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Melanoma patient remains a challenging for the radiologist, due to the difficulty related to the management of a patient more often in an advanced stage of the disease. It is necessary to determine a stratification of risk, optimizing the means, with diagnostic tools that should be optimized in relation to the type of patient, and improving knowledge. Staging and risk assessment procedures are determined by disease presentation at diagnosis. Melanoma staging is a critical tool to assist clinical decision-making and prognostic assessment. It is used for clinical trial design, eligibility, stratification, and analysis. The current standard for regional lymph nodes staging is represented by the sentinel lymph node excision biopsy procedure. For staging of distant metastases, PET-CT has the highest sensitivity and diagnostic odds ratio. Similar trend is observed during melanoma surveillance. The advent of immunotherapy, which has improved patient outcome, however, has determined new issues for radiologists, partly due to atypical response patterns, partly due to adverse reactions that must be identified as soon as possible for the correct management of the patient. The main objectives of the new ir-criteria are to standardize the assessment between different trials. However, these ir-criteria do not take into account all cases of atypical response patterns, as hyperprogression or dissociated responses. None of these criteria has actually been uniformly adopted in routine. The immune-related adverse events (irAEs) can involve various organs from head to toe. It is crucial for radiologists to know the imaging appearances of this condition, to exclude recurrent or progressive disease and for pneumonitis, since it could be potentially life-threatening toxicity, resulting in pneumonitis-related deaths in early phase trials, to allow a proper patient management., (© 2022. Italian Society of Medical Radiology.)

Health related quality of life (HRQoL) is an important recognized health outcome for cancer treatments, but also disease course with slower recovery and increased morbidity. These issues are of implication in melanoma, which maintains a risk of disease progression for many years after diagnosis. This study aimed to explore and weigh factors in the perception of the quality of life and possible relationships with demographic-clinical characteristics in people with melanoma via a machine learning approach. In this observational study, patients with melanoma, without metastatic disease, were recruited from January 2020 to December 2021 with a follow-up of at least one year. Demographic variables and clinics were collected, and the 12-Item Short-Form Health Survey (SF-12) was adopted as the physical and mental aspects of the Health-Related Quality of Life (HRQoL) measure. All the variables were processed in a random forest model to weigh at each node of each tree of this machine learning regression model, their actual weight in SF-12 score. We included 203 melanoma patients, mean aged 59.25 ± 15.1 years: 56 (27%) affecting the upper limbs and 147 (73%) affecting the trunk. The model of 142 patients with no missing value, generating 92 trees (MSE = 0.45, R2 of 0.78), reported that the lesion site was the most influencing variable on HRQoL based on the decrease in Gini impurity in variable weighing at each node intersection in forest generation. In this scenario, we built two distinct models for lesion sites and demonstrated that the variable that most influenced the quality of life in upper limb melanoma was lymphedema, while BMI was in the trunk. Given these results, random forest regressions could play a crucial role in the clinical and rehabilitation approach. The machine-learning model for detecting the HRQoL predictor in melanoma patients indicates that the experienced lymphedema and BMI may influence the HRQoL perception. This study suggests that the prevention and treatment of lymphedema and bodyweight reduction might improve the quality of life in melanoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pinto, Marotta, Caracò, Simeone, Ammendolia and de Sire.)

Background: CD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here, we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy., Methods: Exosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry, also in the presence of selective inhibitors of CD73. Interferon (IFN)-γ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control., Results: Isolated exosomes carried CD73 on their surface, which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-γ release, which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment, expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders, while exosomal CD73 resulted significantly increased in non-responders., Conclusions: CD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment, elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy., Competing Interests: Competing interests: PAA has/had a consultant/advisory role for Bristol Myers Squibb, Roche-Genentech, Merck Sharp & Dohme, Novartis, Array, Merck Serono, Pierre-Fabre, Incyte, Medimmune, AstraZeneca, Syndax, Sun Pharma, Sanofi, Idera, Ultimovacs, Sandoz, Immunocore, 4SC, Alkermes, Italfamaco, Nektar, Boehringer Ingelheim, Eisai, Regeneron, Daiichi Sankyo, Pfizer, Oncosec, Nouscom, Takis and Lunaphore. He also received research funding from Bristol Myers Squibb, Roche-Genentech, Array and Sanofi, and travel support from MSD. All outside the submitted work. The other authors declare no potential competing interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Mucosal melanomas (MM) are rare tumors, being less than 2% of all diagnosed melanomas, comprising a variegated group of malignancies arising from melanocytes in virtually all mucosal epithelia, even if more frequently found in oral and sino-nasal cavities, ano-rectum and female genitalia (vulva and vagina). To date, there is no consensus about the optimal management strategy of MM. Furthermore, the clinical rationale of molecular tumor characterization regarding BRAF, KIT or NRAS, as well as the therapeutic value of immunotherapy, chemotherapy and targeted therapy, has not yet been deeply explored and clearly established in MM. In this overview, focused on anorectal and genital MM as models of rare melanomas deserving of a multidisciplinary approach, we highlight the need of referring these patients to centers with experts in melanoma, anorectal and uro-genital cancers treatments. Taking into account the rarity, the poor outcomes and the lack of effective treatment options for MM, tailored research needs to be promptly promoted.

BCL-2 overexpression has been associated with resistance to chemotherapy and reduced survival in acute myeloid leukemia (AML), but few data are available in elderly patients, a subset accounting for majority of AML cases and with dismal prognosis. We retrospectively analyzed 113 AML patients aged ≥65 years treated with 3 + 7 chemotherapy ( n = 51) or hypomethylating agents (HMAs) ( n = 62), evaluating the role of BCL-2 expression on complete remission (CR) and overall survival (OS). BCL-2 was expressed in 81 patients (72%), more frequently in those with unfavorable cytogenetic-molecular risk. CR was achieved in 34.5% cases, without differences according to BCL-2 expression or induction therapy. In the whole population 1-year OS was 39%, similar in BCL-2+ and BCL-2- cases. In BCL-2 positive patients OS was superior with HMAs (56% vs. 25% with 3 + 7; p = 0.02), while no advantage for HMA was found in BCL-2 negative cases (36% vs. 27% for 3 + 7). Therapy with HMAs was the only factor associated with longer OS in BCL-2+ AML by multivariable analysis. Use of HMAs, possibly in combination with BCL-2 inhibitors, appears to be particularly appealing in BCL2+ AML, where it is associated with superior survival.

The real-life application of immune checkpoint inhibitors (ICIs) may yield different outcomes compared to the benefit presented in clinical trials. For this reason, there is a need to define the group of patients that may benefit from treatment. We retrospectively investigated 578 metastatic melanoma patients treated with ICIs at the Istituto Nazionale Tumori IRCCS Fondazione "G. Pascale" of Napoli, Italy (INT-NA). To compare patients' clinical variables (i.e., age, lactate dehydrogenase (LDH), neutrophil-lymphocyte ratio (NLR), eosinophil, BRAF status, previous treatment) and their predictive and prognostic power in a comprehensive, non-hierarchical manner, a clinical categorization algorithm (CLICAL) was defined and validated by the application of a machine learning algorithm-survival random forest (SRF-CLICAL). The comprehensive analysis of the clinical parameters by log risk-based algorithms resulted in predictive signatures that could identify groups of patients with great benefit or not, regardless of the ICI received. From a real-life retrospective analysis of metastatic melanoma patients, we generated and validated an algorithm based on machine learning that could assist with the clinical decision of whether or not to apply ICI therapy by defining five signatures of predictability with 95% accuracy.

The median overall survival (OS) and local control (LC) of patients with melanoma brain metastases (MBMs) are poor even with immune checkpoint inhibitors and/or radiotherapy (RT). The aims of the study were to evaluate the association and timing of stereotactic radiotherapy (SRT)/radiosurgery (SRS) performed with the CyberKnife ® System and ipilimumab (IPI). A total of 63 MBMs patients were analyzed: 53 received RT+IPI and 10 RT alone. Therefore, the patients were divided into four groups: RT PRE-PI (>4 weeks before IPI) (18), RT CONC-IPI (4 weeks before/between first and last cycle/within 3 months of last cycle of IPI) (20), RT POST-IPI (>3 months after IPI) (15), and NO-IPI (10). A total of 127 lesions were treated: 75 with SRS (one fraction) and 24 with SRT (three to five fractions). The median follow-up was 10.6 months. The median OS was 10.6 months for all patients, 10.7 months for RT+IPI, and 3.3 months for NO-IPI ( p = 0.96). One-year LC was 50% for all patients, 56% for RT+IPI, and 18% for NO-IPI ( p = 0.08). The 1-year intracranial control was 45% for all patients, 44% for RT+IPI, and 51% for NO-IPI ( p = 0.73). IPI with SRS/SRT in MBMs treatment could improve LC. However, the impact and timing of the two modalities on patients' outcomes are still unclear.

