Journal Articles

Cancer Immunobiology

Basic research studies that extend knowledge in the field of cancer immunotherapy

Soluble SLAMF6 receptor induces strong CD8+ T cell effector function and improves anti-melanoma activity in vivo

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SLAMF6, a member of the SLAM (signaling lymphocyte activation molecules) family, is a homotypic-binding immune receptor expressed on NK, T, and B lymphocytes. Phosphorylation variance between T-cell subclones prompted us to explore its role in anti-melanoma immunity. Using a 203-amino acid sequence of the human SLAMF6 (seSLAMF6) ectodomain, we found that seSLAMF6 reduced activation-induced cell death and had an anti-apoptotic effect on tumor infiltrating lymphocytes. CD8+ T cells costimulated with seSLAMF6 secreted more interferon gamma and displayed augmented cytolytic activity. The systemic administration of seSLAMF6 to mice sustained adoptively transferred transgenic CD8+ T cells in comparable numbers to high doses of interleukin-2. In a therapeutic model, lymphocytes activated by seSLAMF6 delayed tumor growth, and when further supported in vivo with seSLAMF6, induced complete tumor clearance. The ectodomain expedites the loss of phosphorylation on SLAMF6 that occurs in response to T-cell receptor triggering. Our findings suggest that seSLAMF6 is a costimulator that could be used in melanoma immunotherapy.

Author Info: (1) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital galiteis@gmail.com. (2) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (3) Sharrett Institute of Oncology, Hadassah

Author Info: (1) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital galiteis@gmail.com. (2) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (3) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (4) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (5) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (6) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (7) Weizmann Institute of Science. (8) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (9) Compugen Ltd. (10) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (11) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital. (12) Sharrett Institute of Oncology, Hadassah Hebrew University Hospital.

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High macrophage PD-L1 expression not responsible for T cell suppression

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Tumors are often comprised of microenvironments (TMEs) with a high proportion of cells and molecules that regulate immunity. Peritoneal cavity (PerC) cell culture reproduces key features of TMEs as lymphocyte proliferation is suppressed by PerC macrophages (Mvarphis). We monitored the expression of T cell stimulatory (Class II MHC, B7) and inhibitory (PD-L1) molecules by PerC APCs before and after culture and report here that IFNgamma-driven PD-L1 expression increased markedly on PerC Mvarphis after TCR ligation, even more so than seen with direct APC activation by LPS. Considering the high APC composition of and pronounced PD-L1 expression by PerC cells, it was surprising that blocking PD-1/PD-L1 interaction by mAb neutralization or genetic ablation did not relieve suppression. This result parallels TME challenges observed in the clinic and validates the need for further study of this culture model to inform strategies to promote anti-tumor immunity.

Author Info: (1) Department of Biology, Rider University, Lawrenceville, NJ, 08648, USA. (2) Department of Biology, Rider University, Lawrenceville, NJ, 08648, USA. (3) Department of Biology, Rider

Author Info: (1) Department of Biology, Rider University, Lawrenceville, NJ, 08648, USA. (2) Department of Biology, Rider University, Lawrenceville, NJ, 08648, USA. (3) Department of Biology, Rider University, Lawrenceville, NJ, 08648, USA. (4) Department of Biology, Rider University, Lawrenceville, NJ, 08648, USA. (5) Department of Biology, Rider University, Lawrenceville, NJ, 08648, USA. (6) Department of Biology, Rider University, Lawrenceville, NJ, 08648, USA. (7) Department of Biology, Rider University, Lawrenceville, NJ, 08648, USA. Electronic address: riggs@rider.edu.

