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Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients’ stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner. Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E. To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide. When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation. Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.

Background. Reports on the expression of CD38 in Sézary syndrome (SS), erythrodermic primary cutaneous T cell lymphoma with leukemic involvement, are limited. The aim of the present study is the analysis of the expression of CD38 by skin-infiltrating mononuclear cells and circulating T lymphocytes in a cohort of SS patients. Methods. SS patients diagnosed since 1985 in our clinic were retrospectively analyzed for CD38 expression in biopsy and blood samples by immunohistochemistry and flow cytometry, respectively. Results. SS patients show a predominant CD38-negative phenotype on both skin and blood. A subgroup of patients was found expressing CD38 (12 cases) in either the skin (>25% cell infiltrate) or blood (CD4+CD38+ >50%), among whom 4 in the blood, 7 in the skin, and 1 in both blood and skin. Conclusion. The implications of these observations may be twofold: the relevance in basic science is related to a potential role in immune defense regulation, whilst in perspective CD38 may become a target for antibody therapy, considering the availability of different anti-CD38 monoclonal antibodies.

Primary cutaneous lymphomas encompass a wide spectrum of rare lymphoproliferative disorders originating in the skin, among which, mycosis fungoides (MF) is the most common subtype. The treatment of this disease is based on skin-directed therapies eventually in association with biologic response modifiers in the early phases, whereas in patients with the advanced stages, several therapeutic strategies can be used including mono and/or poly-chemotherapy and bone marrow transplantation. In recent years, the identification of specific markers (phenotypical, immunological, and molecular) has led to the development of several studies (including two randomized phase III trials). The results of these studies are modifying our therapeutic strategy toward a personalized treatment approach in which the clinical characteristics of the patients and tumor-node-metastasis-blood stage are considered together with the expression of specific markers (i.e., a CD30-positive expression for the use of brentuximab vedotin). This review will provide a comprehensive scenario of the main phenotypical, molecular, and immunological markers related to MF pathogenesis and disease evolution, which could represent the target for the development of innovative effective treatments in this disease.

Aim: A survival benefit was demonstrated by dabrafenib + trametinib for metastatic BRAF-mutated melanoma patients. Best response is a strong prognostic marker for survival. Patients & methods: The specific features associated with complete response (CR) were evaluated. Results: A total of 15/66 patients achieved CR. Median size of lesions was 3 cm (range: 0.5–10). Using that value as cut-off, the CR rate was 39.3% in patients with smaller lesions and 10.5% in patients with bigger size (p = 0.006). The clinical features associated with CR were the number of metastatic sites and the largest diameter of the biggest metastatic site. Conclusion: The number of the metastases and the diameter of the largest metastatic site are associated with a higher CR rate.

Metastatic melanoma is an aggressive and rapidly progressive disease. Heart metastases were shown in over 50% of autopsy. However, these metastases are usually clinically silent and reported in less than 2% of living patients1.2. There are no specific guidelines for the treatment of cardiac metastases as the therapeutic approach varies with the clinical features of the patient. This article is protected by copyright. All rights reserved.

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In the last years the onset of new therapies changed the management of malignant melanoma. Anti CTLA-4 antibody ipilimumab was the first drug to achieve a significant improvement in survival of advanced stage melanoma. This new therapeutic agent is characterized by a number of side effects that are totally different from those of traditional chemotherapy, mainly caused by the immune system activation. The purpose of this paper is to underline the central role of ipilimumab in the treatment of metastatic melanoma and to characterize related adverse events in terms of incidence, duration and severity of presentation. The early recognition of these side effects is crucial in order to ensure an appropriate management of the toxicities, thus reducing the long term clinical sequelae and the inappropriate treatment discontinuation.

Background: Mycosis fungoides (MF) and Sézary syndrome (SS) are the most frequent cutaneous T-cell lymphomas (CTCL). Human endogenous retroviruses (HERVs) were reverse transcribed and integrated into primate chromosomal DNA, becoming noninfectious, although various stimuli may reactivate them. HERV expression seems to be impaired in several human diseases but limited data regarding CTCL are available. Objective: To evaluate the endogenous retroviral transcription profile in CTCL and their expression among disease clinical stages. Methods: Peripheral blood mononuclear cells from 42 MF/SS patients were analyzed. Total RNA was extracted and amplified with reverse transcription polymerase chain reaction. Results were compared with those obtained in a cohort of 20 healthy donors. Results: HERVs were significantly overexpressed in MF/SS patients compared with healthy donors. No differences were found between early and advanced CTCL stages. Conclusion: HERVs can act as promoters in MF/SS pathogenesis. It remains to link HERV hyperexpression to the outcome in CTCL patients.

