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Purpose:Phase II trials have shown encouraging activity with ipilimumab plus fotemustine and ipilimumab plus nivolumab in melanoma brain metastases. We report the primary analysis and 4-year follow-up of the NIBIT-M2 study, the first phase III trial comparing these regimens with fotemustine in patients with melanoma with brain metastases.Patients and Methods:This phase III study recruited patients 18 years of age and older with BRAF wild-type or mutant melanoma, and active, untreated, asymptomatic brain metastases from nine centers, randomized (1:1:1) to fotemustine, ipilimumab plus fotemustine, or ipilimumab plus nivolumab. The primary endpoint was overall survival (OS).Results:From January, 2013 to September, 2018, 27, 26, and 27 patients received fotemustine, ipilimumab plus fotemustine, and ipilimumab plus nivolumab. Median OS was 8.5 months [95% confidence interval (CI), 4.8–12.2] in the fotemustine arm, 8.2 months (95% CI, 2.2–14.3) in the ipilimumab plus fotemustine arm (HR vs. fotemustine, 1.09; 95% CI, 0.59–1.99; P = 0.78), and 29.2 months (95% CI, 0–65.1) in the ipilimumab plus nivolumab arm (HR vs. fotemustine, 0.44; 95% CI, 0.22–0.87; P = 0.017). Four-year survival rate was significantly higher for ipilimumab plus nivolumab than fotemustine [(41.0%; 95% CI, 20.6–61.4) vs. 10.9% (95% CI, 0–24.4; P = 0.015)], and was 10.3% (95% CI, 0–22.6) for ipilimumab plus fotemustine. In the fotemustine, ipilimumab plus fotemustine, and ipilimumab plus nivolumab arms, respectively, 11 (48%), 18 (69%), and eight (30%) patients had treatment-related grade 3 or 4 adverse events, without treatment-related deaths.Conclusions:Compared with fotemustine, ipilimumab plus nivolumab significantly improved overall and long-term survival of patients with melanoma with asymptomatic brain metastases.

Study design and treatment schedule. Patients in either the ipilimumab plus fotemustine arm or the ipilimumab plus nivolumab arm could continue maintenance treatment until disease progression, excessive toxicity, or patient refusal

Maintenance phase treatment exposure

Swimmer plot analysis of NIBIT-M2 patients. Swimmer plot showing by study arm patients who at the time of data cut-off were alive and either still on study therapy (n=7) or off study therapy without having received subsequent treatment (n=6), and all patients who had received subsequent treatment at the time of data cut-off, regardless of whether alive or dead (n=32). Patients excluded from the plot (n=31) died without receiving any subsequent therapy.

Summary Background The NIBIT-MESO-1 study demonstrated the efficacy and safety of tremelimumab combined with durvalumab in patient with unresectable mesothelioma followed up for a median of 52 months [IQR 49–53]. Here, we report 4-year survival and outcomes after retreatment, and the role of tumour mutational burden (TMB) in identifying patients who might have a better outcome in response to combined therapy. Methods NIBIT-MESO-1 was an open-label, non-randomised, phase 2 trial of patients with unresectable pleural or peritoneal mesothelioma who received intravenous tremelimumab (1 mg/kg bodyweight) and durvalumab (20 mg/kg bodyweight) every 4 weeks for four doses, followed by maintenance intravenous durvalumab at the same dose and schedule for nine doses. In this follow-up study, patients with disease progression following initial clinical benefit—ie, a partial repsonse or stable disease—were eligible for retreatment and with the same doses and schedules for tremelimumab and durvalumab as used in the NIBIT-MESO-1 trial. The primary endpoint, immune-related objective response rate, was evaluated per immune-related modified Response Evaluation Criteria in Solid Tumors (RECIST) or immune-related RECIST 1.1 criteria for patients with pleural or peritoneal malignant mesothelioma, respectively. Key secondary endpoints were overall survival and safety, and TMB was also evaluated post hoc in patients who had tumour tissue available before treatment. The intention-to-treat population was used for analysis of all efficacy endpoints. This study is registered with ClinicalTrials.gov , number NCT02588131 . Findings 40 patients were enrolled in the NIBIT-MESO-1 study between Oct 30, 2015, and Oct 12, 2016. At data cut-off, April 30, 2020, five (13%) of 40 patients were alive, and 35 (88%) patients had died of progressive disease. At a median follow-up of 52 months (IQR 49–53), median overall survival was 16·5 months (95% CI 13·7–19·2). Survival was 20% (eight of 40 patients) at 36 months and 15% (six of 40 patients) and 48 months. 17 (43%) of 40 patients met the criteria for enrolment in the retreatment study and were retreated with at least one dose of tremelimumab and durvalumab. No immune-related objective responses were observed in the 17 retreated patients. Seven (41%) of 17 patients achieved immune-related stable disease. From the start of retreatment to a median follow-up of 24 months (22·0–25·0), median overall survival was 12·5 months (95% CI 0·0–25·8), and survival at 12 months was 52·9%, at 18 months was 35·3%, and at 24 months was 23·5%. There were no grade 3–4 immune-related adverse events in the retreatment cohort. In a post-hoc analysis of 28 patients for whom tumour tissue before treatment was available, patients with a TMB higher than the median value of 8·3 mutations per Mb had a higher median overall survival compared with patients with TMB below the median value, but this difference was non-significant. Moreover, when patients were additionally stratified for ICI retreatment (n=13), there was a significant difference in survival between those with a TMB higher than the median of 8·3 mutations per Mb and those with TMB lower than the median in the retreated cohort (41·3 months vs 17·4 months; p=0·02). Interpretation Tremelimumab combined with durvalumab was associated with long-term survival in patients with mesothelioma. Retreatment was safe and resulted in clinically meaningful outcomes, thus suggesting its potential application in the clinical practice of mesothalioma patients. Funding NIBIT Foundation, Fondazione AIRC, AstraZeneca.

