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TPS3160 Background: Immunotherapy with the anti-PD-(L)1 checkpoint inhibitors (CPIs) has been largely ineffective in so-called non-immunogenic “cold tumors”. Facilitating T cell infiltration is necessary to invoke an immune response which may be augmented or complemented by the activity of CPIs like durvalumab. Selective T cell Redirection Proteins (STRs) are fusion proteins that consist of genetically engineered Superantigen (Sag) linked to Fragment antigen binding (Fab) moieties directed to tumor-associated antigens. Nap is a first in class STR compound, recognizing the tumor-associated oncofetal antigen 5T4, whereas the SAg moiety selectively engages the T cell receptor β variable (TRBV) 7-9. Nap has been shown to induce specific T cells expansion, activation and infiltration into the tumor in pre-clinical and clinical studies. Pre-clinical data demonstrated that the combination of STR with CPI may lead to long term durable responses not possible in most patients receiving single agent CPI therapy and suggests that combining CPIs with STR may be a promising therapeutic strategy for patients with solid tumors. Methods: Patients will be treated with the combination of Nap and durvalumab using a flat dose of durvalumab (1120 mg) and the 3+3 design for Nap dose escalation (2, 5, 10, 15 and 20 mcg/kg). The MTD of Nap for the combination treatment will be established based on DLTs occurring during the first treatment cycle. The dose escalation part will be followed by MTD expansion cohort in which 10–15 patients will be treated with MTD of Nap and 1120 mg durvalumab (Clinical trial registry number NCT03983954). Major eligibility criteria include patients with pretreated advanced or metastatic, 5T4 expressing solid tumors, including patients previously progressed on CPI therapy. As of January 2020, enrollment to dose levels 2, 5 and 10 mcg/kg has been completed without DLT, enrollment to dose level 15 mcg/kg will start on February 2020. Clinical trial information: NCT03983954 .

e18016 Background: Cervical cancer is a Human papillomaviruses (HPV)-related disease. T cell infiltration is reported to be associated with a positive prognosis. We evaluated the prognostic value of the tumor-infiltrating dendritic cells (DC). Targeting this immune compartment might be clinically relevant. Methods: We characterized by immunohistochemistry the Dendritic cell lysosomal associated membrane glycoprotein (DC-Lamp) and CD8 T cell infiltration (high/low according to the median density) in patients with locally advanced cervical cancer (LACC) undergoing curative-intent concurrent chemoradiation followed by uterovaginal brachytherapy boost. They were treated in our institution between March 2004 and August 2011. The image acquisition was performed with a Zeiss Axio Scan Z1 microscope. CD8 T cells were detected using an algorithm created in Visiopharm software on manually selected regions of interest (ROI) and DC-Lamp was detected manually. We exported the number of cells and the surface of ROI to calculate the density. Results: A total of 91 patients were identified, with a median follow-up of 4.2 years (range: 0.1-10.3). Patient’s characteristics are listed in the table. DC-Lamphigh (n = 45) LACC patients showed a positive prognosis for overall survival (OS) by univariate (Odds Ratio (OR) = 0.20 [0.06 – 0.72], p = 0.013) and multivariate analyses (incorporating FIGO stage) (OR = 0.17 [0.05 – 0.61], p = 0.006). There was a positive correlation between DC-Lamp and CD8 (R = 0,25 et p = 0,095), which is significant in squamous cell carcinoma patients (R = 0.38, p = 0.039). There was a trend for improved risk stratification for the simultaneous assessment of DC-Lamp and CD8 levels, with DC-Lamphigh/CD8high patients having the best prognosis and DC-Lamplow/CD8low patients having the worst prognosis (p = 0.074). Conclusions: LACC patients with high intratumoral DC density have a favorable outcome. These results underscore the clinical potential of therapeutic strategies that target DC (e.g. Toll like receptor agonists) to render more patients responsive to immune checkpoint blockers. [Table: see text]

