An Inflammatory Response Related Gene Signature Associated with Survival Outcome and Gemcitabine Response in Patients with Pancreatic Ductal Adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors with an extremely low 5-year survival rate. Accumulating evidence has unveiled that inflammatory response promotes tumor progression, enhances angiogenesis, and causes local immunosuppression. Herein, we aim to develop an inflammatory related prognostic signature, and found it could be used to predict gemcitabine response in PDAC.Methods: PDAC cohorts with mRNA expression profiles and clinical information were systematically collected from the four public databases. An inflammatory response related genes (IRRGs) prognostic signature was constructed by LASSO regression analysis. Kaplan–Meier survival analysis, receiver operating characteristic analysis, principal component analysis, and univariate and multivariate Cox analyses were carried out to evaluate effectiveness, and reliability of the signature. The correlation between gemcitabine response and risk score was evaluated in the TCGA-PAAD cohort. The GDSC database, pRRophetic algorithm, and connectivity map analysis were used to predict gemcitabine sensitivity and identify potential drugs for the treatment of PDAC. Finally, we analyzed differences in frequencies of gene mutations, infiltration of immune cells, as well as biological functions between different subgroups divided by the prognostic signature.Results: We established a seven IRRGs (ADM, DCBLD2, EREG, ITGA5, MIF, TREM1, and BTG2) signature which divided the PDAC patients into low- and high-risk groups. Prognostic value of the signature was validated in 11 PDAC cohorts consisting of 1337 PDAC patients from 6 countries. A nomogram that integrated the IRRGs signature and clinicopathologic factors of PDAC patients was constructed. The risk score showed positive correlation with gemcitabine resistance. Two drugs (BMS-536924 and dasatinib) might have potential therapeutic implications in high-risk PDAC patients. We found that the high-risk group had higher frequencies of KRAS, TP53, and CDKN2A mutations, increased infiltration of macrophages M0, neutrophils, and macrophages M2 cells, as well as upregulated hypoxia and glycolysis pathways, while the low-risk group had increased infiltration of CD8+ T, naïve B, and plasma and macrophages M1 cells.Conclusion: We constructed and validated an IRRGs signature that could be used to predict the prognosis and gemcitabine response of patients with PDAC, as well as two drugs (BMS-536924 and dasatinib) may contribute to PDAC treatment.

Local immunosuppression in WZS-pig to rhesus monkey Descemet’s stripping automated endothelial keratoplasty: An innovative method to promote the survival of xeno-grafts

Corneal xenotransplantation is an effective solution for the shortage of human corneas. We investigated the feasibility and efficacy of different postoperative protocols on xeno-Descemet's stripping automated endothelial keratoplasty (DSAEK) grafts. Thirty rhesus monkeys were randomly divided into three groups: control group (C), only Descemet's membrane (DM) stripping; DSAEK 1 (D1) and DSAEK 2 (D2) groups, DM stripping followed by endothelial keratoplasty. Betamethasone 3.5 mg was subconjunctival injected in groups control and D1 postoperatively, while animals in group D2 were treated with topical 0.1% tacrolimus and topical steroids. All groups were evaluated by slit-lamp microscopy, anterior segment OCT and LSCM for at least nine months. A total of 24 monkeys (24 eyes) met the inclusion criteria. Nine months after DSAEK surgery, all xenografts showed good attachment, and most corneas were transparent. Graft rejection occurred in 25% of the cases in group D1 and 28.57% of those in group D2 (P > 0.05). The corneal endothelium density in the DSAEK groups was 2715.83±516.20/mm² (D1) and 2220.00 ± 565.13/mm² (D2) (P > 0.05). Xenogeneic corneal endothelial grafts can survive and function in rhesus monkey eyes for a long time with subconjunctival steroid or topical tacrolimus and steroid treatment.