Background Growing evidence are demonstrating the therapeutic efficacy of immune checkpoint inhibitors (ICI) in mesothelioma; however, a limited percentage of patients benefits from this therapeutic approach. Epigenetic modifications play a relevant role in negatively regulating the cross-talk between neoplastic and immune cells, and in contributing to the highly immunosuppressive mesothelioma microenvironment. A better understanding of mesothelioma epigenetic landscape could open the path to novel and potentially more effective approaches combining ICI and epigenetic drugs. We investigated the immunomodulatory potential of epigenetic agents by comparing the activity of DNA hypomethylating agents (DHA) with histone deacetylases inhibitors (HDACi) and EZH2 inhibitors (EZH2i), alone or combined with DHA, in mesothelioma cells. Methods Four mesothelioma cell lines were treated with the DHA guadecitabine 1μM, or with the HDACi, Valproic Acid (VPA) 1mM, or the EZH2i, EPZ-6438 1μM, alone or combined with guadecitabine. We investigated the expression of HLA class I molecules by flow-cytometry and of PD-L1, cancer testis antigens (CTA: NY-ESO, MAGE-A1), Natural Killer Group 2 member D Ligands (NKG2DLs: MIC-A, MIC-B, ULBP2) and EMT-regulating cadherins (CDH1, CDH2) by quantitative Real-Time PCR. Fold change (FC) expression for each treatment vs untreated cells was reported as mean values (FCm) among investigated cell lines. A positive modulation of the expression was considered if FCm>1.5. Results Guadecitabine upregulated the expression of HLA class I antigens (FCm=1.75), PD-L1 (FCm=2.38), NKG2DLs (MIC-A FCm=1.96, MIC-B FCm=2.57, and ULBP2 FCm=3.56), and upregulated/induced CTA expression. Similarly, VPA upregulated HLA class I antigens (FCm=1.67), PD-L1 (FCm=3.17), NKG2DLs (MIC-A FCm=1.78, MIC-B FCm=3.04, and ULBP2 FCm=3.75) expression; however, CTA expression was modulated only in 1 mesothelioma cell line. Conversely, EPZ-6438 up-regulated only NY-ESO-1 and MIC-B expression in 1 mesothelioma cell line. The addition of both VPA and EPZ-6483 to guadecitabine strengthened its immunomodulatory activity. Specifically, guadecitabine plus VPA or EPZ-6438 upregulated the expression of HLA class I antigens FCm=2.55 or 2.69, PD-L1 FCm=8.04 or 2.65, MIC-A FCm=3.81 or 2.26, MIC-B FCm=8.00 or 3.03, ULBP2 FCm=6.24 or 4.53, respectively. Higher levels of CTA upregulation/induction were observed with combination treatments vs guadecitabine alone. Cadherins modulation was mesothelioma histotype-related: CDH1 expression was induced in the 2 constitutively-negative sarcomatoid mesothelioma cells by guadecitabine alone or combined with VPA or EPZ-6438; CDH2 expression was upregulated by VPA alone (FCm=1.53) or plus guadecitabine (FCm=2.54). Conclusions Combination of DHA-based immunotherapies with other classes of epigenetic drugs could be an effective strategy to be pursued in the mesothelioma clinic.

Malignant mesothelioma (MM) is a highly aggressive form of cancer with limited treatment options. Although the role of NK cells has been studied in many solid tumors, the pattern of NK-cell subsets and their recognition of mesothelioma cells remain to be explored. We used RNA expression data of MM biopsies derived from the cancer genome atlas to evaluate the immune cell infiltrates. We characterized the phenotype of circulating NK and T cells of 27 MM patients before and after treatment with an anti-CTLA-4 antibody (tremelimumab). These immune cell profiles were compared to healthy controls. The RNA expression data of the MM biopsies indicated the presence of NK cells in a subgroup of patients. We demonstrated that NK cells recognize MM cell lines and that IL-15 stimulation improved NK cell-mediated lysis in vitro. Using multivariate projection models, we found that MM patients had a perturbed ratio of CD56bright and CD56dim NK subsets and increased serum concentrations of the cytokines IL-10, IL-8 and TNF-α. After tremelimumab treatment, the ratio between the CD56bright and CD56dim subsets shifted back towards physiological levels. Furthermore, the improved overall survival was correlated with low TIM-3+ CD8+ T-cell frequency, high DNAM-1+ CD56dim NK-cell frequency and high expression levels of NKp46 on the CD56dim NK cells before and after immune checkpoint blockade. Together, our observations suggest that NK cells infiltrate MM and that they can recognize and kill mesothelioma cells. The disease is associated with distinct lymphocytes patterns, some of which correlate with prognosis or are affected by treatment with tremelimumab.