As widely acknowledged, 40-50% of all melanoma patients harbour an activating BRAF mutation (mostly BRAF V600E). The identification of the RAS-RAF-MEK-ERK (MAP kinase) signalling pathway and its targeting has represented a valuable milestone for the advanced and, more recently, for the completely resected stage III and IV melanoma therapy management. However, despite progress in BRAF-mutant melanoma treatment, the two different approaches approved so far for metastatic disease, immunotherapy and BRAF+MEK inhibitors, allow a 5-year survival of no more than 60%, and most patients relapse during treatment due to acquired mechanisms of resistance. Deep insight into BRAF gene biology is fundamental to describe the acquired resistance mechanisms (primary and secondary) and to understand the molecular pathways that are now being investigated in preclinical and clinical studies with the aim of improving outcomes in BRAF-mutant patients.

Background: Studies carried out in vitro and in a mouse model have shown that BRAF inhibitors enhance the effects of IFN-α on BRAFV600E melanoma cells through the inhibition of ERK. Therefore, the combination of vemurafenib and IFN-α in patients with BRAFV600E melanoma may provide therapeutic benefits; MEK inhibition may prevent the reactivation of the MAPK pathway induced by BRAF inhibitor resistance., Patients and Methods: In a phase I study, adult patients with advanced BRAFV600-mutated melanoma were treated with vemurafenib + PEG-IFN-α-2b or vemurafenib + cobimetinib + PEG-IFN-α-2b, to assess the safety of the combination and the upregulation of IFN-α/β receptor-1 (IFNAR1)., Results: Eight patients were treated; 59 adverse events with four serious ones (three related to study treatments) were reported. Patients with a pre-treatment IFNAR1 expression on ≤ 35% melanoma cells had a median progression-free survival of 12.0 months (range: 5.6-18.4 months) and a median overall survival of 31.0 months (range: 19.8-42.2 months), while patients with a pre-treatment IFNAR1 expression on > 35% of melanoma cells had a median progression-free survival of 4.0 months (range: 0-8.8; p = 0.03), and a median overall survival of 5 months (p = 0.02). Following treatment, responders had higher levels of growth-suppressor genes, including GAS1 and DUSP1, and genes involved in a metabolically robust immune response, including FAP., Conclusion: Our study supports the overall safety of the vemurafenib + PEG-IFN-α-2b + cobimetinib combination. IFNAR1 expression levels correlated with response to treatment, including survival. Vemurafenib + PEG-IFN-α-2b + cobimetinib would have difficulty finding a niche in the current treatment scenario for advanced melanoma, but we speculate that our findings may contribute to identify subjects particularly responsive to treatment., Trial Registration: The study was registered at clinicaltrials.gov (NCT01959633). Registered 10 October 2013, https://clinicaltrials.gov/ct2/show/NCT01959633.

Background: The coronavirus disease 2019 (COVID-19) pandemic has overwhelmed the health systems worldwide. Data regarding the impact of COVID-19 on cancer patients (CPs) undergoing or candidate for immune checkpoint inhibitors (ICIs) are lacking. We depicted the practice and adaptations in the management of patients with solid tumors eligible or receiving ICIs during the COVID-19 pandemic, with a special focus on Campania region., Methods: This survey (25 questions), promoted by the young section of SCITO (Società Campana di ImmunoTerapia Oncologica) Group, was circulated among Italian young oncologists practicing in regions variously affected by the pandemic: high (group 1), medium (group 2) and low (group 3) prevalence of SARS-CoV-2-positive patients. For Campania region, the physician responders were split into those working in cancer centers (CC), university hospitals (UH) and general hospitals (GH). Percentages of agreement, among High (H) versus Medium (M) and versus Low (L) group for Italy and among CC, UH and GH for Campania region, were compared by using Fisher's exact tests for dichotomous answers and χ 2 test for trends relative to the questions with 3 or more options., Results: This is the first Italian study to investigate the COVID-19 impact on cancer immunotherapy, unique in its type and very clear in the results. The COVID-19 pandemic seemed not to affect the standard practice in the prescription and delivery of ICIs in Italy. Telemedicine was widely used. There was high consensus to interrupt immunotherapy in SARS-CoV-2-positive patients and to adopt ICIs with longer schedule interval. The majority of the responders tended not to delay the start of ICIs; there were no changes in supportive treatments, but some of the physicians opted for delaying surgeries (if part of patients' planned treatment approach). The results from responders in Campania did not differ significantly from the national ones., Conclusion: Our study highlights the efforts of Italian oncologists to maintain high standards of care for CPs treated with ICIs, regardless the regional prevalence of COVID-19, suggesting the adoption of similar solutions. Research on patients treated with ICIs and experiencing COVID-19 will clarify the safety profile to continue the treatments, thus informing on the most appropriate clinical conducts., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Actinic keratosis (AK) affects one quarter of over 60  year olds in Europe with the risk of transforming into invasive squamous cell carcinoma. Daylight photodynamic therapy (dPDT) is an effective and patient preferred treatment that uses sunlight to clear AK. Currently, there is no standardised method for measuring the light received during treatment., Methods: SmartPDT® is a smartphone-based application and web-portal, developed by siHealth Ltd, enabling remote delivery of dPDT. It uses satellite imagery and computational algorithms to provide real-time determination of exposure to PpIX-effective solar radiation ("light dose"). The application also provides forecast of expected radiant exposures for 24- and 48-hs prior to the treatment period. Validation of the real-time and forecasted radiant exposure algorithms was performed against direct ground-based measurement under all weather conditions in Chilton, UK., Results: Agreement between direct ground measurements and satellite-determined radiant exposure for 2-h treatment was excellent at -0.1 % ± 5.1 % (mean ± standard deviation). There was also excellent agreement between weather forecasted radiant exposure and ground measurement, 1.8 % ± 17.7 % at 24-hs and 1.6 % ± 25.2 % at 48-hs. Relative Root Mean Square of the Error (RMSE r ) demonstrated that agreement improved as time to treatment reduced (RMSE r  = 22.5 % (48 -hs), 11.2 % (24-hs), 5.2 % (real-time))., Conclusion: Agreement between satellite-determined, weather-forecasted and ground-measured radiant exposure was better than any existing published literature for dPDT. The SmartPDT® application and web-portal has excellent potential to assist with remote delivery of dPDT, an important factor in reducing risk in an elderly patient population during the Covid-19 pandemic., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gut microbiota composition influences the clinical benefit of immune checkpoints in patients with advanced cancer but mechanisms underlying this relationship remain unclear. Molecular mechanism whereby gut microbiota influences immune responses is mainly assigned to gut microbial metabolites. Short-chain fatty acids (SCFA) are produced in large amounts in the colon through bacterial fermentation of dietary fiber. We evaluate in mice and in patients treated with anti-CTLA-4 blocking mAbs whether SCFA levels is related to clinical outcome. High blood butyrate and propionate levels are associated with resistance to CTLA-4 blockade and higher proportion of Treg cells. In mice, butyrate restrains anti-CTLA-4-induced up-regulation of CD80/CD86 on dendritic cells and ICOS on T cells, accumulation of tumor-specific T cells and memory T cells. In patients, high blood butyrate levels moderate ipilimumab-induced accumulation of memory and ICOS + CD4 + T cells and IL-2 impregnation. Altogether, these results suggest that SCFA limits anti-CTLA-4 activity.

Introduction : Treatment of extensive-stage SCLC is still a challenge but immunotherapy with checkpoint inhibitors is showing promising results. Nivolumab alone or in combination with ipilimumab has demonstrated a benefit in terms of response and survival in patients with pre-treated extensive-stage disease and has been approved as third-line therapy after failure of chemotherapy. However, data from two phase III trials with nivolumab are negative. In the first trial, nivolumab was administered as a single agent compared to second-line chemotherapy, while in the second it was given alone or in combination with ipilimumab as maintenance treatment after platinum-based chemotherapy. Areas covered : Our review focuses on the role of immunotherapy, and in particular nivolumab, in the treatment of SCLC, describing the results of the main trials and its future perspectives, with reference to clinical trials with other checkpoint inhibitors. Expert opinion : The future of nivolumab in the treatment of SCLC needs to be clarified with further clinical trials, in which improved patient selection and a more specific setting and/or timepoint of the disease may be identified.

