System biology session: immunology
O1 Interferon-gamma (INFG), a predictive factor of response to immune checkpoint blockade (ICB) in melanoma and non-small cell lung cancer (NSCLC)
Maria Gonzalez-Cao1,2, Niki Karachaliou2,3, Guillermo Crespo4, Erika Aldeguer2, Ana Drozdowskyj5, Ana Giménez-Capitán2, Cristina Teixidó2, Miguel Angel Molina-Vila2, Santiago Viteri Ramírez1,2, Salvador Martin Algarra6, Elisabeth Pérez-Ruiz7, Iván Márquez-Rodas8, Delvys Rodriguez-Abreu9, Remei Blanco10, Teresa Puértolas11, Maria Angeles Royo12, Rafael Rosell1,2,3,13, on behalf of the Spanish Melanoma Group (GEM)
1Quirón Salud-Dexeus University Institute, IOR, Medical Oncology Department, Barcelona, Spain; 2Pangaea Oncology, Quirón-Dexeus University Institute, Laboratory of Cellular and Molecular Biology, Barcelona, Spain; 3Hospital Universitari Sagrat Cor, IOR-Grupo Quirón Salud-Oncology Department, Barcelona, Spain; 4Hospital Universitario de Burgos, Burgos, Spain; 5Pivotal, Madrid, Spain; 6Clínica Universitaria de Navarra, Pamplona, Spain; 7Hospital Costa del Sol, Oncology Department, REDISSEC, Marbella, Spain; 8Hospital Gregorio Marañon, Madrid, Spain; 9Hospital Universitario Insular De Gran Canaria, Las Palmas De Gran Canaria, Spain; 10Consorcio Sanitario De Terrassa, Terrassa Barcelona, Spain; 11Miguel Servet University Hospital, Aragon’s Health Research Institute, Medical Oncology Department, Zaragoza, Spain; 12Hospital Dr Peset, Valencia, Spain; 13Catalan Institute of Oncology, Germans Trias i Pujol Health Sciences Institute and Hospital, Barcelona, Spain
Journal of Translational Medicine 2018, 16 (Suppl 1):O1
Background: PD-L1 is up-regulated via INFG in a STAT1- and NFκB-dependent manner. We explored whether INFG expression in pre-treatment tumors is associated with the activity of ICB in NSCLC and melanoma patients. The role of inflammation-associated transcription factors STAT3, IKBKE and STAT1 was also examined.
Methods: Total RNA from 17 NSCLC and 21 melanoma patients, was analyzed by qRT-PCR. STAT3 and Rantes, YAP1 and CXCL5, DNMT1, RIG1 and TET1, EOMES, INFG (encoding for INFγ), PD-L1 and CTLA4, IKBKE and NFATC1 mRNA were examined. PD-L1 protein expression in tumor and immune cells and stromal infiltration of CD8+ T cells were also evaluated.
Results: 17 previously treated NSCLC patients received nivolumab; 71% lung adenocarcinoma, 71% male, 53% smokers, 35% KRAS mutant, 88% EGFR wild-type (wt). 21 previously treated melanoma patients received pembrolizumab; 67% male, 67% BRAF wt. PFS to nivolumab was significantly longer in NSCLC patients with high vs low INFG expression (5.12 vs 2 months, p = 0.0124). PFS to pembrolizumab was significantly longer in melanoma patients with high vs low INFG expression (4.99 vs 1.86 months, p = 0.0099). Significantly longer OS was observed for melanoma patients with high vs low INFG expression (not reached vs 3.10 months, p = 0.0183). There was a trend for longer OS for NSCLC patients with high vs low INFG expression (10.15 vs 4.86 months, p = 0.0687). A survival plateau was only observed for patients with high INFG levels: OS21m 60% vs 0% for melanoma, and 15% versus 0% for NSCLC). Clinical benefit (CB)(objective response or stable disease) was observed in 71.43% of NSCLC patients with high INFG levels versus 0% in patients with low levels; for melanoma patients CB was observed in 71.4% of patients with high IFNG levels versus 20% for patients with low INFG. The in tumor and immune cells did not affect the outcome to ICB. IKBKE was positively correlated with INFG and PD-L1 expression (NSCLC Spearman’s ρ = 0.58 and 0.65; melanoma Spearman’s ρ = 0.61 and 0.59), and STAT3 expression was loosely anticorrelated with PD-L1 expression (NSCLC Spearman’s ρ = − 0.21; melanoma Pearson’s ρ = − 0.01). The rest of the biomarkers explored did not affect the outcome to immunotherapy.
Conclusions: NSCLC and melanoma patients with intermediate/high INFG mRNA expression exhibited longer PFS and OS and higher disease control rates with anti PD-1 therapies even when the levels of PD-L1 expression were low.
O2 Biomarkers for cancer immunotherapy: predicting the immune resistance through gene expression profile
Maria Libera Ascierto1,2
1Medimmune, Gaithersburg, MD, USA; 2The Johns Hopkins University, Baltimore, MD, USA
Journal of Translational Medicine 2018, 16 (Suppl 1):O2
Background: Surgery, radiation and chemotherapy have been long considered the three pillars of cancer care. A fourth pillar, immunotherapy, recently expanded providing an exciting new treatment option for many patients. In this regard, monoclonal antibodies blocking PD-1/PD-L1 immune checkpoint have shown promising clinical results .
Despite these very encouraging results, the majority of patients do not respond to immunotherapy regimens as monotherapy, leading to an urgency to identify biomarkers that accurately predict which patients will benefit from treatment and potentially actionable mechanisms of resistance in order to set successful combinatorial approaches . To this end, gene-expression profiling has been here applied on lesions derived from patients with renal cell carcinoma (RCC), melanoma and classical Hodgkin lymphoma (CHL) thus unveiling novel and paradoxical relations between cancer and immune system leading to immunotherapy resistance.
