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Abstract Background Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor, that is refractory to standard treatment and to immunotherapy with immune-checkpoint inhibitors (ICI). Noteworthy, melanoma brain metastases (MM-BM), that share the same niche as GBM, frequently respond to current ICI therapies. Epigenetic modifications regulate GBM cellular proliferation, invasion, and prognosis and may negatively regulate the cross-talk between malignant cells and immune cells in the tumor milieu, likely contributing to limit the efficacy of ICI therapy of GBM. Thus, manipulating the tumor epigenome can be considered a therapeutic opportunity in GBM. Methods Microarray transcriptional and methylation profiles, followed by gene set enrichment and IPA analyses, were performed to study the differences in the constitutive expression profiles of GBM vs MM-BM cells, compared to the extracranial MM cells and to investigate the modulatory effects of the DNA hypomethylating agent (DHA) guadecitabine among the different tumor cells. The prognostic relevance of DHA-modulated genes was tested by Cox analysis in a TCGA GBM patients’ cohort. Results The most striking differences between GBM and MM-BM cells were found to be the enrichment of biological processes associated with tumor growth, invasion, and extravasation with the inhibition of MHC class II antigen processing/presentation in GBM cells. Treatment with guadecitabine reduced these biological differences, shaping GBM cells towards a more immunogenic phenotype. Indeed, in GBM cells, promoter hypomethylation by guadecitabine led to the up-regulation of genes mainly associated with activation, proliferation, and migration of T and B cells and with MHC class II antigen processing/presentation. Among DHA-modulated genes in GBM, 7.6% showed a significant prognostic relevance. Moreover, a large set of immune-related upstream-regulators (URs) were commonly modulated by DHA in GBM, MM-BM, and MM cells: DHA-activated URs enriched for biological processes mainly involved in the regulation of cytokines and chemokines production, inflammatory response, and in Type I/II/III IFN-mediated signaling; conversely, DHA-inhibited URs were involved in metabolic and proliferative pathways. Conclusions Epigenetic remodeling by guadecitabine represents a promising strategy to increase the efficacy of cancer immunotherapy of GBM, supporting the rationale to develop new epigenetic-based immunotherapeutic approaches for the treatment of this still highly deadly disease.

Abstract Association with hypomethylating agents is a promising strategy to improve the efficacy of immune checkpoint inhibitors-based therapy. The NIBIT-M4 was a phase Ib, dose-escalation trial in patients with advanced melanoma of the hypomethylating agent guadecitabine combined with the anti-CTLA-4 antibody ipilimumab that followed a traditional 3 + 3 design (NCT02608437). Patients received guadecitabine 30, 45 or 60 mg/m2/day subcutaneously on days 1 to 5 every 3 weeks starting on week 0 for a total of four cycles, and ipilimumab 3 mg/kg intravenously starting on day 1 of week 1 every 3 weeks for a total of four cycles. Primary outcomes of safety, tolerability, and maximum tolerated dose of treatment were previously reported. Here we report the 5-year clinical outcome for the secondary endpoints of overall survival, progression free survival, and duration of response, and an exploratory integrated multi-omics analysis on pre- and on-treatment tumor biopsies. With a minimum follow-up of 45 months, the 5-year overall survival rate was 28.9% and the median duration of response was 20.6 months. Re-expression of immuno-modulatory endogenous retroviruses and of other repetitive elements, and a mechanistic signature of guadecitabine are associated with response. Integration of a genetic immunoediting index with an adaptive immunity signature stratifies patients/lesions into four distinct subsets and discriminates 5-year overall survival and progression free survival. These results suggest that coupling genetic immunoediting with activation of adaptive immunity is a relevant requisite for achieving long term clinical benefit by epigenetic immunomodulation in advanced melanoma patients.

IntroductionAbout 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots.Methods and resultsBy using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors’ and patients’ peripheral blood cells. ConclusionOur data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

Abstract Background Improvement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents. Methods Melanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets. Results Epigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset. Conclusions The DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches.

