Immunotherapy Bridge 2017 and Melanoma Bridge 2017: meeting abstracts

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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 [3]. 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 [4]. In CHLs, results revealed a dichotomous cellular and cytokine immune milieu in EBV+ vs EBV− CHL [5]. 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 axis 5 . 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 highthroughput 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).
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 [8]. 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 [1]. 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.

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][4][5][6][7][8][9][10][11][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 . 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. 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. 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 [1]. 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 [2], 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 [3] and Lck activity [4] are required for upregulated CD8 binding to prebound 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 TCRproximal 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.
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 [1]. 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 (4T1 shActA ) or non-silencing (4T1 shNS ) 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.
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, AT 1 R (encoded by AGTR1) and AT 2 R (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 AT 1 R inhibitor Losartan and the highly selective AT 2 R 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 AT 2 R 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 AT 1 R 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 AT 1 R 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 AT 1 R. Treatment of melanoma cell lines expressing endogenous AT 2 R with either AngII or the AT 2 R-specific agonist Y6AII induces proliferation in serum-free conditions. Conversely, the AT 2 R-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 AT 2 R may have therapeutic effects in melanomas expressing this receptor and AGTR1 methylation in serum may serve as a biomarker of metastatic melanoma.
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.

O11
Comparison between new therapies of metastatic and/or unresectable melanoma with B-RAF V600E/K mutations: costbenefit 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. J Transl Med 2018, 16(Suppl 1):4

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.  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. J Transl Med 2018, 16(Suppl 1):4 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. Background: Malignant melanoma is an aggressive cancer associated with high mortality worldwide. Many patients have locally advanced or metastatic disease at diagnosis in which case treatments were limited until a few years ago. Immune checkpoint blockade of programmed death receptor-1 (PD-1) pathway represents a recent valid treatment strategy changing the prognosis in this setting. Case presentation: Here we describe the case of a 83-year-old man with many comorbidities (chronic renal failure, diabetes, hypertension) and with colorectal cancer anamnesis (right hemicolectomy in 2004) and laryngeal neoplasia (total laryngectomy in 1981) who presented initially neoformation of the parotid region and a skin nodule of the temporal region at right side. Results: Histological examination on both samples showed epithelial malignant melanoma, BRAF wild type. At CT imaging there was the parotid neoformation extended in the regional adipose tissue predominantly, into contact with the right masseter muscle and with the lower wall of the outer external conduit cranially. In both lungs there were some parenchymal lesions compatible with secondary localizations. On 8 April 2016 the patient started NIVOLUMAB 300 mg total/2 weeks. After the first administration there was a reduction in skin lesions and after 2 months skin nodule of the temporal region were disappeared as well as neoformation of the parotid region. On October at CT imaging, the alterations to the parotid region were completely disappeared and lung metastases appeared hyperdense and cavitated. Last total body PET/TAC (on February 2017) described a single pulmonary localization to the left basal pyramid. The patient is continuing nivolumab therapy with good tolerability, with the exception of a skin rash G1-2 and he is still being in response at a distance of more than 1 year. Conclusions: This case report confirms the efficacy of nivolumab in metastatic melanoma and the safety in elderly patients with polypathology also. Consent for publication: The authors declare that written informed consent was obtained from the patients for publication. They were evaluated 57 reports and others could not be classified; diarrhea was the main adverse event reported and 23 ADRs were serious. Three cases only were treated with systemic immunosuppressive drugs. Analysis of adverse reactions showed that they are common side effects of three inhibitors of immune checkpoints. Conclusions: The pharmacovigilance of Ipilimumab, Nivolumab and Pembrolizumab has produced an appreciable number of reports which however represent known side effects of recent melanoma immunotherapy that can be properly treated with systemic immunosuppressive drugs if recognized early [3]. Severity of adverse events has generally required hospitalization: recognition and treatment of these reactions may have been inappropriate or later. Authors believe not all health workers were joined by a clear and complete information about adverse events of these drugs, their recognition and treatment. In cooperation with the oncologist, hospital chemist is able to promote informational programs that can guarantee the knowledge necessary for an adequate approach to toxicity associated to use of these drugs. The aim is ensuring an effective and efficient health service; in this case a correct information can produce a higher yield by reducing costs associated to inappropriate therapies and excessive hospitalization.