Reshaping the tumor microenvironment with oncolytic viruses, positive regulation of the immune synapse, and blockade of the immunosuppressive oncometabolic circuitry

Immune-related therapies have revolutionized the management of cancer. Oncolytic viruses are now considered part of the immunotherapy armamentarium and have shown promise in clinical trials of patients with glioblastoma. These studies have suggested that tumor microenvironment remodeling is required to achieve an effective response in solid tumors. Here, we showed that Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus expressing the T cell activator OX40L, triggered antitumor immune responses. However, Delta-24-RGDOX also elicited paradoxical activation of the cytokine-driven immunosuppressive IDO-kynurenine-AhR circuitry. The IDO-kynurenine-AhR cascade had the dual effects of preventing optimum viral replication and decreasing the virus-initiated antitumor immune response. To enhance virotherapy, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors. This combination therapy increased the frequency of activated CD8+ T cells and decreased the frequencies of immunosuppressive MDSC and Treg populations in animal models of gliomas and melanoma. Functional studies demonstrated that the IDO blockade-dependent activation of immune cells against tumor antigens could be reversed by the oncometabolite kynurenine. The concurrent targeting of effectors and suppressors in the tumor immune landscape significantly prolonged the survival of glioma- and melanoma-bearing mice. Our data identified the striking role of immunosuppressive pathways in the resistance of solid tumors to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas and other cancers treated with virotherapy.

Hypoxia Regulates Endogenous Double-Stranded RNA Production via Reduced Mitochondrial DNA Transcription

Hypoxia is a common phenomenon in solid tumours strongly linked to the hallmarks of cancer. Hypoxia promotes local immunosuppression and downregulates type I interferon (IFN) expression and signalling, which contribute to the success of many cancer therapies. Double-stranded RNA (dsRNA), transiently generated during mitochondrial transcription, endogenously activates the type I IFN pathway. We report the effects of hypoxia on the generation of mitochondrial dsRNA (mtdsRNA) in breast cancer. We found a significant decrease in dsRNA production in different cell lines under hypoxia. This effect was HIF1α/2α-independent. mtdsRNA was responsible for induction of type I IFN and significantly decreased after hypoxia. Mitochondrially encoded gene expression was downregulated and mtdsRNA bound by the dsRNA-specific J2 antibody was decreased during hypoxia. These findings reveal a new mechanism of hypoxia-induced immunosuppression that could be targeted by hypoxia-activated therapies.

Blockade of Tigit on AML-Derived M2 Macrophages Results in Reprograming into the M1 Phenotype and Enhances CD47-Mediated Phagocytosis