Purpose: The immunomodulatory activity of DNA hypomethylating agents (DHAs) suggests they may improve the effectiveness of cancer immunotherapies. The phase Ib NIBIT-M4 trial tested this hypothesis using the next-generation DHA guadecitabine combined with ipilimumab. Patients and Methods: Patients with unresectable stage III/IV melanoma received escalating doses of guadecitabine 30, 45, or 60 mg/m2/day subcutaneously on days 1 to 5 every 3 weeks, and ipilimumab 3 mg/kg intravenously on day 1 every 3 weeks, starting 1 week after guadecitabine, for four cycles. Primary endpoints were safety, tolerability, and MTD of treatment; secondary were immune-related (ir) disease control rate (DCR) and objective response rate (ORR); and exploratory were changes in methylome, transcriptome, and immune contextures in sequential tumor biopsies, and pharmacokinetics. Results: Nineteen patients were treated; 84% had grade 3/4 adverse events, and neither dose-limiting toxicities per protocol nor overlapping toxicities were observed. Ir-DCR and ir-ORR were 42% and 26%, respectively. Median CpG site methylation of tumor samples (n = 8) at week 4 (74.5%) and week 12 (75.5%) was significantly (P < 0.05) lower than at baseline (80.3%), with a median of 2,454 (week 4) and 4,131 (week 12) differentially expressed genes. Among the 136 pathways significantly (P < 0.05; Z score >2 or ←2) modulated by treatment, the most frequently activated were immune-related. Tumor immune contexture analysis (n = 11) demonstrated upregulation of HLA class I on melanoma cells, an increase in CD8+, PD-1+ T cells and in CD20+ B cells in posttreatment tumor cores. Conclusions: Treatment of guadecitabine combined with ipilimumab is safe and tolerable in advanced melanoma and has promising immunomodulatory and antitumor activity.

Rationale: Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumour in adults, with a very dismal prognosis and limited effective therapies. Immunotherapy may play a role in treating GBM, though the initial clinical results are still poor, and novel combined treatment(s) could possibly improve its efficacy. Manipulating the epigenome has been considered a therapeutic opportunity in GBM, since epigenetic modifications regulate glioma proliferation, metastasis, invasion, and prognosis. In addition, epigenetic alterations may negatively regulate the cross-talk between GBM and immune cells in GBM microenvironment (TME), thus limiting the efficacy of immunotherapy. We previously demonstrated the immunomodulatory potential of epigenetic drugs in cancer cells, and translated this notion in the clinic to improve efficacy of immunotherapy of melanoma patients (Di Giacomo AM, CCR 2019). Accordingly, this study aimed to evaluate the immunomodulatory potential of epigenetic drugs in GBM, and to provide the scientific rationale for new combinatorial therapeutic approaches in GBM patients. Methods: Patient-derived GBM (pGBM) cell lines (n=10) were established from surgically-removed GBM lesions and treated with the DNA hypomethylating agent guadecitabine (1 µM); commercially available human GBM (cGBM) cell lines (n=3) were treated with guadecitabine (1 µM) and/or with the EZH2-inhibitor GSK126 (10 µM). Expression of HLA class I and ICAM-1 by GBM cells was investigated by flow-cytometry; cancer testis antigens (CTA, i.e., NY-ESO, MAGE-A1 and MAGE-A3) and the epithelial-mesenchymal transition (EMT)-regulating cadherins (i.e., CDH1 and CDH2) expression was analyzed by quantitative Real-Time PCR. Results: A positive modulation of immune profile was observed in all investigated guadecitabine-treated GBM cells. Specifically, in untreated vs guadecitabine-treated cells, MFI mean values ± SD of HLA class I were 402±326 vs 442±303 in pGBM cells, and 277±127 vs 381±138 in cGBM cells; MFI mean values ± SD of ICAM-1 were 53±72 vs 92±116 in pGBM cells, and 5±6 vs 26±17 in cGBM cells. Though a slight increment was induced by GSK126 for HLA class I (312±169) and ICAM-1 (9±11) expression in cGBM, an additive effect was detected with GSK126+guadecitabine (405±261 for HLA class I and 35±22 for ICAM-1) in cGBM cells. Guadecitabine induced NY-ESO-1, MAGE-A1, and MAGE-A3 expression in 9/10, 6/10, and 5/8 pGBM cells, respectively and in all cGBM cells. Levels of CTA expression were increased in cGBM by GSK126+guadecitabine vs guadecitabine with mean Fold Change (FC) of 1.5, 1.6 and 1.6 for NY-ESO-1, MAGE-A1 and MAGE-A3 expression. Finally, CDH1 expression was induced in 1/3 and in 2/3 cGBM cells by guadecitabine and guadecitabine+GSK126, respectively; CDH2-positive expression was upregulated by guadecitabine (mean FC=2.45) but not by guadecitabine+GSK126. Conclusions: Immunomodulatory properties of epigenetic drugs in GBM should be exploited to improve the efficacy of GBM immunotherapy. Citation Format: Maria Fortunata Lofiego, Francesca Piazzini, Carolina Fazio, Ornella Cutaia, Carla Chiarucci, Sara Cannito, Elisabetta Gambale, Monica Valente, Anna Maria Di Giacomo, Sandra Coral, Clelia Miracco, Giuseppe Oliveri, Michele Maio, Alessia Covre. Immunomodulatory activity of epigenetic drugs: Laying the ground for new combined immunotherapeutic strategies for glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4488.