e17542 Background: We sought to analyze the transcriptional landscape of HNSCC in an attempt to identify tumor-intrinsic oncogenic pathways that appear to mediate T-cell infiltration of tumor tissue. In this direction, we employ a methodology that integrates histopathology data of the tumor microenvironment with its corresponding transcriptome. Methods: 32 frozen HNSCCs were subjected to RNA-seq and corresponding FFPE were scored for plasma cells, tertiary lymphoid structures and CD8a+ TILs (center, invasive margin). RNA-seq data were analyzed to identify differentially expressed genes (DEGs) between tumors scored by immunohistochemistry (IHC) as CD8a high and CD8a low. Gene ontology analysis (GO) was performed based on DEGs > 1.5 fold expression change between CD8a high and CD8a low groups. Candidate genes were investigated by hierarchical clustering in TCGA RNA-seq data and further validated by IHC and quantitative RT-PCR in our cohort. Results: 32 HNSCCs were either scored as CD8a high or CD8a low based on IHC detection of CD8a+ cells in invasive margin of tumors. Comparative analysis of mRNA expression data between CD8a high and CD8a low groups in our cohort revealed that Muc1/16 overexpression and glycosylation was highly enriched in T cell infiltrated group of tumors. This finding was further validated using antibodies that detect glycosylated epitopes for both mucins. Analysis of TCGA RNA-seq data indicated that Muc1/16 overexpressing tumors share signatures of early keratinocyte differentiation and stem cell identity and co-express high levels of enzymes that promote Muc1/16 glycosylation. Interestingly, loss of CDH1 and acquisition of epithelial mesenchymal transition (EMT) markers in the cluster of Muc1/16 overexpressing tumors is strongly correlated with elevated CD8a, IDO1, CD274 and CXCL10 mRNA levels (P < 0.0001). Conclusions: Muc1/16 overexpressing tumors represent a very immunogenic HNSCC cluster. Previous studies have shown that mucins 1 and 16 in cancer cells expose glycosylated-specific epitopes that are recognized by T cells as cancer antigens. To this end, MUC1/16 expression may serve as predictive biomarkers for response to immunotherapy and MUC-targeted immunotherapy may function as an attractive partner to checkpoint inhibitors in HNSCC.

118 Background: Preclinical studies suggest that MMP9 inhibition relieves immune suppression and promotes T cell infiltration to potentiate checkpoint blockade. To test this hypothesis, patient tumor samples obtained in a phase 2, open-label, randomized study (NCT02864381) of previously treated advanced gastric cancer comparing the MMP9-specific inhibitor andecaliximab (ADX) plus nivolumab (N) vs N alone were evaluated for T cell biomarkers. Methods: CD8 and PD-L1 (28-8 DAKO) were assessed by immunohistochemistry. IFNg, Teffector (Teff), and activated CD8+ T cell (ActT) gene signatures (GS) were assessed by RNASeq in archival baseline (BL) and biopsies obtained between weeks 5 and 9 (on-treatment; OT). Results: For both N and ADX/N, intratumoral CD8+ cells were significantly increased in OT biopsies relative to BL. The CD8+ OT increase was significantly greater for the ADX/N treatment relative to N in the PD-L1+ subgroup. In the ADX/N group only, IFNg, Teff, and ActT GS were significantly higher in OT biopsies relative to BL. OT change from BL of ≥ 300% vs

e24160Background: The degree of tumor infiltration by CD8 T cells is associated with favorable outcomes to immunotherapy. Biopsies to assess T cell infiltration are invasive and may not capture het...

2556Background: Arginine deprivation with pegylated arginine deiminase (ADI-PEG 20) has been shown to upregulate tumor programmed death-ligand 1 (PD-L1) expression and T cell infiltration. Current ...