Checkpoint inhibitors can cause an imbalance in immune tolerance that may clinically manifest as immune-related adverse events (irAEs). These events may involve many organs and tissues, including the skin, gastrointestinal (GI) tract, liver, endocrine system, kidneys, central nervous system (CNS), eyes and lungs. The incidence of irAEs appears to be lower with anti-programmed death antigen-1/programmed death antigen-ligand-1 agents than with the anti-cytotoxic T-lymphocyte-associated protein-4 antibody ipilimumab. Combined immunotherapy does not appear to be associated with novel safety signals compared with monotherapy, but more organs may be involved. Increased experience and the use of algorithms for the most common irAEs have resulted in severe toxicity and related deaths being reduced. However, continuous vigilance, especially regarding less common events, is needed to better characterize the wide spectrum of clinical manifestations., Competing Interests: Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript., (© 2019 Ester Simeone.)

The therapeutic landscape in metastatic melanoma has changed dramatically in the last decade, with the success of immune checkpoint inhibitors resulting in durable responses for a large number of patients. For patients with BRAF mutations, combinations of BRAF and MEK inhibitors demonstrated response rates and benefit comparable to those from immune checkpoint inhibitors, providing the rationale for sequential treatment with targeted and immunotherapies and raising the question of optimal treatment sequencing.Biomarkers for the selection of anti-PD-1 therapy in BRAF wild type (BRAF WT) and in BRAF mutated (BRAF MUT) patients help development of alternative treatments for patients unlikely to benefit, and might lead to better understanding of the interaction of checkpoint inhibition and targeted therapy. In this paper we evaluate the performance of a previously developed serum proteomic test, BDX008, in metastatic melanoma patients treated with anti-PD-1 agents and investigate the role of BRAF mutation status. BDX008, a pre-treatment proteomic test associated with acute phase reactants, wound healing and complement activation, stratifies patients into two groups, BDX008+ and BDX008-, with better and worse outcomes on immunotherapy.Serum samples were available from 71 patients treated with anti-PD1 inhibitors; 25 patients had BRAF mutations, 39 were wild type. Overall, BDX008+ patients had significantly better overall survival (OS) (HR = 0.50, P = 0.016) and a trend for better progression-free survival (PFS) (HR = 0.61, P = 0.060) than BDX008- patients. BDX008 classification was statistically significant in the analyses adjusted for mutation status, LDH, and line of treatment (P = 0.009 for OS and 0.031 for PFS). BRAF WT BDX008+ patients had markedly long median OS of 32.5 months and 53% landmark 2 years survival, with statistically significantly superior OS as compared to BDX008- patients (HR = 0.41, P = 0.032). The difference between BDX008+ and BDX008- in PFS in BRAF WT patients and in OS and PFS in BRAF MUT patients did not reach statistical significance, though numerically was consistent with overall results. The test demonstrated significant interaction with neutrophil-to-lymphocyte ratio (NLR) (PFS P = 0.041, OS P = 0.004). BDX008 as a biomarker selecting for benefit from immune checkpoint blockade, especially in patients with wild type BRAF and in subgroups with low NLR, warrants further evaluation.

CTLA-4 blockade by means of ipilimumab (IPI) potentiates the immune response and improves overall survival (OS) in a minority of metastatic melanoma (MM) patients. We investigated the role of soluble CTLA-4 (sCTLA-4) as a possible biomarker for identifying this subset of patients. sCTLA-4 levels were analyzed at baseline in sera from 113 IPI-treated MM patients by ELISA, and the median value (200 pg/ml) was used to create two equally sized subgroups. Associations of sCTLA-4 with best overall response (BOR) to IPI and immune-related adverse events (irAEs) were evaluated through logistic regression. Kaplan-Meier and Cox regression methods were used to analyze OS. A remarkable association between sCTLA-4 levels and BOR was found. Specifically, the proportion of patients with sCTLA-4 > 200 pg/ml in irSD or irPD (immune-related stable or progressive disease) was, respectively, 80% (OR = 0.23; 95%CL = 0.03-1.88) and 89% (OR = 0.11; 95%CL = 0.02-0.71) and was lower than that observed among patients in irCR/irPR (immune-related complete/partial response). sCTLA-4 levels increased during IPI treatment, since the proportion of patients showing sCTLA > 200 pg/ml after 3 cycles was 4 times higher (OR = 4.41, 95%CL = 1.02-19.1) than that after 1 cycle. Moreover, a significantly lower death rate was estimated for patients with sCTLA-4 > 200 pg/ml (HR = 0.61, 95%CL = 0.39-0.98). Higher baseline sCTLA-4 levels were also associated with the onset of any irAE (p value = 0.029), in particular irAEs of the digestive tract (p value = 0.041). In conclusion, our results suggest that high sCTLA-4 serum levels might predict favorable clinical outcome and higher risk of irAEs in IPI-treated MM patients.

Background : Tumor microenvironment may have a key role in providing immunological markers that can help predict clinical response to treatment with checkpoint inhibitors. We investigated whether the baseline expression of PD-L1 in advanced melanoma patients treated with ipilimumab may correlate with clinical outcome. Methods : PD-L1 expression was assessed in 114 patients with advanced melanoma treated with ipilimumab and, in a cohort of 77 patients, a comprehensive assessment using multispectral imaging to assess the presence and distribution of CD3+, CD8+, CD163+, FOXP3+ and PD-L1+ cells inside and at periphery of the tumor was performed. Results : PD-L1 status alone was not a predictive biomarker for response or survival. There was an association between clinical benefit from ipilimumab therapy with the coexistence of low densities of CD8+ and high densities of CD163+ PD-L1+ cells at the periphery of the tumor. Conclusions : To explain the association of this peculiar microenvironment with clinical benefit from ipilimumab, we proposed a model where baseline CD8 cells levels are low due to inhibitory effect of Tregs and to pro-tumor activity of TAM M2 (CD163+ PD-L1+ cells). Ipilimumab treatment causes a decrease of Treg cells, mediated by ADCC from macrophages, with a concomitant change in TAM polarization that switches from M2 to M1 with a subsequent attraction of CD8 cells and the increase of antitumor response.

Background: Previous studies have suggested that elevated neutrophil-to-lymphocyte ratio (NLR) is prognostic for worse outcomes in patients with a variety of solid cancers, including those treated with immune checkpoint inhibitors., Methods: This was a retrospective analysis of 97 consecutive patients with stage IV melanoma who were treated with nivolumab. Baseline NLR and derived (d) NLR were calculated and, along with other characteristics, correlated with progression-free survival (PFS) and overall survival (OS) in univariate and multivariate analyses. The best cutoff values for NLR and dNLR were derived using Cutoff Finder software based on an R routine which optimized the significance of the split between Kaplan-Meier survival curves., Results: In univariate analysis, increasing absolute neutrophil count (ANC), NLR, dNLR and lactate dehydrogenase (LDH) (continuous variables) were all significantly associated with OS. Only NLR (hazard ratio [HR] = 2.85; 95% CI 1.60-5.08; p < 0.0001) and LDH (HR = 2.51; 95% CI 1.36-4.64; p < 0.0001) maintained a significant association with OS in multivariate analysis. Patients with baseline NLR ≥5 had significantly worse OS and PFS than patients with NLR < 5, as did patients with baseline dNLR ≥3 versus < 3. Optimal cut-off values were ≥ 4.7 for NLR and ≥ 3.8 for dNLR. Using this ≥4.7 cut-off for NLR, the values for OS and PFS were overlapping to the canonical cut-off for values, and dNLR< 3.8 was also associated with better OS and PFS., Conclusion: Both Neutrophil-to-lymphocyte ratio (NLR) and derived (d) NLR were associated with improved survival when baseline levels were lower than cut-off values. NLR and dNLR are simple, inexpensive and readily available biomarkers that could be used to help predict response to immunotherapy in patients with advanced melanoma.