Background: Bidirectional interactions between the tumor microenvironment (TME) and AML cells lead to disease progression through induction of angiogenesis, migration, cancer stemness and local immunosuppression. Leukemia-associated macrophages (LAM) constitute an important cell population within the TME, but little is known about the phenotype, function, and plasticity of these cells. In the present study we provide an extensive characterization of the macrophage population in patients with AML. Methods: The phenotype and expression of co-regulatory receptors was assessed on different bone marrow-derived CD68 +CD14 + LAM populations, in comparison to corresponding CD3 + T-cells and CD117 +CD34 + AML cells (n=35), as well as peripheral blood monocytes from healthy donors (HD, n=16) using multi-parameter flow cytometry. The expression of surface markers and the distribution of LAM subpopulations was correlated with clinical parameters. The effect of a blocking anti-TIGIT antibody on the in vitro plasticity on primary LAMs and monocyte-derived macrophages from healthy donors was investigated. Furthermore, we analyzed if the treatment with blocking anti-TIGIT and anti-CD47 antibodies could increase the anti-leukemic phagocytosis of AML cell lines and in vitro polarized monocyte-derived M2 macrophages. Results: Phenotypic analysis of M1 and M2 macrophages in AML and HD revealed that the predominant macrophage population in patients with AML is made up of immunosuppressive alternatively activated M2 LAMs defined by expression of CD163 and CD86 (M1 AML vs. HD p<0.01 and M2 AML vs. HD p=0.02). These M2 LAMs contained significantly higher frequencies of cells expressing the immune checkpoint receptors TIGIT and TIM-3 than M1 LAMs (TIGIT + M2 vs. M1 p<0.01 and TIM-3 + M2 vs. M1 p<0.01, respectively). Regarding co-expression of multiple co-inhibitory receptors, the frequency of macrophages co-expressing TIM-3 or LAG-3 with TIGIT was higher in samples from AML patients in comparison to HDs (p=0.01 and p<0.01, respectively). This difference was caused by the significant up-regulation of TIM-3 and LAG-3 on TIGIT + M2 LAMs in comparison to their corresponding M1 LAMs (p<0.01and p<0.01, respectively). Importantly, in vitro blockade of TIGIT in primary LAMs of AML patients or differentiated PB-derived M2 macrophages of HDs resulted in a change in polarization from the M2 towards the M1 phenotype after 24 hours (AML: anti-TIGIT vs. IgG2a p<0.01, n=7 and HD: anti-TIGIT vs. IgG2a p=0.02, n=3). Moreover, the additional blockade of TIGIT on PB-derived M2 macrophages augmented the anti-CD47-mediated phagocytosis of the AML cell lines MOLM-13 and MV4-11 after 4 hours (MOLM-13: anti-CD47 vs. IgG1a 31% vs. 10.9%, p=0.04; anti-CD47 vs. combined anti-CD47 + anti-TIGIT 31% vs. 46.4%, p<0.01 and combined anti-CD47 + anti-TIGIT vs. IgG1a + IgG2a 46.4% vs. 13.6%, p<0.01, n=3 and for MV4-11: anti-CD47 vs. IgG1a 14.4% vs. 7.345%, p=0.03; anti-CD47 vs. combined anti-CD47 + anti-TIGIT 14.4% vs. 28.6%, p=0.03 and combined anti-CD47 + anti-TIGIT vs. IgG1a + IgG2a 28.6% vs. 12.85%, p=0.04, n=2). Next, we correlated the phenotypic data with clinical parameters. AML patients of the intermediate risk group according to ELN criteria exhibited a significantly higher frequency of M2 LAMs co-expressing TIGIT and LAG-3 than those in the favorable group (p=0.04 and p=0.01). Moreover, the frequency of TIM-3 + M2 LAMs was significantly increased in patients with adverse and intermediate risk in comparison to those with a favorable risk (p=0.01, p=0.0053). Furthermore, TIGIT + M2 LAMs were significantly more frequent in patients with the FLT3 ITD mutation in comparison with the wilde type (p=0.03). Conclusions: Our findings suggest that the proven clinical effect of monoclonal antibodies against TIGIT and TIM-3 in cancer may be due in part to their action on macrophages and depend on macrophage polarization. Our study identifies TIGIT + M2 LAMs co-expressing TIM-3 and LAG-3 as a promising effector population in AML. Further experiments should be conducted to investigate macrophage-mediated cytotoxicity in AML. Disclosures Brauneck: Daiichi Sankyo: Consultancy, Honoraria, Other: meeting attendance; Servier: Consultancy, Honoraria, Other: meeting attendance; Jazz Pharmaceuticals: Other: meeting attendance; Novartis: Other: meeting attendance. Bokemeyer: BMS: Honoraria, Other: Travel accomodation, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel accomodation; Merck Serono: Consultancy, Other: Travel accomodation ; Bayer Schering Pharma: Consultancy; GSO: Consultancy; AOK Health insurance: Consultancy; Abbvie: Research Funding; ADC Therapeutics: Research Funding; Agile Therapeutics: Research Funding; Alexion Pharmaceuticals: Research Funding; Amgen: Research Funding; Apellis Pharmaceuticals: Research Funding; Astellas: Research Funding; BerGenBio: Research Funding; Blueprint Medicine: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Eisai: Research Funding; Gilead Sciences: Research Funding; Gylcotope GmbH: Research Funding; GlaxoSmithKline: Research Funding; Inside: Research Funding; IO Biotech: Research Funding; Isofol Medical: Research Funding; Janssen-Cilag: Research Funding; Karyopharm Therapeutics: Research Funding; Lilly: Research Funding; Millenium: Research Funding; MSD: Research Funding; Merck KGaA: Honoraria; Bayer: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Merck Sharp Dohme: Consultancy, Honoraria; AstraZeneca: Honoraria, Research Funding; Lilly/ImClone: Consultancy; Nektar: Research Funding; Rafael Pharmaceuticals: Research Funding; Springworks Therapeutics: Research Funding; Taiho Pharmaceutical: Research Funding; Pfizer: Other. Fiedler: Celgene: Consultancy; Servier: Consultancy, Other: support for meeting attendance; Abbvie: Consultancy, Honoraria; Morphosys: Consultancy; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Other: support for meeting attendance; Jazz Pharmaceuticals: Consultancy, Other: support for meeting attendance; Stemline: Consultancy; Novartis: Consultancy; ARIAD/Incyte: Consultancy; Amgen: Consultancy, Other: support for meeting attendance, Patents & Royalties, Research Funding.