Method: RNA was isolated from (i): 13 formalin-fixed, paraffin-embedded (FFPE) pre-PD-1 treatment tumor biopsies derived from RCC patients; (ii): 10 FFPEs regressing/progressing cutaneous metastases derived from on autopsy case of melanoma; (iii): 24 FFPEs derived from CHL Epstein Barr virus (EBV) positive (+) and negative (−) patients.
RNA was subjected to whole genome microarray and multiplex quantitative (q) RT-PCR.
Results: In renal cell carcinoma, gene expression profile highlighted metabolic and immunologic molecules to be associated with the effective response to immunotherapy with anti- PD-1 blockade . In melanoma, transcriptional signatures mostly associated with epithelial to mesenchymal transition (EMT) and accumulation of neutrophils were found to be associated with PD-1 blockade therapy resistance . In CHLs, results revealed a dichotomous cellular and cytokine immune milieu in EBV+ vs EBV− CHL . Particularly, EBV+ tumors displayed a T helper 1 (Th1) profile while EBV-tumors manifested a pathogenic Th17 profile and ongoing engagement of the interleukin-23 (IL-23)/IL-17 axis5. These findings suggest that drugs blocking the IL-23/IL-17 axis, may enhance the therapeutic impact of immunotherapy in EBV–CHL.
Conclusions: Many pathways might determine the clinical response to immunotherapies in cancer patients, thus suggesting that in the evaluation of biomarkers associated with response to immunotherapy, all intersections between immunological, genetic and tissue specific factors must be evaluated. Merging together the usage of high-throughput screenings, bioinformatic analysis and immune biology assays might be necessary to establish a framework for describing the diversity of these interactions with the aim to focus on features that help guide immunotherapeutic treatment choices on an individual basis (i.e. personalized medicine).
Acknowledgments Studies here described have been conducted at Johns Hopkins University during author’s post-doctoral fellowship. Special thanks goes to S.L. Topalian for post-doctoral mentorship, insight suggestions and continuous support. The author also thanks E.J. Lipson, J.M. Taube, T.L. McMiller, A.E. Berger, A.S. Duffield, C.G. Drake, R.F. Ambinder, and D.M. Pardoll for advices, technical assistance and data analysis.
Ascierto ML, Melero I, Ascierto PA. Melanoma: from incurable beast to a curable bet. The success of immunotherapy. Front Oncol. 2015;5:152.
Bedognetti D, Marincola FM, Wang E, Ascierto ML. Molecular profiling of immunotherapeutic resistance. In: Prendergast GC, Jaffee EM. Cancer immunotherapy: immune suppression and tumor growth. 2nd ed. Elsevier; 2013.
Ascierto ML, McMiller TL, Berger AE, Danilova L, Anders RA, Netto GJ, Xu H, Pritchard TS, Fan J, Cheadle C, Cope L, Drake CG, Pardoll DM, Taube JM, Topalian SL. The intratumoral balance between metabolic and immunologic gene expression is associated with anti-PD-1 response in patients with renal cell carcinoma. Cancer Immunol Res. 2016;4:726–733.
Ascierto ML, Makohon-Moore A, Lipson EJ, Taube JM, McMiller TL, Berger AE, Fan J, Kaunitz GJ, Cottrell TR, Kohutek ZA, Favorov A, Makarov V, Riaz N, Chan TA, Cope L, Hruban RH, Pardoll DM, Taylor BS, Solit DB, Iacobuzio-Donahue CA, Topalian SL. Transcriptional mechanisms of resistance to anti-PD-1 therapy. Clin Cancer Res. 2017;23(12):3168–3180.
Duffield AS, Ascierto ML, Anders RA, Taube JM, Meeker AK et al. Chen S, McMiller TL, Phillips NA, Berger AE, Pardoll DM, Topalian SL, Ambinder RF. Th17 immune microenvironment in Epstein–Barr virus negative Hodgkin lymphoma: implications for immunotherapy. Blood Adv (In press).
O3 4SC-202 induces inflamed tumor microenvironment, strongly enhances tumor infiltration with cytotoxic T cells and primes tumors for anti-PD-1/PD-L1 therapy
Svetlana Hamm, Tanja Wulff, Kerstin Kronthaler, Sabine Schrepfer, Ulrike Parnitzke, Anne Catherine Bretz, Roland Baumgartner
4SC AG, 82152 Planegg, Martinsried, Germany
Journal of Translational Medicine 2018, 16 (Suppl 1):O3
Background: Various HDAC inhibitors were described as beneficially affecting anti-tumoral immune response. Although different HDAC inhibitors were investigated in syngeneic tumor models, the mode of anti-tumoral action is not yet fully understood. Here, we analyzed the anti-tumoral mode-of-action (MOA) of 4SC-202, an orally available clinical stage epigenetic small molecule inhibitor targeting histone deacetylases (HDAC) class I. To ensure that the conclusions would be relevant for the clinical situation we used a clinically equivalent dosage regimen.
Materials and methods: Anti-tumoral efficacy and the impact on tumor microenvironment (TME) were analyzed in syngeneic colorectal CT26 and C38 models in immunocompetent BALB/c or in nude/irradiated BALB/c mice. A broad spectrum HDAC inhibitor was used for comparison. Transcriptome analysis was performed by RNA-Seq, and the composition of immune cell subpopulations was determined by flow cytometry.
Results: 4SC-202 treatment significantly inhibited growth of CT26 and C38 tumors. A competent immune system was apparently necessary for the anti-tumoral effect of 4SC-202 since its tumor-reducing effect was lost in immunocompromised mice. 4SC-202 treatment increased IFN-γ and chemokine expression, and reduced pro-inflammatory IL-1 and IL-23 in the TME of CT26 tumors. Detailed analysis revealed that 4SC-202 increased the number of cytotoxic CD8+ T cells (CTLs) in TME of both, T cell-inflamed C38 as well as of non-T-cell-inflamed CT26 tumors without affecting the number of CTLs in blood. In contrast, a broad-spectrum HDAC inhibitor tested in the same model demonstrated anti-tumoral efficacy but did not affect the number of CTLs in tumors demonstrating that HDAC inhibitors employ different MOAs for their anti-tumoral response and that the effect on CTLs is not attributed to HDAC inhibition in general. Since the T cell abundance is pre-requisite for the efficacy of PD1/PD-L1 blockade, combinations of 4SC-202 with anti-PD-1/anti-PD-L1 antibodies were tested in C38 and CT26 models, respectively. The combined treatment was more efficacious than monotherapies and resulted in significantly longer survival in both models with 55% tumor-free animals in C38 model.