The aim of this prospective clinical trial was to evaluate the efficacy and safety of a combination of Gemtuzumab-Ozogamicin (GO) and FLAI scheme (fludarabine, cytarabine, idarubicin) as a first-line therapy in CD33 positive AML. We treated 130 patients, aged <65, with a median age of 52 years. FLAI-GO induction regimen included fludarabine (30 mg/sqm) and cytarabine (2 g/sqm) on days 1-5; idarubicin (10 mg/sqm) on days 1, 3, and 5; and GO (3 mg/sqm) on day 6. SCT was planned for all high-risk AML patients, after consolidation with intermediate doses of cytarabine and idarubicin and a high dose of cytarabine. CD33 expression exceeded 20% in all cases. Primary endpoints of the study included feasibility, overall response rate (ORR) and toxicity. Secondary endpoints included the evaluation of MRD by WT1 expression, feasibility and outcome of consolidation with SCT, overall survival (OS) and disease-free survival (DFS). After induction with FLAI-GO, complete remission (CR) rate was 82%. Four patients achieved partial remission (PR) and 12% were resistant (ORR 85%); death during induction (DDI) was 3%. The hematological and extra hematological toxicity of FLAI-GO was manageable; 45% of patients experienced transient and reversible GO infusion related adverse events. In the setting of patients who achieved a cytological CR after FLAI-GO, the mean of WT1 copies dropped from 8337±9936 copies/10 4 ABL (diagnosis) to 182 ± 436 copies after induction therapy (p = 0.0001) showing a very good disease debulking. After a median follow-up of 54 months, 67/130 (52%) patients were alive. The probability of 1, 2, and 5-year OS was 80%, 63%, and 52%, respectively. The probability of 1, 2, and 5-year DFS was 77%, 58%, and 52%, respectively. Allogeneic and autologous SCT was performed in 60 (46%) and 23 (18%) patients, respectively. In summary, the final results of this trial confirm that FLAI-GO is an active and safe treatment strategy for CD33-positive AML patients aged ≤ 65 years, allowing a high ORR, a good disease debulking, favorable safety profile, low DDI, and subsequent high SCT rate. The encouraging results of this trial, consolidated by a long follow-up, support the reintroduction of GO in clinical practice., (© 2018 Wiley Periodicals, Inc.)

[This corrects the article on p. 386 in vol. 8, PMID: 28446908.].

The mitogen-activated protein kinase (MAPK) cascade is an intracellular signaling pathway involved in the regulation of cellular proliferation and the survival of tumor cells. Several different mutations, involving BRAF or NRAS, exert an oncogenic effect by activating the MAPK pathway, resulting in an increase in cellular proliferation. These mutations have become targets for new therapeutic strategies in melanoma and other cancers. Selective MEK inhibitors have the ability to inhibit growth and induce cell death in BRAF- and NRAS-mutant melanoma cell lines. MEK inhibitor therapy in combination with a BRAF inhibitor is more effective and less toxic than treatment with a BRAF inhibitor alone, and has become the standard of care for patients with BRAF-mutated melanoma. Trametinib was the first MEK inhibitor approved for the treatment of BRAF-mutated metastatic melanoma not previously treated with BRAF inhibitors, and is also approved in combination with the BRAF inhibitor dabrafenib. Furthermore, cobimetinib is another MEK inhibitor approved for the treatment of BRAF-mutated metastatic melanoma in combination with a BRAF inhibitor, vemurafenib. The MEK inhibitor binimetinib in combination with the BRAF inhibitor encorafenib is in clinical development. The addition of an anti-PD-1/PD-L1 agent, such as pembrolizumab, durvalumab or atezolizumab, to combined BRAF and MEK inhibition has shown considerable promise, with several trials ongoing in metastatic melanoma. Binimetinib has also shown efficacy in NRAS-mutated melanoma patients. Future possibilities for MEK inhibitors in advanced melanoma, as well as other solid tumors, include their use in combination with other targeted therapies (e.g. anti-CDK4/6 inhibitors) and/or various immune-modulating antibodies.

Checkpoint inhibitors have improved survival for patients with melanoma, non-small-cell lung cancer (NSCLC), bladder, head and neck and other cancers. Antibodies against PD-L1, including atezolizumab, avelumab and durvalumab, are also being developed and have been approved for various cancers. Compared with anti-CTLA-4 drugs, studies with anti-PD-1/PD-L1 agents have suggested higher response rates and improved survival. Targeting PD-L1 rather than PD-1 may also theoretically offer further benefit, with the potential for improved efficacy and reduced toxicity, although this has not been clearly shown by clinical experience to date. Anti-PD-L1 agents have shown good efficacy and manageable toxicity in several tumor types.

The inhibitory immune checkpoint PD-L1/PD1 promotes the alternative splicing of the FKBP5 gene, resulting in increased expression of its variant 4 in the peripheral blood mononuclear cells of melanoma patients. The variant 4 transcript is translated into the truncated FKBP51s protein. Given the importance of co-inhibitory signalling in tumour immune escape, here we tested the potential for using FKBP51s expression to predict immunotherapy outcomes. To do this, we immunophenotyped PBMCs from 118 melanoma patients and 77 age- and sex-matched healthy controls. Blood samples were collected before patients underwent ipilimumab treatment. In 64 of the 118 patients, FKBP51s expression was also assessed in regulatory T cells (Tregs). We found that each PBMC subset analysed contained an FKBP51s pos fraction, and that this fraction was greater in the melanoma patients than healthy controls. In CD4 T lymphocytes, the FKBP51s neg fraction was significantly impaired. Tregs count was increased in melanoma patients, which is in line with previous studies. Also, by analyses of FKBP51s in Tregs, we identified a subgroup of ipilimumab nonresponder patients (p = 0.002). In conclusion, FKBP51s-based immunophenotyping of melanoma patients revealed several profiles related to a negative immune regulatory control and identified an unknown Treg subset. These findings are likely to be useful in the selection of the patients that are candidate for immunotherapy.

