PanCancer Immune Profiling Panel Research Articles

Immune profiling and clinical outcomes in patients treated with ramucirumab and pembrolizumab in phase I study JVDF.

3089 Background: In Study JVDF (NCT02443324), we combined ramucirumab (VEGFR2 antagonist) and pembrolizumab (PD-1 antagonist) to simultaneously target the tumor microenvironment and immune checkpoints in patients with advanced non-small cell lung cancer (NSCLC), gastric or gastroesophageal junction adenocarcinoma (G/GEJ), urothelial carcinoma (UC) or biliary tract cancer (BTC). We reported that this combination was associated with increased antitumor activity in patients with PD-L1 positive tumors by immunohistochemistry (IHC) compared with PD-L1 negative tumors.1) Here we explore the association between baseline gene expression profiles and clinical outcomes. Methods: JVDF was a nonrandomized phase 1a/b trial that treated patients with intravenous ramucirumab at 8 mg/kg on days 1 and 8 (G/GEJ, BTC) or 10 mg/kg on day 1 (G/GEJ, NSCLC, UC) plus pembrolizumab (200 mg day 1) every 3 weeks.1 Baseline tumor samples from 53 patients across 7 study cohorts were analyzed with the NanoString PanCancer Immune Profiling Panel for RNA expression and the DAKO PD-L1 IHC 22C3 pharmDx assay for PD-L1 protein expression. Clinical outcomes included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). Results: Across cohorts, PD-L1 gene expression was correlated with increased IFNγ gene expression and immune-related gene signatures (T effector, T cell-inflamed (TIS)), and trended with PD-L1 protein expression. Expression of immune checkpoint-related genes and myeloid-derived suppressor cell /regulatory T cell markers was increased in the NSCLC TPS≥50% PD-L1 IHC subgroup (N=8), while no clear pattern of expression was observed in other cohorts. Higher T effector and TIS scores appeared associated with better survival and response in NSCLC cohorts (mean TIS: 1.21±0.80 in responders (N=7) vs -0.13±0.57 in non-responders (N=7); p=0.004), and a trend was observed in G/GEJ cohorts (mean TIS: -0.18±0.30 (N=5) vs -0.39±0.21 (N=13); p=0.207). Of note, partial responses and stable disease were also observed in NSCLC and G/GEJ patients with a low baseline inflammatory signature score. Additional analyses are ongoing and will be presented. Conclusions: Baseline PD-L1 gene and protein expression tends to correlate with immune-related gene expression, and an inflamed tumor microenvironment may be associated with better clinical outcomes with ramucirumab and pembrolizumab. However, interpretation is limited by lack of control arm and sample size.1) Herbst et al. Lancet Oncol. 2019 Aug; 20 (8):1109-1123. Clinical trial information: NCT02443324 .

Comparison of skin biopsy sample processing and storage methods on high dimensional immune gene expression using the Nanostring nCounter system

BackgroundDigital multiplex gene expression profiling is overcoming the limitations of many tissue-processing and RNA extraction techniques for the reproducible and quantitative molecular classification of disease. We assessed the effect of different skin biopsy collection/storage conditions on mRNA quality and quantity and the NanoString nCounter™ System’s ability to reproducibly quantify the expression of 730 immune genes from skin biopsies.MethodsHealthy human skin punch biopsies (n = 6) obtained from skin sections from four patients undergoing routine abdominoplasty were subject to one of several collection/storage protocols, including: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater (ThermoFisher) for 24 h at room temperature then stored at − 80 °C; iii) formalin fixation with further processing for FFPE blocks; iv) DNA/RNA shield (Zymo) stored and shipped at room temperature; v) placed in TRIzol then stored at − 80 °C; vi) saline without RNAse for 24 h at room temperature then stored at − 80 °C. RNA yield and integrity was assessed following extraction via NanoDrop, QuantiFluor with RNA specific dye and a Bioanalyser (LabChip24, PerkinElmer). Immune gene expression was analysed using the NanoString Pancancer Immune Profiling Panel containing 730 genes.ResultsExcept for saline, all protocols yielded total RNA in quantities/qualities that could be analysed by NanoString nCounter technology, although the quality of the extracted RNA varied widely. Mean RNA integrity was highest from samples that were placed in RNALater (RQS 8.2 ± 1.15), with integrity lowest from the saline stored sample (RQS < 2). There was a high degree of reproducibility in the expression of immune genes between all samples with the exception of saline, with the number of detected genes at counts < 100, between 100 and 1000 and > 10,000 similar across extraction protocols.ConclusionsA variety of processing methods can be used for digital immune gene expression profiling in mRNA extracted from skin that are comparable to snap frozen skin specimens, providing skin cancer clinicians greater opportunity to supply skin specimens to tissue banks. NanoString nCounter technology can determine gene expression in skin biopsy specimens with a high degree of sensitivity despite lower RNA yields and processing methods that may generate poorer quality RNA. The increased sensitivity of digital gene expression profiling continues to expand molecular pathology profiling of disease.

Characterization of the immune profile of oral tongue squamous cell carcinomas with advancing disease.

We investigated whether a unique immune response was instigated with the development of oral tongue squamous cell carcinomas (OTSCC), with/without nodal involvement, with/without recurrent metastatic disease, or within tumor involved nodes. One hundred and ten formalin‐fixed paraffin‐embedded samples were collected from a retrospective cohort of 67 OTSCC patients and 10 non‐cancerous tongue samples. Targets including CD4, CD8, FOXP3, PD‐L1, and PD‐1 were analyzed by immunohistochemistry. The Nanostring PanCancer Immune Profiling Panel was used for gene expression profiling. Data were externally validated in the The Cancer Genome Atlas (TCGA) head and neck (HNSCC), melanoma and lung squamous cell carcinoma (LSCC) cohorts. A 24‐immune gene signature was identified that discriminated more aggressive OTSCC cases, and although not prognostic in HNSCC was associated with survival in other TCGA cohorts (improved survival for melanoma, P < .001 and worse survival for LSCC, P = .038). OTSCC exhibited concordant gene and immunohistochemical (IHC) features characterized by a TH‐2 biased, proinflammatory profile with upregulated B cell and neutrophil gene activity and increased CD4, FOXP3, and PD‐L1 expression (P < .001 for all by IHC). Compared to less advanced disease, nodal involvement and recurrent OTSCC did not induce a different immune response although recurrent disease was characterized by significantly higher PD‐L1 expression (P = .004 by SP263, P = .013 by 22C3, P = .004 for gene expression). Identification of a gene signature associated with different prognostic effects in other cancers highlights common pathways of immune dysregulation that are impacted by the tumor origin. The significant immunosuppressive signaling in OTSCC indicates primary failure of immune system to control carcinogenesis emphasizing the need for early, combination therapeutic approaches.

Prognostic role of PD-L1 and immune-related gene expression profiles in giant cell tumors of bone.

