Association between PD-1 single nucleotide gene variants and the risk of metastatic melanoma.

Previous studies showed an association between single nucleotide gene variants (SNVs) of PD-1 and cancer susceptibility. We analyzed PD1.5 C > T and PD1.7 T > C SNVs to investigate their association with the risk of developing metastatic melanoma (MM). Utilizing a cohort of 125 MM patients treated with anti-PD-1 agents and 84 healthy controls, we examined genotype/allele frequencies through a modified Poisson regression model, adjusted for age and sex. Our findings indicate that the PD1.5 T allele is associated with a reduced risk of MM, showing a significantly lower risk in both codominant (RR = 0.56, 95%CL: 0.37-0.87) and dominant (RR = 0.73 95%CL: 0.59-0.90) models. Conversely, the PD1.7 C allele is linked to an increased risk of MM, with the C/C genotype exhibiting a higher risk in the codominant (RR = 1.65, 95%CL: 1.32-2.05) and allelic (RR = 1.23, 95%CL: 1.06-1.43) models. These results are consistent with previous meta-analyses on other cancer types, mainly highlighting the PD1.5 SNV's potential role in promoting anti-tumor immunity through increased PD1-positive circulating effector T cell activity.

Machine Learning Methods for Gene Selection in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy with a limited five-year survival for metastatic patients. Limited therapeutic treatments are currently available for metastatic disease, even if the genomics of this tumor has been deeply studied using next-generation sequencing (NGS) and functional experiments. The profound knowledge of the molecular features that characterize this tumor has not led to the development of efficacious therapies, and the survival of metastatic patients has not changed for decades. Several bioinformatics methods have been applied to mine NGS tumor data in order to unveil tumor biology and detect possible molecular targets for new therapies. Each application can be single domain based while others are more focused on data integration from multiple genomics domains (as gene expression and methylation data). Examples of single domain approaches include differentially expressed gene (DEG) analysis on gene expression data with statistical methods such as SAM (significance analysis of microarray) or gene prioritization with complex algorithms such as deep learning. Data fusion or integration methods merge multiple domains of information to define new clusters of patients or to detect relevant genes, according to multiple NGS data. In this work, we compare different strategies to detect relevant genes for metastatic disease prediction in the TCGA uveal melanoma (UVM) dataset. Detected targets are validated with multi-gene score analysis on a larger UM microarray dataset.

Characterization of soluble PD-L1 in pleural effusions of mesothelioma patients: potential implications in the immune response and prognosis.

PURPOSE: Programmed death-ligand 1 (PD-L1) protein plays a central role in the antitumor immune response, and appears to be a predictor of prognosis and efficacy for PD-L1 and programmed death 1 (PD-1) blockade therapy. The immunoregulatory role and prognostic impact of PD-L1 soluble form (sPD-L1) have been investigated in biological fluids of patients with different tumors. In malignant pleural mesothelioma (MPM), circulating sPD-L1 has been recently reported in patients' sera, but no data are available in pleural effusions (PE). In our study, we evaluated the baseline expression levels of sPD-L1 in PE from 84 MPM patients and correlated them with PD-L1-status in matched tumors and patients' overall survival (OS). METHODS: sPD-L1 in PE was determined by ELISA and tumor PD-L1 by immunohistochemistry. Association of sPD-L1 with OS was estimated using the Cox regression model. RESULTS: We observed that sPD-L1 was variably expressed in all the PE and tended to be higher (by 30%) in patients with PD-L1-positive tumors (cut-off ≥ 1% stained cells) as compared to patients with PD-L1-negative tumors (geometric mean ratio = 1.28, P value = 0.288). sPD-L1 levels were significantly higher than those of sPD-1 (P value = 0.001) regardless of the MPM histotypes and they were positively correlated (r = 0.50, P value < 0.001). Moreover, high PE sPD-L1 concentrations were associated with a trend towards increased OS (hazard ratio 0.79, 95% CL 0.62-1.01, P value = 0.062). CONCLUSIONS: Our study documents the presence of sPD-L1 in PE of MPM patients, and suggests its possible biological and prognostic role in MPM.