Objective: Overexpression of ABCG2 and CD200 has been independently associated with poor outcome in acute myeloid leukemia (AML). However, no data are available on the role of these two factors in patients with core-binding factor (CBF)-positive or FLT3-negative/NPM1-mutated cytogenetically normal (CN) AML., Methods: We analyzed 65 adult AML patients with CBF+ (n=16) or FLT3-/NPM1+ CN (n=49), evaluating clinical and biological factors associated with complete remission attainment, leukemia-free survival (LFS) and overall survival (OS)., Results: ABCG2 was expressed in 36 (55%) cases, and CD200 was positive in 33 (51%) cases, six at high levels. Both ABCG2 and CD200 positivity have a negative impact on relapse risk: 3-year LFS was 51% vs 82% in ABCG2+ cases (RR 3.3), 49% vs 82% in CD200+ patients (RR=4.4), and 25% in CD200- high cases (RR=17.1). ABCG2 and CD200 affected also OS with 3-year OS of 39% in ABCG2+ (compared to 71% in ABCG2-; RR=2.6) and CD200+ (compared to 68% in CD200-; RR=2.5) patients., Conclusions: Our data confirm a negative impact of ABCG2 and CD200 overexpression also in AML patients considered at favorable risk according to ELN cytogenetic/molecular classification., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Overexpression of CD200, a trans-membrane protein belonging to the immunoglobulin superfamily, has been associated with poor prognosis in patients with acute myeloid leukemia (AML). As few data are available in the subset of cytogenetically-normal (CN) AML, we retrospectively evaluated the correlations between CD200 expression and response to therapy in a series of 139 adults with CN-AML. CD200 was expressed in 67/139 (48%) cases; 18 of them (28%) expressed CD200 at high intensity. No differences in CD200 expression rate were observed according to age, WBC count, type of leukemia, FLT3 or NMP1 mutation, and CD56 expression. A higher incidence of CD200 expression was observed in CD34+ cases (P<0.0001) and in BCL2+ patients (P=0.04). Complete remission (CR) was evaluable achieved in 98 patients (70%): 56/71 (79%) in CD200- and 47/67 (63%) in CD200+ patients (P=0.03), with a lower CR rate in patients with high CD200 intensity (9/18, 50%). CD200 expression had a negative impact on long-term outcome. CD200 expression, per se, did not impact on disease-free survival (DFS), but cases with high CD200 expression had a lower 3-year DFS compared to CD200-negative and low-expressing ones (0% vs 65% vs 68%, P=0.019). Three-year overall survival (OS) was 51% in CD200- and 27% in CD200+ patients (P=0.01), with a significant difference among cases with low or high CD200 expression (35% vs 0%, P=0.001). CD200 high expression defined a group with very poor DFS and OS also among the 37 FLT3-/NPM1+: 3-year DFS and OS were 88% and 60% in CD200-, 50% and 32% in CD200 low and 0% and 0% in CD200 high patients, respectively (P=0.01 for DFS and P=0.05 for OS). Our data suggest a negative impact of CD200 expression in CN-AML, with a further worsening in high-expressing cases, also in the subset of FLT3-/NPM1+ patients., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The FMS-like tyrosine kinase 3 (FLT3) mutation in acute myeloid leukemia (AML) is a negative prognostic factor and, in these cases, allogeneic stem cell transplantation (allo-SCT) can represent an important therapeutic option, especially if performed in complete remission (CR). However, it is increasingly clear that not all cytological CRs (cCRs) are the same and that minimal residual disease (MRD) before allo-SCT could have an impact on AML outcome. Unfortunately, FLT3, due its instability of expression, is still not considered a good molecular MRD marker. We analyzed the outcome of allo-SCT in a population of FLT3-positive AML patients according to molecular MRD at the pretransplantation workup, assessed by the quantitative expression evaluation of the panleukemic marker Wilms' tumor (WT1) gene. Sixty-two consecutive AML FLT3-positive patients received allo-SCT between 2005 and 2016 in our center. The median age at transplantation was 55 years. The quantitative analysis of the WT1 gene expression (bone marrow samples) was available in 54 out of 62 (87%) cases, both at diagnosis (100% overexpressing WT1 with a mean of 9747 ± 8064 copies) and before allo-SCT (33 WT1-negative and 21 WT1-positive cases at the pretransplantation workup). Of these cases, 33/54 (61%) were both in cCR and molecular remission (WT1-negative) at the time of transplantation, 13/54 (24%) were in cCR but not in molecular remission (WT1-positive), and 8/54 (15%) showed a cytological evidence of disease (relapsed or refractory). Both post-allo-SCT overall survival (OS) and disease-free survival (DFS) were significantly better in patients who were WT1-negative (WT1 <250 copies) at the time of transplantation compared with those who were WT1-positive (WT1 >250 copies), with a median OS and DFS not reached in the WT1-negative group and 10.2 and 5.5 months, respectively, in the WT1-positive group (OS log-rank p = 0.0005; hazard ratio [HR] = 3.7, 95% confidence interval [95% CI] = 1.5-9; DFS log-rank p = 0.0001; HR = 4.38, 95% CI = 1.9-10). Patients with cCR who were WT1-positive had the same negative outcome as those with a cytological evidence of disease. The relapse rate after allo-SCT was 9% (3/33) in pre-allo-SCT WT1-negative cases and 54% (7/13) in WT1-positive cases (p = 0.002). At multivariate analysis, WT1 negativity before allo-SCT and grade <2 acute graft versus host disease were the only independent prognostic factors for improved OS and DFS. These data show that pre-allo-SCT molecular MRD evaluation through WT1 expression is a powerful predictor of posttransplantation outcomes (OS, DFS, relapse rate). Patients with both cCR and a WT1-negative marker before allo-SCT have a very good outcome with very low relapse rate; conversely, patients with positive molecular MRD and refractory/relapsed patients have a negative outcome. The WT1 MRD stratification in FLT3-positive AML is a valuable tool with which to identify patients who are at high risk of relapse and that could be considered from post-allo-SCT prophylaxis with FLT3 inhibitors or other strategies (donor lymphocyte infusion, tapering of immunosuppression, azacitidine)., (Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Background: Extensive squamous cell carcinoma has few therapeutic options. In such cases, electrochemotherapy involving electroporation combined with antineoplastic drug appears to be a new potential option and may be considered as an alternative treatment. The aim of this retrospective single-center study was to evaluate electrochemotherapy efficacy in treatment of locally advanced stage III squamous cell carcinoma, in which surgical procedures would have entailed wide tissue sacrifice., Methods: Clinical features, treatment response, and adverse effects were evaluated in 22 patients treated with electrochemotherapy with intravenous injection of bleomycin for extensive stage III cutaneous squamous cell carcinoma. Treatment of cutaneous lesions were performed according to the European Standard Operating Procedures of Electrochemotherapy., Results: Overall response to electrochemotherapy treatment was observed in 18 (81.8%) patients. Clinical response with necrosis of tumor mass was observed from the first session and lasted for all follow up period that ranged between 5 and 48 months with a median of 34 months. Overall the treatment was well tolerated with a very low complication rate., Conclusions: Electrochemotherapy represents a safe and effective therapeutic approach, associated with a good tolerability.

Ipilimumab (IPI) blocks CTLA-4 immune checkpoint resulting in T cell activation and enhanced antitumor immunity. IPI improves overall survival (OS) in 22% of patients with metastatic melanoma (MM). We investigated the association of CTLA-4 single nucleotide variants (SNVs) with best overall response (BOR) to IPI and OS in a cohort of 173 MM patients. Patients were genotyped for six CTLA-4 SNVs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, and CT60G>A). We assessed the association between SNVs and BOR through multinomial logistic regression (MLR) and the prognostic effect of SNVs on OS through Kaplan-Meier method. Both -1577G>A and CT60G>A SNVs were found significantly associated with BOR. In particular, the proportion of responders was higher in G/G genotype while that of stable patients was higher in A/A genotype. The frequency of patients experiencing progression was similar in all genotypes. MLR evidenced a strong downward trend in the probability of responsiveness/progression, in comparison to disease stability, as a function of the allele A "dose" (0, 1, or 2) in both SNVs with reductions of about 70% (G/A vs G/G) and about 95% (A/A vs G/G). Moreover, -1577G/G and CT60G/G genotypes were associated with long-term OS, the surviving patients being at 3 years 29.8 and 30.8%, respectively, as compared to 12.9 and 14.4% of surviving patients carrying -1577G/A and CT60G/A, respectively. MM patients carrying -1577G/G or CT60G/G genotypes may benefit from IPI treatment in terms of BOR and long-term OS. These CTLA-4 SNVs may serve as potential biomarkers predictive of favorable outcome in this subset of patients.

Introduction: Myeloid Sarcoma (MS) is a rare hematologic myeloid neoplasm that can involve any site of the body. It can occur as an exclusively extramedullary form or it can be associated with an acute myeloid leukemia (AML), a chronic myeloproliferative neoplasm (MPN) or a myelodysplastic syndrome (MDS) at onset or at relapse. The rarity of MS does not enable prospective clinical trials and therefore a specific multicenter register can be useful for the clinical and biological studies of this rare disease., Patients and Results: we report the clinical characteristics and outcome of 48 histologically confirmed MS, diagnosed and treated in 9 Italian Hematological Centers in the last 10 years. The patient's median age was 46 years. There were 9/48 de novo extramedullary MS, 24/48 de novo AML-related MS and 15/48 were secondary AML-related MS. The most common extramedullary anatomic sites of disease were: skin, lymph nodes and soft tissues. Forty-three patients (90%) underwent a program of intensive chemotherapy including FLAI, HDAC-IDA, HyperCVAD and MEC schemes, with a DDI of 5% and a CR Rate of 45%. Twenty-two (46%) patients underwent Allogeneic SCT, 13 from a MUD, 8 from an HLA-identical sibling donor and 1 from an haploidentical donor. The median OS of the whole population (48 pts) was 16.7 months. The OS probability at 1, 2 and 5 years was 64%, 39% and 33%, respectively. The OS was better in patients that underwent an intensive therapeutic program (median OS: 18 months vs 5 months). Among the intensively treated patients, in univariate analysis, the OS was better in young patients (P=0,008), in patients that underwent Allo-SCT (P=0,009) and in patients that achieved a CR during treatment (P=0,001), and was worse in pts with secondary AML-related MS (P=0,007). Age, response to intensive chemotherapy and Allo-SCT were the only three variables that significantly influenced DFS (P=0,02, P=0,01 and P=0,04, respectively). In multivariable analysis, Allo-SCT and response to intensive chemotherapy remained significant in predicting a better OS (P=0,04 and P=0,001, respectively), and response to intensive chemotherapy was the only significant variable in predicting DFS (P=0,01). After Allo-SCT we observe a survival advantage in patients who achieved a pre-transplant CR (P=0,008) and in those who developed a chronic GvHD (P=0,05)., Conclusions: Patients with MS, both with de novo and secondary forms, still have a very unfavorable outcome and require an intensive therapeutic program, that includes Allo-SCT whenever possible. The outcome after Allo-SCT is positively influenced by the development of chronic GvHD suggesting a Graft versus MS effect., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Raf-mitogen-activated protein kinase (Raf-MAPK) pathway inhibition with the BRAF inhibitors vemurafenib and dabrafenib, alone or in combination with a MEK inhibitor, has become a standard therapeutic approach in patients with BRAF-mutated metastatic melanoma. Both vemurafenib and dabrafenib have shown good safety and efficacy as monotherapy compared with chemotherapy. However, the duration of response is limited in the majority of patients treated with BRAF inhibitor monotherapy because of the development of acquired resistance. The addition of a MEK inhibitor can improve blockade of the MAPK pathway and may help to overcome resistance and thereby prolong efficacy, as well as reduce cutaneous toxicity. Combinations of BRAF inhibitors and MEK inhibitors (dabrafenib plus trametinib and vemurafenib plus cobimetinib) have been approved for the treatment of BRAF-mutant metastatic melanoma and may become a new standard of care. However, acquired resistance is still a significant concern with BRAF and MEK inhibitor combination therapy, and other strategies are being investigated, including the use of sequential and intermittent schedules. The combination of BRAF or MEK inhibitors with immunotherapy has been shown to hold considerable promise, with several combinations being evaluated in clinical trials. Preliminary results from clinical trials involving triple combination therapy with BRAF-MEK inhibitors and anti-PD-L1 antibodies appear promising and may indicate a new strategy to treat patients with BRAF-mutated metastatic melanoma. Biomarkers are needed to help identify patients with BRAFV600 mutations most likely to benefit from first-line BRAF/MEK inhibitor therapy rather than immunotherapy and vice versa.