Giant cell tumor of bone (GCTB) is a locally aggressive and rarely metastatic tumor, with a relatively unpredictable clinical course. A retrospective series of 46 GCTB and a control group of 24 aneurysmal bone cysts (ABC) were selected with the aim of investigating the PD-L1 expression levels and immune-related gene expression profile, in correlation with clinicopathological features. PD-L1 and Ki67 were immunohistochemically tested in each case. Furthermore, comprehensive molecular analyses were carried out using NanoString technology and nCounter PanCancer Immune Profiling Panel, and the gene expression results were correlated with clinicopathological characteristics. PD-L1 expression was observed in 13/46 (28.3%) GCTB (and in 1/24, 4.2%, control ABC, only) and associated with a shorter disease free interval according to univariate analysis. Moreover, in PD-L1-positive lesions, three genes (CD27, CD6 and IL10) were significantly upregulated (p < 0.01), while two were downregulated (LCK and TLR8, showing borderline significance, p = 0.06). Interestingly, these genes can be related to maturation and immune tolerance of bone tissue microenvironment, suggesting a more immature/anergic phenotype of giant cell tumors. Our findings suggest that PD-L1 immunoreactivity may help to select GCTB patients with a higher risk of recurrence who could potentially benefit from immune checkpoint blockade.

NanoString Gene Expression Profiling in Chronic Lung Allograft Dysfunction (CLAD)

CLAD, mainly manifest as bronchiolitis obliterans syndrome (BOS), is the leading cause of late death in lung recipients. BOS develops despite routine immunosuppression and its pathogenesis is poorly understood. The histological correlate of BOS is bronchiolitis obliterans (BO), or airways fibrosis. We quantified over 700 immune-related genes in BO affected airways to better understand immune responses active in end-stage BOS. Formalin fixed paraffin embedded lung tissue was procured at retransplantation for BOS (n=6). Unused donor tissue was used as normal control (n=5). Histologies of interest were isolated using laser capture microdissection (LCMD). Within each BOS lung, airways histologically affected by BO pathology, defined as intraluminal fibrous expansion, were isolated. Additionally, airways unaffected by evident fibrosis were isolated. Within each donor lung, histologically normal airways were isolated. Cellular RNA was extracted and multiplex gene expression analysis was performed using the NanoString PanCancer Immune Profiling Panel. Linear regression was used to compare gene expression among groups. The primary comparison was differential expression in BO affected vs. normal donor airways. We also compared expression in BO affected vs. unaffected airways within BOS explant lungs. P-values were corrected for multiple comparisons using Benjamini-Hochberg. Sixty-seven genes were significantly different between BO affected vs. normal donor airways, of which 52 had a fold change (FC) <0.5 or >2. Along with genes in adaptive immunity, several genes related to innate and humoral activation were significantly increased in BO affected airways. Among the innate immune genes with increased expression in BO were toll-like receptor 1 (FC 2.31) and its adaptor molecule CD14 (FC 5.26) in addition to the complement subcomponent C1s (FC 3.06), while those in humoral immunity included bone marrow stromal antigen 2 (FC 3.41), important to the growth and development of B-cells. No genes were significantly differentially expressed between BO affected vs. unaffected airways within BOS explant lungs. Our results suggest innate and humoral immune responses are active in airways affected by BO pathology, and also in airways without evident fibrosis. Ongoing analyses of BO lesions through LCMD will provide new insights into CLAD pathogenesis.

Immune Profiling of Deficient Mismatch Repair Colorectal Cancer Tumor Microenvironment Reveals Different Levels of Immune System Activation

To understand the immune landscape of deficient mismatch repair colorectal cancer (dMMR CRC) tumor microenvironment, gene expression profiling was performed by the nCounter PanCancer Immune Profiling Panel. This study was conducted retrospectively on 89 dMMR-CRC samples. The expression of CD3, CD8, programmed death-1, and programmed death ligand-1 protein was evaluated on a subset ofsamples by immunohistochemistry, and lymphocyte density was calculated. A subset of deregulated genes was identified. Functional clustering analysis performed on these genes generated four main factors: antigen processing and presentation, with its major histocompatibility complex-II-related genes; genes correlated with the cytotoxic activity of immune system; T-cell chemotaxis/cell adhesion genes; and T-CD4+ regulator cell-related genes. A deregulation score (DS)was calculated for each sample. On the basis of their DS, tumors were then classified as COLD (DS≤-3) to select the samples with a strong down-regulation of the immune system and NOT COLD (DS≥-2). The COLD group of patients showed a worse prognosis in terms of survival considering all patients (P=0.0172) and patients with metastatic disease (P=0.0031). These results confirm that dMMR-CRCs do not constitute a homogeneous group as concerns the immune system activity of tumor microenvironment. In particular, the distinction between COLD and NOT COLD tumors may improve the management of these two subsets of patients.

Abstract P5-02-10: Overexpression of Indoleamine 2,3-dioxygenase 1 in HER2 positive breast cancer correlates with increase of tumor-infiltrating lymphocytes level and worse overall survival

