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Abstract Therapy of melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPKi) and immunotherapies. However, drug resistance continues to limit the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanomas. Among them, a pivotal role is played by the oncosuppressor miR-579-3p. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF, is able to stabilize MITF protein thus inducing its own transcription. From biological points of view, early exposure to MAPKi or, alternatively miR-579-3p transfection, induce block of proliferation and trigger senescence programs in BRAF-mutant melanoma cells. Finally, the long-term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to therapies in BRAF-mutant melanomas.

Basal cell carcinoma (BCC) is the most common malignant tumour in white populations. Multiple studies demonstrated that the aberrant activation of Hedgehog signaling is a driver of BCC development, and its blockade represents a potential therapeutic target. In Italy, clinicians can prescribe Hedgehog inhibitors (HhIs) Vismodegib and Sonidegib. To highlight the treatment choice of clinicians, we conducted an online survey between November 1 and November 18, 2020 with 33 Italian clinicians from 27 reference hospitals, in which each participant received an anonymous survey consisting of two multiple-choice questions on clinical efficacy and safety profile of Sonidegib and Vismodegib. Respondents reported their opinions on which efficacy and tolerability data of the pivotal phase-II BOLT trial were more relevant in the treatment choice of patients with locally advanced BCC (laBCC). This survey shows that overall response rate (ORR) and the duration of response (DoR) are the most expected across dermatologists and oncologists. The different pharmacokinetic profile of the two HhIs are behind their diverse toxicity spectrum, dose and schedule modification seem to address the choice between vismodegib and sonidegib among dermato-oncology prescribers.

Abstract Background The current therapeutic algorithm for Advanced Stage Melanoma comprises of alternating lines of Targeted and Immuno-therapy, mostly via Immune-Checkpoint blockade. While Comprehensive Genomic Profiling of solid tumours has been approved as a companion diagnostic, still no approved predictive biomarkers are available for Melanoma aside from BRAF mutations and the controversial Tumor Mutational Burden. This study presents the results of a Multi-Centre Observational Clinical Trial of Comprehensive Genomic Profiling on Target and Immuno-therapy treated advanced Melanoma. Methods 82 samples, collected from 7 Italian Cancer Centres of FFPE-archived Metastatic Melanoma and matched blood were sequenced via a custom-made 184-gene amplicon-based NGS panel. Sequencing and bioinformatics analysis was performed at a central hub. Primary analysis was carried out via the Ion Reporter framework. Secondary analysis and Machine Learning modelling comprising of uni and multivariate, COX/Lasso combination, and Random Forest, was implemented via custom R/Python scripting. Results The genomics landscape of the ACC-mela cohort is comparable at the somatic level for Single Nucleotide Variants and INDELs aside a few gene targets. All the clinically relevant targets such as BRAF and NRAS have a comparable distribution thus suggesting the value of larger scale sequencing in melanoma. No comparability is reached at the CNV level due to biotechnological biases and cohort numerosity. Tumour Mutational Burden is slightly higher in median for Complete Responders but fails to achieve statistical significance in Kaplan–Meier survival analysis via several thresholding strategies. Mutations on PDGFRB, NOTCH3 and RET were shown to have a positive effect on Immune-checkpoint treatment Overall and Disease-Free Survival, while variants in NOTCH4 were found to be detrimental for both endpoints. Conclusions The results presented in this study show the value and the challenge of a genomics-driven network trial. The data can be also a valuable resource as a validation cohort for Immunotherapy and Target therapy genomic biomarker research.

Abstract Background An observational study involving patients recovered from COVID-19 was conducted in order to evaluate the presence/absence of vein wall thickness increasing, according to the severity of pulmonary involvement quantified with a CT-scoring system. Methods The venous wall thickness (VWT) of 31 patients (23 males and 8 females) with COVID 19 previously admitted to Federico II University Hospital of Naples was evaluated through ultrasound measurement of the common femoral Vein 1 cm proximal to the saphenous-femoral junction and the popliteal Vein 1 cm distal to the confluence of gemellary veins. Measurements were taken with an automated tool to avoid human error. All patients were evaluated in the supine position. Patients were then stratified into two groups, VWT > 1 mm and VWT 1 mm group was 7.4 (S.D. 4.83), whilst the mean value of the COVID score in the VWT

Abstract Background BRAF-mutant melanoma patients benefit from the combinatorial treatments with BRAF and MEK inhibitors. However, acquired drug resistance strongly limits the efficacy of these targeted therapies in time. Recently, many findings have underscored the involvement of microRNAs as main drivers of drug resistance. In this context, we previously identified a subset of oncomiRs strongly up-regulated in drug-resistant melanomas. In this work, we shed light on the molecular role of two as yet poorly characterized oncomiRs, miR-4443 and miR-4488. Methods Invasion and migration have been determined by wound healing, transwell migration/invasion assays and Real Time Cell Analysis (RTCA) technology. miR-4488 and miR-4443 have been measured by qRT-PCR. Nestin levels have been tested by western blot, confocal immunofluorescence, immunohistochemical and flow cytometry analyses. Results We demonstrate that the two oncomiRs are responsible for the enhanced migratory and invasive phenotypes, that are a hallmark of drug resistant melanoma cells. Moreover, miR-4443 and miR-4488 promote an aberrant cytoskeletal reorganization witnessed by the increased number of stress fibers and cellular protrusions-like cancer cell invadopodia. Mechanistically, we identified the intermediate filament nestin as a molecular target of both oncomiRs. Finally, we have shown that nestin levels are able to predict response to treatments in melanoma patients. Conclusions Altogether these findings have profound translational implications in the attempt i) to develop miRNA-targeting therapies to mitigate the metastatic phenotypes of BRAF-mutant melanomas and ii) to identify novel biomarkers able to guide clinical decisions. Graphical Abstract