Conclusions: 4SC-202 already demonstrated a favorable safety profile in a phase I clinical trial with relapsed or refractory hematological malignancies with two objective responses (1 CR, 1 PR) and disease stabilizations in several patients. 4SC-202’s immune priming capacity offers further options for clinical development of 4SC-202 in combination with various cancer immunotherapy approaches. Combination of 4SC-202 with PD-1 blockade will be evaluated in a Phase Ib/II clinical study in advanced cutaneous melanoma patients refractory/non-responding to treatment with anti-PD-1 antibodies (‘SENSITIZE’, NCT03278665).
Immunotherapy beyond melanoma
O4 A new triple negative breast cancer (TNBC) murine model for in vivo preclinical immunotherapies
Veronica Ferrucci1,2,3, Francesco Paolo Pennino1,2, Luisa Dassi2, Fatemeh Asadzadeh2, Roberto Siciliano2, Marianeve Carotenuto1,2, Daniela Spano1,2, Cristina Maria Chiarolla1,2, Adelaide Greco1,2, Monica Cantile4, Maurizio Di Bonito4, Gerardo Botti4, Vandenbussche Jonathan5, Gevaert Kris5, Massimo Zollo1,2,3
1Dipartimento di Medicina Molecolare e Biotecnologie Mediche, DMMBM, Naples, Italy; 2CEINGE, Biotecnologie Avanzate, Naples, Italy; 3European School of Molecular Medicine, SEMM, Milan, Italy; 4Pathology Unit, Istituto Nazionale Tumori Fondazione “G. Pascale”, Naples, Italy; 5Department of Medical Protein Research, VIB, Department of Biochemistry, Ghent University, Ghent, Belgium
Journal of Translational Medicine 2018, 16 (Suppl 1):O4
Background: Triple Negative Breast Cancers (TNBCs), lacking hormone receptors and HER2, are highly metastatic and chemoresistant and metastatic events (mBC) are the most common cause of death in women. Tumour microenvironment (TME) is a complex network of cells that supports tumorigenesis and metastatic spread. Among the immune cells of TME, Tumour-Associated-Macrophages (TAMs) are the most abundant in BC by regulating invasion, metastases and chemoresistance. TAMs can acquire distinct phenotypes in response to different signals. M2-polarized-TAMs have immunosuppressive activities by expressing inflammatory molecules.
Prune-1 belongs to DHH (Asp-His-His) phosphoesterase superfamily with an exopolyphosphatase activity [2, 8]. The overexpression of Prune-1 is correlated with metastases and poor prognosis in several tumours including BC . Prune-1 has been also found to induce Epithelial-Mesenchymal-Transition (EMT) and metastatic dissemination through the enhancement of canonical TGF-β signalling by counterbalancing its inhibition operated by NM23-H1 [3, 4]. Furthermore, we also have evidences that lung cancer progression is driven by Prune-1 via canonical WNT signalling in autocrine and paracrine manner via Wnt3a secretion .
Results: We identified Prune-1 with the ability to recruit and polarize TAMs toward a pro-tumorigenic M2-phenotype within the TME of TNBC using a double Genetically Engineered Mouse (GEM) model of TNBC over-expressing both Prune-1 and Wnt-1 in mammary glands (MMTV-Prune1/Wnt1; generated through the use of vectors construct containing the human transgene cDNAs under the control of Mouse Mammary Tumour Virus [MMTV] promoter). These novel Genetically Engineered Mouse (GEMs) model of TNBC (MMTV-Prune1-WNT1) develop BCs with 100% penetrance between months 2–3 of life (starting after the mammary gland is fully developed) and importantly they always generate lung metastases, while the single transgenic MMTV-Wnt1 TNBC models [5, 7] is not able to make them. These GEMs were then crossed with the receptor 2 of VEGF (VEGFR2) promoter driving firefly luciferase gene expression. Results are indicating that once these recombinant animals MMTV-Wnt1 or MMTV-Prune1/Wnt1 develop tumours activating VEGF then these initiating tumorigenic cells can be visible by in vivo bioluminescence imaging (BLI) luciferase technology. Studies in primary cells derived from the BC generated by these GEM models, indicate that the over-expression of Prune-1 is responsible for the activation of intracellular pathways (i.e. TGF-β, FAK and NF-κB) and for both the activation and polarization of macrophages in vitro shown by the activation of JAK-STAT3 and NF-κB signalling cascades and the increase of inflammatory cytokines (e.g. Arg1, iNOS, MMP9 and IL1β in those macrophages treated with conditioned media derived from MMTV-Prune-1/Wnt-1 primary cells. This thus confirms Prune-1 able to polarize TAMs toward an M2-phenotype.
Conclusions: We generate a TNBC murine model with lung metastases which can be monitored by in vivo imaging (BLI) technology. This GEM model can be an useful source for immunotherapy trials being a model of enhancement of M2-TAMs polarized cells within the TME in primary tumour and lung metastases.
These results are of impact for immunotherapy for studies with new check-points inhibitors with activities against these specialized cells.
Acknowledgements: This study was supported by Associazione per la Ricerca sul Cancro IG: 11963 (AIRC-MZ), PRIN (E5AZ5F) 2008 (M.Z.), FP7-Tumic HEALTH-F2-2008- 201662 (M.Z.), Fondazione Adolfo Volpe e Associazione Pediatri di famiglia (M.Z.), POR Rete delle Biotecnologie in Campania Movie (M.Z.), Regione Campania legge n.5 (M.Z.), Wellcome Trust (WT098051) and European School of Molecular Medicine SEMM for the fellowship (V.F.).