Abstract Introduction: HER2+ breast cancer (BC) shows a large amount of mutation burden and high lymphocyte infiltration in the tumor. Tumor-infiltrating lymphocytes (TILs) reflect the host immune reaction against tumor cells, and TILs levels have been studied as prognostic and predictive factors in breast cancers, particularly in the triple-negative and HER2+ subtype. Conversely, tumor cells develop immunosuppressive tumor microenvironment by change the gene expression. This study evaluated the differences in expression of immune-related genes particularly involving immune evasion mechanism according to TILs level and confirmed protein expression in the tumor tissue. Materials and Methods: This study included 167 patients with primary HER2+ BC who underwent surgery. All hematoxylin and eosin slides of the tumors were reviewed and their TILs level was recorded. All tumors were divided into high and low TILs groups with a cutoff value of 60%. RNA was extracted from formalin-fixed paraffin embedded (FFPE) block of 12 cases to determine their immune-related gene RNA expression levels using the NanoString nCounter® PanCancer Immune Profiling Panel. Among the expressed genes with significance, indoelamine-2,3-dioxygenase 1 (IDO1)expression was further evaluated by immunohistochemistry (IHC) in tissue microarray. The IDO1 expression on tumor cells and TILs was categorized as low or high. Results: The IDO1 gene expression level was 7.1-fold higher in high-TILs group compared to low-TILs group (p=0.024). High IDO1 expression in the tumor cells and TILs by IHC analysis were related to high-TILs group (Table 1). Tumors with high IDO1 expression showed a high nuclear grade and related to worse overall survival (OS), particularly in hormone receptor-/HER2+ patients (all, p &amp;lt; 0.05). Multivariate analysis indicated that high IDO1 expression in tumor cells was an independent poor prognostic factor for OS (p=0.045, Table 2) Conclusion: This study demonstrated that HER2+ BC with high TILs level overexpressed IDO1 in tumor tissue. High IDO1 expression in the tumor cell is related to higher nuclear grade and poor OS in patients with HER2+ BC. In conclusion, this study showed that HER2-positive breast cancer has high immunogenicity and is related to the IDO1 immunosuppressive mechanism and related with poor OS. This result indicates that IDO1 targeted therapy may be effective in patients with HER2-positive breast cancer patients. Table 1. Clinicopathologic features according to IDO1 expression level in HER2+ BCFactorsIDO1TC expressionIDO1TIL expressionLowHighP-value†LowHighP-value‡Age (mean, yr)51.6753.680.20254.0751.780.163Size (median, cm)3.103.350.3763.213.210.997Histologic grade (n, %)0.0540.740Low40 (44.0)22 (28.9)23 (39.0)39 (36.1)High51 (56.0)54 (71.1)36 (61.0)69 (63.9)Nuclear grade (n, %)0.040*0.523Low21 (23.1)8 (10.5)12 (20.3)17 (15.7)High70 (76.9)68 (89.5)47 (79.7)91 (84.3)Lymphovascular invasion(n, %)0.7580.257Absent48 (52.7)42 (55.3)28 (47.5)62 (57.4)Present43 (47.3)34 (44.7)31 (52.5)46 (42.6)Node metastasis(n, %)0.3500.630Absent46 (50.5)32 (42.1)26 (44.1)52 (48.1)Present45 (49.5)44 (57.9)33 (55.9)56 (51.9)AJCC 7th Stage (n, %)0.4710.853Early71 (78.0)55 (72.4)44 (74.6)82 (75.9)Advanced20 (22.0)21 (27.6)15 (25.4)26 (24.1)HR expression (n, %)0.2810.517Positive52 (57.1)37 (48.7)29 (49.2)60 (55.6)Negative39 (42.9)39 (51.3)30 (50.8)48 (44.4)TILs level (n, %)0.047*0.002*Low (≤60%)79 (86.8)56 (73.7)55 (93.2)80 (74.1)High (&amp;gt;60%)12 (13.2)20 (26.3)4 (6.8)28 (25.9)TLSs in DCIS (n, %)0.5240.067Absent58 (63.7)44 (57.9)42 (71.2)60 (55.6)Present33 (36.3)32 (42.1)17 (28.8)48 (44.4)TLSs in invasive component (n, %)0.7440.308Absent58 (63.7)51 (67.1)42 (71.2)67 (62.0)Present33 (36.3)25 (32.9)17 (28.8)41 (38.0)Abb: IDO1TC, IDO1 in tumor cells; IDO1TIL, IDO1 in TILs; TLS, tertiary lymphoid structure Table 2. Univariate and multivariate analysis of IDO expression for OS in HER2+ BCUnivariateMultivariateFactorsHR95% CIp-valueHR95% CIp-valueIDO1TC expression0.022*0.045*Low(ref)(ref)(ref)(ref)High3.7411.206-11.6023.2161.028-10.060IDO1TIL expression0.771Low(ref)(ref)High0.8610.313-2.368TLSs in DCIS0.036*0.079Absent(ref)(ref)(ref)(ref)Present0.2060.047-0.9050.2620.059-1.169TLSs in invasive0.364Absent(ref)(ref)Present0.5920.191-1.836Abb: OS, overall survival; HR, hazard ratio; CI, confidence interval; IDO1TC, IDO1 in tumor cells; IDO1TIL, IDO1 in TILs; TLS, tertiary lymphoid structure Citation Format: Jeeyeon Kim, Ahrong Kim, So Jeong Lee. Overexpression of Indoleamine 2,3-dioxygenase 1 in HER2 positive breast cancer correlates with increase of tumor-infiltrating lymphocytes level and worse overall survival [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-10.

Comparison of immune-related gene expression between primary and metastatic site in advanced gastric cancer patients with peritoneal dissemination.

423 Background: Immune checkpoints inhibitor (ICI) is effective and approved in some solid tumors including advanced gastric cancer (GC). Peritoneal dissemination is known as a poor prognostic factor and was reported to be associated with the resistance to ICI according to the previous reports. The aim of our study is to compare the immune-related gene expression between primary site and peritoneal lesion in advanced GC patients. Methods: Among advanced GC patients, we selected those who underwent surgical resection for both primary and peritoneal lesions simultaneously. Formalin-fixed paraffin-embedded (FFPE) tumor tissues of primary and peritoneal lesions were prepared and RNA was extracted by Maxwell RSC RNA FFPE kit (Promega). Immune-related gene expression was evaluated by using nCounter Max Analysis System (NanoString). We used nCounter PanCancer Immune Profiling Panel Kit which includes 770 immune-related genes. Results: Immune-related gene expression was evaluated by using twenty-four FFPE tumor tissues in twelve GC patients. Scatter plot and hierarchical clustering analyses showed that the pattern of immune-related gene expression was not much different between primary and peritoneal lesions beyond the individual differences. Regarding the T cell function, high expression of Immune-related genes was widely detected in patients with EB virus-positive (n = 2) and HER2-positive (n = 1). Gene expressions such as CD70, FAS, MAF and IL-3 were higher in peritoneal lesion compared with primary lesion (p &lt; 0.05). Whereas, expressions of F2RL1 and IL-11 were lower in peritoneal lesion compared with primary lesion (p &lt; 0.05). Conclusions: Our study indicated that there was not much difference of Immune-related gene expression between primary and peritoneal lesion in advanced GC patients. Positive status of EB virus and HER2 may be associated with high expression of immune-related genes. Further analysis to evaluate immune-related gene expression between primary and metastatic site may contribute the further understanding of cancer immunity in advanced GC.

Flow cytometry and targeted immune transcriptomics identify distinct profiles in patients with chronic myeloid leukemia receiving tyrosine kinase inhibitors with or without interferon-\u03b1

BackgroundTumor cells have evolved complex strategies to escape immune surveillance, a process which involves NK cells and T lymphocytes, and various immunological factors. Indeed, tumor cells recruit immunosuppressive cells [including regulatory T-cells (Treg), myeloid-derived suppressor cells (MDSC)] and express factors such as PD-L1. Molecularly targeted therapies, such as imatinib, have off-target effects that may influence immune function. Imatinib has been shown to modulate multiple cell types involved in anti-cancer immune surveillance, with potentially detrimental or favorable outcomes. Imatinib and other tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) have dramatically changed disease course. Our study aimed to characterize the different populations of the immune system in patients with CML affected by their treatment.MethodsForty-one patients with CML [33 treated with TKIs and 8 with TKIs plus interferon (IFN)-α] and 20 controls were enrolled in the present study. Peripheral blood populations of the immune system [referred to as the overview of immune system (OVIS) panel, Treg cells and MDSCs] and PD-1 expression were evaluated by flow cytometry. The immunological profile was assessed using the mRNA Pan-Cancer Immune Profiling Panel and a NanoString nCounter FLEX platform.ResultsPatients receiving combination therapy (TKIs + IFN-α) had lower numbers of lymphocytes, particularly T cells [838/µL (95% CI 594–1182)] compared with healthy controls [1500/µL (95% CI 1207 – 1865), p = 0.017]. These patients also had a higher percentage of Treg (9.1%) and CD4+PD-1+ cells (1.65%) compared with controls [Treg (6.1%) and CD4+/PD-1+(0.8%); p ≤ 0.05]. Moreover, patients treated with TKIs had more Mo-MDSCs (12.7%) whereas those treated with TKIs + IFN-α had more Gr-MDSC (21.3%) compared to controls [Mo-MDSC (11.4%) and Gr-MDSC (8.48%); p ≤ 0.05]. CD56bright NK cells, a cell subset endowed with immune-regulatory properties, were increased in patients receiving TKIs plus IFN-α compared with those treated with TKIs alone. Interestingly, serum IL-21 was significantly lower in the TKIs plus IFN-α cohort. Within the group of patients treated with TKI monotherapy, we observed that individuals receiving 2nd generation TKIs had lower percentages of CD4+ Treg (3.63%) and Gr-MDSC (4.2%) compared to patients under imatinib treatment (CD4+ Treg 6.18% and Gr-MDSC 8.2%), but higher levels of PD-1-co-expressing CD4+ cells (1.92%).ConclusionsOur results suggest that TKIs in combination with IFN-α may promote an enhanced immune suppressive state.