Abstract Background Identifying response markers is highly needed to guide the treatment strategy in patients with metastatic melanoma. Methods A retrospective study was carried out in patients with unresectable/metastatic melanoma (stage IIIb–IV), treated with anti-PD-1 in the first line setting, to better explore the role and the timing of neutrophil/lymphocyte ratio (NLR) as potential biomarker of response. The relationship of NLR with inflammation-immune mediators and the underlying negative effect of raising NLR during immunotherapy, have been investigated with transcriptomic gene analysis. Results The results confirmed previous findings that a high baseline NLR is associated with a poorer prognosis and with higher serum level of lactate dehydrogenase (LDH), regardless of the presence of brain metastases. The transcriptomic analysis showed that high baseline NLR is associated with a characteristic gene signature CCNA1, LDHA and IL18R1, which correlates with inflammation and tumorigenesis. Conversely, low baseline NLR is associated with the signature CD3, SH2D1A, ZAP70 and CD45RA, linked to the immune-activation. The genes positively associated with NLR (CD39 (ENTPD1), PTEN, MYD88, MMP9 and LDH) are involved in processes of immunosuppression, inflammation and tumor-promoting activity. Increased expression of CD39 correlated with TGFβ2, a marker of the N2 neutrophils with immunosuppressive activity. Conclusions These results suggest that increasing NLR is associated with an increased neutrophil population, with polarization to the N2 phenotype, and this process may be the basis for the negatively prognostic role of NLR.

The innate immune system, composed of neutrophils, basophils, eosinophils, myeloid-derived suppressor cells (MDSCs), macrophages, dendritic cells (DCs), mast cells (MCs), and innate lymphoid cells (ILCs), is the first line of defense. Growing evidence demonstrates the crucial role of innate immunity in tumor initiation and progression. Several studies support the idea that innate immunity, through the release of pro- and/or anti-inflammatory cytokines and tumor growth factors, plays a significant role in the pathogenesis, progression, and prognosis of cutaneous malignant melanoma (MM). Cutaneous melanoma is the most common skin cancer, with an incidence that rapidly increased in recent decades. Melanoma is a highly immunogenic tumor, due to its high mutational burden. The metastatic form retains a high mortality. The advent of immunotherapy revolutionized the therapeutic approach to this tumor and significantly ameliorated the patients’ clinical outcome. In this review, we will recapitulate the multiple roles of innate immune cells in melanoma and the related implications for immunotherapy.

Summary: Background: Both dabrafenib/trametinib (D/T) and anti-PD-1 monotherapy (PD-1) are approved adjuvant therapies for patients with stage III BRAF V600-mutant melanoma. However, there is still a lack of head-to-head comparative data. We aimed to describe efficacy and toxicity outcomes for these two standard therapies across melanoma centers. Methods: This multicenter, retrospective cohort study was conducted in 15 melanoma centers in Australia, China, Germany, Italy, Japan, UK, and US. We included adult patients with resected stage III BRAF V600-mutant melanoma who received either adjuvant D/T or PD-1 between Jul 2015 and Oct 2022. The primary endpoint was relapse-free survival (RFS). Secondary endpoints included overall survival (OS), recurrence pattern and toxicity. Findings: We included 598 patients with stage III BRAF V600-mutant melanoma who received either adjuvant D/T (n = 393 [66%]) or PD-1 (n = 205 [34%]) post definitive surgery between Jul 2015 and Oct 2022. At a median follow-up of 33 months (IQR 21–43), the median RFS was 51.0 months (95% CI 41.0-not reached [NR]) in the D/T group, significantly longer than PD-1 (44.8 months [95% CI 28.5-NR]) (univariate: HR 0.66, 95% CI 0.50–0.87, P = 0.003; multivariate: HR 0.58, 95% CI 0.39–0.86, P = 0.007), with comparable OS with PD-1 (multivariate, HR 0.90, 95% CI 0.48–1.70, P = 0.75). Similar findings were observed using a restricted-mean-survival-time model. Among those who experienced recurrence, the proportion of distant metastases was higher in the D/T cohort. D/T had a higher incidence of treatment modification due to adverse events (AEs) than PD-1, but fewer persistent AEs. Interpretation: In patients with stage III BRAF V600-mutant melanoma post definitive surgery, D/T yielded better RFS than PD-1, with higher transient but lower persistent toxicity, and comparable OS. D/T seems to provide a better outcome compared with PD-1, but a longer follow-up and ideally a large prospective trial are needed. Funding: Dr. Xue Bai was supported by the Beijing Hospitals Authority Youth Programme (QMS20211101) for her efforts devoted to this study. Dr. Keith T. Flaherty was funded by Adelson Medical Research Foundation for the efforts devoted to this study.

Abstract Background Prognostic factors for initial response of advanced cutaneous squamous cell carcinoma to cemiplimab treatment are lacking. Il-6 has been found to affect immune cell populations which impact tumor development. The aim was to investigate the prognostic significance of IL-6 serum levels before and during treatment. Methods Serum levels of IL-6 were correlated with clinical outcomes in a retrospective study. Results Overall, 39 patients were enrolled. High serum levels of IL-6 (> 5.6 pg/ml) were associated with poorer survival (45.1% vs 0 deaths; OS: 16.1 ± 1.5 vs 20.8 ± 0 months, 95% CI 13,046 to 19,184) and shorter PFS (10.3 ± 1.9 vs 18.9 ± 1.5 months; 95% CI 3433 to 10,133) in patients with advanced CSCC treated with cemiplimab. In addition, patients whose IL-6 level increased after treatment with cemiplimab, independently of the basal level, had a poorer response to treatment than patients whose level was reduced or stable after immunotherapy. Conclusions Serum levels of IL-6 at baseline and changes after cemiplimab immunotherapy may have a prognostic significance in patients with advanced cutaneous squamous cell carcinoma.