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NF-kappaB and the Transcriptional Control of Inflammation

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The NF-kappaB transcription factor was discovered 30 years ago and has since emerged as the master regulator of inflammation and immune homeostasis. It achieves this status by means of the large number of important pro- and antiinflammatory factors under its transcriptional control. NF-kappaB has a central role in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and autoimmunity, as well as diseases comprising a significant inflammatory component such as cancer and atherosclerosis. Here, we provide an overview of the studies that form the basis of our understanding of the role of NF-kappaB subunits and their regulators in controlling inflammation. We also describe the emerging importance of posttranslational modifications of NF-kappaB in the regulation of inflammation, and highlight the future challenges faced by researchers who aim to target NF-kappaB transcriptional activity for therapeutic benefit in treating chronic inflammatory diseases.

Author Info: (1) Rheumatoid Arthritis Pathogenesis Centre of Excellence, Centre for Immunobiology, Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom. (2) Centre for

Author Info: (1) Rheumatoid Arthritis Pathogenesis Centre of Excellence, Centre for Immunobiology, Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom. (2) Centre for Immunobiology, Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, United Kingdom. Electronic address: ruaidhri.carmody@glasgow.ac.uk.

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Immune Consequences of in vitro Infection of Human Peripheral Blood Leukocytes with Vesicular Stomatitis Virus

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BACKGROUND: Oncolytic vesicular stomatitis virus (VSV) can be delivered intravenously to target primary and metastatic lesions, but the interaction between human peripheral blood leukocytes (PBLs) and VSV remains poorly understood. Our study aimed to assess the overall immunological consequences of ex vivo infection of PBLs with VSV. METHODS: Phenotypic analysis of lymphocyte subsets and apoptosis were evaluated with flow cytometry. Caspase 3/7 activity was detected by luminescence assay. Virus release was evaluated in a murine cell line (L929). Gene expression and cytokine/chemokine secretion were assessed by real-time PCR and multiplex assay, respectively. RESULTS: Ex vivo infection of PBLs with VSV elicited upregulated expression of RIG-I, MDA-5, tetherin, IFITM3, and MxA. VSV infection triggered rapid differentiation of blood monocytes into immature dendritic cells as well as their apoptosis, which depended on caspase 3/7 activation. Monocyte differentiation required infectious VSV, but loss of CD14+ cells was also associated with the presence of a cytokine/chemokine milieu produced in response to VSV infection. CONCLUSIONS: Systemic delivery is a major goal in the field of oncolytic viruses. Our results shed further light on immune mechanisms in response to VSV infection and the underlying VSV-PBL interactions bringing hope for improved cancer immunotherapies, particularly those based on intravenous delivery of oncolytic VSV.

Author Info: (1) Laboratory of Virology, Institute of Immunology and Experimental Therapy (IIET), Polish Academy of Sciences, Wroclaw, Poland. (2) (3) (4) (5) (6) (7)

Author Info: (1) Laboratory of Virology, Institute of Immunology and Experimental Therapy (IIET), Polish Academy of Sciences, Wroclaw, Poland. (2) (3) (4) (5) (6) (7)

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p53-reactive T cells are associated with clinical benefit in patients with platinum-resistant epithelial ovarian cancer after treatment with a p53 vaccine and gemcitabine chemotherapy

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PURPOSE: To conduct a Phase I trial of a Modified Vaccinia Ankara vaccine delivering wild type human p53 (p53MVA) in combination with gemcitabine chemotherapy in patients with platinum-resistant ovarian cancer. EXPERIMENTAL DESIGN: Patients received gemcitabine on days 1 and 8 and p53MVA vaccine on day 15, during the first 3 cycles of chemotherapy. Toxicity was classified using the NCI Common Toxicity Criteria and clinical response assessed by CT scan. Peripheral blood samples were collected for immunophenotyping and monitoring of anti-p53 immune responses. RESULTS: 11 patients were evaluated for p53MVA/gemcitabine toxicity, clinical outcome and immunological response. TOXICITY: There were no DLTs but 3/11 patients came off study early due to gemcitabine-attributed adverse events (AEs). Minimal AEs were attributed to p53MVA vaccination. Immunological and Clinical Response: Enhanced in vitro recognition of p53 peptides was detectable after immunization in both the CD4+ and CD8+ T cell compartments in 5/11 and 6/11 patients respectively. Changes in peripheral T regulatory cells (Tregs) and myeloid derived suppressor cells (MDSC) did not correlate significantly with vaccine response or progression free survival (PFS). Patients with the greatest expansion of p53-reactive T cells had significantly longer PFS than patients with lower p53-reactivity post therapy. Tumor shrinkage or disease stabilization occurred in 4 patients. CONCLUSIONS: p53MVA was well tolerated, but gemcitabine without steroid pre-treatment was intolerable in some patients. However, elevated p53-reactive CD4+ and CD8+T cell responses post therapy correlated with longer PFS. Therefore, if responses to p53MVA could be enhanced with alternative agents, superior clinical responses may be achievable.