Abstract B37: The complex immune-microenvironment heterogeneity in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, resulting in more than 3,000 deaths per year in The Netherlands. The tumor microenvironment (TME) contributes largely to the treatment failure of the disease. TME consists of the stromal compartment, extracellular matrix, and tumor-infiltrative lymphocytes (TILs). Types and numbers of TILs are associated with clinical outcome, i.e., highCD8+: FOXP3 ratio is linked to a favorable prognosis. However, the intratumor immune variation in pancreatic cancer has not been studied so far. This study aims at investigating the differences of the immune infiltration within various areas of the same pancreatic cancer samples (intratumor variation) and comparing to healthy controls (extratumor variation). Such differences may be used for microenvironment remodeling, which may open new therapeutic approaches for PDAC patients. Materials and Methods: Formalin-fixed, paraffin-embedded (FFPE) samples of 26 PDAC patients and 8 healthy controls were selected. RNA was isolated separately from the tumor core and from the adjacent normal-like pancreatic tissue of the same sample. Immune profiling was carried out using the PanCancer Immune Profiling Panel of Nanostring technology. Subsequently, the gene expression data were analyzed using nSolver basic software and the advanced module. Results: The immune infiltration in the core of PDAC samples was different from that in the adjacent normal-like areas of the same patients. Tumor samples exhibited the lowest CD8+, Th1, neutrophil, and Natural Killer cell infiltration as compared to normal-like areas. In addition, the normal-like areas of PDAC samples showed a different immune profile as compared to the healthy controls. B lymphocytes were significantly higher in healthy controls compared to normal-like PDAC samples. Discussion and Conclusion: Our results present the variation in the immune microenvironment between the normal-like pancreatic tissue and the healthy pancreas, indicating that changes at the immune levels precede morphologic changes, tumorigenesis, and tumor cell infiltration. Additionally, specific changes in TILs occur in a gradient fashion from adjacent normal-like tissue to the tumor core. Understanding the differences of the immune microenvironment in the same tissue samples will enable developing an accurate microenvironment remodeling therapeutic approaches to treat PCDA patients. Citation Format: Frederieke Grevers, Diba Latifi, Willem de Koning, Casper H.J. van Eijck, Dana A. Mustafa. The complex immune-microenvironment heterogeneity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B37.

Endoscopic ultrasound may be used to deliver gene expression signatures using digital mRNA detection methods to immunophenotype pancreatic ductal adenocarcinoma to facilitate personalized immunotherapy

Background & objectivesBiomarkers are increasingly required to molecularly characterize pancreatic ductal adenocarcinoma (PDAC) subgroup populations, to determine who may benefit from immune based targeted therapy. We evaluated the feasibility of gene expression signature detection and the respective landscape of specific tumor infiltrating lymphocytes (TILs), cancer/testis (CT) antigens, and immune checkpoints for possible future personalized immunotherapy eligibility. MethodsDedicated digital mRNA oncologic immune profiling of 770 genes using a Nanostring nCounter® PanCancer Immune Profiling Panel was performed using archived endoscopic ultrasound fine needle biopsy (EUS FNB) PDAC specimens as a case series in a tertiary care setting. ResultsThe spectrum of mRNA gene expression within the tumor specimens revealed that 44.8%, 10.0% and 50.7% of evaluated genes had a ≥ 2-fold increase, a ≤ 2-fold reduction or between <2 and >2 change of mRNA expression, when compared to normal controls. The corresponding landscape of TILs, CT antigens, and immune checkpoints highlighted several possibilities that could potentially be amenable to targeted personalized immunotherapy. This includes members of the Tumor Associated Macrophage family (CD68, CXCL5, and MARCO), members of the CT antigen family (PRAME, TTK and PBK) and the “second generation” checkpoints TIM3 and BTLA. ConclusionsOur study represents the ability to successfully perform digital mRNA expression profile analyses to immunophenotype PDAC EUS FNB specimens by evaluating the expression of >730 genes within the tumor immune microenvironment. This may facilitate the search for novel therapeutic targets, offering the opportunity to go beyond immune monotherapy, but perhaps to use combined immunomodulatory agents.

Long Term Responses to Venetoclax and Ibrutinib in Mantle Cell Lymphoma Are Associated with Immunological Recovery and Prognostic Changes in Inflammatory Biomarkers