Abstract Background Melanoma is the deadliest form of skin cancer and metastatic disease is associated with a significant survival rate drop. There is an urgent need for consistent tumor biomarkers to scale precision medicine and reduce cancer mortality. Here, we aimed to identify a melanoma-specific circulating microRNA signature and assess its value as a diagnostic tool. Methods The study consisted of a discovery phase and two validation phases. Circulating plasma extracellular vesicles (pEV) associated microRNA profiles were obtained from a discovery cohort of metastatic melanoma patients and normal subjects as controls. A pEV-microRNA signature was obtained using a LASSO penalized logistic regression model. The pEV-microRNA signature was subsequently validated both in a publicly available dataset and in an independent internal cohort. Results We identified and validated in three independent cohorts a panel of melanoma-specific circulating microRNAs that showed high accuracy in differentiating melanoma patients from healthy subjects with an area under the curve (AUC) of 1.00, 0.94 and 0.75 respectively. Investigation of the function of the pEV-microRNA signature evidenced their possible immune suppressive role in melanoma patients. Conclusions We demonstrate that a blood test based on circulating microRNAs can non-invasively detect melanoma, offering a novel diagnostic tool for improving standard care. Moreover, we revealed an immune suppressive role for melanoma pEV-microRNAs.

Background Chemoimmunotherapy represents the standard of care for patients with advanced non-small cell lung cancer (NSCLC) and programmed death-ligand 1 (PD-L1)

Abstract Microenvironment‐related immune and inflammatory markers, when combined with established Ki‐67 and morphology parameters, can improve prognostic prediction in gastro‐entero‐pancreatic neuroendocrine neoplasms (GEP‐NENs). Therefore, we evaluated the prognostic value of microenvironment and tumor inflammatory features (MoTIFs) in GEP‐NENs. For this purpose, formalin‐fixed paraffin‐embedded tissue sections from 350 patients were profiled by immunohistochemistry for immune, inflammatory, angiogenesis, proliferation, NEN‐, and fibroblast‐related markers. A total of 314 patients were used to generate overall survival (OS) and disease‐free survival (DFS) MoTIFs prognostic indices (PIs). PIs and additional variables were assessed using Cox models to generate nomograms for predicting 5‐year OS and DFS. A total of 36 patients were used for external validation of PIs and nomograms' prognostic segregations. From our analysis, G1/G2 versus G3 GEP‐NENs showed phenotypic divergence with immune‐inflammatory markers. HLA, CD3, CD8, and PD‐1/PD‐L1 IHC expression separated G3 into two sub‐categories with high versus low adaptive immunity‐related features. MoTIFs PI for OS based on COX‐2Tumor(T) > 4, PD‐1Stromal(S) > 0, CD8S < 1, and HLA‐IS < 1 was associated with worst survival (hazard ratio [HR] 2.50; 95% confidence interval [CI], 2.12–2.96; p < 0.0001). MoTIFs PI for DFS was based on COX‐2T > 4, PD‐1S > 4, HLA‐IS < 1, HLA‐IT < 2, HLA‐DRS < 6 (HR 1.77; 95% CI, 1.58–1.99; p < 0.0001). Two nomograms were developed including morphology (HR 4.83; 95% CI, 2.30–10.15; p < 0.001) and Ki‐67 (HR 11.32; 95% CI, 5.28–24.24; p < 0.001) for OS, and morphology (PI = 0: HR 10.23; 95% CI, 5.67–18.47; PI = 5: HR 2.87; 95% CI, 1.21–6.81; p < 0.001) and MoTIFs PI for DFS in well‐differentiated GEP‐NENs (HR 6.21; 95% CI, 2.52–13.31; p < 0.001). We conclude that G1/G2 to G3 transition is associated with immune‐inflammatory profile changes; in fact, MoTIFs combined with morphology and Ki‐67 improve 5‐year DFS prediction in GEP‐NENs. The immune context of a subset of G3 poorly differentiated tumors is consistent with activation of adaptive immunity, suggesting a potential for responsiveness to immunotherapy targeting immune checkpoints.