Abstract Background The clinical observation showed a potential additive effect of anti-PD-1 agents and cetirizine in patients with advanced melanoma. Methods Clinical outcomes of concomitant cetirizine/anti-PD-1 treatment of patients with stage IIIb–IV melanoma were retrospectively collected, and a transcriptomic analysis was performed on blood samples obtained at baseline and after 3 months of treatment. Results Patients treated with cetirizine concomitantly with an anti-PD-1 agent had significantly longer progression-free survival (PFS; mean PFS: 28 vs 15 months, HR 0.46, 95% CI: 0.28–0.76; p = 0.0023) and OS (mean OS was 36 vs 23 months, HR 0.48, 95% CI: 0.29–0.78; p = 0.0032) in comparison with those not receiving cetirizine. The concomitant treatment was significantly associated with ORR and DCR (p

Checkpoint inhibitors represent an effective treatment approach for a variety of cancers through their inhibition of immune regulatory pathways within the tumor microenvironment (TME). Unfortunately only a minority of patients with cancer achieve clinical benefit from immunotherapy, with the TME emerging as an important predictor of outcomes and sensitivity to therapy. The extent and pattern of T-cell infiltration can vary prominently within/across tumors and represents a biological continuum. Three immune profiles have been identified along this continuum: ‘immune-desert’ or ‘T-cell cold’ phenotype, ‘immune-active’, ‘inflamed’, or ‘T-cell hot’ phenotype, and ‘immune excluded’ phenotype. Of the three profiles, immune excluded remains the most ill-defined with no clear, universally accepted definition even though it is commonly associated with lack of response to immune checkpoint inhibitors and poor clinical outcomes. To address this, 16 multidisciplinary cancer experts from around the world were invited to participate in a symposium using a three-round modified Delphi approach. The first round was an open-ended questionnaire distributed via email and the second was an in-person discussion of the first round results that allowed for statements to be revised as necessary to achieve a maximum consensus (75% agreement) among the rating committee (RC). The final round questionnaire was distributed to the RC via email and had a 100% completion rate. The Delphi process resulted in moving us closer to a consensus definition for immune exclusion that is practical, clinically pertinent, and applicable across a wide range of cancer histologies. A general consensus of the role of immune exclusion in resistance to checkpoint therapy and five research priorities emerged from this process. Together, these tools could help efforts designed to address the underlying mechanisms of immune exclusion that span cancer types and, ultimately, aid in the development of treatments to target these mechanisms to improve patient outcomes.

Abstract Background To evaluate the capability of basal and one-month differed white blood cells (WBC), neutrophil, lymphocyte and platelet values and their ratios (neutrophils-to-lymphocytes ratio, NLR, and platelets-to-lymphocytes ratio, PLR) in predicting the response to immune checkpoint inhibitors (ICI) in metastatic melanoma (MM). Methods We performed a retrospective study of 272 BRAF wild-type MM patients treated with first line ICI. Bivariable analysis was used to correlate patient/tumor characteristics with clinical outcomes. Variations between time 1 and time 0 (Δ) of blood parameters were also calculated and dichotomized using cut-off values assessed by ROC curve. Results At baseline, higher neutrophils and NLR negatively correlated with PFS, OS and disease control rate (DCR). Higher PLR was also associated with worse OS. In multivariable analysis, neutrophils (p = 0.003), WBC (p = 0.069) and LDH (p = 0.07) maintained their impact on PFS, while OS was affected by LDH (p

Abstract Immunotherapy with checkpoint inhibitors (CPIs) strongly improved the outcome of metastatic melanoma patients. However, not all the patients respond to treatment and identification of prognostic biomarkers able to select responding patients is currently of outmost importance. Considering that development of vitiligo-like depigmentation in melanoma patients represents both an adverse event of CPIs and a favorable prognostic factor, we analyzed soluble biomarkers of vitiligo to validate them as early indicators of response to CPIs. Fifty-seven metastatic melanoma patients receiving CPIs were enrolled and divided according to the best overall response to treatment. Patient sera were evaluated at pre-treatment and after 1 and 3 months of therapy. We found that basal CD25 serum levels were higher in stable and responding patients and remained higher during the first 3 months of CPI therapy compared to non-responders. CXCL9 was absent in non-responding patients before therapy beginning. Moreover, an increase of CXCL9 levels was observed at 1 and 3 months of therapy for all patients, although higher CXCL9 amounts were present in stable and responding compared to non-responding patients. Variations in circulating immune cell subsets was also analyzed, revealing a reduced number of regulatory T lymphocytes in responding patients. Altogether, our data indicate that a pre-existing and maintained activation of the immune system could be an indication of response to CPI treatment in melanoma patients.

Background Nivolumab is an anti-PD-1 antibody approved for treating metastatic melanoma (MM), for which still limited evidence is available on the correlation between drug exposure and patient outcomes.Methods In this observational retrospective study, we assessed whether nivolumab concentration is associated with treatment response in 88 patients with MM and if the patient’s genetic profile plays a role in this association.Results We observed a statistically significant correlation between nivolumab serum concentration and clinical outcomes, measured as overall and progression-free survival. Moreover, patients who achieved a clinical or partial response tended to have higher levels of nivolumab than those who reached stable disease or had disease progression. However, the difference was not statistically significant. In particular, patients who reached a clinical response had a significantly higher concentration of nivolumab and presented a distinct genetic signature, with more marked activation of ICOS and other genes involved in effector T-cells mediated proinflammatory pathways.Conclusions In conclusion, these preliminary results show that in patients with MM, nivolumab concentration correlates with clinical outcomes and is associated with an increased expression of ICOS and other genes involved in the activation of T effectors cells.

Melanoma displays a rising incidence, and the mortality associated with metastatic form remains high. Monoclonal antibodies that block programmed death (PD-1) and PD Ligand 1 (PD-L1) network have revolutionized the history of metastatic disease. PD-L1 is expressed on several immune cells and can be also expressed on human neutrophils (PMNs). The role of peripheral blood PMNs as predictive biomarkers in anti-PD-1 therapy of melanoma is largely unknown. In this study, we aimed to determine activation status and PD-L1 expression on human neutrophils as possible novel biomarkers in stage IV melanoma patients (MPs). We found that PMNs from MPs displayed an activated phenotype and increased PD-L1 levels compared to healthy controls (HCs). Patients with lower PD-L1+ PMN frequencies displayed better progression-free survival (PFS) and overall survival (OS) compared to patients with high PD-L1+ PMN frequencies. Multivariate analysis showed that PD-L1+ PMNs predicted patient outcome in BRAF wild type MP subgroup but not in BRAF mutated MPs. PD-L1+ PMN frequency emerges as a novel biomarker in stage IV BRAF wild type MPs undergoing anti-PD-1 immunotherapy. Our findings suggest further evaluation of the role of neutrophil subsets and their mediators in melanoma patients undergoing immunotherapy.