Targeting of the programmed cell death protein (PD)-1/programmed death-ligand 1 (PD-L1) axis has shown a significant clinical impact in several tumor types. Accordingly, our phase II NIBIT-MESO-1 study demonstrated an improved clinical efficacy in mesothelioma patients treated with the anti-PD-L1 durvalumab combined with the anti-cytotoxic T-lymphocyte antigen (CTLA)-4 tremelimumab, as compared to tremelimumab alone. Due to the promising therapeutic activity of immune check-point inhibitors (ICIs) in mesothelioma patients, the identification of biomarkers predictive of response to treatment is of crucial relevance. The prognostic role of soluble PD-L1 (sPD-L1) proposed in cancer patients prompted us to investigate this protein in sera from mesothelioma patients (n = 40) enrolled in the NIBIT-MESO-1 study. A significant (p &lt, 0.001) increase in sPD-L1 levels was detected in patients after the first cycle and during therapy vs. baseline. A longer overall survival (OS) was observed in patients with sPD-L1 concentrations below (at baseline, d1C2, d1C5 (p &lt, 0.01)) or FC values above (p &lt, 0.05 at d1C2, d1C3, d1C5) their statistically calculated optimal cut-offs. On the basis of these initial results, the specific role of CTLA-4-, PD-L1-, or PD-1-targeting on sPD-L1 release was then investigated in sera from 81 additional ICI-treated solid cancer patients. Results showed a significant (p &lt, 0.001) increase of sPD-L1 levels during therapy compared to baseline only in anti-PD-L1-treated patients, supporting the specific involvement of PD-L1 targeting in the release of its soluble form. Our findings suggest that sPD-L1 represents a predictive biomarker of clinical response to anti-PD-L1 cancer immunotherapy.

DNA hypomethylating agents (DHAs) play a well-acknowledged role in potentiating the immunogenicity and the immune recognition of neoplastic cells. This immunomodulatory activity of DHAs is linked to their ability to induce or to up-regulate on neoplastic cells the expression of a variety of immune molecules that play a crucial role in host-tumor immune interactions. To further investigate the clinical potential of diverse epigenetic compounds when combined with immunotherapeutic strategies, we have now compared the tumor immunomodulatory properties of the first generation DHAs, azacytidine (AZA) and decitabine (DAC) and of the next generation DHA, guadecitabine. To this end, human melanoma and hematological cancer cells were treated in vitro with 1 μM guadecitabine, DAC or AZA and then studied by molecular and flow cytometry analyses for changes in their baseline expression of selected immune molecules involved in different mechanism(s) of immune recognition. Results demonstrated a stronger DNA hypomethylating activity of guadecitabine and DAC, compared to AZA that associated with stronger immunomodulatory activities. Indeed, the mRNA expression of cancer testis antigens, immune-checkpoint blocking molecules, immunostimulatory cytokines, involved in NK and T cell signaling and recruiting, and of genes involved in interferon pathway was higher after guadecitabine and DAC compared to AZA treatment. Moreover, a stronger up-regulation of the constitutive expression of HLA class I antigens and of Intercellular Adhesion Molecule-1 was observed with guadecitabine and DAC compared to AZA. Guadecitabine and DAC seem to represent the optimal combination partners to improve the therapeutic efficacy of immunotherapeutic agents in combination/sequencing clinical studies.

Glioblastoma (GB) is the most common and aggressive primary tumor of the central nervous system. The current standard of care for GB consists of surgical resection, followed by radiotherapy combined with temozolomide chemotherapy. However, despite this intensive treatment, the prognosis remains extremely poor. Therefore, more effective therapies are urgently required. Recent studies indicate that SRC family kinases (SFKs) could represent promising molecular targets for GB therapy. Here, we challenged four GB cell lines with a new selective pyrazolo[3,4-d]pyrimidine derivative SFK inhibitor, called SI221. This compound exerted a significant cytotoxic effect on GB cells, without significantly affecting non-tumor cells (primary human skin fibroblasts), as evaluated by MTS assay. We also observed that SI221 was more effective than the well-known SFK inhibitor PP2 in GB cells. Notably, despite the high intrinsic resistance to apoptosis of GB cells, SI221 was able to induce this cell death process in all the GB cell lines, as observed through cytofluorimetric analysis and caspase-3 assay. SI221 also exerted a long-term inhibition of GB cell growth and was able to reduce GB cell migration, as shown by clonogenic assay and scratch test, respectively. Moreover, through in vitro pharmacokinetic assays, SI221 proved to have a high metabolic stability and a good potential to cross the blood brain barrier, which is an essential requirement for a drug intended to treat brain tumors. Therefore, despite the need of developing strategies to improve SI221 solubility, our results suggest a potential application of this selective SFK inhibitor in GB therapy. J. Cell. Biochem. 116: 856–863, 2015. © 2014 Wiley Periodicals, Inc.

The introduction of immune checkpoint blockade into the clinical practice resulted in improvement of survival of a significant portion of melanoma patients. Consequently, predictive biomarkers of response are needed to optimize patient's stratification and the development of combination therapies. The aim of this study was to determine whether levels of soluble NKG2D ligands (MICA, MICB, ULBP1, 2 and 3; sNKG2DLs) in the serum of melanoma patients can serve as useful predictors of response to the treatment with immune checkpoint blockade. sNKG2DLs were measured by ELISA in baseline and post-treatment serum and these results were correlated with the clinical outcome of melanoma patients (N = 194). The same determinations were performed also in a cohort of patients (N = 65) treated with either chemotherapy, radiotherapy, or mutated BRAF inhibitors (BRAFi). Absence of soluble MICB and ULBP-1 in baseline serum correlated with improved survival (OS = 21.6 and 25.3 mo and p = 0.02 and 0.01, respectively) of patients treated with immunological therapies while detectable levels of these molecules were found in poor survivors (OS = 8.8 and 12.1 mo, respectively). Multivariate analysis showed that LDH (p