e14543 Background: Poor infiltration of activated lymphocytes into solid tumors can be a fundamental factor limiting their efficacy. The purpose of this study was to determine whether a cycled tumor-penetrating peptide, iRGD, with a well-defined role in delivering drugs into extravascular tumor tissues, could facilitate lymphocytes infiltration into tumors in both the iRGD combination regimen and conjugated pattern. Methods: We used polyethylene glycol-conjugated phospholipid (PEG-lipid) derivatives, a time-efficient and versatile platform, to immobilize iRGD on T cell membrane, and examined the ability of iRGD to improve lymphocytes infiltration into tumors in multicellular spheroids and two xenograft mouse models. Furthermore, the synergy effect of iRGD modification and PD-1 gene knock out in adoptive T cell transfer immunotherapy was evaluated in a xenograft model of EBV associated gastric cancer. Results: In this study, we first showed that systematic injection of iRGD could double the number of CD3+ T cells in 4T1 tumor in BALB/C mouse and the synthetic iRGD-PEG-lipid, which could spontaneously transfer from solution to cell surfaces, had no effect to cell vitality, proliferation, phenotype and function. In adoptive transfer studies, the infiltration of T cells could be promoted by the co-administration with iRGD while T cells modified with iRGD could spread more extensively throughout both the multicellular spheroids and tumor mass. Furthermore, We demonstrated that iRGD modified T cells had superior antitumor efficiency to iRGD combination regimen due to increased T cell infiltration, and exhibited robust synergy effect with PD-1 gene knock out. Conclusions: Our study offers a simple method to target immune cells into tumors and may have the potential to enhance the clinical efficacy of adoptive T cell immunotherapy.

// Shenying Jin 1, 2 , Bo Xu 1 , Lixia Yu 1 , Yao Fu 3 , Hongyan Wu 3 , Xiangshan Fan 3 , Jia Wei 1 and Baorui Liu 1 1 The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing 210008, China 2 The Comprehensive Cancer Center of Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing 210008, China 3 Department of Pathology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China Correspondence to: Baorui Liu, email: baoruiliu@nju.edu.cn Jia Wei, email: weijia01627@hotmail.com Keywords: programmed cell death protein 1, programmed cell death ligand 1, CD3+ T cell, microsatellite instability, gastric signet-ring cell carcinoma Received: January 09, 2017 Accepted: February 24, 2017 Published: March 21, 2017 ABSTRACT Recent data supports a potentially significant role for immune checkpoint inhibitors in the treatment of gastric cancer. However, there are few data on the clinical implications of immunotherapy markers in gastric signet-ring cell carcinoma (SRCC). We evaluated the expression of programmed cell death protein-1 (PD-1), programmed cell death ligand 1(PD-L1), infiltration by CD3+ T cell, microsatellite instability (MSI), and Epstein-Barr Virus (EBV), and the relationship of each factor to survival in 89 advanced SRCC patients. All patients received 5-FU-based first-line chemotherapy. PD-L1 and PD-1 were expressed in 40.4% and 18.0% of the patients, respectively. There was a significant correlation between PD-L1 and PD-1 expression (r=0.363, p

e14608 Background: Recent research suggests an important role for Wnt/β-catenin signaling in mediating cancer immune evasion and resistance to immune checkpoint therapy. The mechnism is believed to involve blocking of specific cytokines which trigger immune cell recruitment to the tumor, resulting in the phenomenon of T-cell exclusion and rendering the tumor to a non-inflamed state. Inhibition of β-catenin may be an effective strategy for increasing the low response rate to these effective medicines in numerous cancer populations. DCR-BCAT is an advanced preclinical development candidate that has a potent and specific chemically-optimized RNA interference (RNAi) trigger targeting CTNNB1, the gene that encodes β-catenin, formulated in a tumor-selective lipid nanoparticle. Methods: Syngeneic murine models and transgenic MMTV-Wnt1 mouse models were used in this study. In both cases, a sequential dose regimen was employed where, in each dosing cycle, animals received DCR-BCAT, followed by a combination of anti-PD-1 and anti-CTLA-4 on subsequent days. Pharmacodynamic endpoints included CTNNB1 (β-catenin), CCL4, PD-1, PD-L1 mRNA measurement by quantitative PCR, as well as β-catenin, perforin, and granzyme B immunohistochemistry. Results: In syngeneic models, β-catenin inhibition with DCR-BCAT significantly improved the T-cell infiltration. The combination of DCR-BCAT and immune checkpoint blockade yielded significant tumor growth inhibition compared to monotherapy in B16F10, 4T1, Neuro2A and Renca tumors. The combination therapy was associated with high levels of granzyme B and perforin, strongly suggesting that the mechanism of sensitization to checkpoint therapy was a sharp increase in T-cell mediated cytotoxicity. Finally, when DCR-BCAT was combined with anti-PD-1/CTLA-4 antibodies in mice which develop spontaneous Wnt-driven mammary tumors, checkpoint therapy potentiation yielded complete tumor regressions. Conclusions: These data offer proof-of-concept for conversion of non-inflamed tumors to inflamed tumors by β-catenin inhibition, and support clinical evaluation of this combination approach using a first-in-class RNAi-based agent.