Phenotypic characterization of tumor CTLA-4 expression in melanoma tissues and its possible role in clinical response to Ipilimumab.

The expression of the immune checkpoint molecule CTLA-4 has been almost exclusively studied in the T cell lineage, but increasing evidence has shown its expression on tumors with implications for immunotherapy. To date, the degree of expression of CTLA-4 on tumor cells as a predictive biomarker of response to immune checkpoint inhibitors has not been studied. In this report, we analyzed this issue in melanoma patients treated with CTLA-4 inhibitor Ipilimumab (IPI). We show that the level of CTLA-4 expression on melanoma cells is higher than that on tumor infiltrating lymphocytes (TIL) and it is associated with clinical response to IPI therapy supporting the idea of its possible role as a predictive biomarker.

Response to ipilimumab therapy in metastatic melanoma patients: potential relevance of CTLA-4+ tumor infiltrating lymphocytes and their in situ localization.

Immune checkpoint inhibitors, including ipilimumab (IPI), achieve a clinical benefit in a small proportion of melanoma patients highlighting the need to investigate predictive biomarkers. In this study, we characterized tumor infiltrating lymphocytes (TILs), focusing on the CTLA-4+ subset, and evaluated their possible predictive significance. We characterized TIL density, cell type, and localization in 40 melanoma lesions from 17 patients treated with IPI. Associations of TILs with IPI timing, tissue localization, and response to IPI were estimated using a linear mixed-effects modelling approach. We found that most of TIL subsets increased in situ upon IPI therapy, with particular reference to FoxP3+ cells. TILs and TIL subsets, such as CD3+, CD45RO+, CTLA-4+, CD4+, CD8+ T cells, CD20+ B cells, and NKp46+ NK cells, showed significantly different spatial distributions in the tumor microenvironment being higher at the invasive margin (IM) as compared to the tumor center (TC) (P value < 0.001 for TIL score and P value < 0.05 for all subsets). Remarkably, high TIL score and density of CD3+, CD8+ T cells, and CTLA-4+ immune cells were significantly associated with a better response to IPI (P values = 0.002, 0.023, 0.007, and 0.001, respectively, for responders vs non-responders). In conclusion, we provide a detailed analysis of CTLA-4+ TIL distribution in melanoma tissues taking into account localization, relationship with CD3+/CD8+ TILs, and changes in response to IPI treatment. We identified that CTLA-4+ TILs may represent a marker of IPI response, alone or with CD3+/CD8+ subsets, although this requires confirmation in larger studies.

Soluble CTLA-4 as a favorable predictive biomarker in metastatic melanoma patients treated with ipilimumab: an Italian melanoma intergroup study.

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.

Immune Checkpoints and Innovative Therapies in Glioblastoma.

Targeting the Immune Checkpoint molecules (ICs; CTLA-4, PD-1, PD-L1/2, and others) which provide inhibitory signals to T cells, dramatically improves survival in hard-to-treat tumors. The establishment of an immunosuppressive environment prevents endogenous immune response in glioblastoma; therefore, manipulating the host immune system seems a reasonable strategy also for this tumor. In glioma patients the accumulation of CD4+/CD8+ T cells and Treg expressing high levels of CTLA-4 and PD-1, or the high expression of PD-L1 in glioma cells correlates with WHO high grade and short survival. Few clinical studies with IC inhibitors (ICis) were completed so far. Notably, the first large-scale randomized trial (NCT 02017717) that compared PD-1 blockade and anti-VEGF, did not show an OS increase in the patients treated with anti-PD-1. Several factors could have contributed to the failure of this trial and must be considered to design further clinical studies. In particular the possibility of targeting at the same time different ICs was pre-clinically tested in an animal model were inhibitors against IDO, CTLA-4 and PD-L1 were combined and showed persistent and significant antitumor effects in glioma-bearing mice. It is reasonable to hypothesize that the immunological characterization of the tumor in terms of type and level of expressed IC molecules on the tumor and TIL may be useful to design the optimal ICi combination for a given subset of tumor to overcome the immunosuppressive milieu of glioblastoma and to efficiently target a tumor with such high cellular complexity.