2549 Background: DNA hypomethylating agents show broad immuno-modulatory activity in neoplastic cells, and may improve the effectiveness of cancer immunotherapies. The phase 1b NIBIT-M4 trial investigated a previously unexplored therapeutic strategy using the next-generation DNA hypomethylating agent guadecitabine sequenced with ipilimumab for the treatment of advanced melanoma. Methods: Patients with unresectable Stage III/IV melanoma received escalating doses of guadecitabine 30, 45 or 60 mg/m2 subcutaneously on Days 1–5 every three weeks, and ipilimumab 3 mg/kg intravenously on Day 1 every three weeks, starting one week after guadecitabine, for four cycles. Primary endpoints were the safety, tolerability and maximum tolerated dose of treatment; secondary endpoints included immune-related disease control and objective response. Genome-wide methylation, RNA sequencing, and immunohistochemistry analyses were performed on tumor samples collected at baseline, W4 and W12. (NCT02608437). Results: 19 patients were treated and evaluable for safety and efficacy. The most common treatment-related adverse events of any grade were myelotoxicity (n = 17; 89%) and immune-related adverse events (n = 12; 63%). Grade 3 or 4 myelotoxicity occurred in 15 (79%) patients. There were no dose limiting toxicities. Rates of immune-related disease control and objective response were 8/19 (42%) and 5/19 (26%), respectively. Exploratory analyses of tumour samples (n = 8) showed that median CpG site methylation at Week 4 (74.5%) and Week 12 (75.5%) was significantly lower (p < 0.05) than at baseline (80.3%), with a median of 2454 (Week 4) and 4131 (Week 12) differentially expressed genes identified compared to baseline; among the 136 pathways significantly modulated by treatment, the most frequently activated were immune-related. Tumour immune contexture analysis (n = 11) demonstrated up-regulation of Human Leukocyte Antigen (HLA) class I molecules on melanoma cells, and an increase in CD8+, PD-1+ T cells and in CD20+ B cells in post-treatment tumour core specimens. Conclusions: Sequential guadecitabine and ipilimumab is safe and tolerable in patients with metastatic melanoma, and has promising immunological and anti-tumour activity. Clinical trial information: NCT02608437.

Compelling experimental evidences point to monitoring of the absolute number of circulating ICOS-positive T cells as an early predictive marker of clinical activity of anti-CTLA-4 antibodies in cancer patients. Here, we report available data focusing on this issue and operative procedures to detect ICOS expression on circulating peripheral blood mononuclear cells during CTLA-4 blockade therapy.

Compelling experimental evidences point to monitoring of the absolute number of circulating ICOS-positive T cells as an early predictive marker of clinical activity of anti-CTLA-4 antibodies in cancer patients. Here, we report available data focusing on this issue and operative procedures to detect ICOS expression on circulating peripheral blood mononuclear cells during CTLA-4 blockade therapy.

Introduction: The anti-CTLA-4 monoclonal antibody (mAb) ipilimumab (Ipi) has firstly shown the therapeutic potential of targeting immune checkpoints in metastatic melanoma (MM) patients (pts), though with limited objective clinical responses, ranging from 10 to 13% (Hodi, NEJM 2010; Ascierto, TLO 2017). Targeting of CTLA-4 is of interest due to its role in the priming phase of the immune response; thus, anti-CTLA-4 mAb are being tested in different combinations to improve their clinical efficacy also by overcoming tumor resistance to treatment. We have thoroughly demonstrated that epigenetic remodelling of cancer cells with DNA hypomethylating agents (DHA) improves tumor immunogenicity and immune recognition (Maio M, CCR 2015). These findings identified DHA as highly intriguing “partner agents” to improve the therapeutic index of checkpoint blockers, including CTLA-4 mAb. This hypothesis is being tested in the ongoing NIBIT-M4 study (NCT02608437). Methods: The NIBIT-M4 is a phase 1b, dose-escalation study sequencing treatment-naïve/pre-treated, unresectable Stage III/IV MM pts, with the next generation DHA guadecitabine followed by Ipi. The dose escalation follows a 3+3 design: guadecitabine (at 30, 45 or 60 mg/m2) is administered s.c. on Week (W) 0, 3, 6, 9, days 1-5 and Ipi i.v. at 3 mg/kg on W1, 4, 7 and 10 q21d. Tumor assessment per immune-related response criteria is performed at screening (W0), W12, 18, 24 and q12 weeks. Patients-wise genome-wide methylation, RNA sequencing, whole-exome sequencing, and immunohistochemistry (IHC) analyses were performed on tumor samples collected at W0, 4 and 12. Canonical pathways and upstream regulators were investigated with IPA. Preliminary results: Fourteen out of the 19 planned pts have been enrolled so far, with no dose limiting toxicities. Among the 11 evaluable pts, the objective response rate is 27%. A reduction in tumor CpG sites methylation, from W0 (median 81.18%) to W4 (median 76.54%) and W12 (median 76.89%), was identified in the initial 8 pts enrolled. Transcriptomic analyses identified 3,897 ± 3,577 and 5,414 ± 3,569 differentially expressed genes (DEG) (q 1.0) at W4 and W12 compared to W0, respectively. Among the 137 pathways that were significantly modulated in all tumor samples at either W4 and/or W12, the most frequently activated ones were immune related (i.e., Th1 and Th2, iCOS-iCOSL and CD28 Signaling in T Helper Cells, and Interferon (IFN) Signaling). In addition, the top predicted upstream regulators were genes involved in the interferon IFN pathway (i.e., IFNG, STAT1, IRF7, IFNA2, IRF1). Though limited by the current sample size investigated, a greater number of significantly (p Conclusions: Epigenetic tumor immune-remodeling seems to be a promising strategy to improve the efficacy of CTLA-4 blockade, to be further pursued also with other immunomodulating mAb. *AMDG and AC equally contributed. Citation Format: Anna M. Di Giacomo, Alessia Covre, Francesca Finotello, Dietmar Rieder, Luca Sigalotti, Carolina Fazio, Ornella Cutaia, Christoph Bock, Florent Petitprez, Laetitia Lacroix, James N. Lowder, Wolf H. Fridman, Catherine Sautès-Fridman, Zlatko Trajanoski, Michele Maio. Epigenetic tumor remodelling to improve the efficacy of immune checkpoint blockade: the NIBIT-M4 clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT059.