3599 Background: Pre-operative CRT followed by total mesorectal excision (TME) is nowadays the standard of care for patient with LARC (cT3-T4N0 or cTxN+). Currently, pathologic complete response occurs in +/- 15% after CRT. Colorectal cancer T-cell infiltration is a strong prognostic factor for survival after primary tumor resection. Our aim was to determine whether T-cell infiltration in PTB could be predictive of tumor response and relapse after CRT + TME. Methods: Between 1999 and 2012, patients with LARC who underwent CRT + TME and with available clinical follow-up and PTB (with sufficient tumor cells density) were identified at the Cliniques universitaires St-Luc. The density of CD3 (T cells) and CD8 (cytotoxic) was quantified on immunostained PTB slides and analyzed with a dedicated image analysis software on whole-slide imaging. Comparisons were made using the Wilcoxon-Mann-Whitney test. Cumulative disease-free survival (DFS) was performed using the Kaplan-Meier estimator and compared by log-rank tests. Cox regression we used for uni- and multi-variate analysis. P value of less than 0.05 was considered statistically significant. Results: 154 patients (sex ratio M/F 1.8; mean age 65 years-old; upper (20%), mid (29%) and low rectum (51%), synchronous metastases (11%)) were analyzed. High CD3 and CD8 PTB densities were significantly associated with a higher pathological response (Dworak 3-4) and lower ypTNM stage after CRT +TME (p < 0,05). Higher CD3 and CD8 PTB densities were associated with higher patient DFS (CD3: HR = 2,30 (CI95%:1,15-4,59) p = 0,02; CD8: HR = 1,95 (CI95%: 1,01-3,75) p = 0,04). These results were confirmed in uni and multivariate analysis. CD3 and CD8 PTB densities added to pathological response (ypTNM/Dworak) but also clinical response (ycTNM) after CRT + TME increases significantly the accuracy prediction of tumor relapse. Conclusions: Pretherapeutic T-cell infiltration of LARC is predictive of tumor response and relapse after CRT +TME. This biomarker could be helpful for patient treatment decision. It must be validated in larger patient cohorts.

3053 Background: Activation of the Wnt-β-catenin signaling pathway is associated with poor T cell infiltration of tumors. We have previously demonstrated that paracrine Wnt5a-β-catenin signaling is a critical trigger of dendritic cell (DC) tolerization and regulatory T cell (Treg) differentiation in the melanoma microenvironment. In a transgenic BRAFV600EPTEN-/- model, the genetic silencing of melanoma-derived Wnt5a potently enhances infiltrating CD8+T cell effector function and promotes responses to anti-PD-1 Ab therapy. Ipafricept (IPA) is a recombinant Wnt decoy receptor and Vantictumab (VAN) is a Fzd receptor monoclonal Ab. Both molecules inhibit Wnt-β-catenin signaling and have been well-tolerated in ongoing phase I/Ib clinical trials. We explored the ability of IPA/VAN to reverse tumor-mediated immune tolerance and enhance the efficacy of anti-PD-1 Ab immunotherapy in a pre-clinical model that closely recapitulates human melanoma. Methods: Both IPA and VAN were utilized to investigate Wnt-β-catenin inhibition as a strategy for suppressing melanoma-induced DC indoleamine 2,3-dioxygenase (IDO) expression and Treg differentiation in vitro. These agents were further tested for their ability to enhance anti-tumor T cell responses and to augment the efficacy of anti-PD-1 Ab therapy in syngeneic and autochthonous models of BRAFV600EPTEN-/- melanoma. Results: IPA and VAN effectively inhibit Wnt5a and melanoma-induced DC IDO expression and Treg differentiation in vitro. Further studies demonstrate that IPA and VAN significantly augment anti-PD-1 Ab-mediated suppression of primary and metastatic tumor progression in both syngeneic and autochthonous BRAFV600EPTEN-/- melanoma models. These anti-tumor effects correlated with suppressed IDO enzymatic activity, enhanced tumor-infiltrating CD8+T cell/Treg ratios, and increased activation of TRP2 antigen-specific effector T cells. Conclusions: The pharmacological inhibition of paracrine Wnt-β-catenin signaling with IPA and VAN augment the anti-tumor efficacy of anti-PD-1 Ab therapy and represent a promising strategy for further phase I testing in melanoma and other solid tumors.