EXTH-13. LOCAL DELIVERY OF AN IL-15 SUPERAGONIST USING A REPLICATING RETROVIRUS SIGNIFICANTLY IMPROVES SURVIVAL AND LYMPHOCYTE INFILTRATION IN POORLY IMMUNOGENIC MURINE GLIOBLASTOMA MODELS

Glioblastoma (GBM) leads to severe systemic and local immunosuppression, and immunotherapies have had limited clinical success. Here, we evaluated the treatment efficacy of RLI, a superagonist of T-cell activator IL-15, delivered to tumor cells using a tumor-selective retroviral replicating vector (RRV) in the syngeneic murine SB28 and Tu2449 GBM models, which are both engineered to be poorly immunogenic with low-mutational burden and known resistance to immunotherapy, and hence more accurate biomimetic models of human GBM. RRV-RLI replicated and spread effectively in cultured murine GBM cells with robust production of functional RLI (165.4 ± 5.3 ng/mL). Stereotactic injection of RRV-RLI into pre-established intracerebral SB28 tumors significantly reduced tumor growth on bioluminescent imaging, and increased median survival compared to control mice (55 vs. 19 days, p=0.002), leading to long-term survival in 12% of treated mice. In the Tu2449 model, imaging results showed complete eradication of intracerebral tumors after RRV-RLI treatment, with long-term survival (median not reached) in > 85% of treated mice, compared to a median survival of 12.5 days in control mice (p=0.001). RRV-RLI treated tumors showed significantly increased CD8 T-cell infiltration, without altering immunosuppressive cell populations. Similarly, broad anti-tumor inflammatory changes, including increased expression of genes involved in T-cell activation and killing, were observed in the NanoString nCounter platform using a 770-gene panel representing various immune cell types. Notably, RLI was not detected in the blood of treated mice, and tumor-localized RRV-RLI gene delivery showed no adverse systemic immune effects in either model. In summary, RRV-mediated RLI immunotherapy results in immunostimulatory and pro-inflammatory changes to the tumor microenvironment and achieves a significant survival benefit in two poorly immunogenic syngeneic murine models of GBM. This tumor-localized immunomodulatory gene therapy has the potential to safely reverse the T-cell depleted immunophenotype of GBM.