The optimal sequencing of targeted treatment and immunotherapy in the treatment of advanced melanoma is a key question and prospective studies to address this are ongoing. Previous observations suggest that treating first with targeted therapy may select for more aggressive disease, meaning that patients may not gain full benefit from subsequent immunotherapy. In a single-center retrospective analysis, we investigated whether response to pembrolizumab was affected by previous BRAF inhibitor therapy. A total of 42 patients with metastatic cutaneous or mucosal melanoma who had received previous treatment with ipilimumab were treated with pembrolizumab as part of the Italian expanded access program. Sixteen of these patients had BRAF-mutated melanoma and had also been previously treated with a BRAF inhibitor (vemurafenib or dabrafenib), while 26 had BRAF wild-type melanoma (no previous BRAF inhibitor). Patients with BRAF-mutant melanoma who were previously treated with BRAF inhibitors had a significantly lower median progression-free survival (3 [2.3-3.7] versus not reached [2-8+] mo; p = 0.001) and disease control rate (18.6% versus 65.4%; p = 0.005) than patients with BRAF wild-type, while there was also a trend toward a lower response rate (assessed using immune-related response criteria) although this was not significantly different between groups (12.5% versus 36.4%; p = 0.16). These data are consistent with previous reports that BRAF inhibitor therapy may affect subsequent response to immunotherapy.

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 - CD56 dim 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.

Purpose: Biomarkers for outcome after immune-checkpoint blockade are strongly needed as these may influence individual treatment selection or sequence. We aimed to identify baseline factors associated with overall survival (OS) after pembrolizumab treatment in melanoma patients., Experimental Design: Serum lactate dehydrogenase (LDH), routine blood count parameters, and clinical characteristics were investigated in 616 patients. Endpoints were OS and best overall response following pembrolizumab treatment. Kaplan-Meier analysis and Cox regression were applied for survival analysis., Results: Relative eosinophil count (REC) ≥1.5%, relative lymphocyte count (RLC) ≥17.5%, ≤2.5-fold elevation of LDH, and the absence of metastasis other than soft-tissue/lung were associated with favorable OS in the discovery (n = 177) and the confirmation (n = 182) cohort and had independent positive impact (all P < 0.001). Their independent role was subsequently confirmed in the validation cohort (n = 257; all P < 0.01). The number of favorable factors was strongly associated with prognosis. One-year OS probabilities of 83.9% versus 14.7% and response rates of 58.3% versus 3.3% were observed in patients with four of four compared to those with none of four favorable baseline factors present, respectively., Conclusions: High REC and RLC, low LDH, and absence of metastasis other than soft-tissue/lung are independent baseline characteristics associated with favorable OS of patients with melanoma treated with pembrolizumab. Presence of four favorable factors in combination identifies a subgroup with excellent prognosis. In contrast, patients with no favorable factors present have a poor prognosis, despite pembrolizumab, and additional treatment advances are still needed. A potential predictive impact needs to be further investigated. Clin Cancer Res; 22(22); 5487-96. ©2016 AACR., (©2016 American Association for Cancer Research.)

The anti-PD-1 agent, nivolumab, has been approved both as monotherapy and in combination with ipilimumab for the treatment of unresectable or metastatic melanoma in the USA and European Union. Here we present the case of a patient with treatment-naive, metastatic mucosal melanoma and baseline LDH approximately seven times the upper limit of normal. The patient was enrolled in a clinical trial (CheckMate 066) and achieved a partial response, followed by a durable complete response with nivolumab treatment. The patient's LDH levels were documented in each cycle and dropped markedly within 2 months, when partial response to treatment was already evident. LDH levels remained low for the rest of follow-up, consistent with the ongoing complete response to treatment. The patient experienced only mild immune-related adverse events (grade 1-2), which included vitiligo and rash. This exceptional response suggests that patients with high LDH levels at baseline should be considered for nivolumab treatment. LDH levels, however, should not serve as a predictive marker of response to nivolumab. Moreover, this case suggests the need to identify patients who will achieve the greatest benefit from nivolumab monotherapy.

Background: BRAF inhibitor vemurafenib achieves high response rate and an improvement in survival in patients with BRAF-mutated metastatic melanoma. However, median progression-free survival is only 6.9 months in the phase 3 study. Retrospective analyses suggest that treatment with BRAF inhibitors beyond initial progression might be associated with improved overall survival. We aimed to prospectively investigate the activity of prolonged treatment with vemurafenib and the addition of fotemustine in patients with systemic progression on prior single-agent BRAF inhibitor., Patients and Methods: In this two-centres, single-arm Phase 2 trial, we enrolled patients with systemic progressive disease during single-agent vemurafenib treatment. Participants received vemurafenib 960 mg twice daily or dose administered at time of disease progression with vemurafenib previous treatment and fotemustine 100 mg/m2 intravenously every three weeks. The primary endpoint was PFS., Results: Thirty-one patients were enrolled in the study; 16 patients had brain metastases at baseline. Median PFS was 3.9 months and 19 patients (61.3%) achieved disease control (1 CR, 4 PR, 14 SD). For patients achieving disease control, median duration of treatment was 6 months. Median OS was 5.8 months from enrolment and 15.4 months from start of previous vemurafenib. Five patients (16.1%) had a G3-4 AE, the most common being thrombocytopenia, which occurred in 3 patients.This trial is registered with ClinicalTrials.gov number NCT01983124., Conclusion: The combination of vemurafenib plus fotemustine has clinical activity and an acceptable safety profile in BRAF-refractory patients., Competing Interests: CONFLICTS OF INTEREST P. Queirolo served as advisor to Bristol-Myers Squibb, Roche-Genentech, MSD, GlaxoSmithKline and Novartis; F. Spagnolo received lecture fees from Bristol-Myers Squibb, GlaxoSmithKline, Novartis and Roche; V. Picasso received lecture fees from Bristol-Myers Squibb; P.A. Ascierto served as paid consultant or advisor from Bristol-Myers Squibb, Roche-Genentech, GlaxoSmithKline, Amgen, Ventana, Novartis and Merck Sharp and Dohme, and received research funds by Roche, Ventana and BMS. M. Del Vecchio served as paid consultant or advisor, and received research funds from Bristol-Myers Squibb, Roche-Genentech, GlaxoSmithKline and Merck Sharp and Dohme.