Glioblastoma (GB) is the most common and aggressive primary tumor of the central nervous system. The current standard of care for GB consists of surgical resection, followed by radiotherapy combined with temozolomide chemotherapy. However, despite this intensive treatment, the prognosis remains extremely poor. Therefore, more effective therapies are urgently required. Recent studies indicate that SRC family kinases (SFKs) could represent promising molecular targets for GB therapy. Here, we challenged four GB cell lines with a new selective pyrazolo[3,4-d]pyrimidine derivative SFK inhibitor, called SI221. This compound exerted a significant cytotoxic effect on GB cells, without significantly affecting non-tumor cells (primary human skin fibroblasts), as evaluated by MTS assay. We also observed that SI221 was more effective than the well-known SFK inhibitor PP2 in GB cells. Notably, despite the high intrinsic resistance to apoptosis of GB cells, SI221 was able to induce this cell death process in all the GB cell lines, as observed through cytofluorimetric analysis and caspase-3 assay. SI221 also exerted a long-term inhibition of GB cell growth and was able to reduce GB cell migration, as shown by clonogenic assay and scratch test, respectively. Moreover, through in vitro pharmacokinetic assays, SI221 proved to have a high metabolic stability and a good potential to cross the blood brain barrier, which is an essential requirement for a drug intended to treat brain tumors. Therefore, despite the need of developing strategies to improve SI221 solubility, our results suggest a potential application of this selective SFK inhibitor in GB therapy.

Summary Background CTLA4 blockade by tremelimumab 15 mg/kg every 90 days provided preliminary evidence of activity in patients with pretreated malignant mesothelioma; however, retrospective exposure–response analysis of data from patients with melanoma suggested that this schedule could result in underexposure to tremelimumab. We therefore investigated the efficacy and safety of an intensified schedule of tremelimumab in patients with advanced malignant mesothelioma. Methods In this open-label, single-arm, phase 2 study, participants aged 18 years or older with unresectable, advanced malignant mesothelioma (measurable in accordance with the Response Evaluation Criteria in Solid Tumors [RECIST]), a life expectancy of 3 months or more, an Eastern Cooperative Oncology Group performance status of 2 or less, and who had failed a first-line platinum-based regimen were enrolled at the University Hospital of Siena, Siena, Italy. Participants received tremelimumab 10 mg/kg once every 4 weeks for six doses, then every 12 weeks until disease progression, unacceptable toxic effects, or refusal to continue treatment. The primary endpoint was the proportion of patients achieving an immune-related objective response (complete or partial), assessed in all patients who received at least one dose of the study drug. This study is registered with the European Union Clinical Trials Register, number 2012-002762-12, and ClinicalTrials.gov, number NCT01655888. Findings Between July 30, 2012, and July 15, 2013, we enrolled 29 patients with a median age of 65 years (range 42–78), stage III (n=11) or IV (n=18) disease, and an Eastern Cooperative Oncology Group performance status of 0–1 (n=23) or 2 (n=6). Malignant mesothelioma histology was epithelioid (n=21, including one peritoneal), biphasic (n=6), sarcomatoid (n=1), or undefined (n=1). Patients received a median of six doses of tremelimumab (range 1–13). After a median follow-up of 21·3 months (IQR 18·7–25·9), four immune-related-partial responses were recorded, one at the first tumour assessment (after about 12 weeks) and three at the second tumour assessment (about 24 weeks), with two responses occurring after initial progressive disease and one response after initial stable disease. 15 (52%) of patients achieved disease control, with a median duration of 10·9 months (95% CI 8·2–13·6). According to modified RECIST, one patient (3%) achieved a partial response and 11 (38%) patients achieved disease control rate. Grade 1–2 treatment-related adverse events occurred in 26 (90%) patients and grade 3–4 adverse events in two (7%) patients. The most common treatment-related adverse events were gastrointestinal, dermatological, and fever. Interpretation Our results suggest that the intensified schedule of tremelimumab investigated seems to have clinical and immunological activity in patients with advanced malignant mesothelioma, and a good safety profile. The same intensified schedule is now being investigated in an ongoing randomised, double-blind, placebo-controlled, phase 2b study. Funding Associazione Italiana per la Ricerca sul Cancro, Istituto Toscano Tumori, and MedImmune.

The intratumoral injection of cytokines, in particular IL2, has shown promise for cutaneous melanoma patients with unresectable disease or continuous recurrence despite surgery. We recently reported that the intralesional injection of L19-IL2, an immunocytokine combining IL2 and the human monoclonal antibody fragment L19, resulted in efficient regional control of disease progression, increased time to distant metastasis and evidence of effect on circulating immune cell populations. We have also shown in preclinical models of cancer a remarkable synergistic effect of the combination of L19-IL2 with L19-TNF, a second clinical-stage immunocytokine, based on the same L19 antibody fused to TNF. Here, we describe the results of a phase II clinical trial based on the intralesional administration of L19-IL2 and L19-TNF in patients with stage IIIC and IVM1a metastatic melanoma, who were not candidate to surgery. In 20 efficacy-evaluable patients, 32 melanoma lesions exhibited complete responses upon intralesional administration of the two products, with mild side effects mainly limited to injection site reactions. Importantly, we observed complete responses in 7/13 (53.8 %) non-injected lesions (4 cutaneous, 3 lymph nodes), indicating a systemic activity of the intralesional immunostimulatory treatment. The intralesional administration of L19-IL2 and L19-TNF represents a simple and effective method for the local control of inoperable melanoma lesions, with a potential to eradicate them or make them suitable for a facile surgical removal of the residual mass.