Author Info: (1) Experimental Therapeutics, City of Hope. (2) Department of Information Sciences, City of Hope. (3) Information Sciences, City of Hope National Medical Center. (4) Department

Author Info: (1) Experimental Therapeutics, City of Hope. (2) Department of Information Sciences, City of Hope. (3) Information Sciences, City of Hope National Medical Center. (4) Department of Medical Oncology and Therapeutics Research, City Of Hope National Medical Center. (5) Hematology/Hematopoietic Cell Transplantation, City of Hope Comprehensive Cancer Center. (6) Department of Experimental Therapeutics, Beckman Research Institute of the City of Hope. (7) Department of Experimental Therapeutics, Beckman Research Institute of the City of Hope. (8) Ps Medical Oncology, City of Hope. (9) Department of Medical Oncology and Therapeutics Research, City Of Hope National Medical Center. (10) Department of Medical Oncology and Therapeutics Research, City of Hope Comprehensive Cancer Center. (11) Clinical Trials Office, City of Hope National Medical Center. (12) Clinical Trials Office, City Of Hope National Medical Center. (13) Antatomy Pathology, City of Hope National Medical Center. (14) General & Oncologic Surgery, City of Hope National Medical Center. (15) Department of Experimental Therapeutics, Beckman Research Institute of the City of Hope ddiamond@coh.org. (16) Medical Oncology and Therapeutic Research, City of Hope.

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Exosomes associated with human ovarian tumors harbor a reversible checkpoint of T cell responses

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Nano-sized membrane-encapsulated extracellular vesicles isolated from the ascites fluids of ovarian cancer patients are identified as exosomes based on their biophysical and compositional characteristics. We report here that T cells pulsed with these tumor-associated exosomes during TCR-dependent activation inhibit various activation endpoints including translocation of NFkB and NFAT into the nucleus, upregulation of CD69 and CD107a, production of cytokines and cell proliferation. Additionally, the activation of virus-specific CD8+ T cells that are stimulated with the cognate viral peptides presented in the context of class I MHC is also suppressed by the exosomes. The inhibition occurs without loss of cell viability, and coincidentally with the binding and internalization of these exosomes. This exosome-mediated inhibition of T cells was transient and reversible: T cells exposed to exosomes can be reactivated once exosomes are removed. We conclude that tumor-associated exosomes are immunosuppressive, and represent a therapeutic target blockade of which would enhance the antitumor response of quiescent tumor-associated T cells and prevent the functional arrest of adoptively transferred tumor-specific T cells or chimeric antigen receptor (CAR) T cells.

Author Info: (1) Microbiology and Immunology, School of Medicine, University at Buffalo. (2) Microbiology and Immunology, School of Medicine, University at Buffalo. (3) Flow and Image Cytometry

Author Info: (1) Microbiology and Immunology, School of Medicine, University at Buffalo. (2) Microbiology and Immunology, School of Medicine, University at Buffalo. (3) Flow and Image Cytometry Shared Resource, Roswell Park Cancer Institute. (4) Pharmaceutical Sciences, University at Buffalo. (5) Microbiology and Immunology, School of Medicine, University at Buffalo. (6) Flow and Image Cytometry Shared Resource, Roswell Park Cancer Institute. (7) Flow Cytometry, Roswell Park Cancer Institute. (8) Gynecologic Oncology, Roswell Park Cancer Institute. (9) Pharmaceutical Sciences, University at Buffalo. (10) Microbiology and Immunology, School of Medicine, University at Buffalo rbankert@buffalo.edu.