Carotenuto M, De Antonellis P, Liguori L, Benvenuto G, Magliulo D, Alonzi A, Turino C, Attanasio C, Damiani V, Bello AM, Vitiello F, Pasquinelli R, Terracciano L, Federico A, Fusco A, Freeman J, Dale TC, Decraene C, Chiappetta G, Piantedosi F, Calabrese C, Zollo M. H-Prune through GSK-3β interaction sustains canonical WNT/β-catenin signaling enhancing cancer progression in NSCLC. Oncotarget. 2014;5(14):5736–49.
D’Angelo A, Garzia L, André A, Carotenuto P, Aglio V, Guardiola O, Arrigoni G, Cossu A, Palmieri G, Aravind L, Zollo M. Prune cAMP phosphodiesterase binds nm23-H1 and promotes cancer metastasis. Cancer Cell. 2004;5(2):137–49.
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Li Y, Hively WP, Varmus HE. Use of MMTV-Wnt-1 transgenic mice for studying the genetic basis of breast cancer. Oncogene. 2000;19(8):1002–9.
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O5 Characterization of melanoma patients with brain metastases diagnosed between 2014–2016, in one center
Teresa Amaral, Ioanna Tampouri, Ulrike Keim, Thomas Eigentler, Claus Garbe, Andrea Forschner
Center for Dermatooncology, Department of Dermatology, Liebermeisterstr. 25, University Hospital Tuebingen, 72076 Tuebingen, Germany
Journal of Translational Medicine 2018, 16 (Suppl 1):O5
Background: The treatment of patients suffering from melanoma brain metastasis is challenging. Combination of local and systemic therapies is under evaluation in several ongoing clinical trials. Moreover, treatment sequence needs further optimization and therefore standards for the management of brain metastases in melanoma patients do not exist so far.
Methods: After approval of the ethical commission, we conducted a retrospective study including 168 patients diagnosed with melanoma brain metastases between 2014 and 2016 and treated with local and/or systemic therapies. The cut-off date for data collection was April 2017. Overall survival was analyzed using a Kaplan–Meier estimator.
Results: The median follow-up since the first melanoma diagnosis was 61.8 months [23–80.75] and 8.59 months [3–12] since the first diagnosis of brain metastasis. As of the date cut-off 39% of the patients were still alive. The median melanoma specific survival, defined as the time between melanoma diagnosis and last observation or death, was 63 months [25–129]. The median overall survival (OS) for the all population, defined as the time between brain metastasis diagnosis and last observation or death, was 9 months [4.0–22.0].
For patients treated with immunotherapy as first systemic therapy, the median OS was 13 months (95% CI 7.65–18.35 months) and 11 months (95% CI 6.55–15.46 months; p = 0.005) for those treated with targeted therapy. When the type of first line local therapy is analyzed, the median OS was 22 months (95% CI 11.24–31.76 months) for patients treated with surgery or stereotaxic radiotherapy and 6 months (95% CI 4.36–7.64 months; p = 0.0001) for patients treated with whole brain radiation. The best results were obtained when both systemic and local therapies were combined in a 4 weeks interval, but this was not significant (p = 0.061). In patients with BRAF mutation, longer median OS was observed in patients treated with immunotherapy as first systemic therapy when compared to targeted therapy. The median OS was not reached in the first group and was 11 months (95% CI 6.54–15.45 months; p = 0.004) in the second group.
Conclusion: The availability of new therapies increased OS of patients with brain metastases, in comparison with historical controls (9 months vs 5 months). Immunotherapy as first systemic therapy was associated with the best outcomes, including in patients harboring BRAF mutation.
O6 A novel translational research tool for the development of predictive signatures of immunotherapy response
Alessandra Cesano, Sarah Warren
NanoString Technologies, Inc., Seattle, WA, USA
Journal of Translational Medicine 2018, 16 (Suppl 1):O6
The Tumor Inflammation Signature (TIS) is an 18 gene biomarker of a suppressed adaptive immune response within tumor which measures four key areas of biology—antigen presentation, T/NK cell abundance, IFN signaling, and T cell exhaustion. The TIS is currently under evaluation in three clinical trials to predict immune response to pembrolizumab, and may have broad utility to predict response to other immune checkpoint inhibitors. The TIS has been embedded into the NanoString® IO 360 panel—a 770 gene expression panel allows for the parallel assessment of additional mechanisms of immune-evasion in the RUO setting using a single 5 μm FFPE tissue section. The panel contains content to characterize evasion in the context of an inflamed tumor phenotype (such as additional checkpoints inhibitors or suppressive immune cell populations) as well as in the context of an “immune excluded” or “immune desert” tumor microenvironment phenotype (such as activation of oncogenic pathway affecting immune cell trafficking or intrinsic alteration of the antigen presentation process). The IO 360 panel enables the development of diagnostic tests that will select populations that respond to novel and existing immunotherapies as well as combination therapies based on the parallel assessment and integration of multiple mechanisms of immune evasion in a single assay.
SITC session—evolving topics in cancer immunotherapy
O7 Mechanisms of primary resistance to cancer immunotherapies
Duane Moogk1, Kaitao Li2, Zhou Yuan3, Shi Zhong1, Zhiya Yu4, Ivan Liadi5, William Rittase3, Victoria Fang1, Janna Dougherty1, Arianne Perez-Garcia1, Iman Osman1,7, Navin Varadarajan5, Nicholas P. Restifo4, Alan Frey6, Cheng Zhu2,3, Michelle Krogsgaard1,8
1Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA; 2Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA; 3George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA; 4Center for Cancer Research, NCI, NIH, Bethesda, MD, USA; 5Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, USA; 6Perlmutter Cancer Center, Departments of Cell Biology, New York University, New York, NY, USA; 7Perlmutter Cancer Center, Departments of Dermatology, New York University, New York, NY, USA; 8Perlmutter Cancer Center, Departments of Pathology, New York University, New York, NY, USA
Journal of Translational Medicine 2018, 16 (Suppl 1):O7
Background: Although much clinical progress has been made in harnessing the immune system to recognize and target cancer, there is still a significant lack of an understanding of how tumors evade immune recognition and the mechanisms that drive tumor resistance to both T cell and checkpoint blockade immunotherapy. Our objective is to understand how tumor-mediated signaling through inhibitory receptors, including PD-1, combine to affect the process of T cell recognition of tumor antigen and activation signaling, with the goal of understanding the basis of resistance to PD-1 blockade and the potential identification of new molecular targets to enable T cells to overcome dysfunction mediated by multiple inhibitory receptors.