Meeting abstracts Ipilimumab (IPI) in combination with fotemustine (FTM) has shown a promising clinical activity in metastatic melanoma (MM) patients (pts) enrolled in the NIBIT-M1 trial (Di Giacomo, et al., Lancet Oncology, 2012). This study investigated changes in immunological parameters in the

Summary Background Monoclonal antibodies to cytotoxic T-lymphocyte antigen 4 (CTLA4) have therapeutic activity in different tumour types. We aimed to investigate the efficacy, safety, and immunological activity of the anti-CTLA4 monoclonal antibody, tremelimumab, in advanced malignant mesothelioma. Methods In our open-label, single-arm, phase 2 study, we enrolled patients aged 18 years or older with measurable, unresectable malignant mesothelioma and progressive disease after a first-line platinum-based regimen. Eligible patients had to have a life expectancy of 3 months or more, an Eastern Cooperative Oncology Group performance status of 2 or less, and no history of autoimmune disease. Patients received tremelimumab 15 mg/kg intravenously once every 90 days until progressive disease or severe toxicity. The primary endpoint was the proportion of patients who achieved an objective response (complete or partial response), with a target response rate of 17% according to the modified Response Evaluation Criteria in Solid Tumors (RECIST) for pleural malignant mesothelioma or standard RECIST 1.0 for peritoneal malignant mesothelioma. Analyses were done according to intention to treat. This trial is registered with EudraCT, number 2008-005171-95, and ClinicalTrials.gov, number NCT01649024. Findings Between May 27, 2009, and Jan 10, 2012, we enrolled 29 patients. All patients received at least one dose of tremelimumab (median two doses, range one to nine). No patients had a complete response and two patients (7%) had a durable partial response (one lasting 6 months and one lasting 18 months); one partial response occurred after initial progressive disease. Thus, the study did not reach its primary endpoint. However, we noted disease control in nine (31%) patients and a median progression-free survival of 6·2 months (95% CI 1·3–11·1) and a median overall survival of 10·7 months (0·0–21·9). 27 patients (93%) had at least one grade 1–2 treatment-emergent adverse event (mainly cutaneous rash, pruritus, colitis, or diarrhoea), and four patients (14%) had at least one grade 3–4 treatment-emergent adverse event (two gastrointestinal, one neurological, two hepatic, and one pancreatic). Interpretation Although the effect size was small in our phase 2 trial, tremelimumab seemed to have encouraging clinical activity and an acceptable safety and tolerability profile in previously treated patients with advanced malignant mesothelioma. Funding Associazione Italiana per la Ricerca sul Cancro, Istituto Toscano Tumori, Pfizer, and Fondazione Buzzi Unicem.

Ipilimumab can result in durable clinical responses among patients with advanced melanoma. However, no predictive marker of clinical activity has yet been identified. We provide preliminary data describing the correlation between immunological parameters and response/survival among patients with advanced melanoma who received ipilimumab 10 mg/kg in an expanded access programme.Patients received ipilimumab 10 mg/kg every 3 weeks (Q3W) for four doses (induction) and Q12W from week 24 (W24) as maintenance therapy. Tumor assessments were conducted Q12W. Expression of inducible T cell costimulator (ICOS) on CD4(+) and CD8(+) T cells was assessed at baseline, W7, W12 and W24, and the ratio between absolute neutrophils (N) and lymphocytes (L) determined at baseline, W4, W7 and W10.Median overall survival among 27 patients was 9.6 months (95 % CI 3.2-16.1), with 3- and 4-year survival rates of 20.4 %. Five patients survived4 years. Patients with an increase in the number of circulating ICOS(+) T cells at W7 were more likely to experience disease control and have improved survival. An N/L ratio below the median at W7 and W10 was also associated with better survival compared with an N/L ratio above the median.Ipilimumab can induce long-term survival benefits in heavily pretreated patients with metastatic melanoma. Changes in the number of circulating ICOS(+) T cells or N/L ratio during ipilimumab treatment may represent early markers of response. However, given the limited sample size, further investigation is required.

No treatment prolongs the survival of malignant mesothelioma (MM) patients. Since MM elicits anti-tumor host's immune responses, immunotherapy represents a promising strategy for its control. Immunomodulatory antibodies against components of the B7 family of immunomodulatory molecules that regulate T cell activation are being investigated in human malignancies including MM. The expression of B7-H3, a new component of the B7 family was investigated in primary cultures of human mesothelial cells (HMC) and in MM cell lines by flow cytometry and molecular analyses, and in MM tissues by immunohistochemistry. The role of DNA hypomethylating agents in modulating levels of B7-H3 expression in MM cells was also studied. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that B7-H3 mRNA was consistently detectable in mesothelial and MM cells investigated; however, real-time quantitative RT-PCR analyses showed highly heterogeneous levels of B7-H3 mRNA among investigated MM cells. The analysis of B7-H3 protein expression indicated that comparable levels of B7-H3 were expressed on both cell types. Treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine did not significantly affect the expression of B7-H3 mRNA in MM cells. In vivo, while B7-H3 was expressed in all 13 tumor biopsies of the epithelial variant, with high levels in 54% of cases, it was rarely detectable in spindle type MM in which 1/5 biopsies weakly expressed B7-H3. These findings suggest that B7-H3 is a promising target for new immunotherapeutic strategies in MM, with particular emphasis in the epithelial variant.