P2 Pharmacovigilance in melanoma immunotherapy
Background: Anti-programmed cell death protein-1 (PD-1)/ligand-1 (PD-L1) antibodies can induce an immune-related bullous pemphigoid (BP) [1,2]. About its pathogenesis, it has been hypothesized that the blockade of the PD-1/PD-L1 pathway may increase autoantibody production against the hemidesmosomal protein BP180 [1]. Here we report a case of BP during treatment with nivolumab. Case report: A 68-year-old man was admitted to our Oncology Unit in March 2015 with the diagnosis of BRAF wild-type melanoma. A CT scan performed on May 2016 showed lung metastases. Patient's medical history included hypertension. Long-standing medications included omeprazole and zofenopril. He had no relevant history of skin or autoimmune disorders and no new medications. On June 2016, nivolumab was started. On June 2017, the patient began to develop erosions and vesicles on the buccal mucosa, especially with the involvement of the lower lip. The severity of the patient's bullous dermatitis ranged from grade 1-2. A biopsy of these lesions showed eosinophilic spongiosis and a mixed dermal inflammatory infiltrate with eosinophils. Direct immunofluorescence showed linear deposition of IgG and C3 at the basement of the dermoepidermal junction, establishing a diagnosis of BP. A treatment with oral prednisone dosed according to severity and topic clobetasol was started. He improved within about 2 weeks and the steroid was tapered (Fig. 1). Nivolumab was restarted. Total IgE level was elevated and complete blood count revealed an increased absolute eosinophil count compared to pretreatment levels, but it remained within normal ranges. The patient experienced a near-complete response to nivolumab, how shown by CT scan performed on August 2017. At this time, he presents only some lesions in the lower lip and he continues treatment with prednisone 5 mg bid and intermittent topical steroids.

Fig. 1 Discriminant analysis of healthy donors and melanoma patients
Conclusion: Although we cannot be sure that BP is due to nivolumab, the timing of cancer diagnosis (24 months prior) compared with nivolumab initiation (12 months prior) and the fact that the other medications were tolerated for years without similar cutaneous lesions argue against a paraneoplastic BP or BP related to another medication, respectively [1,2]. About treatment of BP during anti-PD-1/PD-L1, our research in the literature found a case report of BP during nivolumab with elevated IgE level and successfully treated with omalizumab, thus sparing steroids that could interfere with the antitumor activity of nivolumab [2,3]. Finally, all but few BP cases in the setting of anti-PD-1/PD-L1 therapy were associated with partial/complete response or stable disease: is there a relationship between development of BP and anti-cancer responses [1,2]? Consent for publication: The authors declare that written informed consent was obtained from the patients for publication. Two major phenotypes of human melanoma metastases have been observed based on gene expression profiling and confirmatory assays. One subgroup of patients has a T cell-inflamed phenotype that includes expression of chemokines, T cell markers, and a type I IFN signature. In contrast, the other major subset lacks this phenotype and appears to display immune "exclusion". The mechanisms of immune escape are likely distinct in these two subsets, and therefore the optimal immunotherapeutic interventions necessary to promote clinical responses may be different. The T cell-inflamed tumor microenvironment subset shows the highest expression of negative regulatory factors, including PD-L1, IDO, and FoxP3+ Tregs. Deep analysis of tumor antigen-specific T cells in the tumor microenvironment has identified additional mechanisms of immune dysfunction and new potential therapeutic targets. Treatment strategies targeting several pathways have been translated back into the clinic, including anti-PD-1/PD-L1 mAbs and IDO inhibitors, and combinations of these agents also look promising. In contrast to the T cell-inflamed melanomas, non-T cellinflamed tumors are largely immunotherapy resistant with current approaches. Natural innate immune sensing of tumors appears to occur via the host STING pathway, type I IFN production, and crosspriming of T cells via CD8+ DCs, and these factors are absent in non-T cell-inflamed tumors. New strategies are being developed to engage or mimic this pathway as a therapeutic endeavor, including STING agonists. The molecular mechanisms that mediate the absence of the T cell-inflamed tumor microenvironment in patients are being elucidated using parallel genomics platforms. The first oncogene pathway identified that mediates immune exclusion is the Wnt/β-catenin pathway, which argues that new pharmacologic strategies to target this pathway should be developed to restore immune access to the tumor microenvironment. Recent evidence has indicated that host factors, including the intestinal microbiota, are also critical. We recently have identified commensal bacteria in mouse models that augment spontaneous anti-tumor immunity and increase efficacy of anti-PD-L1 therapy. Similar analyses in human melanoma patients have been performed, and commensal bacteria have similarly been identified that correlate with anti-PD-1 efficacy. These results have prompted the development of new probiotics as potential therapeutics, to improve spontaneous immune infiltration and expand immunotherapy efficacy in the clinic.