Background Patients with V600BRAF mutant metastatic melanoma have higher rates of progression-free survival (PFS) and overall survival (OS) with first-line anti-PD1 (PD1]+anti-CTLA-4 (IPI) versus PD1. Whether this is also true after BRAF/MEKi therapy is unknown. We aimed to determine the efficacy and safety of PD1 versus IPI +PD1 after BRAF/MEK inhibitors (BRAF/MEKi).Methods Patients with V600BRAF mutant metastatic melanoma treated with BRAF/MEKi who had subsequent PD1 versus IPI+PD1 at eight centers were included. The endpoints were objective response rate (ORR), PFS, OS and safety in each group.Results Of 200 patients with V600E (75%) or non-V600E (25%) mutant metastatic melanoma treated with BRAF/MEKi (median time of treatment 7.6 months; treatment cessation due to progressive disease in 77%), 115 (57.5%) had subsequent PD1 and 85 (42.5%) had IPI+PD1. Differences in patient characteristics between PD1 and IPI+PD1 groups included, age (med. 63 vs 54 years), time between BRAF/MEKi and PD1±IPI (16 vs 4 days), Eastern Cooperative Oncology Group Performance Status (ECOG PS) of ≥1 (62% vs 44%), AJCC M1C/M1D stage (72% vs 94%) and progressing brain metastases at the start of PD1±IPI (34% vs 57%). Median follow-up from PD1±IPI start was 37.8 months (95% CI, 33.9 to 52.9). ORR was 36%; 34% with PD1 vs 39% with IPI+PD1 (p=0.5713). Median PFS was 3.4 months; 3.4 with PD1 vs 3.6 months with IPI+PD1 (p=0.6951). Median OS was 15.4 months; 14.4 for PD1 vs 20.5 months with IPI+PD1 (p=0.2603). The rate of grade 3 or 4 toxicities was higher with IPI+PD1 (31%) vs PD1 (7%). ORR, PFS and OS were numerically higher with IPI+PD1 vs PD1 across most subgroups except for females, those with 3 years OS (area under the curve, AUC=0.74), while ECOG PS ≥1, progressing brain metastases and presence of bone metastases predicted primary progression (AUC=0.67).Conclusions IPI+PD1 and PD1 after BRAF/MEKi have similar outcomes despite worse baseline prognostic features in the IPI+PD1 group, however, IPI+PD1 is more toxic. A combination of clinical factors can identify long-term survivors, but less accurately those with primary resistance to immunotherapy after targeted therapy.

The Sonic hedgehog (Shh) signaling pathway is an essential pathway in the human body that plays an important role in embryogenesis and tissue homeostasis. Aberrant activation of this pathway has been linked to the development of different diseases, ranging from cancer to immune dysregulation and infections.Uncontrolled activation of the pathway through sporadic mutations or other mechanisms is associated with cancer development and progression in various malignancies, such as basal cell carcinoma, medulloblastoma, pancreatic cancer, breast cancer and small-cell lung carcinoma. Targeted inhibition of the pathway components has therefore emerged as an attractive and validated therapeutic strategy for the treatment of a wide range of cancers. Currently, two main components of the pathway, the smoothened receptor and the glioma-associated oncogene homolog transcriptional factors, have been investigated for the development of targeted drugs, leading to the marketing authorization of three smoothened receptor inhibitors for the treatment of basal cell carcinoma and acute myeloid leukemia.The Shh pathway also seems to be involved in regulating the immune response, possibly playing a role in immune system evasions by tumors, development of autoimmune diseases, such as rheumatoid arthritis and Crohn’s disease, airway inflammation, and diseases related to aberrant activation of T-helper 2 cellular response, such as allergy, atopic dermatitis, and asthma.Finally, the Shh pathway is involved in pathogen-mediated infection, including influenza-A and, more recently, SARS-CoV-2 viruses. Therefore, agents that inhibit the Shh signaling pathway might be used to treat pathogenic infections, shifting the therapeutic approach from strain-specific treatments to host-based strategies that target highly conserved host targets.

Abstract Coronavirus disease 2019 (COVID-19) global pandemic has created unique challenges to healthcare systems throughout the world. Ensuring subjects’ safety is mandatory especially in oncology, in consideration of cancer patients’ particular frailty. We examined the proportion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM and/or IgG positive subjects in three different groups from Istituto Nazionale Tumori – IRCCS “Fondazione G. Pascale” in Naples (Campania region, Italy): cancer patients treated with Innovative Immunotherapy (Immune Checkpoint Inhibitors, ICIs), cancer patients undergoing standard Chemotherapies (CHTs) and healthcare providers. 9 out of 287 (3.1%) ICIs patients resulted positive, with a significant lower percentage in respect to CHTs patients (39 positive subjects out of 598, 6.5%) (p = 0.04). There was no statistically significant difference between ICIs cohort and healthcare providers, 48 out of 1050 resulting positive (4.6%). Performing a Propensity Score Matching based on gender and tumor stage, the effect of treatment on seropositivity was analyzed through a regression logistic model and the ICIs treatment resulted to be the only protective factor significantly (p = 0.03) associated with positivity (odds ratio—OR: 0.41; 95% confidence interval—CI 0.18–0.91). According to these preliminary data, ICIs would appear to be a protective factor against the onset of COVID-19 infection.