This case report shows, for the first time, a patient experiencing a complete response after one dose of avelumab following extensive disease progression with prior electrochemotherapy (ECT) treatment. We suggest that ECT may help to establish a tumor microenvironment favorable to immunotherapy. Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with seldom durable chemotherapy responses. ECT has recently emerged as a potential treatment option for several malignancies, including MCC. Avelumab, an anti-programmed cell death-ligand 1 (PD-L1) monoclonal antibody, became the first approved treatment for patients with metastatic MCC. ECT has been shown to activate the immune response, but it is still unknown how ECT may affect patient’s response to subsequent immunotherapy. We report a case of a patient with MCC who presented with a rapidly growing skin nodule of the right cheek and experienced extensive disease progression following surgical debulking and ECT treatment. The patient received a flat dose of 800 mg avelumab intravenously every 2 weeks showing complete tumor regression after only one dose. Immunohistochemical analysis of surgical and post-ECT biopsies collected from the primary lesion revealed tumor expression of programmed cell death protein-1 (PD-1), but not PD-L1. Analysis of the tumor samples also revealed no expression of Merkel cell polyomavirus (MCPyV). Comparison of the biopsies showed a decrease in myeloid and T-cell markers after ECT but an increase in major histocompatibility complex (MHC) class I expression on tumor cells. Additionally, the patient experienced an increase in neutrophil-to-lymphocyte ratio and lactate dehydrogenase values post-ECT, which subsequently decreased with avelumab treatment. As of 30 October 2019, the patient was still receiving avelumab treatment and had an ongoing complete response. In this case report, a patient with PD-L1-negative and MCPyV-negative MCC who had disease progression following ECT experienced complete tumor regression with avelumab treatment, suggesting, for the first time to our knowledge, that ECT may help to establish a tumor microenvironment favorable to immunotherapy via a potential abscopal effect. Tumor-intrinsic PD-1 expression and modulation of MHC class I antigens after ECT may contribute to the clinical efficacy of avelumab in this context.

We previously published the results of a pilot study showing that vaccination with tumor-loaded dendritic cells (DCs) induced both T and B cell response and produced clinical benefit in the absence of toxicity in patients with relapsed, indolent non-Hodgkin lymphoma (iNHL). The purpose of the present short report is to provide a 15-year follow-up of our study and to expand the biomarker analysis previously performed. The long-term follow-up highlighted the absence of particular or delayed toxicity and the benefit of active immunization with DCs loaded with autologous, heat-shocked and UV-C treated tumor cells in relapsed iNHL (5-year and 10-year progression-free survival (PFS) rates: 55.6% and 33.3%, respectively; 10-year overall survival (OS) rate: 83.3%). Female patients experienced a better PFS (p=0.016) and a trend towards a better OS (p=0.185) compared with male patients. Of note, we observed a non-negligible fraction of patients (22%) who experienced a long-lasting complete response. In a targeted gene expression profiling of pre-treatment tumor biopsies in 11 patients with available formalin-fixed, paraffin-embedded tissue, we observed that KIT, ATG12, TNFRSF10C, PBK, ITGA2, GATA3, CLU, NCAM1, SYT17 and LTK were differentially expressed in patients with responder versus non-responder tumors. The characterization of peripheral monocytic cells in a subgroup of 14 patients with available baseline blood samples showed a higher frequency of the subset of CD14++CD16+ cells (intermediate monocytes) in patients with responding tumors. Since in patients with relapsed iNHL the available therapeutic options are often incapable of inducing a long-lasting complete remission and can be sometimes characterized by intolerable toxicity, we think that the encouraging results of our long-term follow-up analysis represent a stimulus to further investigate the role of active vaccination in this specific setting and in earlier lines of therapy and to explore novel combinatorial strategies encompassing other innovative immunotherapy agents, such as immune-checkpoint inhibitors.

Abstract Background Melanoma, the most aggressive form of skin cancer, is characterized by high rates of metastasis, drug resistance and mortality. Here we investigated the role of Semaphorin 5A (Sema5A) on the properties associated with melanoma progression and the factors involved in Sema5A regulation. Methods Western blotting, qRT-PCR, Chromatin immunoprecipitation (ChIP) assay, immunohistochemistry of melanoma patient specimens and xenograft tissues, in vitro Transwell assay for cell migration and invasion evaluation, in vitro capillary-like structure formation analysis. Results A significant correlation of Sema5A mRNA expression and melanoma progression was observed by analyzing GEO profile dataset. Endogenous Sema5A protein was detected in 95% of human melanoma cell lines tested, in 70% of metastatic specimens from patients affected by melanoma, and 16% of in situ melanoma specimens showed a focal positivity. We demonstrated that Sema5A regulates in vitro cell migration and invasion and the formation of vasculogenic structures. We also found an increase of Sema5A at both mRNA and protein level after forced expression of Bcl-2. By use of transcriptional and proteasome inhibitors, we showed that Bcl-2 increases the stability of Sema5A mRNA and protein. Moreover, by ChIP we demonstrated that Sema5A expression is under the control of the transcription factor c-Myb and that c-Myb recruitment on Sema5A promoter is increased after Bcl-2 overexpression. Finally, a concomitant decrease in the expression of Sema5A, Bcl-2 and c-Myb proteins was observed in melanoma cells after miR-204 overexpression. Conclusion Overall our data provide evidences supporting the role of Sema5A in melanoma progression and the involvement of Bcl-2, miR-204 and c-Myb in regulating its expression.