Background: Acute myeloid leukaemia not responsive to first induction chemotherapy (PIF-AML) still remains a challenge, and there are only few recent epidemiological data regarding the outcome of these patients. In this multicentre survey, we evaluate the prognosis and outcome of patients with PIF-AML, who were diagnosed and treated in the last 5 yrs in four Italian institutions., Results: One hundred PIF-AML were recorded, 57 males and 43 females, with a median age of 63 yrs (19-79), 42% were younger than 60 yrs; 42% had a secondary AML and 40% had an adverse karyotype. According to cytogenetic/molecular risk stratification at diagnosis, 33% of patients were classified as favourable/intermediate-1 risk and 56% as intermediate-2/adverse risk. After a median follow-up of 11 months (1-49), 77% of patients died, while 23% were alive (with 12/23 in cCR). Thirty-six patients underwent allogeneic SCT, and of these, 11 of 36 (31%) were alive at last follow-up. The 12- and 24-month OS probability of the whole population was 45% and 21%, respectively. In multivariate analysis, the probability of OS of the whole population was significantly improved by Allo-SCT procedure (12-month OS probability 60% vs. 35%; P < 0.0001) and was better in patients with favourable/intermediate-1 risk at diagnosis (12-month OS probability 58% vs. 40%; P = 0.028). In transplanted cases, a pretransplant responsive disease was the only significant factor to predict a favourable outcome after Allo-SCT (P = 0.006)., Conclusion: Treatment options of PIF-AML still are limited and the prognosis, even recently, remains extremely poor. This survey shows that PIF-AML is still rarely cured without Allo-SCT and confirms the importance of initiating an urgent unrelated donor search in cases without a matched sibling donor. Moreover, the outcome of Allo-SCT is better in patients who achieve a good AML debulking before transplant. To reach this goal, new predictive scores and new protocols of salvage therapy (with target drugs or combinations) need to be explored urgently in PIF-AML., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: BRAF(V600E)-mediated MAPK pathway activation is associated in melanoma cells with IFNAR1 downregulation. IFNAR1 regulates melanoma cell sensitivity to IFNα, a cytokine used for the adjuvant treatment of melanoma. These findings and the limited therapeutic efficacy of BRAF-I prompted us to examine whether the efficacy of IFNα therapy of BRAF(V600E) melanoma can be increased by its combination with BRAF-I., Methods: BRAF/NRAS genotype, ERK activation, IFNAR1, and HLA class I expression were tested in 60 primary melanoma tumors from treatment-naive patients. The effect of BRAF-I on IFNAR1 expression was assessed in three melanoma cell lines and in four biopsies of BRAF(V600E) metastases. The antiproliferative, pro-apoptotic and immunomodulatory activity of BRAF-I and IFNα combination was tested in vitro and in vivo utilizing three melanoma cell lines, HLA class I-MA peptide complex-specific T-cells and immunodeficient mice (5 per group for survival and 10 per group for tumor growth inhibition). All statistical tests were two-sided. Differences were considered statistically significant when the P value was less than .05., Results: The IFNAR1 level was statistically significantly (P < .001) lower in BRAF(V600E) primary melanoma tumors than in BRAF wild-type tumors. IFNAR1 downregulation was reversed by BRAF-I treatment in the three melanoma cell lines (P ≤ .02) and in three out of four metastases. The IFNAR1 level in the melanoma tumors analyzed was increased as early as 10 to 14 days following the beginning of the treatment. These changes were associated with: 1) an increased susceptibility in vitro of melanoma cells to the antiproliferative (P ≤ .04), pro-apoptotic (P ≤ .009) and immunomodulatory activity, including upregulation of HLA class I antigen APM component (P ≤ .04) and MA expression as well as recognition by cognate T-cells (P < .001), of BRAF-I and IFNα combination and 2) an increased survival (P < .001) and inhibition of tumor growth of melanoma cells (P < .001) in vivo by BRAF-I and IFNα combination., Conclusions: The described results provide a strong rationale for the clinical trials implemented in BRAF(V600E) melanoma patients with BRAF-I and IFNα combination., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

CD200, a protein belonging to the immunoglobulin superfamily, has been associated with a poor prognosis in lymphoproliferative disorders and in acute leukemia. We studied the expression of CD200 in a series of 244 patients with diagnosis of acute myeloid leukemia (AML), to evaluate its impact on outcome and its possible association with other known prognostic factors. CD200 was found in 136/244 (56%) patients, in 41 of whom (30%) with high intensity of expression (MFI ≥ 11). CD200 was more frequent in secondary compared to de novo leukemia (p = 0.0006), in CD34 positive cases (p = 0.00001), in Bcl2 overexpressing cases (p = 0.01), in those wild-type Flt3 (p = 0.004) and with favorable or unfavorable compared to intermediate karyotype (p = 0.0003). CD200+ patients have a two-fold lower probability to attain complete remission, both in univariate (p = 0.006) and multivariate (p = 0.04) analysis. The negative impact of CD200 was found also in overall survival (p = 0.02) and was correlated with the intensity of expression of the molecule (p = 0.024). CD200 has an additive negative impact on survival in patients with unfavorable cytogenetic (p = 0.046) and in secondary leukemia (p = 0.05), and is associate with a worsening of outcome in patients with favorable biological markers, such as mutated NPM (p = 0.02), wild-type Flt3 (p = 0.034), negativity of CD34 (p = 0.03) and of CD56 (p = 0.03). In conclusion, CD200 is emerging as both a prognostic factor and a potential target of novel therapeutic approaches for AML, aiming to reverse the "do not eat me" signal of CD200 or to manipulate the suppressive immune microenvironment induced by CD200 binding to its receptor.

The concomitant inhibition of both BRAF and MEK can produce a more durable and greater tumor response than BRAF monotherapy while reducing BRAF inhibitor-related toxicity. Further evidence of the benefits of combined MEK and BRAF inhibition have been provided by the CoBRIM trial in which median progression-free survival was significantly increased with vemurafenib plus cobimetinib compared with vemurafenib alone (9.9 vs 6.2 months; hazard ratio for death or progression: 0.51; 95% CI: 0.39-0.68; p < 0.001) in 495 patients with advanced BRAF -mutated melanoma. Overall survival data in the CoBRIM trial were immature at time of final progression-free survival analysis but showed an hazard ratio for death of 0.65 (95% CI: 0.42-1.00; p = 0.046; boundary p < 0.0000037). Combination therapy was well tolerated with a reduced incidence of cutaneous squamous-cell carcinoma/keratoacanthoma. This combination may be a starting point for novel combination strategies with immunotherapies and other targeted therapies., Competing Interests: Financial & competing interests disclosure PA Ascierto had/has a consultant/advisory role for Bristol-Myers Squibb, Roche-Genentech, Merck Sharp & Dohme, GlaxoSmithKline, Ventana and Novartis. He received research funds from Bristol-Myers Squibb and Ventana. He also receive honoraria from Bristol-Myers Squibb, Roche-Genentech, GlaxoSmithKline. AM Grimaldi and E Simeone received honoraria from Bristol-Myers Squibb, GlaxoSmithKline and Novartis. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

FKBP51 (gene FKBP5) is an immunophilin capable of immunosuppression expressed in melanoma and lymphocytes. We found increased levels of a spliced FKBP5 variant in the PBMCs of 124 patients with melanoma. This variant encodes for an unknown isoform (FKBP51s). We hypothesized that FKBP51s resulted from tumour interaction with immune cells, through PDL-1/PD-1. To address this issue, we performed melanoma/PBMC cocultures. Furthermore, the immunohistochemistry of 76 melanoma specimens served to investigate whether FKBP51s stained tumour infiltrating lymphocytes. Our results showed that PBMCs expressed FKBP51s when cocultured with melanoma. Tumour PDL-1 knockdown or anti-PD-1 reduced FKBP51s expression in cocultured PBMCs. IHC showed a strong FKBP51s signal in tumour infiltrating lymphocytes, and lymphocytes of the invasion front of the tumour, along with melanoma PDL-1 expression. When overexpressed in melanoma, FKBP51s facilitated PDL-1 expression on the cell surface. In conclusion, our study shows that FKBP51s marks the PBMCs of patients with melanoma and is exploited by the tumour to immunomodulate through PDL-1., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The development of novel treatments that selectively inhibit the RAS-RAF-MAPK pathway represents a milestone in the history of melanoma treatment. BRAF mutations occur in approximately 45% of cutaneous melanomas, while mutations in NRAS occur in 15-25%. Vemurafenib was the first BRAF inhibitor to be approved in 2011, based on the results of a phase III trial (BRIM-3) that showed higher progression-free survival and overall survival compared with dacarbazine chemotherapy in metastatic BRAF-mutated melanoma. Dabrafenib, another BRAF inhibitor, has shown similar results and was approved in 2013. Preclinical studies suggested that another novel group of agents, the MEK inhibitors, showed stronger inhibition of both mutated BRAF and NRAS cell cultures than vemurafenib. Trametinib was the first MEK inhibitor approved in 2014, both as a single agent and in combination with dabrafenib for the treatment of advanced BRAF-mutated melanoma. Other MEK inhibitors are also in development. Concomitant inhibition of both MEK and BRAF has shown more durable and greater tumor response than BRAF monotherapy, by overcoming the multiple genetic mechanisms of escape. Combined therapy prevents the development of acquired resistance as well as decreasing cutaneous toxicity secondary to paradoxical activation of the MAPK pathway induced by BRAF inhibitors. Various combinations of BRAF and MEK inhibitors have shown promising results. Moreover, triple combination therapies involved other agents with novel mechanisms of action are also being evaluated. These and other combination strategies involving immunotherapies and targeted therapies offer the hope of improving outcomes beyond those already achieved with anti-BRAF treatments.