Methods and results: We show that Lck activity affects T cell sensitivity and influences the probability of inducing effector function . Under non-activating conditions, Lck and Shp-1 phosphorylation and activity vary based on CD8+ memory T cell phenotype. Shp-1 interaction with Lck under non-activation conditions can also vary, as suggested by our results showing decreased Shp-1 S591 phosphorylation, which affects Shp-1 localization and correlates with increased Shp-1 colocalization with Lck. Further, we showed that Shp-1 directly influences Lck activity under non-activating conditions, as inhibition of Shp-1 leads to increased Lck activity. Importantly, inhibition of Shp-1/2, a major mediator of PD-1 signaling, targeting CD28 and Lck , prior to activation leads to increased T cell cytotoxic effector function. Our proteomics-based analysis of patient T cells identified both mediators of PD-1 signaling and signaling components and pathways associated with blockade resistance. It has generally been thought that TCR and CD8 binding depend mainly on their ectodomain interactions with pMHC. We have shown, however, that Lck-CD8 binding  and Lck activity  are required for upregulated CD8 binding to pre-bound TCR-pMHC complex. Therefore, the cytoplasmic associations of Lck with CD8 and Zap-70, as well as CD3 with Zap-70 may influence formation and stability of the TCR–pMHC–CD8 complex. To determine the mechanistic basis of PD-1 inhibition of TCR–-pMHC–CD8 binding we utilized 2D affinity combined with Biomembrane Force Probe (BFP) measurements[5, 6] and showed that PD-1 directly suppresses TCR–pMHC–CD8 binding. Our data also revealed that TCR-pMHC binding was independent of PD-1-PD-L1, but TCR–pMHC–CD8 binding was suppressed by PD-1-PD-L1 binding demonstrating negative cooperativity, as fewer bonds formed than the sum of bonds formed by each interaction alone.
Conclusions: Together, our results show that the activities of TCR-proximal signaling components affect T cell mechanosensing and sensitivity at the earliest stages of antigen recognition and are influenced by PD-1. Targeting these interactions may enhance tumor-specific T cell sensitivity for cancer immunotherapy and understanding the basis of resistance to PD-1 blockade to potentially allow identification of new molecular targets to enable T cells to overcome dysfunction mediated by multiple inhibitory receptors.
Moogk D, et al. Constitutive Lck activity drives sensitivity differences between CD8+ memory T cell subsets. J Immunol. 2016 (In press).
Hui E, et al. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017;355(6332):1428–1433.
Casas J, et al. Ligand-engaged TCR is triggered by Lck not associated with CD8 co-receptor. Nat Commun. 2014;5:5624.
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O8 Role of TGFβ superfamily members in hindering the pro-immunogenic effects of radiotherapy
Claire Vanpouille-Box1, Silvia C. Formenti1 and Sandra Demaria1
1Department of Radiation Oncology, Weill Cornell Medicine, New York, NY, USA
Journal of Translational Medicine 2018, 16 (Suppl 1):O8
Background: Transforming Growth Factor-beta (TGFβ) and activin A (actA) are TGFβ superfamily members with overlapping functions in many processes including regulation of inflammation and immunity. We have recently shown that in situ vaccination by local tumor irradiation is hindered by activation of latent TGFβ . Intriguingly, TGFβ blockade enhanced activation of dendritic cells and T-cell priming, but it increased (rather than reduced) intratumoral regulatory T cells (Tregs). We have recently found that actA release by breast cancer cells is enhanced by radiotherapy (RT). Interestingly, prolonged exposure to TGFβ inhibitors also resulted in actA upregulation, consistent with a previously described compensatory mechanism. Here we hypothesized that actA and TGFβ regulate RT-induced anti-tumor immunity.
Methods: Secretion of actA by untreated and irradiated 4T1 mouse carcinoma cells was quantified by ELISA. Transwell co-culture was used to assess the ability of cancer cell-derived actA to promote the conversion of naïve CD4+ T cells into Tregs. 4T1 cell derivatives engineered to express a tetracycline-inducible shRNA specific for actA (4T1shActA) or non-silencing (4T1shNS) were generated and injected s.c. to syngeneic BALB/c mice (day 0). ActA knockdown was induced by systemic doxycycline administration at day 8. TGFβ-neutralizing 1D11 or isotype control antibodies were given i.p. every other day starting on day 12. RT was delivered to the primary tumor in 6 Gy fractions on 5 consecutive days beginning on day 13. Mice were followed for tumor growth or euthanized at day 22 for analysis.
Results: TGFβ blockade improved RT-mediated tumor control, an effect mediated by T cells. However, tumor recurred. Notably, ActA KD or 1D11 increased intratumoral Tregs (Control: 11%; 1D11: 26%, shActA: 21%) and enhanced Tregs infiltration induced by RT (RT: 15%; RT+1D11: 27%; RT+shActA: 30%). When both TGFβ and actA were blocked Tregs significantly decreased in both untreated (1D11+shActA: 13%) and RT-treated tumors (RT+1D11+shActA: 8% of Tregs). Tumor-specific IFNγ production by CD8+ T cells was significantly higher in RT+1D11+shActA-treated mice compared to RT+1D11 (*p) and RT+shActA (**p). ActA KD in mice treated with RT+1D11 reduced tumor recurrence and improved survival (RT+1D11 vs RT+1D11+shActA **p; RT+shActA vs RT+1D11+shActA ***p).