System biology session in melanoma
Background: Natural Killer (NK) cells selectively recognize lymph node associated melanoma metastasis cells [1]. Peripheral blood frequencies of CD56bright and CD56dim NK cell subsets are subverted in stage III melanoma patients, while a high frequency of CCR7+KIR+CD57+CD56dim NK cells in melanoma colonized lymph nodes directly correlates with the patients survival [2]. Here, we validate and identify additional changes in the NK cells repertoire characterizing the transition from the different stages of melanoma that can improve the patients diagnosis, and dissected CCR7 potential role in metastatic process by measuring it on both melanoma and NK cells. Results: Immuno-profile of 42 healthy donors and 103 melanoma patients (stage II, III, IV), together with biographical variables, was used to create an OPLS-DA multivariate model. The model gave a good separation between healthy donors and the three groups of patients (Fig. 1A). Immuno profile of both stage II and III melanoma patients showed an increased CXCR2 percentage, as previously observed [2], and a reduced CD57 frequency and NKp46 expression on the NKdim cells, that correlated with lack of responsiveness to K562 cells pulsing ( Figure 2). Instead, stage IV melanoma patients showed high frequencies of, CCR7+CD56bright NK cells, which displayed a longitudinal steady increase during the disease evolution.

Fig. 2 NK and T cells function analysis in healthy donors and melanoma patients
Cytokine profile analysis showed a progressive sera accumulation of MCP-1, IL-6, IL-8 IL-15 and CCL19 (Table 1), with the first three that have been demonstrated to be produced by primary melanoma cells and found in TILN milieu [2], while IL-15 was involved in phenotypic changes of NK cells from melanoma patients [3]. CCL19 longitudinal increase over the clinical evolution, perfectly matched with the accumulation of circulating CD56brightCCR7+ cells subset (Table 1). CCR7 analysis on melanoma cells showed that it is expressed by a small fraction (1-5%), which is further characterized by a selective coexpression between CCR7 and the inhibitory ligands PD-L1 and Galectin-9 (Fig. 3). Background: MM is the most aggressive skin cancer, its incidence doubled over the past 10 years and its mortality is still around 80%, in the advanced disease. The goal of the present study is to find new more effective screening methods, and to identify MM at high risk of recurrence [1,2]. In more details, the aim of the project is to investigate the role of specific miRNAs, as a screening tool of MM [3][4][5][6]. Materials and methods: Individuals who joined the melanoma screening at the "Lega italiana per la lotta contro i tumori" (LILT), and with a suspect skin lesion, after signing informed consent, underwent a peripheral blood sample collection before the surgical removal. We compared the plasma level of 15  Till now we have analysed the role of miRNAs as potential tools for screening, while their prognostic role is not yet evaluated. The analysis showed a significant reduction in the expression of miR-199a-5p (p = 0.0027) and miR-122-5p (p = 0.05) in the MM (N = 97) group compared to the controls (N = 100). Multivariate analysis is underway in order to associate miRNA expression profiling with clinical-pathological characteristics.