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Notch1 signaling in melanoma cells promoted tumor-induced immunosuppression via upregulation of TGF-beta1

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BACKGROUND: The receptors of Notch family play an important role in controlling the development, differentiation, and function of multiple cell types. The aim of this study is to investigate the role of Notch1 signaling upon immune suppression induced by melanoma cells. METHODS: Melanoma cell line B16 cells were transfected by lentivirus containing mouse Notch1 gene or Notch1 shRNA to generate B16 cell line that highly or lowly expressed Notch1. Notch1 in anti-tumor immune response was comprehensively appraised in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice. The ratios of CD3(+)CD8(+) cytotoxic T cells, CD49b(+)NK cells, CD4(+)CD25(+)FoxP3(+) Tregs and Gr1(+)CD11b(+) MDSCs in tumor-DLN or spleen were examined by flow cytometry. After the co-culture of B16 cells and CD8(+) T cells, the effects of Notch1 on the proliferation and activation of T cells were assessed by CCK8 assay, CFSE dilution and Chromium-release test. The mRNA expression and supernatant secretion of immunosuppressive cytokines, TGF-beta1, VEGF, IL-10 and IFN-gamma were measured by RT-PCR and ELISA, respectively. RESULTS: Downregulation or overexpression of Notch1 in B16 melanoma cells inhibited or promoted tumor growth in immunocompetent mice, respectively. Notch1 expression in B16 melanoma cells inhibited the infiltration of CD8+ cytotoxic T lymphocytes and NK cells and reduced IFN-gamma release in tumor tissue. It could also enhance B16 cell-mediated inhibition of T cell proliferation and activation, and upregulate PD-1 expression on CD4(+) and CD8(+) T cells. The percentage of CD4(+)CD25(+)FoxP3(+) Tregs and Gr1(+)CD11b(+)MDSCs were significantly increased in tumor microenvironment, and all these were attributed to the upregulation of TGF-beta1. CONCLUSION: These findings suggested that Notch1 signaling in B16 melanoma cells might inhibit antitumor immunity by upregulation of TGF-beta1.

Author Info: (1) Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, No.13, Shiliugang Road, Haizhu District, Guangzhou, 510315, Guangdong Province, People's Republic of China

Author Info: (1) Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, No.13, Shiliugang Road, Haizhu District, Guangzhou, 510315, Guangdong Province, People's Republic of China. (2) Cancer Center, The First People's Hospital of Huaihua City, Huaihua, 418000, Hunan Province, People's Republic of China. (3) Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, No.13, Shiliugang Road, Haizhu District, Guangzhou, 510315, Guangdong Province, People's Republic of China. (4) Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, No.13, Shiliugang Road, Haizhu District, Guangzhou, 510315, Guangdong Province, People's Republic of China. (5) Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, No.13, Shiliugang Road, Haizhu District, Guangzhou, 510315, Guangdong Province, People's Republic of China. (6) Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, No.13, Shiliugang Road, Haizhu District, Guangzhou, 510315, Guangdong Province, People's Republic of China. (7) Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, No.13, Shiliugang Road, Haizhu District, Guangzhou, 510315, Guangdong Province, People's Republic of China. (8) Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, No.13, Shiliugang Road, Haizhu District, Guangzhou, 510315, Guangdong Province, People's Republic of China. luorc02@vip.163.com. (9) Oncology Department, Nanfang Hospital, Southern Medical University, No.1838, North of Guangzhou Avenue, Baiyun District, Guangzhou, Guangdong Province, 510515, People's Republic of China. kangshijunlb@163.com.