101 Background: Cancers of the head and neck (HNC) represent some of the most debilitating and aggressive tumors. Radiotherapy (RT) is the primary treatment modality for locally advanced HNC, but less than 50% of patients treated with RT survive to 5 years. Clinical trials with the immune checkpoint inhibitor PD-L1 have shown considerable promise, but on its own has only yielded a 20% response rate. RT has the potential to transform the tumor microenvironment promoting infiltration of T-cells in poorly immunogenic tumors and activating T-cells in tumors with pre-existing T-cell populations. We hypothesized that the combination of PD-L1 inhibition with radiation can enhance therapeutic efficacy through re-invigoration of exhausted T-cells. Methods: Tumor cells were injected into the right buccal mucosa. Treatment was initiated when established tumors were observed. Forty mice per tumor model were randomized to IgG2b control, anti PD-L1, RT alone or RT+anti PD-L1. Anti PD-L1 was started 3 days before RT (single dose of 10Gy) and maintained for 3 weeks. For mechanistic studies, mice received the same treatment but were euthanized 72 hours after RT. Flow cytometric analysis of T-cell expression of PD-1, CTLA-4, FOXP3, CD44 and IFNG was performed on freshly harvested tumors and regional lymph nodes. Results: Tumor-bearing mice formed aggressive tumors with regional lymph node metastasis. Mice treated with RT or anti PD-L1 had slightly improved survival whereas mice treated with RT+anti-PD-L1 had significantly improved survival (p = 0.0007). Tumor control was significantly improved in the RT+anti-PD-L1 group compared to other groups. Anti PD-L1 alone did not improve tumor control. Average tumor volume on day 23 was 175±60.1 mm3 in the RT+anti-PD-L1 group compared to 623±91.0 mm3 in the PD-L1 group. Flow cytometric analysis revealed that this is mediated by enhancement in T-cell infiltration. Analysis of T-cell activation showed increased expression of CD44 and IFNG. Conclusions: Our results show a critical role for RT in mediating sensitivity to PD-L1 inhibition in HNC. In combination with anti PD-L1, RT significantly increased survival and tumor control compared to either modality alone.

23 Background: The success of immunotherapy has raised new issues regarding the selection of patients, design of combination strategies, and sequencing of various regimens. Sarcomas have poor outcomes in the metastatic setting but may be amenable to immune therapies. However, we currently have limited knowledge of the immunologic profiles of different soft tissue sarcoma (STS) subtypes. Methods: We identified patients with the relatively common STS subtypes: leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS) and liposarcoma. Formalin fixed paraffin embedded (FFPE) tumor samples from 81 patients underwent gene expression analysis, immunohistochemistry for PD-1 and PD-L1, and sequencing of the T cell receptor Vβ region. Differences in liposarcoma subsets were also evaluated. Results: UPS and LMS had high expression levels of genes related to antigen presentation and T cell infiltration. UPS had higher levels of PD-L1 (p ≤ 0.001) and PD-1 (p ≤ 0.05) on IHC. UPS also had the highest T cell infiltration based on TCR sequencing, significantly more than SS, which had the lowest (p ≤ 0.05). UPS and LMS both had higher clonality compared with SS and liposarcoma (p ≤ 0.05). A model adjusted for STS histologic subtype found that for all sarcoma T cell infiltration and clonality were highly correlated with PD-1 and PD-L1 staining levels (p ≤ 0.01). Conclusions: In a model adjusted for sarcoma histologic subtypes, T cell infiltration and clonality were highly correlated with PD-1 and PD-L1 expression, consistent with the emerging view of tumor immunity that highly inflamed tumors acquire inhibitory ligands to evade tumor-specific T cells. UPS, which is a more highly mutated STS subtype, provokes a strong immune response evidenced by multiple inflammatory features suggesting that it may be well-suited to checkpoint inhibitor based approaches. SS and liposarcoma subsets are less highly mutated but do express immunogenic self-antigens therefore strategies to improve antigen presentation and T cell infiltration may be valuable for allowing immunotherapeutic success in these tumor types.