Melanoma is responsible for most skin cancer-related deaths and is one of the most common cancers diagnosed in young adults. In melanoma, tumors can become established by activation of the negative regulator of cytotoxic T lymphocytes (CTLs), CTL antigen-4 (CTLA-4). Ipilimumab blocks the interaction of CTLA-4 with CD80/CD86 and augments T-cell activation and proliferation. In electrochemotherapy (ECT), local application of short high-voltage pulses renders cell membranes transiently permeable to chemotherapeutic drugs. The combination of ipilimumab and ECT may be beneficial for the treatment of metastatic melanoma; however, no prospective data are available to date. Here, we report the retrospective analysis of patients treated with ipilimumab in an expanded access program (EAP) who also received ECT. Fifteen patients with previously treated metastatic melanoma who received ipilimumab 3 mg/kg every three weeks for four cycles and underwent ECT for local disease control and/or palliation of cutaneous lesions with bleomycin 15 mg/m 2 after the first ipilimumab infusion were included in the analysis. Over the study period, a local objective response was observed in 67% of patients (27% complete response [CR] and 40% partial response [PR]). According to immune-related response criteria, a systemic response was observed in nine patients (five PR and four stable disease [SD]), resulting in a disease control rate of 60%. Evaluation of circulating T-regulatory (T-reg) cells demonstrated significant differences between responders and non-responders. Overall, treatment was well-tolerated and without notable toxicity. In conclusion, the combination of ipilimumab and ECT appears to be beneficial to patients with advanced melanoma, warranting further investigation in prospective trials.

Aim: This study was undertaken to update the outcome of the treated lesions with electrochemotherapy (ECT) plus intravenous injection of bleomycin in patients with in-transit disease or distant cutaneous metastases in melanoma patients., Patients & Methods: 89 patients with relapsed and refractory cutaneous melanoma metastases or in-transit disease were submitted to 126 treatments of ECT., Results: 34 patients (38.2%) had a partial response and 43 had a complete response (48.3%). 12 patients (13.5%) had no change or progressive disease. The objective response rate was 67.5%., Conclusion: The favorable outcome obtained in the present study demonstrates that ECT is a reliable, easy, fast and effective procedure showing benefits in terms of curative and palliative treatment for unresectable cutaneous lesions respecting the quality of life., Competing Interests: Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Background: Primary umbilical melanoma is an uncommon tumor that is poorly described in the medical literature. The umbilical region is a particular anatomic site owing to the presence of embryonal remnants, which can be a potential metastatic pathway, as well as the braided lymphatic network drainage. Hence, primary malignant neoplasms affecting the umbilicus require a different and more radical surgical approach compared with other melanomas., Case Presentation: In this report, we describe a series of three patients of Caucasian ethnicity who presented with primary umbilical melanoma at the National Cancer Institute of Naples, Italy. All patients underwent wide excision of the tumor including the underlying peritoneum. No surgical complications, either immediate or delayed, were observed in any of the patients. Sentinel lymph node biopsy was negative in two cases. Two of the patients developed metastatic disease and died after systemic medical therapy. The other patient is currently in follow-up, and remains disease-free after 21 months., Conclusions: The umbilicus has vascular and embryological connections with the underlying peritoneum, so that early visceral involvement is more likely to occur with primary umbilical melanomas. As such, tumor resection including the underlying peritoneum is required to avoid local relapse, whilst sentinel lymph node biopsy appears to be of poor diagnostic value.

Interferon (IFN) and PEG-IFN are the only drugs approved as adjuvant therapy in patients with melanoma at high-risk of recurrence after surgical resection. Several clinical trials of adjuvant IFN, using different doses and durations of therapy, have been conducted in these patients. Results generally suggest relapse-free survival and overall survival benefits; however, questions over the optimal dose and duration of treatment and concerns over toxicity have limited its use. IFN exerts its biological activity in melanoma via multiple mechanisms of action, most of which can be considered as indirect immunomodulatory effects. As such, IFN may also be of benefit in the neoadjuvant setting, where it may have a role in melanoma patients with locally advanced disease for whom immediate surgical excision is not possible. However, this has not been well studied. The use of IFN in patients with metastatic melanoma is controversial, with limited data and no convincing evidence of a survival benefit. However, IFN therapy combined with novel biological and immunotherapies offers the potential for a synergistic effect and improved clinical outcomes. Predictive and prognostic factors to better select melanoma patients for IFN treatment have been identified (e.g. disease stage, ulceration, various cytokines) and may also enhance its therapeutic efficacy, but their incorporation into the clinical decision-making process requires validation in prospective trials. In conclusion, the modest efficacy of IFN shown in clinical trials is largely a reflection of differences in response between patients. Despite advancements in the understanding of its biological mechanisms of action, the huge potential of IFN remains to be fully explored and utilized in patients with melanoma., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The prognosis of metastatic melanoma has changed markedly in recent years because of the advent of newer targeted therapies such as BRAF inhibitors. However, the response to BRAF inhibitor therapy is frequently nondurable in patients with advanced melanoma. Novel approaches are thus needed to overcome resistance to these agents and to improve the management of advanced melanoma patients after disease progression. Here, we present the case of a 44-year-old man diagnosed with advanced melanoma in July 2010, harboring a BRAF mutation. Melanoma progressed during first-line chemotherapy with dacarbazine, but showed significant benefit after the initiation of vemurafenib on August 2011. Six months later, the patient experienced disease progression in left-obturator lymphadenopathy; still, anti-BRAF treatment was continued together with stereotactic radiotherapy, and was interrupted only shortly for intestinal occlusion secondary to melanoma metastasis of the bowel. When his conditions were stable, after 1 month of vemurafenib treatment discontinuation, anti-BRAF therapy was reinitiated, with a positive outcome. Vemurafenib treatment was definitively discontinued for disease progression in the brain, peritoneum, lymph node, intestine, and skin in March 2013, after about 20 months from initiation, and the patient died a few weeks later. The clinical case presented here shows that treatment beyond progression with vemurafenib can yield a survival benefit in melanoma patients whose disease progresses in a few sites, which can be treated with locoregional therapies. This clinical strategy needs further validation in prospective clinical trials.

Programmed cell death receptor-1 (PD-1) and programmed cell death-1 ligand-1 (PD-L1) represent promising novel targets in immunotherapy. PD-1 is an inhibitory receptor involved in T-cell regulation that is expressed by activated T cells. Nivolumab and pembrolizumab are anti-PD-1 antibodies that have shown antitumor activity and acceptable tolerability in patients with metastatic melanoma in preclinical development and Phase I/II clinical trials. Several ongoing Phase III studies are further investigating the efficacy and safety of anti-PD-1 therapy in melanoma. Initial data on the combination of anti-PD-1 and anti-cytotoxic T-lymphocyte-associated antigen 4 blockade with nivolumab and ipilimumab also appear promising. Monoclonal antibodies to blockade PD-L1 may also be an effective immunotherapy strategy in melanoma and several anti-PD-L1 antibodies are in development., Competing Interests: Financial & competing interests disclosure PA Ascierto has/had an advisory/consultant role for Bristol Myers Squibb, Roche-Genentech, Merck Sharp & Dohme, GlaxoSmithKline, Ventana, Amgen and Novartis. He also received research funds from Bristol Myers Squibb, Roche-Genentech and Ventana. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.