Conclusions:
The data available today do not allow to identify a miRNA that can be used as a complementary tool for the screening of early-stage MM. Significant reduction of miR-122-5p and miR199-5p in MM compared to controls, however, leads us to deepen their biological role in the pathogenesis of MM and their possible association with clinical-pathological features of subjects under screening. Multivariate analysis data will be available and presented at the meeting.
Background: Anti-PD1 agents are successfully used in therapy to treat patients with advanced melanoma. Here, we retrospectively analysed the CD73 enzyme activity in the peripheral blood of in patients with metastatic melanoma receiving nivolumab. CD73 is an ectonucleotidase able to generate adenosine from AMP. Adenosine in the tumor microenvironment is a potent immune-suppressive mediator, so that inhibition of CD73-generating enzyme or blockade of adenosine receptors is a promising therapeutic strategy to fight cancer. Materials and methods: CD73 enzyme activity was retrospectively analysed in the plasma of patients before receiving nivolumab. Levels of CD73 enzyme activity was correlated with the survival and progression-free survival of the patients analysed in this study and a multivariate analysis was performed to evaluate the prognostic value of this factor. Results: 70% of the patients analysed in this study presented detectable CD73 activity in the plasma. High basal levels of sCD73 enzyme activity in serum were significantly associated with poor overall survival and progression-free survival in melanoma patients. In multivariate analysis, levels of CD73 significantly impact on both, overall survival and progression-free survival. Interestingly, we found that low levels of CD73 in the peripheral blood determined before treatment, were significantly associated with disease control rate to nivolumab. Patients who do not respond to nivolumab therapy instead presented higher levels of CD73 enzyme activity in the blood. Conclusion: Although our results need to be confirmed and validated, they suggest that the activity of CD73 in the peripheral blood of patients with metastatic melanoma might be useful as prognostic factor and potentially as predictor of response to nivolumab treatment.
We also postulate that increased levels of CD73 may contribute to affect the response of immunotherapeutic agents in cancer patients. Consent to publish: All the patients provided written informed consent. Background: Anti-programmed death (PD)-1 monoclonal antibodies have changed the prognosis of metastatic melanoma, improving overall survival [1]. However, still a proportion of patients is unresponsive to these compounds, indicating the presence of other immunosuppressive mechanisms. Thus, the identification of reliable biomarkers to predict the response to checkpoint blockades is still an unmet need. Recent findings showed a tumor-induced immunosuppressive pathway, in which the extracellular adenosine produced by tumor-derived enzyme CD73 (5′-ectonucleotidase) promotes tumor growth by inhibiting immunosuppressive T-cell action [2]. Targeting adenosine generation by blockade of CD73 significantly enhances anti-tumor activity of anti-PD-1 drugs, inducing full regression in some tumor models [3]. The aim of the study was to investigate whether baseline levels of CD73+ on circulating CD8+, CD4+ and MDSCs cells could be considered as potential biomarkers in stage IV melanoma patients treated with nivolumab. Materials and methods: Blood samples from 36 advanced melanoma patients were taken before nivolumab treatment; blood populations were measured in frozen peripheral blood mononuclear cells (PBMCs) that were thawed and then rested briefly, and subjected to flow cytometry analysis for myeloid-derived suppressor cells (MDSCs: CD14+ CD33+ CD11b+ HLA-DR-/low), CD8+ and CD4+, alone or in association with PD-1 and CD73+. Results: Our data demonstrated that patients with lower baseline values of CD8+/PD-1+/CD73+ had high OS and PFS (34.8 and 19.2 months, respectively); conversely, patients with higher baseline frequency of these cells experienced lower OS and PFS (9.5 and 2.8 months, respectively; OS p < 0.003, PFS p < 0.007) ( Tables 1, 2). In addition, increasing number of total CD8+ cells (p < 0.05) and especially of CD8+/PD-1+ cells (p < 0.04) were negatively correlated with survival (Table 1).  Background: Data from the literature indicate that the perception of patients with melanoma regarding the support received from healthcare services is generally inadequate with respect to the patients' need. Approximately 30% of melanoma patients face significant physical and psychological issues related to the diagnosis, treatment and follow-up [1][2][3]. The aim of this study is to evaluate the patients' specific clinical and communication needs in order to maximize their quality of health care. Materials and methods: A predefined questionnaire was submitted to 130 patients with new diagnoses of melanoma (stage I-IV), followed at San Raffaele Hospital (HSR) from January 2015 to January 2017. The study was approved by the Ethical Committee. The participants were recruited using non-probability purposing sampling techniques. The questionnaires were administered by email or telephone. It included 34 items, evaluating patients' demographics, education and employment status, basic knowledge on melanoma, and the understanding of diagnosis, stage, treatment details, prognosis, screening behaviors, quality of medical-patient communication and the perception for healthcare personnel support. The questionnaires were adapted from those used in Australia and New Zealand [4][5]. The reliability of the instrument was tested using test-retest reliability in IBM SPSS version 18. Statistical analysis was carried out using excel and PASW version 18 (SPPSS, Inc, II), adjusted by age, gender, education and stage of tumor. Results: One hundred and thirty patients accessed the questionnaire. Fifty-two have been already completed. Here we present preliminary date. In Table 1 patients' characteristics. Patients were grouped according to age (< 50, 50-70 and > 70 years) and the level of education (high school or less and vocational training). Sixty-three per cent of patients were satisfied about the support received, and 51.4% received enough information regarding diagnosis, prevention, treatment options and recurrence of the disease. Patients underline that like to have a single healthcare consultant.

O19
Conclusions: This is a deepened patient-centred approach conceived for melanoma patients that would provide a perspective on melanoma care, highlighting the areas that require the definition of a new protocol based on patients and their caregivers' needs. In Table 1 patients' characteristics.