Conclusion: Data indicate that both TGFβ and actA impair RT-induced anti-tumor immune responses. Concomitant inhibition of actA and TGFβ is required for optimal in situ vaccination by RT.
Vanpouille-Box C, et al. TGFβ is a master regulator of radiation therapy-induced anti-tumor immunity. Cancer Res 2015;75(11):2232–42.
O9 The renin angiotensin system (RAS) mediates bifunctional growth regulation in melanoma and is a target for therapeutic manipulation
Cristiana Lo Nigro1, Alexander Renziehausen2, Andreas G. Tzakos3, Hexiao Wang4, Bhavya Rao5, Rubeta Matin6, Catherine Harwood6, Daniela Vivenza1, Federica Tonissi1, Marcella Occelli1, Lynda Weir4, Su Li7, Van Ren Sim8, Kevin O’Neill9, Alan Evans10, Alastair Thompson11, Peter Szlosarek12, Colin Fleming13, Charlotte Proby6, Nelofer Syed1, Marco Merlano1, Tim Crook14
1Department of Oncology, S. Croce & Carle Teaching Hospital, Cuneo, Italy; 2John Fulcher Neuro-Oncology Laboratory, Division of Brain Sciences, Imperial College London, London, UK; 3Department of Chemistry, University of Ioannina, Ioannina, Greece; 4Department of Dermatology, The First Hospital of China Medical University, Shenyang, China; 5Medical Research Institute, Ninewells Hospital & Medical School, Dundee, UK; 6Barts and the London School of Medicine and Dentistry, London, UK; 7Royal Marsden Hospital, Fulham Road, London, UK; 8Kent Oncology Centre, Maidstone Hospital, Maidstone, UK; 9Department of Neurosurgery, Charing Cross Hospital, London, UK; 10Department of Pathology, Ninewells Hospital, Dundee, UK; 11Breast Surgical Oncology, MD Anderson Cancer Center, Houston, Texas, USA; 12Department of Medical Oncology, Bart’s Cancer Centre, London, UK; 13Department of Dermatology, Ninewells Hospital, Dundee, UK; 14Department of Oncology, Royal Surrey County Hospital, Guildford, UK
Journal of Translational Medicine 2018, 16 (Suppl 1):O9
Background: Despite the emergence of active new systemic therapies, metastatic melanoma remains a clinically challenging form of skin cancer. The renin-angiotensin system (RAS) is a major physiological regulatory pathway mediated by angiotensin II (AngII) via two receptor subtypes, AT1R (encoded by AGTR1) and AT2R (by AGTR2) (1) The role of the RAS is unexplored in melanoma.
Materials and methods: We investigated the involvement of the two principal angiotensin receptors in a panel of melanoma cell lines, grown as described previously (2). Primary cultures of brain metastatic melanomas were established from fresh tumour surgical tissues. The selective AT1R inhibitor Losartan and the highly selective AT2R agonist Y6AII were developed as described (3). Demethylation experiments using azacytidine and trichostatin were done as described (4). TaqMan® probes were used for gene expression analysis. Transfectants with AGTR1 ORF were analysed for knockdown of AGTR1 by qPCR and WB. Cell proliferation and clonogenic assays were assessed by standard twchniques. The role of AT2R in tumour angiogenesis was investigated in hCMEC/D3 grown in CM collected from PMWK cells treated with AngII alone or in combination with Losartan and PD123319.
Results: Antagonism of AT1R using the Losartan or shRNA-mediated knock-down in melanoma cell lines expressing AGTR1 resulted in acquisition of the ability to proliferate in serum-free conditions, implying that AT1R has a negative growth-regulatory function in melanoma. Consistent with this, ectopic expression of AGTR1 in cell lines lacking endogenous expression inhibits proliferation irrespective of the presence of AngII implying a ligand-independent suppressor function for AT1R. Treatment of melanoma cell lines expressing endogenous AT2R with either AngII or the AT2R-specific agonist Y6AII induces proliferation in serum-free conditions. Conversely, the AT2R-specific antagonists PD123319 and EMA401 inhibit melanoma growth and angiogenesis and potentiate inhibitors of BRAF and MEK.
Consistent with a negative growth regulatory function, we showed that: (i) decreasing expression and increasing CpG island methylation of AGTR1 in metastatic vs primary melanoma; (ii) detection in serum of AGTR1 methylated genomic DNA is associated with metastatic disease.
Conclusions: Our results demonstrate that the RAS has both oncogenic and tumour suppressor functions in melanoma. Pharmacological inhibition of AT2R may have therapeutic effects in melanomas expressing this receptor and AGTR1 methylation in serum may serve as a biomarker of metastatic melanoma.
Karnik SS, Unal H, Kemp JR, Tirupula KC, Eguchi S, Vanderheyden PM, et al. Angiotensin receptors: interpreters of pathophysiological angiotensinergic stimuli. Pharmacol Rev. 2015;67:754–819.
Hoshimoto S, Kuo CT, Chong KK, Takeshima TL, Takei Y, Li MW, et al. AIM1 and LINE-1 epigenetic aberrations in tumor and serum relate to melanoma progression and disease outcome. J Invest Dermatol. 2012;132:1689–97.
Magnani F, Pappas CG, Crook T, Magafa V, Cordopatis P, Ishiguro S, et al. Electronic sculpting of ligand-GPCR subtype selectivity: the case of angiotensin II. ACS Chem Biol 2014;9:1420–425.
Miura S, Karnik SS, Saku K. Constitutively active homo-oligomeric angiotensin II type 2 receptor induces cell signalling independent of receptor conformation and ligand stimulation. Journal Biol Chem 2005;280:18237–18244.