Abstract Background The incidence of Merkel cell carcinoma (MCC), a rare form of skin cancer with a poor prognosis, has increased in Italy in recent decades. Avelumab, an anti-programmed death ligand 1 monoclonal antibody, is approved for the treatment of metastatic MCC (mMCC) based on the results of the phase 2 JAVELIN Merkel 200 trial. The global avelumab expanded access program (EAP) was designed to provide compassionate use of avelumab prior to approval for patients with mMCC who had limited treatment options. We report findings from a subgroup of Italian patients enrolled in the avelumab EAP. Methods Eligible patients had mMCC and progressive disease following ≥ 1 prior line of chemotherapy or were ineligible for chemotherapy or clinical trial participation. Patients received avelumab 10 mg/kg intravenously every 2 weeks. Treating physicians were provided with an initial 3-month supply of avelumab; resupply was permitted if the patient achieved a complete response, partial response, stable disease, or other clinical benefit per physician assessment. Safety and efficacy data for the EAP were reported at the treating physician’s discretion. Results Between April 1, 2016, and September 14, 2018, 109 requests for avelumab were received from Italy, and 102 were approved. All but 1 of the approved patients had received ≥ 1 prior line of therapy. At data cutoff (March 22, 2019), 95 patients had been supplied with avelumab and response data were available for 55 patients. The objective response rate in response-evaluable patients was 29.1%, including 6 patients (10.9%) who achieved a complete response and 10 patients (18.2%) who achieved a partial response; in the total population supplied with avelumab (n = 95), the proportion who had an objective response was 16.8%. The median duration of treatment in responding patients was 9.7 months (range, 3.5–41.7 months). The most frequently reported treatment-related adverse events were infusion-related reaction (single preferred term; n = 3 [3.2%]) and pyrexia (n = 2 [2.1%]). Conclusions Results from Italian patients enrolled in the avelumab EAP are consistent with the findings of the JAVELIN Merkel 200 trial and confirm the efficacy and safety of avelumab treatment in this population.

Abstract Background Tocilizumab blocks pro-inflammatory activity of interleukin-6 (IL-6), involved in pathogenesis of pneumonia the most frequent cause of death in COVID-19 patients. Methods A multicenter, single-arm, hypothesis-driven trial was planned, according to a phase 2 design, to study the effect of tocilizumab on lethality rates at 14 and 30 days (co-primary endpoints, a priori expected rates being 20 and 35%, respectively). A further prospective cohort of patients, consecutively enrolled after the first cohort was accomplished, was used as a secondary validation dataset. The two cohorts were evaluated jointly in an exploratory multivariable logistic regression model to assess prognostic variables on survival. Results In the primary intention-to-treat (ITT) phase 2 population, 180/301 (59.8%) subjects received tocilizumab, and 67 deaths were observed overall. Lethality rates were equal to 18.4% (97.5% CI: 13.6–24.0, P = 0.52) and 22.4% (97.5% CI: 17.2–28.3, P

The gut microbiota has been reported to regulate the efficacy of cancer therapy. Here, the authors show that short-chain fatty acids, which are generated through bacterial fermentation, increases immune tolerance leading to resistance to anti-CTLA-4 immunotherapy in mice and patients with metastatic melanoma.

Monoclonal antibodies targeting immune checkpoints improved clinical outcome of patients with malignant melanoma. However, the mechanisms are not fully elucidated. Since immune check-point receptors are also expressed by helper innate lymphoid cells (ILCs), we investigated the capability of immune checkpoints inhibitors to modulate ILCs in metastatic melanoma patients as well as melanoma cells effects on ILC functions. Here, we demonstrated that, compared to healthy donors, patients showed a higher frequency of total peripheral ILCs, lower percentages of CD117+ ILC2s and CD117+ ILCs as well as higher frequencies of CD117- ILCs. Functionally, melanoma patients also displayed an impaired TNFα secretion by CD117- ILCs and CD117+ ILCs. Nivolumab therapy reduced the frequency of total peripheral ILCs but increased the percentage of CD117- ILC2s and enhanced the capability of ILC2s and CD117+ ILCs to secrete IL-13 and TNFα, respectively. Before Nivolumab therapy, high CCL2 serum levels were associated with longer Overall Survival and Progression Free Survival. After two months of treatment, CD117- ILC2s frequency as well as serum concentrations of IL-6, CXCL8 and VEGF negatively correlated with both the parameters. Moreover, melanoma cells boosted TNFα production in all ILC subsets and increased the number of IL-13 producing ILC2s in vitro. Our work shows for the first time that PD-1 blockade is able to affect ILCs proportions and functions in melanoma patients and that a specific subpopulation is associated with the therapy response.

Immunotherapy denotes an exemplar change in an oncological setting. Despite the effective application of these treatments across a broad range of tumors, only a minority of patients have beneficial effects. The efficacy of immunotherapy is affected by several factors, including human immunity, which is strongly correlated to genetic features, such as intra-tumor heterogeneity. Classic imaging assessment, based on computed tomography (CT) or magnetic resonance imaging (MRI), which is useful for conventional treatments, has a limited role in immunotherapy. The reason is due to different patterns of response and/or progression during this kind of treatment which differs from those seen during other treatments, such as the possibility to assess the wide spectrum of immunotherapy-correlated toxic effects (ir-AEs) as soon as possible. In addition, considering the unusual response patterns, the limits of conventional response criteria and the necessity of using related immune-response criteria are clear. Radiomics analysis is a recent field of great interest in a radiological setting and recently it has grown the idea that we could identify patients who will be fit for this treatment or who will develop ir-AEs.

As of November 17, 2021, SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2), the causative agent of COVID-19 (COronaVIrus Disease 19), has infected ~250 million people worldwide, causing around five million deaths. Titers of anti-SARS-CoV-2 neutralizing antibodies were relatively stable for at least 9 months in a population-based study conducted in Wuhan, China, both in symptomatic and in asymptomatic individuals. In the mass screening campaign conducted in the town of Ariano Irpino (Avellino, Italy) in May, 2020, 5.7% (95% CI: 5.3-6-1) of the 13,444 asymptomatic citizens screened were positive for anti-nucleocapsid antibodies against SARS-CoV-2. Among these, 422 citizens were re-tested for anti SARS-CoV-2 antibodies in January, 2021 and/or in April, 2021 and enrolled in this longitudinal observational study. Median (interquartile range) age of the study cohort was 46 years (29–59), with 47 (11.1%) participants of minor age, while 217 (51.4%) participants were females. There was no evidence of re-infection in any of the subjects included. Presence of anti-nuclear antibodies antibodies (Elecysis, Roche) was reported in 95.7 and 93.7% of evaluable participants in January and April, 2021. Multiple logistic regression analysis used to explore associations between age, sex and seroprevalence showed that adults vs. minors had significantly lower odds of having anti-S1 antibodies (Biorad) both in January, 2021 and in April, 2021. Our findings showed that antibodies remained detectable at least 11.5 months after infection in >90% of never symptomatic cases. Further investigation is required to establish duration of immunity against SARS-CoV-2.