Combination Ibrutinib (IB) plus Venetoclax (Ven) for the treatment of Mantle Cell Lymphoma (MCL) has demonstrated efficacy in the relapsed/refractory setting. The AIM trial treated 24 patients with IB monotherapy for 4 weeks (560mg/day) with Ven added (stepwise to 400mg/day) thereafter resulting in an overall response rate of 71% at 16 weeks (Tam et al, NEJM 2018). 5 patients discontinued treatment within 6 months due to resistant disease and 3 patients relapsed. 16 patients remained on treatment for more than 1 year. In this study we examined the longitudinal changes in peripheral blood (PB) immunology in patients treated in the AIM trial in order to identify immunological and inflammatory biomarkers associated with response and to examine if stable control of MCL without further chemotherapy exposure was associated with immunological recovery. Multiparameter flow cytometry of cryopreserved PBMCs collected at enrolment (baseline), and weeks 4, 16, 56 and 110 of treatment was used to assess the proportions of immune subsets (CD4 and CD8 memory subsets, γδ T cells and CD16+ NK cells) in AIM patients and compared to 13 age matched healthy donor (HD) samples. At baseline samples from MCL patients exhibited alterations in multiple immune subsets - most notably skewing in CD8+ memory subsets (Figure 1). Despite reduction of circulating B cell numbers consistent with clinical responses, there were no significant changes to PB immune subset distributions between baseline and 16 weeks. However at the later time points of 56 and 110 weeks there was evidence in responding patients of PB immunology normalisation to HD levels in T cell subsets including a shift in CD8 T cells from Naïve and Central Memory (CM) to Effector Memory (EM) and Terminally differentiated EM (TEMRA) phenotype and a shift in CD4 T cells from Naïve and CM to EM phenotype (Figure 2). Increased proportions of γδ T cells (Baseline 2.8 ± 3.0% week 56 4.4 ± 3.4% P&lt;0.05) and CD16+ NK cells (Baseline 40.1 ± 28.0% week 56 79.1 ± 21.0% P&lt;0.01) were also observed. T cell function assessed by intracellular IFNγ, TNFα and IL-2 in CD4 and CD8 T cells in response to T cell stimulation beads showed no change in IFNγ or TNFα production by CD4 or CD8 cells following therapy. In patients with elevated IL2+ CD4 and CD8 populations at early time points, these normalised by week 56 to HD levels. To further examine changes in immune function pathways, targeted multiplex gene expression profiling using 770 gene NanoString nCounter® PanCancer Immune Profiling Panel was performed on PBMCs at baseline and weeks 4, 16 and 56 and analyzed using nSolver advanced analysis module. Consistent with the flow cytometric findings there were minimal changes in gene expression at week 16 compared to baseline. The greatest changes were observed at week 56 with alterations in multiple pathways including increased signalling through complement, adhesion, TNF superfamily and transporter pathways in responding patients. The top upregulated genes at week 56 were CTSS (Cathepsin S), FCER1G (Fc fragment of IgE), S100A8 (S100 calcium binding protein A8) and CD14 while the top downregulated gene was CD22 (reflecting the change in B cell burden). Increased expression of C1QA and C1QB at week 16 was significantly associated with disease non-response or relapse on IB + Ven and warrants further investigation in an independent cohort to assess its applicability as a predicative biomarker for patient response. Our findings show that long term treatment with combination IB and Ven in MCL is associated with immune recovery and changes in the expression of inflammatory biomarkers. This study demonstrates that while short term assessments can be used to examine clinical disease responses, long term assessments are required to determine the immunological consequences of small molecule inhibitors. Restoration of immune system function in these patients may allow responses to subsequent immunotherapies and suggests that, in contrast to conventional chemotherapy-based regimens, this combination targeted therapy may result in beneficial impacts on immunological recovery. Disclosures Handunnetti: Gilead: Honoraria. Anderson:Walter and Eliza Hall Institute: Employment, Patents &amp; Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; BeiGene: Research Funding; Janssen: Research Funding; Walter and Eliza Hall Institute: Patents &amp; Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees. Seymour:AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy. Tam:Janssen: Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie company: Honoraria; Roche: Honoraria; AbbVie: Honoraria, Research Funding; BeiGene: Honoraria; Novartis: Honoraria. Ritchie:Sanofi: Honoraria; Novartis: Honoraria; Imago: Research Funding; Beigene: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding. Koldej:NanoString Technologies: Other: Travel grant.

Over-Expression of Transferrin Receptor (TFRC/CD71) and Low Expression of Innate and Adaptive Immune Cell Subsets in HIV-Associated, GCB-DLBCL By Digital Gene Expression Profiling

Introduction: HIV infected individuals are 17x more likely to receive a diagnosis of Diffuse Large B cell Lymphoma (DLBCL) compared to their uninfected counterparts. Moreover, DLBCL is more aggressive in HIV infected individuals. However, the molecular pathology driving the aggressive nature of HIV related DLBCL is poorly understood. Previously, we demonstrated that HIV-associated [HIV(+)] germinal center B-cell (GCB) DLBCL tumors are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes compared to their HIV-not associated [HIV(-)] counterparts (Maguire et al, Int J Cancer,2019). Given the immunocompromised nature of these patients, herein we assessed whether specific immunological signaling pathways are also altered in HIV(+) GCB DLBCL tumors, that may work in conjunction with, or independently of, increased DNA repair, to drive the enhanced aggressive nature of HIV related DLBCL. Methods: We used our previously studied cohort of patient samples, which included a total of 39 molecularly defined GCB DLBCL cases. Of these, 18 were HIV(+) cases from the AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 21 were HIV(-) institutional cases. Samples underwent hematopathologist review for diagnosis confirmation and tumor content assessment. Samples with &lt;60% tumor content were macrodissected, with a total of 4x 5µm FFPE sections per sample used for RNA/DNA extraction. Digital gene expression profiling was used to assess differential expression of 579 immune related genes using the NanoString PanCancer Immune Profiling panel. The data was normalized using nSolver, and differential expression analysis was performed using the R statistical software package NanoStringDiff. The resulting expression data were then analyzed using Gene Set Enrichment Analysis (GSEA), and GSEA Molecular Signatures Database (MSigDB) overlap analyses. Results: A total of 110 genes were differentially expressed between the HIV(+) and HIV(-) cohorts at p&lt;0.05. As expected due to immunosuppression in HIV(+) individuals, 100 genes (91%) were significantly reduced and 10 genes (9%) were significantly increased in the HIV(+) cohort compared to the HIV(-) cohort. GSEA and MSigDB analyses revealed that the genes with reduced expression in the HIV(+) cohort were associated with losses in both adaptive and innate immune signaling including cytokine interactions, the IL1 pathway and B-cell receptor signaling amongst others. MSigDB overlap analyses of the 10 genes upregulated in the HIV(+) cohort identified 3 Gene Ontology molecular functions, all of which were receptor signaling related; including Receptor Binding (CD8A, CD160, CCL8, C1QBP, MIF), Receptor Activity (CD8A, CD160, CCRL1, TFRC, KLRC2), and Cargo Receptor Activity (CCRL1, TFRC). Gene overlaps between the subsets include genes associated with NK and cytolytic T cells (CD8A, CD160), as well as the transferrin receptor TRFC/CD71, which with a fold increase of 3.9, was the most significantly increased gene in the HIV(+) cohort (p=0.002, FDR=0.0025). Conclusions: As expected, the results demonstrate a loss in both adaptive and innate immune signaling in the HIV(+) cohort compared to the HIV negative cohort as well as alterations in receptor signaling. In line with our previous observation that HIV(+) GCB DLBCL tumors have higher expression of proliferation genes and the Ki67 antigen than their HIV(-) counterparts, the results of this study also reveal overexpression of TRFC/CD71 mRNA in HIV(+) GCB DLBCL tumors compared to the HIV(-) cases. TFRC/CD71 is well known as a mediator of iron uptake in erythroid cells. Iron uptake through TFRC/CD71 is also necessary for, and positively regulates T and B cell proliferation; and is commonly expressed on aggressive B cell lymphomas. We suggest that TFRC/CD71 may play a role in the enhanced proliferative capabilities of HIV(+) GCB DLBCL. These effects may be mediated through the HIV protein Nef, which has known effects on TFRC/CD71 protein recycling in lymphoid populations (Madrid et al, J Biol Chem 2005), and has been shown to be transferred from HIV infected macrophages to uninfected B-cells via contact-dependent cellular conduits (Xu et al, Nat Immunol 2009). Nef transfer between cells has also been described to occur via microvesicle transfer and trogocytosis (reviewed in Ellwanger et al, Infect Genet Evol 2017). Further analysis of these pathways is ongoing. Disclosures Rimsza: NanoSting: Patents &amp; Royalties: Named inventor on a patent licensed to NanoSting [Institution].