Corrigendum: Association of CTLA-4 Gene Variants with Response to Therapy and Long-term Survival in Metastatic Melanoma Patients Treated with Ipilimumab: An Italian Melanoma Intergroup Study.

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

MicroRNA in Glioblastoma: An Overview.

Glioblastoma is the most aggressive brain tumor and, even with the current multimodal therapy, is an invariably lethal cancer with a life expectancy that depends on the tumor subtype but, even in the most favorable cases, rarely exceeds 2 years. Epigenetic factors play an important role in gliomagenesis, are strong predictors of outcome, and are important determinants for the resistance to radio- and chemotherapy. The latest addition to the epigenetic machinery is the noncoding RNA (ncRNA), that is, RNA molecules that are not translated into a protein and that exert their function by base pairing with other nucleic acids in a reversible and nonmutational mode. MicroRNAs (miRNA) are a class of ncRNA of about 22 bp that regulate gene expression by binding to complementary sequences in the mRNA and silence its translation into proteins. MicroRNAs reversibly regulate transcription through nonmutational mechanisms; accordingly, they can be considered as epigenetic effectors. In this review, we will discuss the role of miRNA in glioma focusing on their role in drug resistance and on their potential applications in the therapy of this tumor.

Association of CTLA-4 Gene Variants with Response to Therapy and Long-term Survival in Metastatic Melanoma Patients Treated with Ipilimumab: An Italian Melanoma Intergroup Study.

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.

Comparison of the Diagnostic Performance of Fibulin-3 and Mesothelin in Patients with Pleural Effusions from Malignant Mesothelioma.

BACKGROUND: In the literature, there exist conflicting data on the value of fibulin-3 (FBLN3) for the diagnosis of pleural effusion (PE) in malignant pleural mesothelioma (MPM). Therefore we compared the diagnostic performance of FBLN3 against that of soluble mesothelin-related peptide (SMRP) in a cohort of Italian patients. MATERIALS AND METHODS: FBLN3 and SMRP were detected in PE from 33 patients with MPM, 64 with pleural benign lesions and 23 with non-MPM pleural metastases using a commercial enzyme-linked-immunosorbent(ELISA)-assay kit according to manufacturers' instructions. RESULTS: Levels of FBLN3 were similar in PE from MPM and PE from other pathologies (geometric mean=68.1 vs. 66.2 ng/ml; p=0.872) in contrast to SMRP levels, which were significantly higher in PE from MPM (geometric mean=14.6 vs. 3.2 nM; p<0.001). Receiver operating characteristic analysis confirmed that SMRP showed a good performance (area under the curve=0.79, p<0.001), whereas FBLN3 was not able to discriminate MPM from other pathologies (area under the curve=0.44, p=0.838). CONCLUSION: FBLN3 detection in PE, in contrast to SMRP detection, is not useful as a biomarker for the diagnosis of PE from MPM.

Analysis of the Expression and Single-Nucleotide Variant Frequencies of the Butyrophilin-like 2 Gene in Patients With Uveal Melanoma.