Immune checkpoint inhibitors (ICIs) are the standard of care for the treatment of several cancers. While these immunotherapies have improved patient outcomes in many clinical settings, they bring accompanying risks of toxicity, specifically immune-related adverse events (irAEs). There is a need for clear, effective guidelines for the management of irAEs during ICI treatment, motivating the Society for Immunotherapy of Cancer (SITC) to convene an expert panel to develop a clinical practice guideline. The panel discussed the recognition and management of single and combination ICI irAEs and ultimately developed evidence- and consensus-based recommendations to assist medical professionals in clinical decision-making and to improve outcomes for patients.

Abstract Background Recently the 8th version of the American Joint Committee on Cancer (AJCC) classification has been introduced, and has attempted to define a more accurate and precise definition of prognosis in line with the major progresses in understanding the biology and pathogenesis of melanoma. This new staging system introduces major changes in the stage III staging system. Indeed, surgical practice is changing in stage III patients, since, according to recent evidence, there is no survival benefit in radical lymph node dissection following a positive sentinel lymph node dissection. Therefore, some patients currently staged IIIB-C after dissection could be downgraded to IIIA (as in the case of patients with metastatic non-sentinel lymph nodes) since many completion lymph node dissections will no longer be performed. Moreover, new and effective targeted and immune strategies are being introduced in the pharmacological armamentarium in the adjuvant setting, showing major efficacy. Conclusions This article provides the authors’ personal view on the above-mentioned topics.

Abstract Introduction Multiple primary melanomas (MPM) occur up to 8% of patients with cutaneous malignant melanoma (CMM). They are often sporadic harbouring several somatic mutations, but also familial cases harbouring a CDKN2A germline mutation have been describe in Caucasian populations. The aim of this study was to investigate the incidence, the distribution patterns and the impact of known and unknown germline and somatic mutations in patients with MPM from Italy. Materials and methods One-hundred and two MPM patients were enrolled for germline mutation analysis, and five patients with at least four MPMs were identified for somatic mutation analysis. The demographic, pathologic and clinical features were retrieved from medical records. Molecular analysis for both germline and somatic mutations was performed in genomic DNA from peripheral blood and tissue samples, respectively, through a next generation sequencing approach, using a specific multiple-gene panel constructed by the Italian Melanoma Intergroup for somatic analysis and a commercial cancer hotspot panel for somatic analysis. Results CDKN2A mutations were detected in 6/16 (37.5%) and 3/86 (3.5%) MPM cases with and without family history for melanoma, respectively. Furthermore, multiple MC1R and, to a lesser extent, ATM variants have been identified. BAP1 variants were found only in MPM patients from southern Italy. The most frequent somatic variants were the pathogenic BRAF V600E and TP53, followed by KIT, PIK3CA, KDR, and NRAS. Single APC, ERBB4, MET, JAK3 and other variants with unknown function were also detected. Conclusions CDNK2A mutation is the most relevant susceptibility mutation in Italian patients with MPM, especially those with a family history for CMM. The prevalence of this mutation and other sequence variants identified in this study varies among specific sub-populations. Furthermore, some heterogeneity in driver somatic mutations between sporadic MPMs has been observed, as well as in a number of associated sequence variants the clinical impact of which needs to be further elucidated.

Abstract Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host’s response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14–15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual’s recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4–5, 2019.

Abstract Background Recent evidence suggested a potential correlation between overweight and the efficacy of immune checkpoint inhibitors (ICIs) in cancer patients. Patients and methods We conducted a retrospective study of advanced cancer patients consecutively treated with anti-PD-1/PD-L1 inhibitors, in order to compare clinical outcomes according to baseline BMI levels as primary analysis. Based on their BMI, patients were categorized into overweight/obese (≥ 25) and non-overweight (

Cancer is the second leading cause of mortality in EU countries, and the needs to tackle cancer are obvious. New scientific understanding, techniques and methodologies are opening up horizons for significant improvements in diagnosis and care. However, take-up is uneven, research needs and potential outstrip currently available resources, manifestly beneficial practices—such as population-level screening for lung cancer—are still not generalised, and the quality of life of patients and survivors is only beginning to be given attention it merits. This paper, mainly based on a series of multistakeholder expert workshops organised by the European Alliance for Personalised Medicine (EAPM), looks at some of those specifics in the interest of planning a way forward. Part of this exercise also involves taking account of the specific nature of Europe and its constituent countries, where the complexities of planning a way forward are redoubled by the wide variations in national and regional approaches to cancer, local epidemiology and the wide disparities in health systems. Despite all the differences between cancers and national and regional resources and approaches to cancer care, there is a common objective in pursuing broader and more equal access to the best available care for all European citizens.

The Italian municipality of Ariano Irpino (Avellino, Campania, Italy) was locked down by the regional authorities from March until April 2020 after several citizens tested positive for SARS coronavirus 2 (SARS-CoV-2). A serological mass screening campaign targeting the Ariano Irpino population using the Roche Cobas Elecsys anti-SARS-CoV-2 assay was organized by the Zoo-Prophylactic Institute of Southern Italy (Portici, Italy) and conducted in cooperation with the Local Health Unit (Azienda Sanitaria Locale – ASL – Avellino, Avellino, Italy), the Department of Public Health of University Federico II (Naples, Italy) and Department of Health Services of Azienda Ospedaliera dei Colli-Cotugno and Monaldi Hospital (Naples, Italy) in May 2020. A total of 13,218 asymptomatic individuals were reviewed in this analysis. A total of 738 citizens tested positive for anti-SARS-CoV-2 antibodies (398 females, 340 males). The overall prevalence in the sample was 5.6% (95% CI: 5.2–6.0). Among seropositive citizens, 101 cases tested positive on RT-PCR (0.76% of the overall population). Among citizens aged 14–18, 18–65 and >65 years, the seroprevalence was equal to 6.1 (95% CI: 4.1–8.7), 5.6 (95% CI: 5.1–6.1) and 4% (95% CI: 3.3–4.8), respectively. In the pediatric cohort (