Digital Immune-Related Gene Expression Signatures In High-Grade Serous Ovarian Carcinoma: Developing Prediction Models For Platinum Response.

PurposeResponse to platinum-based therapy is a major prognostic factor in high-grade serous ovarian cancer (HGSOC). While the exact mechanisms of platinum-resistance remain unclear, evidence is accumulating for a connection between the organism’s immune-response and response to platinum. However, predictive tools are missing. This study was performed to examine the putative role of the genetic tumor immune-microenvironment in mediating differential chemotherapy response in HGSOC patients.Patients and methodsExpression profiling of 770 immune-related genes was performed in tumor tissues from 23 HGSOC cases. Tumors were screened for prognostic and predictive biomarkers using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Immune Profiling panel. As validation cohort, gene expression data (RNA Seq) of 303 patients with epithelial ovarian carcinoma (EOC) were retrieved from the The Cancer Genome Atlas (TCGA) database. Different scoring-systems were computed for prediction of risk-of-resistance to cisplatin, disease-free survival (DFS) and overall survival (OS).ResultsValidated on the TCGA-dataset, the developed scores identified 11 significantly differentially expressed genes (p <0.01**) associated with platinum response. HSD11B1 was highly significantly associated with lower risk of recurrence and 7 targets were found highly significantly influencing OS time (p <0.01**).ConclusionOur results suggest that response to platinum-based therapy and DFS in ovarian HGSOC is associated with distinct gene-expression patterns related to the tumor immune-system. We generated predictive scoring systems which proved valid when applied to a set of 303 EOC patients.

1715P - Soft tissue sarcomas express a distinct mRNA immune profile

BackgroundThere is a need for novel therapies in metastatic STS, rendering checkpoint inhibitors (CPI) of interest in STS. Immune cell infiltrates are thought as a prerequisite for CPI efficacy and its role remains vague in STS. We analyzed whether the immune profile differed for STS treated within the phase II EPAZ study (NCT01861951). MethodsRNA samples were measured using the PanCancer Immune Profiling Panel from the nCounter® Analysis System. FCF1, HDAC3, ZKSCAN5, MRPS5 and EIF2B4 were used as housekeeping genes. Samples were categorized in three groups based on their overall mRNA expression via unsupervised random forest analysis. Differences in gene expression were tested by T-tests or ANOVA, with correction for multiple testing. Ingenuity Pathway Analysis (IPA) were performed for activity prediction. K-M plots were used for survival estimates and log-rank analyses for comparisons. ResultsSpecimens were available in 70/120 patients and 31 were assessable by immune profiling. 12 pts. received doxorubicin (DOX) and 18 pazopanib (PAZ). Median progression free survival (PFS) and median overall survival (OS) for the total cohort were 2.6mo. and 11.6mo., respectively. Patients were clustered in high (n=4)/mixed (n=20)/low (n=7) mRNA profile expressions. While OS varied numerically between clusters (11.1/9.0/28.9mo.), values were not significant (P=0.5573). A similar pattern was detected for PFS (4.2/1.5/8.9mo.; P=0.4127). ConclusionsOur study indicates that STS express a differential mRNA immune profile. However, clusters were not associated with outcome measures. A major limitation is the small sample size. Clinical trial identificationNCT01861951. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureV. Grünwald: Honoraria (self), Research grant / Funding (institution): Novartis; Honoraria (self), Advisory / Consultancy: Lilly; Honoraria (self): PharmaMar. M. Schuler: Research grant / Funding (institution): Novartis. P. Schoeffski: Honoraria (institution), Advisory / Consultancy: Daiichi; Honoraria (institution), Advisory / Consultancy: Eisai; Honoraria (institution), Advisory / Consultancy: Lilly; Honoraria (institution), Advisory / Consultancy: Medpace; Honoraria (institution), Travel / Accommodation / Expenses: Novartis; Honoraria (institution): Biovitrium; Honoraria (institution): 6th element capital; Advisory / Consultancy, Travel / Accommodation / Expenses: Adaptimmune; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bayer; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Blueprint; Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Advisory / Consultancy, Travel / Accommodation / Expenses: BoBoehringer Ingelheim; Advisory / Consultancy, Travel / Accommodation / Expenses: Cristal Therapeutics; Advisory / Consultancy, Travel / Accommodation / Expenses: Epizyme; Advisory / Consultancy, Travel / Accommodation / Expenses: Genzyme; Advisory / Consultancy, Travel / Accommodation / Expenses: Ipsen; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Loxo Oncology; Advisory / Consultancy, Travel / Accommodation / Expenses: Nektar; Advisory / Consultancy, Travel / Accommodation / Expenses: Novartis. H. Kopp: Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Advisory / Consultancy, Travel / Accommodation / Expenses: Sanofi; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Novartis; Speaker Bureau / Expert testimony: LeoPharma; Speaker Bureau / Expert testimony: Pfizer; Travel / Accommodation / Expenses: Lilly; Travel / Accommodation / Expenses: Amgen. B. Kasper: Honoraria (self), Advisory / Consultancy: Bayer; Honoraria (self), Advisory / Consultancy: Lilly; Honoraria (self), Advisory / Consultancy: Novartis; Advisory / Consultancy: Eisai ; Honoraria (self), Research grant / Funding (institution): PharmaMar. L. Lindner: Advisory / Consultancy: Novartis; Honoraria (self): Lilly; Honoraria (self): Eisai; Honoraria (self): EZ Medconsultant; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: PharmaMar; Research grant / Funding (institution): Sennewald. J.M. Chemnitz: Honoraria (self): Ablynx; Advisory / Consultancy: Amgen; Advisory / Consultancy: Ablynx; Travel / Accommodation / Expenses: Amgen; Travel / Accommodation / Expenses: PharmaMar. G. Egerer: Honoraria (self): MSD; Advisory / Consultancy: MSD; Honoraria (self): PharmaMar; Travel / Accommodation / Expenses: MSD; Travel / Accommodation / Expenses: Astellas. All other authors have declared no conflicts of interest.