IMPORTANCE: Chromosome 6p amplification is associated with more benign behavior for uveal melanomas (UMs) with an otherwise high risk of metastasis conferred by chromosome 3 monosomy. Chromosome 6p contains several members of the B7 family of immune regulator genes, including butyrophilin-like 2 (BTNL2; OMIM, 606000), which is associated with prostate cancer risk and autoimmune diseases. OBJECTIVE: To investigate the expression and variant allele frequencies of BTNL2, a candidate gene for chromosome 6 amplification, in patients with UM. DESIGN, SETTING, AND PARTICIPANTS: In this case-control study, we analyzed the expression of BTNL2 in UM cell lines and human macrophages in patients with UM. Variants of BTNL2 were analyzed using probes for polymerase chain reaction and high-resolution melting. The association of missense variants rs28362679 and rs41441651 with tumor risk was analyzed in 209 patients with UM and 116 matched control patients as well as 12 UM and 64 other tumor cell lines. Genes that were differentially expressed in M1- and M2-polarized macrophages were identified by microarray analysis of 111 patients with UM, and the association of the expression of these genes with disease-free survival was analyzed by Cox regression analysis. Data were collected from September 2013 to November 2015. MAIN OUTCOMES AND MEASURES: Butyrophilin-like 2 single-nucleotide variants were associated with UM risk; M1 and M2 macrophage-specific gene expression was associated with disease-free survival. RESULTS: We genotyped a total of 325 patients. Of the 209 patients with UM, 124 (59.3%) were male, 114 (54.5%) were Italian, and 95 (45.5%) were German; the mean (range) age was 65 (27-94) years. Of the 116 Italian control patients, 67 (57.8%) were female, and the mean (range) age was 39 (21-88) years. Butyrophilin-like 2 is expressed in patients with UM and macrophages. The frequency of the rs28362679 variant was higher in patients with UM (16 of 209 [7.7%]; 95% CI, 4.7-12.2) than frequencies from European Variation Archive and Exome Aggregation Consortium data (2134 of 118 564 [1.8%]; 95% CI, 1.7-1.9) and Exome Sequencing Project data (100 of 4540 [2.2%]; 95% CI, 1.8-2.7) but were not higher compared with Italian control patients (10 of 116 [8.6%]; 95% CI, 4.6-15.4). The rs41441651 variant was present in 5 patients with UM (2.4%; 95% CI, 0.9-5.7), 2 Italian control patients (1.7%; 95% CI, 0.1-6.5), 2846 patients from European Variation Archive and Exome Aggregation Consortium data (2.4%; 95% CI, 2.3-2.5), and 23 patients from Exome Sequencing Project data (0.5%; 95% CI, 0.3-0.8). Human UM cells express M1 and M2 macrophage-specific genes, whose expression is associated with disease-free survival. CONCLUSIONS AND RELEVANCE: Butyrophilin-like 2, expressed at various levels by UM cells and macrophages, might interfere with the immune control of the tumor. Butyrophilin-like 2 variants showed highly variable frequencies among ethnically related cohorts. There was no enrichment of BTNL2 variants in patients with UM compared with control patients.

CTLA-4 in mesothelioma patients: tissue expression, body fluid levels and possible relevance as a prognostic factor.

CTLA-4 function as a negative regulator of T cell-mediated immune response is well established, whereas much less is known about the immunoregulatory role of its soluble isoform (sCTLA-4). No data are available on CTLA-4 expression and prognostic impact in malignant pleural mesothelioma (MPM). We investigated, by immunohistochemistry, CTLA-4 expression in tumor tissues and, by ELISA, sCTLA-4 levels in sera and matched pleural effusions from 45 MPM patients. Prognostic effect of CTLA-4 expression on overall survival (OS) was assessed through Cox regression and prognostic significance expressed as death rate ratio (HR). We found that 56.0 % of MPM tissues expressed CTLA-4 with variable intensity and percentage of positive cells estimated by the immunoreactive score. sCTLA-4 levels were significantly higher in sera (S-sCTLA-4) than in pleural effusions (PE-sCTLA-4) (geometric mean ratio = 2.70, P value = 0.020). CTLA-4 expression at the tissue level was higher in the epithelioid histological subtype than in the sarcomatoid, whereas at the serum level, it was higher in the sarcomatoid subtype. A homogeneous favorable prognostic effect was found for CTLA-4 overexpression in tissue, serum and pleural effusion. Interestingly, only the PE-sCTLA-4 was found to be a statistically significant positive prognostic factor (HR = 0.37, 95 % CI = 0.18-0.77, P value = 0.007). Indeed, PE-sCTLA-4 correlated with CTLA-4 expression in tissues, whereas this latter expression showed a weak association with OS. To confirm our findings, further experimental evidences obtained from a larger cohort of MPM patients are required. However, our results would indicate a positive correlation of PE-sCTLA-4 levels and OS in MPM patients.