568 Tumor-derived GDF-15 prevents therapy success of checkpoint inhibitors by blocking T-lymphocyte recruitment

BackgroundImmune checkpoint blockade (ICB) can achieve durable responses in a subgroup of patients with metastatic cancer, only. Poor immune effector cell infiltration into the tumor microenvironment is a major obstacle to successful therapy. Growth and differentiation factor 15 (GDF-15) is a divergent member of the TGF-β superfamily and has been linked to feto-maternal tolerance, anorexia but recently also to potent local immunosuppression under physiologic and pathophysiologic conditions. GDF-15 is overexpressed in a wide variety of tumors and may be key factor produced by tumors to prevent effective immune cell infiltration into the tumor and to potently block checkpoint inhibitor activity.MethodsEffects of recombinant GDF-15 and a proprietary GDF-15 neutralizing antibody (CTL-002) on immune cell trafficking and activation were analyzed by adhesion and interaction assays and in melanoma-bearing humanized mouse models. The impact of GDF-15 overexpression was tested in subcutaneously implanted, GDF-15-transgenic MC38 cells. Additionally, patient GDF-15 serum levels were correlated with immune infiltration and OS in cutaneous melanoma. Associations between GDF-15 serum levels, response to PD-1-based ICB and corresponding OS were assessed in two independent cohorts of melanoma patients.ResultsGDF-15 impairs adhesion of T and NK cells on activated endothelia. In HV18-MK bearing humanized mice, inhibition of GDF-15 strongly enhances infiltration of activated myeloid and lymphoid cells. In MC38 tumors, GDF-15 overexpression can abrogate tumor rejection upon anti-PD-1 therapy. 50% of the mice with GDF-15 overexpressing tumors were, however, rescued when anti-PD-1 was combined with anti-GDF-15 (CTL-002). Likewise, anti-GDF-15 improved responses to anti-CD40 + poly(I:C) in the same tumor model. Clinically, inverse correlations of GDF-15 levels with CD8+ T cell infiltration were shown for melanoma brain metastases. In two independent melanoma patient cohorts, low baseline serum GDF-15 levels predicted clinical response to anti-PD1 treatment and superior OS. Bivariate analysis including LDH indicates that GDF-15 is an independently predictor for poor survival in anti-PD-1 treated melanoma patients.ConclusionsTumor-derived GDF-15 blocks the infiltration of immune effector cells into tumor tissues. Neutralizing GDF-15 with CTL-002 restores the ability of immune cells to extravasate blood vessels and enter the tumor microenvironment in vivo. GDF-15 thus represents a promising target for cancer immunotherapy. Antibodies against GDF-15 may support treatments with anti-PD-1 and other immunotherapeutic agents. A clinical trial combining anti-GDF-15 (CTL002) with anti-PD-1 (NCT04725474, submitted Abstract ID 15073) is ongoing.Ethics ApprovalUse of patient samples for this study had been approved by the institutional ethics committee Tübingen (ethic vote 125/2015BO2). Use of surplus sera collected in the University of Zurich Hospital (USZ) Biobank during routine blood draws from consenting metastatic melanoma patients was performed according to IRB approval (KEK.Zh- 647/800) and followed the Declaration of Helsinki on Human Rights.ConsentAll patients had given written informed consent to have clinical data recorded by the Central Malignant Melanoma Registry (CMMR) database.

High PANX1 Expression Leads To Neutrophil Recruitment And Formation of A High Adenosine Immunosuppressive Tumor Microenvironment In Triple-Negative Breast Cancer

High adenosine levels are an important characteristic of the tumor microenvironment (TME) in triple-negative breast cancer (TNBC). Pannexin 1 (PANX1) can release intracellular ATP to the extracellular space and elevate extracellular ATP (exATP) levels under physiological conditions. We performed public database bioinformatics analysis, surgical specimen histological validation, RNA sequencing, and exATP/extracellular adenosine (exADO) assays in vitro and in vivo to reveal the role of PANX1 in regulating the immune microenvironment of TNBC. Our results revealed that PANX1 was highly expressed and acted as a poor prognostic factor in TNBC. The PANX1 expression level was positively correlated with exATP and exADO levels in the TNBC TME. PANX1 promoted infiltration of tumor-associated neutrophils (TANs) through exATP, and TANs highly expressed ENTPD1 (CD39) / NT5E (CD73). This study suggests that high PANX1 expression could promote TAN accumulation and adenosine production in the TNBC TME which have been shown to induce local immunosuppression.

Non-invasive Fungal Sinusitis as a Complication of a Steroid-Eluting Stent Following Endoscopic Sinus Surgery: A Case Report