Background In ambulatory patients with cancer with asymptomatic or pauci-symptomatic SARS-CoV-2 infection, the safety of targeted therapies (TTs), chemotherapy (CT) or immune checkpoint inhibitors (ICIs) therapy is still unknown.Material and methods From the start of the first epidemic wave of SARS-CoV-2 in Bergamo, Italy, we have prospectively screened all consecutive outpatients who presented for treatment to the Oncology Division of the Papa Giovanni XXIII Hospital, Bergamo for SARS-CoV-2 antigen expression. We identified patients treated with ICIs and compared these to patients with the same cancer subtypes treated with TTs or CT.Results Between March 5 and May 18, 293 consecutive patients (49% melanoma, 34% non-small cell lung cancer, 9% renal cell carcinoma, 8% other) were included in this study: 159 (54%), 50 (17%) and 84 (29%) received ICIs, CT or TTs, respectively. Overall 89 patients (30.0%) were SARS-CoV-2 positive. Mortality of SARS-CoV-2-positive patients was statistically significantly higher compared with SARS-CoV-2 negative patients (8/89 vs 3/204, respectively, Fisher’s exact test p=0.004). All deaths were due to COVID-19. Serious adverse events (SAEs) were more frequent in SARS-CoV-2-positive patients compared with SARS-CoV-2-negative cases (Cochran-Mantel-Haenszel (CMH) test p=0.0008). The incidence of SAEs in SARS-CoV-2 positive compared with SARS-CoV-2 negative patients was similar in ICI and CT patients (17.3% and 3.7% for positive and negative patients in ICIs and 15.4% and 2.7% in CT, Breslow-Day test p=0.891). No COVID-19-related SAEs were observed in the TTs patients.Conclusions The incidence of SAEs was higher for SARS-CoV-2-positive patients treated with ICIs and CT, mostly in advanced disease. No SAEs were observed in patients treated with TTs. SAEs were COVID-19 related rather than treatment related. Treatment with ICIs does not appear to significantly increase risk of SAEs compared with CT. This information should be considered when determining treatment options for patients.

Background Patients with cancer who are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to develop severe illness and die compared with those without cancer. The impact of immune checkpoint inhibition (ICI) on the severity of COVID-19 illness is unknown. The aim of this study was to investigate whether ICI confers an additional risk for severe COVID-19 in patients with cancer.Methods We analyzed data from 110 patients with laboratory-confirmed SARS-CoV-2 while on treatment with ICI without chemotherapy in 19 hospitals in North America, Europe and Australia. The primary objective was to describe the clinical course and to identify factors associated with hospital and intensive care (ICU) admission and mortality.Findings Thirty-five (32%) patients were admitted to hospital and 18 (16%) died. All patients who died had advanced cancer, and only four were admitted to ICU. COVID-19 was the primary cause of death in 8 (7%) patients. Factors independently associated with an increased risk for hospital admission were ECOG ≥2 (OR 39.25, 95% CI 4.17 to 369.2, p=0.0013), treatment with combination ICI (OR 5.68, 95% CI 1.58 to 20.36, p=0.0273) and presence of COVID-19 symptoms (OR 5.30, 95% CI 1.57 to 17.89, p=0.0073). Seventy-six (73%) patients interrupted ICI due to SARS-CoV-2 infection, 43 (57%) of whom had resumed at data cut-off.Interpretation COVID-19–related mortality in the ICI-treated population does not appear to be higher than previously published mortality rates for patients with cancer. Inpatient mortality of patients with cancer treated with ICI was high in comparison with previously reported rates for hospitalized patients with cancer and was due to COVID-19 in almost half of the cases. We identified factors associated with adverse outcomes in ICI-treated patients with COVID-19.

Objectives: The aim of this study was to estimate the total annual direct costs incurred by the National Health Service for the management and treatment of CSCC and advanced CSCC patients in Italy. Methods: An incidence-based cost of illness (COI) model was developed to estimate direct costs associated with the treatment and management of CSCC patients in Italy. The identified treatment pathway was validated with a team of clinical experts and was distinguished between resectable CSCC and locally advanced CSCC or metastatic CSCC. Treatments costs were obtained through the analysis of the National Hospital Discharge Database (HDD) for the years 2015-2018; monitoring and terminal care costs were obtained from national tariffs of outpatient care service and from the literature respectively. Results: Associating the estimated costs for each phase of the treatment pathway with the proportion of patients present in each phase, the COI model estimated an annual economic burden of about € 25.9 million for the management and treatment of patients with CSCC in Italy, € 2.7 million of which were associated to patients with advanced CSCC. The average cost per patient with advanced CSCC was higher compared to that of patient with resectable CSCC (€ 4,490 vs € 2,236 respectively). Conclusions: Our analysis showed that advanced CSCC patients are associated with a higher average cost than patients with resectable CSCC.

Background In the KEYNOTE-022 study, pembrolizumab with dabrafenib and trametinib (triplet) improved progression-free survival (PFS) versus placebo with dabrafenib and trametinib (doublet) without reaching statistical significance. Mature results on PFS, duration of response (DOR), and overall survival (OS) are reported.Methods The double-blind, phase 2 part of KEYNOTE-022 enrolled patients with previously untreated BRAFV600E/K-mutated advanced melanoma from 22 sites in seven countries. Patients were randomly assigned 1:1 to intravenous pembrolizumab (200 mg every 3 weeks) or placebo plus dabrafenib (150 mg orally two times per day) and trametinib (2 mg orally one time a day). Primary endpoint was PFS. Secondary endpoints were objective response rate, DOR, and OS. Efficacy was assessed in the intention-to-treat population, and safety was assessed in all patients who received at least one dose of study drug. This analysis was not specified in the protocol.Results Between November 30, 2015 and April 24, 2017, 120 patients were randomly assigned to triplet (n=60) or doublet (n=60) therapy. With 36.6 months of follow-up, median PFS was 16.9 months (95% CI 11.3 to 27.9) with triplet and 10.7 months (95% CI 7.2 to 16.8) with doublet (HR 0.53; 95% CI 0.34 to 0.83). With triplet and doublet, respectively, PFS at 24 months was 41.0% (95% CI 27.4% to 54.2%) and 16.3% (95% CI 8.1% to 27.1%); median DOR was 25.1 months (95% CI 14.1 to not reached) and 12.1 months (95% CI 6.0 to 15.7), respectively. Median OS was not reached with triplet and was 26.3 months with doublet (HR 0.64; 95% CI 0.38 to 1.06). With triplet and doublet, respectively, OS at 24 months was 63.0% (95% CI 49.4% to 73.9%) and 51.7% (95% CI 38.4% to 63.4%). Grade 3–5 treatment-related adverse events (TRAEs) occurred in 35 patients (58%, including one death) receiving triplet and 15 patients (25%) receiving doublet.Conclusion In BRAFV600E/K-mutant advanced melanoma, pembrolizumab plus dabrafenib and trametinib substantially improved PFS, DOR, and OS with a higher incidence of TRAEs. Interpretation of these results is limited by the post hoc nature of the analysis.