Analysis of in vitro ADCC and clinical response to trastuzumab: possible relevance of FcγRIIIA/FcγRIIA gene polymorphisms and HER-2 expression levels on breast cancer cell lines.

BACKGROUND: Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab. PATIENTS AND METHODS: Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients' peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by (51)Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab. RESULTS: We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006). CONCLUSIONS: Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory.

A novel multiplex pyrosequencing assay for genotyping functionally relevant CTLA-4 polymorphisms: potential applications in autoimmunity and cancer.

CTLA-4 expression/function can be affected by single nucleotide polymorphisms (SNPs) of CTLA-4 gene, which have been widely associated with susceptibility or progression to autoimmune diseases and cancer development. In this study, we analyzed six CTLA-4 SNPs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, CT60G>A) in 197 DNA samples from 43 B-lymphoblastoid cell lines (B-LCLs), 40 systemic sclerosis (SSc) patients, 14 pre-analyzed melanoma patients and 100 Italian healthy subjects. Genotyping of -1661A>G, -1577G>A, -658C>T and CT60G>A was performed by newly developed multiplex pyrosequencing (PSQ) assays, whereas -319C>T and +49A>G by T-ARMS PCR and direct sequencing. Genotype/allele frequency were analyzed using χ(2) or Fisher exact test. Our study provides the first multiplex PSQ method that allows simultaneous genotyping of two CTLA-4 SNP pairs (i.e. -1661A>G/-658C>T and -1577G>A/CT60G>A) by two multiplex PSQ reactions. Herein, we show the CTLA-4 genotype distribution in the B-LCLs providing the first and best characterized cell line panel typed for functionally relevant CTLA-4 SNPs. We also report the significant association of the -1661A/G genotype, -1661 & -319 AC-GT diplotype and -319 & CT60 TG haplotype with susceptibility to SSc without Hashimoto's thyroiditis occurrence. Furthermore, we confirmed previous genotyping data referred to melanoma patients and provided new genotyping data for Italian healthy subjects.

Mesothelin is more useful in pleural effusion than in serum in the diagnosis of pleural mesothelioma.

BACKGROUND/AIM: Soluble mesothelin-related peptide (SMRP) is regarded as a biomarker of malignant pleural mesothelioma (MPM). Herein, we compared the diagnostic performances of SMRP in matched pleural effusion (PE-SMRP) and serum (S-SMRP). MATERIALS AND METHODS: Diagnosis on pleural biopsies was performed for all patients including 43 with MPM, 23 with non-MPM pleural metastases (MTS) and 36 with benign (BNG) pleural diseases. SMRP was measured by a MesoMark ELISA (Cis-Bio International Gif/Yvette; France). RESULTS: Using the receiver operating characteristic (ROC) analysis, 12.70 and 1.08 nM were detected as cut-off values to optimal discrimination for PE-SMRP and S-SMRP, respectively. PE-SMRP showed a better diagnostic accuracy than S-SMRP in MPM vs. MTS+BNG (area under the ROC curve=81.6 vs. 70.5; sensitivity=69.8% vs. 46.5%; specificity=88.1% vs. 84.7%; diagnostic odds ratio (DOR)=17.1 vs. 4.8). In S-SMRP-negative patients, PE-SMRP maintained an acceptable performance (Sensitivity=47.8%; DOR=8.3; p=0.001), whereas in PE-SMRP-negative patients, S-SMRP performed very poorly (Sensitivity=15.4%; DOR=1.2; p=0.858). CONCLUSION: PE-SMRP detection has a superior diagnostic accuracy than S-SMRP detection in the diagnosis of MPM.

The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.

BACKGROUND: CTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma. METHODS: CTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student's t-test. RESULTS: All melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity.Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts. CONCLUSIONS: Our studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.