Objective: Steroid eluting stents have proven to be a highly useful adjunctive therapy for chronic rhinosinusitis (CRS) and play an important role in the treatment of many inflammatory diseases of the sinuses. Few reports of adverse events were reported in clinical trials and are described in the literature. However, we describe the first known case of an immunocompetent patient developing non-invasive fungal tissue infection as a sequelae of stent-related tissue necrosis requiring surgical debridement. Methods: A 69-year-old immunocompetent male with CRS had Propel™ stents placed in the bilateral frontal sinus outflow tracts during revision endoscopic sinus surgery. He presented 2 weeks post-operatively with severe facial pain without vision changes, fevers, mental status changes, or evidence of cranial neuropathies. On rigid nasal endoscopy, necrotic tissue and gross fungal elements were visualized in the left frontal sinus outflow tract at the area of previous steroid stent position. Results: The patient was taken for urgent endoscopic sinus surgery and debridement given significant symptoms and concern for invasive fungal infection. A revision left maxillectomy, ethmoidectomy, and draf 2b frontal sinus drillout were performed, with healthy bleeding tissue encountered beneath necrotic tissue. Pathology revealed tissue necrosis, exudative lumenal debris, and extensive fungal elements with no evidence of tissue invasion, and cultures yielded growth of aspergillus niger. The patient’s symptoms improved significantly on post-operative day 1, he had normal post-operative changes at 2 weeks following debridement, and had no recurrence of fungal infection with complete healing at 4 months. Conclusion: While likely rare, steroid-eluting stents may pose a risk of saprophytic tissue infection as a result of tissue necrosis and local immunosuppression. Caution should be taken in using these devices in immunocompromised patients.

Safety and efficacy of the anti-CD73 monoclonal antibody (mAb) oleclumab ± durvalumab in patients (pts) with advanced colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), or EGFR-mutant non-small cell lung cancer (EGFRm NSCLC).

9047 Background: Upregulation of CD73 in multiple cancers increases adenosine production, leading to local immunosuppression. Oleclumab, a human IgG1λ mAb, inhibits CD73 function and may increase antitumor immunity. Initial data from a Phase I, first-in-human, dose-escalation and expansion study showed that oleclumab ± durvalumab had manageable safety and encouraging clinical activity in pts with advanced CRC or PDAC. We report updated safety and activity in these cohorts and the first results in an expansion cohort of pts with advanced EGFRm NSCLC. Methods: Previously treated pts with histologically or cytologically confirmed microsatellite stable CRC, PDAC, or EGFRm NSCLC received oleclumab 5–40 mg/kg (escalation) and 40 mg/kg (expansion) IV Q2W, alone (escalation only) or with durvalumab 10 mg/kg IV Q2W. The primary objective was safety; secondary efficacy objectives included objective response (OR) per RECIST v1.1 and duration of response (DoR). Results: 66 pts were enrolled in the escalation phase (35 CRC, 31 PDAC) and 126 in the expansion phase (42 CRC, 42 PDAC, 42 EGFRm NSCLC). At data cutoff (DCO; June 9, 2020), the median number of oleclumab doses was 4 in pts on monotherapy (range 1–26) and 4 in pts on combination therapy across both phases (range 1–76). In the escalation phase, there were no DLTs in pts on monotherapy or combination therapy; treatment-related adverse events (TRAEs) occurred in 54.8% of pts on monotherapy (Grade 3–4 in 7.1%) and 54.2% of pts on combination therapy (Grade 3–4 in 20.8%); fatigue was the most common TRAE with both regimens. No TRAEs resulted in death. In previous interim analyses before this DCO, no ORs were reported in the escalation phase. In the expansion phase, 5 pts were treated for ≥12 mos; 6 pts were ongoing at DCO. TRAEs occurred in 54.0% (Grade 3–5 in 15.1%); the most common TRAEs were fatigue (15.1%), diarrhea (9.5%), and rash (7.1%). One pt had a TRAE resulting in death (systemic inflammatory response syndrome). ORs were seen in 1 CRC pt (DoR 35.9+ mos [+ = ongoing response]), 2 PDAC pts (DoR 22.1+ and 28.6+ mos), and 4 EGFRm NSCLC pts (DoR range 5.6 to 15.7+ mos, median not reached; only 1 of the 4 pts had ≥25% programmed cell death ligand-1 [PD-L1]+ tumor cells). Nine CRC pts, 8 PDAC pts, and 9 EGFRm NSCLC pts had SD. Of 6 pts with matched biopsies who received combination therapy, 5 had increases in CD8+ T cells, PD-L1, and granzyme B. Baseline tumor CD73 expression and association with clinical response will be presented. Conclusions: Oleclumab ± durvalumab had a tolerable safety profile and combination therapy showed promising antitumor activity in EGFRm NSCLC. ORs and SD were durable, even in tumor types that are generally immunotherapy-resistant. Clinical trial information: NCT02503774.