BackgroundWe performed a multicenter retrospective observational study to investigate the impact of clinical–pathological features and therapeutic strategies on both the complications and survival of patients with bone metastases (BMs) from malignant melanoma.Patients and MethodsA total of 305 patients with melanoma and radiological evidence of BMs were retrospectively enrolled from 19 Italian centers. All patients received conventional treatments in accordance with each own treating physician’s practice. Both univariate and multivariate models were used to explore the impact of melanoma features, including skeletal-related events (SREs), and different treatments on both overall survival (OS) and time-to-SREs. The chi-squared test evaluated the suitability of several parameters to predict the occurrence of SREs.ResultsEighty-three percent of patients had metachronous BMs. The prevalent (90%) bone metastatic site was the spine, while 45% had involvement of the appendicular skeleton. Forty-seven percent experienced at least one SRE, including palliative radiotherapy (RT) in 37% of cases. No melanoma-associated factor was predictive of the development of SREs, although patients receiving early treatment with bone-targeted agents showed 62% lower risk and delayed time of SRE occurrence. Median OS from the diagnosis of bone metastasis was 10.7 months. The multivariate analysis revealed as independent prognostic factors the number of BMs, number of metastatic organs, baseline lactate dehydrogenase levels, and treatment with targeted therapy or immunotherapy. Subgroup analyses showed the best OS (median = 16.5 months) in the subset of patients receiving both immunotherapy and palliative RT.ConclusionBased on our results, patients undergoing immunotherapy and palliative RT showed an OS benefit suggestive of a possible additive effect. The apparent protective role of bone targeting agent use on SREs observed in our analysis should deserve prospective evaluation.

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.

Colorectal cancer (CRC) is a high incidence disease, characterized by high morbidity and mortality rates. Early diagnosis remains challenging because fecal occult blood screening tests have performed sub-optimally, especially due to hemorrhoidal, inflammatory, and vascular diseases, while colonoscopy is invasive and requires a medical setting to be performed. The objective of the present study was to determine if serum metabolomic profiles could be used to develop a novel screening approach for colorectal cancer. Furthermore, the study evaluated the metabolic alterations associated with the disease. Untargeted serum metabolomic profiles were collected from 100 CRC subjects, 50 healthy controls, and 50 individuals with benign colorectal disease. Different machine learning models, as well as an ensemble model based on a voting scheme, were built to discern CRC patients from CTRLs. The ensemble model correctly classified all CRC and CTRL subjects (accuracy = 100%) using a random subset of the cohort as a test set. Relevant metabolites were examined in a metabolite-set enrichment analysis, revealing differences in patients and controls primarily associated with cell glucose metabolism. These results support a potential use of the metabolomic signature as a non-invasive screening tool for CRC. Moreover, metabolic pathway analysis can provide valuable information to enhance understanding of the pathophysiological mechanisms underlying cancer. Further studies with larger cohorts, including blind trials, could potentially validate the reported results.

Abstract 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

Abstract Background Accumulating evidence demonstrates that the Urokinase Receptor (uPAR) regulates tumor cell migration through its assembly in composite regulatory units with transmembrane receptors, and uPAR88–92 is the minimal sequence required to induce cell motility through the Formyl Peptide Receptor type 1 (FPR1). Both uPAR and FPR1 are involved in melanoma tumor progression, suggesting that they may be targeted for therapeutic purposes. In this study, the role of the uPAR-FPR1 cross-talk to sustain melanoma cell ability to invade extracellular matrix and cross endothelial barriers is investigated. Also, the possibility that inhibition of the uPAR mediated FPR1-dependent signaling may prevent matrix invasion and transendothelial migration of melanoma cells was investigated. Methods Expression levels of uPAR and FPR1 were assessed by immunocytochemistry, Western Blot and qRT-PCR. Cell migration was investigated by Boyden chamber and wound-healing assays. Migration and invasion kinetics, trans-endothelial migration and proliferation of melanoma cells were monitored in real time using the xCELLigence technology. The agonist-triggered FPR1 internalization was visualized by confocal microscope. Cell adhesion to endothelium was determined by fluorometer measurement of cell-associated fluorescence or identified on multiple z-series by laser confocal microscopy. The 3D–organotypic models were set up by seeding melanoma cells onto collagen I matrices embedded dermal fibroblasts. Data were analyzed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons. Results We found that the co-expression of uPAR and FPR1 confers to A375 and M14 melanoma cells a clear-cut capability to move towards chemotactic gradients, to cross extracellular matrix and endothelial monolayers. FPR1 activity is required, as cell migration and invasion were abrogated by receptor desensitization. Finally, melanoma cell ability to move toward chemotactic gradients, invade matrigel or fibroblast-embedded collagen matrices and cross endothelial monolayers are prevented by anti-uPAR84–95 antibodies or by the RI-3 peptide which we have previously shown to inhibit the uPAR84–95/FPR1 interaction. Conclusions Collectively, our findings identify uPAR and FPR1 as relevant effectors of melanoma cell invasiveness and suggest that inhibitors of the uPAR84–95/FPR1 cross-talk may be useful for the treatment of metastatic melanoma.