T-cell-based immunotherapy strategies have profoundly improved the clinical management of several solid tumors and hematological malignancies. A recently developed and promising immunotherapy approach is to redirect polyclonal MHC-unrestricted T lymphocytes toward cancer cells by bispecific antibodies (bsAbs) that engage the CD3 complex and a tumor-associated antigen (TAA). The TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) is an attractive immunotherapy target, frequently expressed by neoplastic cells, that we decided to exploit as a TAA. We found that a TRAIL-R2xCD3 bsAb efficiently activates T cells and specifically redirect their cytotoxicity against cancer cells of different origins in vitro, thereby demonstrating its potential as a pan-carcinoma reagent. Moreover, to mimic in vivo conditions, we assessed its ability to retarget T-cell activity in an ex vivo model of ovarian cancer patients' ascitic fluids containing both effector and target cells—albeit with a suboptimal effector-to-target ratio—with remarkable results.

Despite the success of immune checkpoint blockade in melanoma, the majority of patients do not respond. We hypothesized that the T and NK cell subset frequencies and expression levels of their receptors may predict responses and clinical outcome of anti-CTLA-4 treatment. We thus characterized the NK and T cell phenotype, as well as serum levels of several cytokines in 67 melanoma patients recruited in Italy and Sweden, using samples drawn prior to and during treatment. Survival correlated with low expression of the inhibitory receptor TIM-3 on circulating T and NK cells prior to and during treatment and with the increased frequency of mature circulating NK cells (defined as CD3−CD56dim CD16+) during treatment. Survival also correlated with low levels of IL-15 in the serum. Functional experiments in vitro demonstrated that sustained exposure to IL-15 enhanced the expression of PD-1 and TIM-3 on both T and NK cells, indicating a causative link between high IL-15 levels and enhanced expression of TIM-3 on these cells. Receptor blockade of TIM-3 improved NK cell-mediated elimination of melanoma metastasis cell lines in vitro. These observations may lead to the development of novel biomarkers to predict patient response to checkpoint blockade treatment. They also suggest that induction of additional checkpoints is a possibility that needs to be considered when treating melanoma patients with IL-15.

The Raf/MEK/ERK pathway is an importantmediator of tumor cell proliferation and angiogenesis. Here, weinvestigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.

Background Adoptive cell therapy with ex vivo expanded autologous antitumor cytotoxic T lymphocytes represents an important therapeutic option as an anticancer strategy. In order to identify a reliable method for producing adequate amounts of functional antitumor cytotoxic T lymphocytes with a potentially long in vivo lifespan, we tested the T-cell expansion efficiency of a new artificial antigen-presenting cell-based system.Design and Methods Our artificial antigen-presenting cells were generated with activating (anti-CD3), co-stimulating (anti-CD28) and adhesion (anti-LFA-1) biotinylated monoclonal antibodies preclusterted in microdomains held on a liposome scaffold by neutravidin rafts. The co-localization of T-cell ligands in microdomains and the targeting of an adhesion protein, increasing the efficiency of immunological synapse formation, represent the novelties of our system. The activity of our artificial antigen-presenting cells was compared with that of anti-CD3/-CD28 coated immunomagnetic microbeads and immobilized anti-CD3 monoclonal antibody (OKT3 clone), the only two commercially available artificial systems.Results Our artificial antigen-presenting cells expanded both polyclonal T cells and MART-1-specific CD8+ T cells in a more efficient manner than the other systems. Stimulation with artificial antigen-presenting cells allows for the generation of viable T cells displaying an immunophenotype consistent with in vivo potential for persistence, without increasing the frequency of regulatory T cells. The starting specificity of anti MART-1 CD8+ T cells was preserved after stimulation with artificial antigen-presenting cells and it was statistically greater when compared to the activity of the same cells expanded with the other systems. Finally, our artificial antigen-presenting cells proved to be suitable for large-scale application, minimizing the volume and the costs of T-cell expansion.Conclusions Our artificial antigen-presenting cells might represent an efficient tool to rapidly obtain a sufficient number of functional T cells for adoptive immunotherapy in patients with cancer.