Anti-CD40 antibodies are in clinical development in patients with metastatic melanoma, a cancer that has been reported earlier to express CD40. The antitumor activity of anti-CD40 antibodies may be mediated by direct cytotoxic effects on CD40-positive melanoma cells or indirectly through modulation of host cells. In these studies, biopsies of patients with primary and metastatic melanoma, short-term cultures, and established melanoma cell lines were analyzed for CD40 expression using a combination of methods including immunohistochemistry, flow cytometry, and gene expression profiling, and the cytotoxic effects of the anti-CD40 antibody CP-870,893 on melanoma cell lines were tested using cell viability assays. CD40 was expressed at a higher frequency in metastatic melanoma lesions compared with primary melanomas. There was a variable expression of CD40 in synchronous and metachronous melanoma metastases. Expression of CD40 was present in slightly over half of a large panel of short-term primary melanoma cultures, with a wide range of expression levels by flow cytometry. Similar results were obtained in established melanoma cell lines when analyzed at the mRNA level or by surface protein staining. In approximately one-third of cell lines, the expression of CD40 could be up-regulated with a histone deacetylase inhibitor. Treatment with increasing concentrations of CP-870,893 had no antitumor activity against either CD40-positive or negative melanoma cell lines. In conclusion, approximately one-third to one-half of melanomas expresses CD40 at variable levels. Direct exposure to a CD40-activating antibody does not lead to antitumor activity in melanoma cell lines, suggesting that the antitumor effects of these antibodies in the clinic may be indirectly mediated.

Purpose: Tremelimumab is a fully human IgG2 monoclonal antibody specific for cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4/CD152). Tremelimumab blocks the interaction of CTLA-4 with its ligands B7.1 and B7.2, and enhances human T-cell activation, as demonstrated by increased interleukin-2 production in in-vitro/in-vivo studies. The primary objectives of this analysis were to assess the preliminary population pharmacokinetics (PK) of tremelimumab in mesothelioma and identify the impact of potential patient/disease characteristics on PK variability. Methods: Tremelimumab serum concentration-time data were collected from a second-line, single-arm phase-2 study (NCT01649024/NCT01655888) designed to evaluate clinical activity along with safety and tolerability of multiple IV doses of tremelimumab in patients with malignant mesothelioma. Two dosing regimens were tested: 15 mg/kg every 90 days (Q90D) up to 4 doses in a 12 month period until progressive disease or unacceptable toxicity and, 10 mg/kg every 4 weeks (Q4W) for 6 months followed by 10 mg/kg every 12 weeks (Q12W) until progressive disease or unacceptable toxicity. A total of 40 subjects provided evaluable PK data at various prespecified time points following both dosing regimens. Tremelimumab serum concentrations were determined using a validated enzyme-linked immunosorbent assay (ELISA) with a lower limit of quantitation (LLOQ) of 0.156 µg/mL. PK analysis was performed using a non-linear mixed effects modeling approach with NONMEM 7.2 software. Results: Tremelimumab PK exposure in mesothelioma subjects was similar to previous melanoma studies following 15 mg/kg Q90D. Following the 15 mg/kg Q90D, PK exposure was below the target trough level of ∼30 µg/mL for about half of the dosing interval with almost all subjects below the LLOQ at the end of the 90-day dosing interval. Following more frequent dosing of 10 mg/kg Q4W, PK exposure was increased and maintained at or above the target level in the majority of subjects over the entire dosing interval. Tremelimumab PK was best described using a 2-compartment linear model with first order elimination. Following IV dosing, the typical clearance (CL) and central volume of distribution (Vc) were 0.2 L/day and 3.5 L, respectively. The between-subject variability for CL and Vc were 22% and 7%, respectively. The estimated typical PK parameters were similar to other monoclonal antibodies without target mediated elimination. The baseline body weight and Eastern Cooperative Oncology Group (ECOG) performance status were identified as significant covariates for CL, whereas only baseline body weight was significant covariate for volume of distribution. Conclusions: A 2-compartment population PK model adequately described tremelimumab PK in subjects with mesothelioma. PK parameter estimates were similar in both mesothelioma and melanoma populations. Tremelimumab 10 mg/kg Q4W yielded higher exposure than 15 mg/kg Q90D with a majority of subjects maintaining concentrations above the target trough level of ∼30 µg/mL over the dosing interval. Preliminary covariate screening identified body weight and ECOG as influential factors for PK parameters. These correlations will be further validated using data from larger clinical trials. Citation Format: Luana Calabro', Rajesh Narwal, Paul B. Robbins, Alessandra Di Pietro, Ornella Cutaia, Ester Fonsatti, Diego Annesi, Carolina Fazio, Ramy Ibrahim, Michele Maio. Pharmacokinetics of Tremelimumab, a fully human anti-CTLA-4 monoclonal antibody, in subjects with unresectable malignant mesothelioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-228. doi:10.1158/1538-7445.AM2014-LB-228

7531 Background: We reported encouraging clinical activity of tremelimumab (treme), a fully human IgG 2 monoclonal antibody specific for cytotoxic T lymphocyte-associated antigen 4, at 15 mg/kg q3 ...

TPS9103^ Background: Preclinical studies in three different murine models of subcutaneous tumors have shown that a single intratumoral injection of a combination of two immunocytokines, L19-IL2 and...