity and self-renewal. High-throughput PCR arrays were used to examine gene expression. Flow-cytometry was used to measure the expression of immuno-checkpoints. HMGB1 and ATP release, and calreticulin exposure were used as parameters of immunogenic cell death in response to chemotherapy known for inducing immunogenic effects, such as doxorubicin and cisplatin. MPM SCs had higher endogenous expression of immunosuppressive cytokines such as IL-10 and IL-4, and higher expression of JAK/STAT- related genes, such as JAK2−3 and STAT3, than non SCs. Unexpectedly, they had lower expression of the immuno-checkpoint ligand PD-L1, resulting poorly responsive to the immunotherapy targeting PD1/PD-L1 system. Chemotherapy induced immunogenic cell death in non SCs MPM cells but not in MPM SCs. Our study suggests that MPM SCs have immunoevasive properties, resist to the immunogenic cell death induced by chemotherapy and promote tumor-induced immunosuppression by activating the JAK/STAT axis and producing immnosuppressive cytokines. Targeting this axis may open new therapeutic possibilities in overcoming MPM immunoresistance. No conﬂict of interest. 275 Poster (Board P101) Evaluation of immune checkpoint marker co-expression proﬁles in the tumor microenvironment K. Wilkens 1 , E. Park 2 , J. Kim 2 , X.J. Ma 2 , L. Na 2 . 1 Advanced Cell Diagnostics, Segrate, Italy; 2 Advanced Cell Diagnostics, Newark, USA Background: Immunotherapy strategies that target checkpoint pathways have proved promising in recent years. In particular, blocking the immunosuppressive programmed cell death-1 (PD-1) pathway either by targeting PD-1 or one of its ligands, PD-L1, has shown durable efﬁcacy in patients with different cancer types including melanoma and lung cancers. Beyond the PD1/PD-L1 blockade, numerous other checkpoint inhibitors are being developed and various combinatorial approaches are currently being evaluated for clinical efﬁcacy. Thus, measuring biomarkers to predict treatment responses is a desirable ability. While various methods have been employed to measure biomarker expression, spatially mapped information at the single-cell level to provide pivotal perception regarding the cellular organization and cell-to-cell interactions in complex tissue has been lacking. Material and Methods: In this study, we analyzed the tumor mi- croenvironment of 60 archived formalin-ﬁxed parafﬁn-embedded (FFPE) non-small cell lung cancer (NSCLC) specimens for single-cell gene expression of immune checkpoint makers by applying the RNAscope ® in- situ hybridization assay. Results: The PD-L1 gene expression proﬁle of 56 tumor samples and four adjacent non-tumor tissues presented diverse and heterogeneous expression patterns in both tumor and stromal cells, with PD-L1 positivity in >50% of tumor samples. Co-expression proﬁles of PD1 or PD-L1 with various other checkpoint markers including PD-L2, LAG3, TIM3, IDO, CTLA4, OX40 revealed complex and differing co-expression proﬁles in the same cell or tumor environment. Importantly, each tumor sample showed unique co-expression proﬁles of checkpoint markers. Conclusions: The ﬁndings in this study may provide insight into therapeutic approaches for selecting patients for various different checkpoint inhibitors and combination therapies. No conﬂict of interest. 276 Poster (Board P102) Development of the anti-IL-10 mAb MK-1966 in combination with in situ vaccination of a TLR9 agonist SD-101 for cancer immunotherapy U. Phan 1 , R. Ueda 1 , R. Mangadu 1 , M. Sathe 2 , E. Rimmer 2 , F. Vives 2 , G. Ayanoglu 2 , Y. Yu 2 , J. Wong 2 , S. Sadekova 2 , T. McClanahan 2 , B. Bhagwat 2 , A. Willingham 2 , R. Raubertas 1 , R. Kastelein 1 . 1 Merck & Co. Inc., Early Development and Discovery Sciences, Kenilworth, USA; 2 Merck & Co. Inc., Biologics & Vaccines, Kenilworth, USA MK-1966 is a humanized IgG1/kappa neutralizing monoclonal antibody (mAb) against interleukin-10 (IL-10). IL-10 is an anti-inﬂammatory cytokine that inhibits secretion of cytokines from activated macrophages, production of CC and CXC chemokines, and a TH1 response, down-regulates MHC and costimulatory molecules on dendritic cells (DCs), and induces regulatory T cells. The immunosuppressive properties of IL-10 support targeting IL-10 in combination with in situ vaccination of toll-like receptor 9 (TLR9) agonists that induce IL-10 production. SD-101 is a potent TLR9 agonist that is being developed by Dynavax Technologies. SD-101 is a CpG C-class oligodeoxynucleotide that both activates plasmacytoid DCs (pDCs) and B cells and induces secretion of interferon alpha (IFNa) from pDCs. SD-101 also induces secretion of immunosuppressive molecules including IL-10 that may dampen an anti-tumor immune response. We show in the TC-1 syngeneic mouse bilateral tumor model that SD-101, administered intratumorally into a single tumor in combination with a surrogate anti-mouse IL-10 mAb administered intraperitoneally, resulted in robust anti-tumor activity of not only the injected tumor but the non- injected tumor, demonstrating abscopal effect. The combination also induced gene expression of T cell markers, inﬂammatory cytokines, and IFNa-inducible genes in both the injected and non-injected tumors in the TC-1 model. SD-101 induced IL-10 and IFNa2a in human PBMCs, and SD-101 in combination with MK-1966 strongly induced IFNg in human PBMCs. In addition, we have demonstrated that SD-101 induced expression of IFNa-inducible genes, cytokines including IL-10, and immune cell activation markers in various human tumor specimens