999PD - Immune-related gene expression signatures (irGES) in patients (pts) with ovarian clear cell carcinomas (OCCC): A multicenter analysis

BackgroundWe previously showed that OCCC can be classified into 4 molecular subgroups based on their irGES profiles with distinct clinicopathological characteristics and prognostic outcomes. A combined analysis of samples from 3 independent centres was performed to investigate the prognostic relevance of the irGES profiles in a larger cohort of OCCC patients. MethodsImmune-related gene profiling was performed on 255 FFPE OCCC samples collected from 3 centres (NUH, Singapore, Saitama, Japan, and Edinburgh, UK) using the nanoString nCounter PanCancer Immune Profiling Panel. Unsupervised hierarchical clustering analysis was performed and correlated with clinical outcome. MMR protein levels were assessed by immunohistochemistry. ResultsTotal of 255/264 samples from 3 independent cohorts were successfully profiled. Median age at diagnosis was 57 yrs. 137 (53.7%) were stage 1 (Singapore 46.9%, Saitama 65.8%, Edinburgh 50.6%) with 113 (44.3%) Stages 2-4 (Singapore 47.9%, Saitama 34.2%, Edinburgh 49.4%) and 5 (1.9%) missing data. 68.8%, 80.3% and 80.7% of pts from Singapore, Saitama and Edinburgh respectively, received adjuvant treatment. Median PFS for OCCC pts from Singapore, Saitama and Edinburgh were 27, 29.2 and 31.9 mths, respectively. Median OS were 33.5, 35.1 and 41.6 mths respectively for pts from Singapore, Saitama and Edinburgh. Based on irGES, 4 distinct molecular subgroups of OCCCs were consistently identified and reproducible. The PD-1 high subgroup was observed to have poorer 3yr OS rate (52% vs 57% vs 73% vs 74%, p=0.05) and a trend towards poorer 3 year PFS rate (42% vs 58.7% vs 51% vs 65%, p=0.12) compared to the CTLA4, ProA and AP subgroups respectively. A similar trend was observed when pts were stratified based on stage and whether they received adjuvant treatment. MMR expression levels were noted to be deficient in 6.3% and 5.1% of pts from Singapore and Saitama, respectively and were not significantly associated with any irGES group. ConclusionsClinical outcomes, based on irGES profiles, appeared to be similar across Asian and Caucasian OCCC pts. 4 molecular subgroups based on their irGES profiles were consistently identified between 3 independent cohorts with distinct prognostic outcomes. Legal entity responsible for the studyThe authors. FundingNUHS Clinician Scientist Programme-Residency (NCSP-R) Grant NMRC -Centre grant scheme Yong Siew Yoon fellowship grant Clinician Scientist Award (IMAC) CSA-NMRC grant Nicola Murray Foundation. DisclosureC. Gourley: Honoraria (self), Advisory / Consultancy: Roche, AstraZeneca, Tesaro, Nucana, Clovis, Foundation One, Sierra Oncology, Cor2Ed; Research grant / Funding (self): AstraZeneca, Novartis, Aprea, Nucana, Tesaro; Licensing / Royalties: PCT/US2012/040805, PCT/GB2013/053202, 1409479.1, 1409476.7 and 1409478.3. D.S. Tan: Honoraria (self): Roche, Bayer, MSD, Genmab, AstraZeneca, Merck Serono, Tessa Therapeutics, Novartis; Research grant / Funding (institution): Bayer, Karyopharm, AstraZeneca. R. Huang: Spouse / Financial dependant: ACT genomics Singapore. All other authors have declared no conflicts of interest.

Abstract 582: Tumor immunoediting in a lung cancer mouse model harboring EGFR mutations

Abstract Background: Anti-Programed cell death 1 (PD-1) antibodies such as nivolumab or pembrolizumab show promising treatment effects in smoking-related lung cancer. However, anti-PD-1 antibodies have very little effect on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). EGFR mutations are the most frequent oncogenic drivers in non-smoking related NSCLC. Therefore, we aimed to explore the mechanism underlying tumor immunoediting in a lung cancer mouse model harboring EGFR mutations. Methods: We previously established two strains of transgenic lung cancer mouse (C57BL/6) harboring a mouse Egfr exon 19-deletion (mDEL) or a human EGFR L858R (hLR) mutation, driven by a surfactant protein (SP)-C promoter (Ohashi, Cancer Sci. 2008; Ohashi, Cancer Res. 2009). These mice developed EGFR-dependent multifocal lung adenocarcinomas from type II pneumocytes. We modified the two models by transplanting lung tumors subcutaneously into C57BL/6 (Ohashi, AACR. 2018). Evaluation of mice tumor lymphocyte profile was done through flow cytometry or immunohistochemistry (IHC). Effects of T cell depletion or anti-mouse PD-1 antibody (4H2) in vivo were also assessed. Comprehensive analysis of mRNA expression of immuno-checkpoint molecules in the tumors was performed using PanCancer Immune Profiling Panel (NanoString Technologies). Results: CD4+ T cells and/or CD8+ T cells were depleted in the subcutaneously transplanted tumor model followed by treatment with gefitinib (50 mg/kg/day, 5 days/week) for 2 weeks. As a result, after gefitinib discontinuation, rapid regrowth was seen in the tumors from groups with depletion of CD8+ T cells; depletion of CD4+ T cells had little effect on tumor regrowth, and tumor inhibition continued for 2 weeks after cessation of gefitinib. IHC showed relatively increased numbers of CD4+ T cells in the mice tumors whereas a decrease in the number of CD8+ T cells or Foxp3 positive cells was noted. PD-L1 expression was not observed in the tumors. Consistently, 4H2 showed little effect on tumor growth in vivo. The cancer immune panel showed increased expression of immune mediators such as Ccl6, Clec4n, and Lcn2. Conclusions: Our results suggest that CD8+ T cells can recognize mice tumors in vivo. New immuno-checkpoint molecules regulating CD8+ T cells from PanCancer Immune Profiling Panel were evaluated. Further, we plan to evaluate the efficacy of various immuno-checkpoint inhibitors in vivo. Citation Format: Kazuya Nishii, Kadoaki Ohashi, Kiichiro Ninomiya, Go Makimoto, Hiromi Watanabe, Hirohisa Kano, Naofumi Hara, Heiichiro Udono, Katsuyuki Kiura. Tumor immunoediting in a lung cancer mouse model harboring EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 582.

Abstract 2267: Therapeutic synergy of the chimeric, oncolytic virus VSV-GP with immune modulators

Abstract Introduction: Oncolytic viruses (OVs), which selectively replicate in and destroy tumor cells, represent a highly promising class of cancer therapeutics. In recent years, the focus in the OV field has significantly shifted from a pure oncolytic mode of action to their strong immune stimulatory potential. Tumor cells lysed by the viruses release pro-inflammatory signals, tumor associated antigens as well as display hallmarks of immunogenic cell death. VSV-GP, a chimeric VSV pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) is a potent tumor cell-lysing agent and capable of jump-starting α-tumor immunity. The current study explores the immune promoting properties of VSV-GP and investigates clinically relevant combination therapies with checkpoint inhibition (α-PD-1) as well as a “second mitochondria-derived activator of caspases“ mimetic (SMACm). Experimental Procedures: Immune competent mice bearing established LLC1-IFNARKO, B16-F1-OVA or CT26.CL25-IFNARKO tumors were subject to treatments comprising VSV-GP, α-PD-1, a SMACm or combinations of VSV-GP with either of the latter therapies. α-tumor effects were determined by tumor growth inhibition and/or changes in overall survival. VSV-GP's tumor cell selective replication, resulting in immune activation, was assessed using FFPE based IHC staining for the viral N-protein, PD-L1 and different immune cell subsets as well as the NanoString platform (nCounter Pan Cancer Immune Profiling Panel). Data Summary: Here we present data on VSV-GP, which displays potent α-tumor efficacy and upon systemic delivery increases the local T-cell infiltration, expression of chemokines, T-cell and myeloid cell specific activation markers as well as expression of immune checkpoints, such as PD-1 and PD-L1 within the tumor microenvironment. Building on the observed therapeutic potential of VSV-GP as monotherapy, its immune promoting properties and the delineated compensatory mechanisms, such as PD-1/-L1 upregulation, we explored therapeutic combinations with a PD-1 blocking antibody, which resulted in a strong improvement of VSV-GP-mediated α-tumor efficacy as well as overall survival. Cured, tumor free animals were protected from tumor rechallange indicating immunological memory formation. We further explored the therapeutic combination of VSV-GP with a SMACm, which also resulted in a strongly improved therapeutic benefit and increased overall survival. Conclusion: In summary VSV-GP induces a pro-inflammatory microenvironment within infected tumors, increases immune cell infiltration and synergizes with α-PD-1 as well as a SMACm. VSV-GP is a potent OV with a strong α-tumor immunity promoting potential. Preclinical data justify further testing of VSV-GP and the combinations with PD-1 blockade or a SMACm in clinical trials. Citation Format: Philipp Mueller, Patrik Erlmann, Monika Petersson, Tobias Nolden, Carles Urbiola, Liesa-Marie Schreiber, Bart Spiesschaert, Klaus Erb, Birgit Stierstorfer, Maria Antonietta Impagnatiello, Eric Borges, Knut Elbers, Guido Wollmann, John Park. Therapeutic synergy of the chimeric, oncolytic virus VSV-GP with immune modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2267.