O10 Functional genomics to identify germline markers of melanoma immunotherapy efficacy and toxicity
Robert Ferguson, Danny Simpson, Carlos Martinez, Matjaz Vogelsang, Esther Kazlow, Melissa Wilson, Anna Pavlick, Jeffrey Weber, Ryan Sullivan, Keith Flaherty, Antoni Ribas, Iman Osman, Tomas Kirchhoff
Laura and Issac Perlmutter Cancer Center, New York University School of Medicine, New York City, NY, USA
Journal of Translational Medicine 2018, 16 (Suppl 1):O10
Background: Approximately 40% of metastatic cutaneous melanoma (CM) patients do not respond to the current immunotherapy (IT) regimens, pointing to other, yet unknown factors conferring IT resistance. In addition, > 60% of patients from single-line or combined treatment (COMBO) regimens present severe immune related adverse events (irAEs). In this study we have developed a novel genomic approach interrogating expression quantitative trait loci (eQTLs) to explore weather germline genetic variation can serve as novel personalized determinant of immunotherapy response and toxicity.
Methods: By interrogating the genome wide expression data and SNP array datasets of healthy twin cohort (MuTHER), we have identified 85 eQTLs most significantly associated with the expression of 265 immune genes. Using the MassARRAY system, the 85 SNPs were genotyped in 138 anti-CTLA-4 treated patients, 87 PD-1 treated patients, and 69 patients from combined (COMBO) treatments, collected from multi-institutional collaborations. To test the association of SNPs with IT response and irAEs, logistic regression analyses were performed for each treatment group adjusting by demographic and clinical covariates.
Results: We found significant associations with COMBO IT resistance for and eQTL in IL10/IL19 (OR = 4.249, p = 0.0167), which we have recently identified for association with melanoma survival and which, interestingly, is an established locus associated with the risk of several autoimmune diseases. Additionally, we also identified eQTLs that are associated with IT sensitivity; IL1-β with resistance to anti-CTLA-4 and SPI1 with resistance to anti-PD-1. Interestingly, genomic scan of 85 eQTLs has identified novel loci predictive of severe autoimmunity and site specific irAEs in patients treated with COMBO or single-line anti-CTLA4 IT.
Conclusions: In this study, we report that eQTLs from IL19/IL10 locus, previously shown to predict autoimmunity risk and CM survival, is also a surrogate marker of response to COMBO IT, indicating a strong relationship between interleukin pathways and tumor immunogenicity. Novel loci have been found as predictive markers for autoimmune toxicity, in patients treated with COMBO and anti-CTLA4 IT. This is a first evidence that immunomodulatory pathways modulated by germline genetic variation can impact susceptibility to irAEs as well as IT efficacy. Currently, a large scan is underway using genome-wide genetic screens to further test the functional validity of these findings in a large collaborative setting.
Focus on health cost assessments and pharmacovigilance in immunotherapy
O11 Comparison between new therapies of metastatic and/or unresectable melanoma with B-RAF V600E/K mutations: cost–benefit assessment
Antonio D’Avino, Licia Guida, Augusto Cosacco, Roberta D’Aniello, Piera Maiolino
IRCCS Istituto Nazionale Tumori Fondazione “G. Pascale”, Napoli, Italy
Journal of Translational Medicine 2018, 16 (Suppl 1):O11
Background: Incidence of melanoma continues to rise, and the mortality associated with unresectable or metastatic melanoma remains high. B-RAF targeted therapy has been established as a treatment standard for patients who have metastatic melanoma with activating BRAF mutations and recently a regimen combining a B-RAF inhibitor with a MEK inhibitor has been associated with a higher response rate and longer duration of response, as compared with anti-BRAF monotherapies. These drugs are subject monitoring by Italian Medicines Agency (AIFA) and submitted to negotiating agreements between AIFA and Pharmaceutical companies, known as Managed Entry Agreements (MEAs). The objective of our study is to evaluate the cost–benefit evaluation of anti-BRAF and anti-MEK agents alone or in combination to calculate the average of pharmacological cycles performed, the number of progressions and the percentage of partial and/or complete remission percentage Treatments That Meet MEAs.
Methods: The clinical, therapeutic and diagnostic data of each patient were obtained from the hospital databases and the monitoring register by Italian Medicines Agency, that contains data for monitoring patients who are receiving this therapies. The report of this register include the medicines that are registered and some of the outcomes that were being monitored, for example, the number of treated patients, the patients that have finalized the treatment and the reasons for stopping the treatment. In these monitoring, we are included all patients with advanced melanoma with B-RAF V600E/K mutation treated between 2012 and 2016.
Result: 112 patients with non-resectable metastatic melanoma were treated from 2012 to 2016, of which 68 were treated with monotherapy (65 with Vemurafenib and 3 with Dabrafenib), 44 with BRAF and MEK-inhibitor associations (12 Vemu/Cob and 32 Dab/Tram). Patients performed on average 12 cycles of Vemurafenib, 3 cycles of Dabrafenib, 5 cycles of Vemu/Cob and 6 cycles of Dab/Tram. Compared to the MEAs, over half of the therapies, on average over 60%, monotherapy or association, meet the expectations described.
Conclusion: Higher PfS therapies are monotherapy with Vemurafenib (4,4) and the Dabr/Tram (3,26) association, which on average patients take the drug for more time following the Vemurafenib monotherapy (12 cycles), exceeding the MEAs expectations.
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American Cancer Society. Cancer facts and figures 2014 (http://www.cancer.org.acs/groups/content/@search/documents/webcontent/acspc-042154.pdf).
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Maio M, Ascierto P, Testori A, et al. The cost of unresectable stage III or stage IV melanoma in Italy. J ExpClinCancer Res. 2012;31:91. https://doi.org/10.1186/1756-9966-31-91.
Il farmacista nella gestione delle sperimentazioni cliniche: analisi delle sperimentazioni farmacologiche condotte Dal 2011 AL 2014 ALL’ INT. Pascale G, D’Avino A, Maiolino P, D’Aniello R, Sarno MR—Bollettino SIFO 01/2016.
Clinical and economic evaluation of the introduction of the combinazion trametinib + dabrafenib in the management of advanced melanoma in the Italian market Lorenzo Pradelli, Paolo Ascierto.
Robert C, Karaszewska B, Schachter J, et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med. 2015;372:30–9.