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Immune Evasion in Pancreatic Cancer: From Mechanisms to Therapy

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Pancreatic ductal adenocarcinoma (PDA), the most frequent type of pancreatic cancer, remains one of the most challenging problems for the biomedical and clinical fields, with abysmal survival rates and poor therapy efficiency. Desmoplasia, which is abundant in PDA, can be blamed for much of the mechanisms behind poor drug performance, as it is the main source of the cytokines and chemokines that orchestrate rapid and silent tumor progression to allow tumor cells to be isolated into an extensive fibrotic reaction, which results in inefficient drug delivery. However, since immunotherapy was proclaimed as the breakthrough of the year in 2013, the focus on the stroma of pancreatic cancer has interestingly moved from activated fibroblasts to the immune compartment, trying to understand the immunosuppressive factors that play a part in the strong immune evasion that characterizes PDA. The PDA microenvironment is highly immunosuppressive and is basically composed of T regulatory cells (Tregs), tumor-associated macrophages (TAMs), and myeloid-derived suppressive cells (MDSCs), which block CD8(+) T-cell duties in tumor recognition and clearance. Interestingly, preclinical data have highlighted the importance of this immune evasion as the source of resistance to single checkpoint immunotherapies and cancer vaccines and point at pathways that inhibit the immune attack as a key to solve the therapy puzzle. Here, we will discuss the molecular mechanisms involved in PDA immune escape as well as the state of the art of the PDA immunotherapy.

Author Info: (1) Cancer Research Program, Hospital del Mar Medical Research Institute (IMIM), Barcelona 08003, Spain. nmartinez@imim.es. (2) Cancer Research Program, Hospital del Mar Medical Research Institute

Author Info: (1) Cancer Research Program, Hospital del Mar Medical Research Institute (IMIM), Barcelona 08003, Spain. nmartinez@imim.es. (2) Cancer Research Program, Hospital del Mar Medical Research Institute (IMIM), Barcelona 08003, Spain. jvinaixa@imim.es. (3) Cancer Research Program, Hospital del Mar Medical Research Institute (IMIM), Barcelona 08003, Spain. pnavarro@imim.es. Institute of Biomedical Research of Barcelona (IIBB-CSIC), Barcelona 08036, Spain. pnavarro@imim.es.

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Antigen-specific antitumor responses induced by OX40 agonist are enhanced by IDO inhibitor indoximod

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Although an immune response to tumors may be generated using vaccines, so far, this approach has only shown minimal clinical success. This is attributed to the tendency of cancer to escape immune surveillance via multiple immune suppressive mechanisms. Successful cancer immunotherapy requires targeting these inhibitory mechanisms along with enhancement of antigen-specific immune responses to promote sustained tumor-specific immunity. Here we evaluated the effect of indoximod, an inhibitor of the immunosuppressive indoleamine-(2,3)-dioxygenase (IDO) pathway, on antitumor efficacy of anti-OX40 agonist in the context of vaccine in the IDO- TC-1 tumor model. We demonstrate that although the addition of anti-OX40 to the vaccine moderately enhances therapeutic efficacy, incorporation of indoximod into this treatment leads to enhanced tumor regression and cure of established tumors in 60% of treated mice. We show that the mechanisms by which the IDO inhibitor leads to this therapeutic potency include (i) an increment of vaccine-induced tumor-infiltrating effector T cells that is facilitated by anti-OX40, and (ii) a decrease of IDO enzyme activity produced by non-tumor cells within the tumor microenvironment that results in enhancement of the specificity and the functionality of vaccine-induced effector T cells. Our findings suggest a translatable strategy to enhance the overall efficacy of cancer immunotherapy.