Abstract 2259: Tumor abscopal responses induced by the TLR9 agonist, SD-101, are strongly potentiated by a HDAC class I inhibitor, domatinostat

Abstract In cancer settings severe T cell exhaustion is one major road block limiting immunotherapy efficacy. Epigenetic changes happening during T cell effector differentiation lock T cells in a non-programmable state which limits the efficacy of checkpoint blockade. In addition, histone modification has been reported to critically modulate MHC I and II expression on tumor cells and influx of T cells into tumor. We have previously shown that intratumoral administration of a TLR9 agonist, SD-101, induces T cell responses capable of attacking tumor lesions throughout the body. SD-101, combined with the PD-1 inhibitor pembrolizumab, is currently being investigated in patients with metastatic melanoma and head and neck cancer with encoraging results. Domatinostat (4SC AG) is a well-tolerated orally available selective class I histone deacetylase inhibitor HDACi) with demonstrated anti-tumor activity in hematological cancers as well as in numerous animal models of solid tumors as monotherapy and in combination with checkpoint blockade. Domatinostat is currently being investigated in patients with metastatic melanoma and gastrointestinal cancers in combination with PD(L)-1 antibodies. This study addresses whether the addition of domatinostat to the SD-101 regimen would potentiate the T cell response induced by SD-101. Studies were conducted in multiple syngeneic models in which tumors grow in multiple site of the body but only one is treated intratumorally with SD-101 and domatinostat is given systemically (oral). SD-101 and domatinostat were given alone or in combination, at clinically relevant dosages. The combination of intratumoral SD-101 and domatinostat induced substantial regression of the primary tumor (injected) and distant site cutaneous tumors, as well as lung metastases The combination was superior to either agent given alone and led to increased tumor specific cytotoxic CD8 T cells (CTL) infiltrating distant site lesions. CTL’s were characterized by a low exhaustion phenotype, increased proliferative capacity and increased co-expression of cytolytic markers, CD107 and GranzymeB. Gene expression profiling was performed on abscopal tumors using the PanCancer immune profiling panel from Nanostring. The combination of SD-101 and domatinostat led to upregulation of multiple immune related signatures indicating increased T, NK and dendritic cell functions. Notably, the combination of SD-101 and domatinostat was more efficacious than SD-101 combined with various other epigenetic compounds. The addition of PD-1 blockade to SD-101 and domatinostat further boosted T cell responses leading to rejection in mice with high metastatic burdens. These results suggest that domatinostat acts on SD-101 primed tumor specific CD8+ T cells to expand them, render them more cytolytic and facilitate their infiltration in abscopal tumors. Citation Format: Émilie Degagné, Jose Romo, Marilena Gallotta, Shravan Kannan, Robert L. Coffman, Cristiana Guiducci. Tumor abscopal responses induced by the TLR9 agonist, SD-101, are strongly potentiated by a HDAC class I inhibitor, domatinostat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2259.

Abstract 1174: Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer

Abstract Background: Given the success of both targeted and immunotherapies against cancer, there is increasing utility for identifying targeted agents that also promote anti-tumor immunity. EphA2 is a receptor tyrosine kinase that contributes to tumor growth and metastasis in various cancer types and has been identified as a viable target for non-small cell lung cancer (NSCLC). Here, we examine how tumor-specific EphA2 affects the infiltration of different immune populations, the activation or inhibition of those populations, and the cytokine and chemokine milieu in mouse models of NSCLC. Methods: We generated two murine NSCLC cell lines, one derived from Lewis Lung Carcinoma and the other derived from a primary lung tumor harboring a KRAS mutation, as well as p53 and LKB1 loss of function alterations created using CRISPR-Cas9 gene editing. Effects of EphA2 overexpression in these cells were evaluated in both in vitro assays and in vivo models via subcutaneous tumor implantation and tail vein-injected orthotopic tumor formation in immunocompetent, syngeneic mice. Tumor immune infiltrate was assessed by flow cytometry, and cytokine and chemokine RNA expression levels were evaluated using Nanostring’s PanCancer Immune Profiling Panel. Results: Although EphA2 overexpression in these cell lines did not display significant proliferative advantage in vitro in MTT and colony formation assays, it did confer a growth advantage in vivo. Analysis of lung tumor immune infiltrate revealed decreased NK and T cells in the EphA2-overexpressing tumors, as well as increased myeloid populations, such as macrophages and monocytes. Furthermore, T cell activation markers, such as CD25, CD44, and CD69, in both CD4 and CD8 T cells were decreased in the EphA2-overexpressing tumors, while the percentage of regulatory T cells was increased. These changes in T cell activity were accompanied by increased presence of monocyte-derived CD11b+ Ly6C+ Ly6G- cells and Gr1- F4/80+ MHCII+ tumor-associated macrophages (TAMs). Higher infiltration of these myeloid populations coincided with increased monocyte-attracting chemokine and receptor expression, including CCL2, CCL7, CCL8, CCL12, and CCR2 and increased expression of immunosuppressive proteins, including TGFβ and arginase 1. Conclusion: Our studies suggest tumor-specific EphA2 inhibits infiltration and activation of key lymphocytic populations, especially T cells, while recruiting monocytes and likely promoting their transformation into myeloid-derived suppressor cells (MDSCs) and TAMs. Further functional studies are needed to validate the role of these myeloid cells. Elucidation of EphA2’s role in regulating immune recruitment and function will advance our understanding of tumor immune evasion and the potentially beneficial consequences of targeting EphA2 in cancer on the tumor microenvironment. Citation Format: Eileen Shiuan, Wenqiang Song, Shan Wang, Mark Boothby, Jin Chen. Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1174.