Larkin J, Ascierto PA, Dreno B, et al. Combined vemurafenib and Cobimetinib in BRA-mutated melanoma. N Engl J Med. 2014;371:1867–76.
Long GV, Stroyakovskiy D. Gogas H, et al. Combined BRAF inhibition along in melanoma. N Engl Med. 2014;371:1877–88.
ClinPharmacokinet. 2017. https://doi.org/10.1007/s40262-017-0523-7. Clinical Pharmacokinetics of Vemurafenib.Zhang W1, Heinzmann D2, Grippo JF3 (Epub ahead of print).
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Flaherty KT, Infante JR, Daud A, et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med. 2012;367:1694–703.
Long GV, Stroyakovskiy D, Gogas H, et al. Combined BRAF and MEK inhibition versus BRAF inhibition alone in melanoma. N Engl J Med. 2014;371:1877–88.
Robert C, Karaszewska B, Schachter J, et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med. 2015;372:30–9.
O12 The drug day an effective strategy for the containment of immunotherapy costs
Teresa Tramontano, Maria R. Sarno, Ida Palazzo, Angela Di Napoli, Gianclaudio Acunzo, Piera Maiolino
S.C. Farmacia, IRCCS Fondazione “G. Pascale”, Napoli, Italy
Journal of Translational Medicine 2018, 16 (Suppl 1):O12
Background: The Centralized Unit for Handling Antineoplastic of National Cancer Institute “G. Pascale” of Naples planned a strategy for the use of high cost cancer drug Opdivo®. In order to reduce the therapy cost we decide to dedicate for Opdivo® a drug day to optimize the use of drug vials with reduction of waste and/or optimal use of the residues. The aim of this work was to report the analysis of drug consumption and cost from March 2016 when it was officially authorized as a hospital drug and it was introduced into clinical practice to August 2017.
Materials and methods: The drug day was organized in accord with Opdivo® prescribed physicians. The number of patients treated, the individual patient’s treatment line, consumption and cost data relating to the period under review, were collected from the hospital database. In primis, it was performed an assessment of the total consumption and cost per year and then a comparison exercise was made between the number of drug vials really used and those that should be used without the drug day and, the comparison between the real cost incurred and the hypothetical cost out of drug day.
Results: From March 2016 to August 2017 the pharmacy staff set 1872 preparation for 211 patients of Opdivo®. The drug was administered for 68 patients as first-line treatment, for 116 patients as second-line treatment and finally for 27 patients as third-line treatment. Given the considerable number of patients and considering that Opdivo® stability is 24 h, 2 consecutive days per week were dedicated for treatment as drug days with a range of 15–20 preparations per day. Comparing the used drug in drug day to a hypothetical daily preparation we saved 36.860 mg (approximately 367 vials of 100 mg) with an economy of €435.943,22. Noteworthy, the overfill of injecting drug vials, corresponding to about 10–12 mg over the declared amount of drug, as prescribed by F.U. XII Edition (2.9.17) and by FDA guidelines on the filling volume in excess of vials, allowed us to rescue 13.167 mg with an economy of €155.887,67.
Conclusions: Programming cancer therapies in a drug day was very complex and involved close cooperation between prescribed physicians, pharmacists and patients. However this strategy allows to reduce at minimum drug waste residues and, furthermore, to use overfill of the samples, which should became waste. In conclusion the drug day resulted a very effective tool for the containment of pharmaceutical costs.
O13 Adverse effects related to PD-1 inhibitor immunotherapy: an overview
Mariarosanna De Fina
Department Science of Life, Magna Graecia University, Catanzaro, Italy
Journal of Translational Medicine 2018, 16 (Suppl 1):O13
Background: Immunotherapies, like nivolumab (Opdivo®) and pembrolizumab (Keytruda®), have changed the cancer treatment landscape. They are inhibitors of PD-1 protein, which cancer cells use to elude the immune system. Post-marketing surveillance of these drugs have revealed severe adverse drug reactions (ADRs). The occurrence of ADRs has high morbidity and mortality, accounting for the fifth leading cause of death in industrialised countries. EudraVigilance.org is European Medicine Agency’s (EMA) web-based Monitor System for reporting and evaluating suspected ADRs.
Aim: A retrospective observational study was done. The aim of this study is to investigate the ADRs occurred in patients treated with PD-1 inhibitors.
Methods: All ADRs associated with Opdivo® and Keytruda® reported in EudraVigilance up to 24 September 2017 were analysed for overall numbers, age, gender and geographic origin. A quantitative measurement (proportional reporting ratio [PRRs]) was developed for signal generation from large databases of spontaneous ADRs reports to find a statistical link between each PD-1 inhibitor and ADRs.
Results: A total of 10600 ADRs related to the PD-1 Inhibitors therapy have been reported and analysed in this study. Opdivo (65.87%), Keytruda (31.5%) and nivolumab (2.63%). 55.25% occurred in males (male/female ratio = 1.73). Only in 12.8% of ADRs reported, sex is not specified. Most ADRs occurred in patients with age between 18 and 65 years (41.6%). The higher incidence of ADRs occurred in patients of non-European Economic Area than European Economic Area, respectively 73.7% vs 26.3%.
The “Neoplasm benign malignant and unspecified” is the most reported ADRs category for both Opdivo® and Keytruda® (18.9% vs 27.7%) with a PRR = 0.67, ADRs occurred in males show PRR = 8.76. Serious but rare ADRs reported in database is Uveitis, that shows PRR = 0.61. The Uveitis incidence rate, reported in database, is 0.70 vs 0.33, respectively for Keytruda®-treated patients than Opdivo®-treated patients.
Conclusions: Analysis of Pharmacovigilance database is important to understand the safety of drugs in post-marketing and in real clinical practice. Patient safety is priority in care management. For this reason, oncologist, pharmacists and multidisciplinary team have to be careful about adverse reactions that could be occurred in patients treated.
Limitations of this study was cases were based on spontaneous reporting which clearly suffered from underreporting. Clinical data were not available.