3042Background: Mutations in DNA repair genes are common in cancer, and may induce a state of hypermutability. Importantly, mutational burden is linked to enhanced response to immunotherapies, as p...

5543 Background: CD8 positive intra-epithelial tumor infiltrating T cells (TIL) are associated with improved outcomes in ovarian cancer, particularly in HGSC. Radiotherapy (XRT) has immunologic eff...

e22129 Background: Ovarian cancer is diagnosed in more than 190,000 new patients every year and is known to have the highest mortality rate among gynaecologic cancers. The type of immune cells that are present within the tumor microenvironment can play a crucial role in the survival of patients. However, little is known about the dynamics of the tumor-infiltrating immune cells during disease progression. Methods: We studied the immune cells infiltrating the tumor tissue of ovarian cancer patients at different stages of disease. We analysed the patterns of T lymphocytes in fresh tumor tissue as well as blood samples of 44 newly diagnosed ovarian cancer patients by flow cytometry. To evaluate whether regulatory T cells (Tregs) develop in situ or migrate to tumor tissue, we measured a concentration of chemokine CCL22 in tumor cell culture supernatants. We also determined the expression of CCR4 on circulating as well as tumor-infiltrating Tregs by flow cytometry. Results: The early stages of development of ovarian carcinomas were characterized by a strong Th17 immune response, whereas in stage II patients, recruitment of high numbers of Th1 cells was observed. In disseminated tumors (stage III-IV), we detected a dominant population of Helios+ activated regulatory T cells along with high numbers of macrophages and immature myeloid dendritic cells. Tumor-infiltrating Tregs had markedly lower expression of CCR4 than circulating Tregs, and the numbers of tumor-infiltrating Tregs significantly correlated with the levels of CCL22 in ovarian tumor cell culture supernatants, suggesting their recruitment via a CCR4/CCL22 interaction. CCL22 was mainly produced by tumor cells, macrophages and mDCs in the primary ovarian tumors, and its expression markedly increased in response to IFNgamma. Conclusions: Taken together, the specific recruitment of Tregs, probably triggered by inflammatory stimuli, leads to a significant immune suppression in the advanced stages of ovarian cancer.

8501 Background: Despite frequent induction of tumor antigen-specific T cells in melanoma patients following vaccination, tumor regressions remain rare. This observation prompted systematic analysis of the melanoma tumor microenvironment to identify factors that may influence the effector phase of the anti-tumor immune response. Methods: Gene expression profiling using the Affymetrix platform was performed on a series of melanoma metastases, melanoma cell lines, and primary melanocyte cell lines. Confirmatory assays were done by real-time RT-PCR, protein array, immunohistochemistry (IHC), and in vitro chemokine migration assays. Results: Non- supervised hierarchical clustering revealed 3 major subsets of tumors, with the main clustering based on differential expression of T cell-derived transcripts. The presence of CD8+ T cells was confirmed by IHC. Tumors that contained T cells uniquely expressed high levels of multiple chemokines. Protein array confirmed high expression of CCL2, CCL4, and CCL5; real-time RT-PCR additionally confirmed relatively high levels of CXCL9, CXCL10, and CCL3 transcripts. Transwell assays confirmed that each of these 6 chemokines recruited CD8+ effector cells in vitro. Conclusions: We have identified a set of 6 chemokines that likely regulates recruitment of activated T cells into melanoma metastases. Tumors that lack such chemokines might not be capable of supporting the effector phase of the anti-tumor immune response. We suggest that chemokine profiling of tumor sites should be performed in clinical trials of active immunotherapy. No significant financial relationships to disclose.