Author Info: (1) Georgia Cancer Center, Augusta University. (2) Georgia Cancer Center, Augusta University. (3) Georgia Cancer Center, Augusta University. (4) Georgia Cancer Center, Augusta University. (5)

Author Info: (1) Georgia Cancer Center, Augusta University. (2) Georgia Cancer Center, Augusta University. (3) Georgia Cancer Center, Augusta University. (4) Georgia Cancer Center, Augusta University. (5) Georgia Cancer Center, Augusta University. (6) Georgia Cancer Center, Augusta University. (7) Georgia Cancer Center, Augusta University. (8) Georgia Cancer Center, Augusta University. (9) The University of Aberdeen Dental School & Hospital, The Institute of Medicine, Medical Sciences & Nutrition, The University of Aberdeen. (10) Medimmune Inc. (11) Georgia Cancer Center, Augusta University. (12) Georgia Cancer Center, Augusta University skhleif@augusta.edu.

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T regulatory cells mediate immunosuppresion by adenosine in peripheral blood, sentinel lymph node and TILs from melanoma patients

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T regulatory cells (Tregs), involved in tumour tolerance, can generate Adenosine by CD39/CD73 surface enzymes, which identify four Tregs subsets: CD39(+)CD73(-) nTregs, CD39(+)CD73(+) iTregs, CD39(-)CD73(+) oTregs and CD39(-)CD73(-) xTregs. In melanoma patients, increased Tregs levels are detected in peripheral blood (PB), sentinel lymph node (SLN) and tumour infiltrating lymphocytes (TILs), but Adenosine role was not investigated yet. We examined total Tregs and Adenosine subsets in PB, SLN and TILs from melanoma patients (n = 32) and PB from healthy donors (HD; n = 10) by flow cytometry. Total Tregs significantly increased in stage III-IV patients PB, in SLN and TILs, as compared to HD/stage I-II patients. Tregs subsets analyses showed that: 1) PB nTregs significantly increased in SLN and decreased in TILs; 2) iTregs significantly increased in stage III-IV patients PB and further significantly increased in SLN and TILs; 3) PB oTregs and xTregs significantly decreased in SLN and TILs. Patients clinical features did not significantly influence total Tregs, except SLN excision order. Results confirmed Tregs role in melanoma progression and indicate Adenosine generation as a novel escape mechanism, being nTregs and iTregs increased in PB/SLN/TILs.

Author Info: (1) Plastic and Reconstructive Surgery Unit - Regional Melanoma Referral Center and Melanoma & Skin Cancer Unit, Tuscan Tumour Institute (ITT) - Santa Maria Annunziata

Author Info: (1) Plastic and Reconstructive Surgery Unit - Regional Melanoma Referral Center and Melanoma & Skin Cancer Unit, Tuscan Tumour Institute (ITT) - Santa Maria Annunziata Hospital, Florence, Italy. Electronic address: paola.digennaro@unifi.it. (2) Plastic and Reconstructive Surgery Unit - Regional Melanoma Referral Center and Melanoma & Skin Cancer Unit, Tuscan Tumour Institute (ITT) - Santa Maria Annunziata Hospital, Florence, Italy. (3) Central Laboratory, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy. (4) Plastic and Reconstructive Surgery Unit - Regional Melanoma Referral Center and Melanoma & Skin Cancer Unit, Tuscan Tumour Institute (ITT) - Santa Maria Annunziata Hospital, Florence, Italy. (5) Plastic and Reconstructive Surgery Unit - Regional Melanoma Referral Center and Melanoma & Skin Cancer Unit, Tuscan Tumour Institute (ITT) - Santa Maria Annunziata Hospital, Florence, Italy. (6) Dept. Anatomic Pathology - Dermatopathology Section, Santa Maria Annunziata Hospital, Florence, Italy. (7) Dept. Surgery and Translational Medicine, Dermatology Section, University of Florence, Italy. (8) Plastic and Reconstructive Surgery Unit - Regional Melanoma Referral Center and Melanoma & Skin Cancer Unit, Tuscan Tumour Institute (ITT) - Santa Maria Annunziata Hospital, Florence, Italy.

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