Background Advanced-stage mycosis fungoides (MF)/Sezary syndrome (SS) patients are weighted by an unfavorable prognosis and share an unmet clinical need of effective treatments. International guidelines are available detailing treatment options for the different stages but without recommending treatments in any particular order due to lack of comparative trials. The aims of this second CLIC study were to retrospectively analyze the pattern of care worldwide for advanced-stage MF/SS patients, the distribution of treatments according to geographical areas (USA versus non-USA), and whether the heterogeneity of approaches has potential impact on survival. Patients and methods This study included 853 patients from 21 specialist centers (14 European, 4 USA, 1 each Australian, Brazilian, and Japanese). Results Heterogeneity of treatment approaches was found, with up to 24 different modalities or combinations used as first-line and 36% of patients receiving four or more treatments. Stage IIB disease was most frequently treated by total-skin-electron-beam radiotherapy, bexarotene and gemcitabine; erythrodermic and SS patients by extracorporeal photochemotherapy, and stage IVA2 by polychemotherapy. Significant differences were found between USA and non-USA centers, with bexarotene, photopheresis and histone deacetylase inhibitors most frequently prescribed for first-line treatment in USA while phototherapy, interferon, chlorambucil and gemcitabine in non-USA centers. These differences did not significantly impact on survival. However, when considering death and therapy change as competing risk events and the impact of first treatment line on both events, both monochemotherapy (SHR = 2.07) and polychemotherapy (SHR = 1.69) showed elevated relative risks. Conclusion This large multicenter retrospective study shows that there exist a large treatment heterogeneity in advanced MF/SS and differences between USA and non-USA centers but these were not related to survival, while our data reveal that chemotherapy as first treatment is associated with a higher risk of death and/or change of therapy and thus other therapeutic options should be preferable as first treatment approach.

The prognosis of stage IV metastatic melanoma is poor. An overall 1-year survival of 25.5% and a median survival of 6.2 months were reported without any significant improvement during the last 30 years before the introduction of new drugs (immune checkpoint inhibitors and targeted therapies) which completely modified the therapeutic approach and induced an overwhelming improvement on the survival rates of these patients. This review will analyze the therapeutic tools available for the treatment of patients with metastatic melanoma, including adjuvant interferon and locoregional therapies (surgery, radiotherapy and electrochemotherapy) and will mainly focus on the presentation of results obtained by the new treatments (checkpoint inhibitors and targeted therapies).

Background Ipilimumab (Ip) is an option in Metastatic Melanoma (MM) patients (pt) in case of disease progression after antiPD1 (AP) treatment and BRAF+MEK inhibitors (BMi) administration (for BRAF mutated melanoma). Clinical trial are evaluating potential Ip-based combinations in 2nd/3rd line setting. Many studies underline the role of some parameters (as LDH, ECOG PS, Neutrophile/Leucocyte ratio) as progostic factors for immunotherapy used in first-line. We evaluate the prognostic role of some relevant clinical or laboratoristic parameters for Ip used in late line after AP, Bmi, in order to define pt that benefit most from Ip monotherapy in this setting. Methods A retrospective multicenter study was conducted in 8 Italian Oncology Centers, evaluating MM pt treated with Ip after AP and/or BMi. Endpoints were OS and PFS, Kaplan Mayer and Cox regression were applied for survival analysis. Results Among 200 pt that received AP or Bmi, 48 were eligible for Ip administration in 2nd/3rd line. Before Ip treatment, ECOG PS was 0 in 21 pt, number of metastatic sites was less then 3 in 14 pt, LDH was within normal range in 19 pt, NLR ratio (= baseline neutrophils/total leukocytes) was less then 0.7 in 28 pt: in univariate analysis, only ECOG PS and NLR resulted significantly associated with better PFS and OS. For pt with ECOG PS 0 or 1 medianPFS was 3.2, 2.3 month respectively (p value 0.0066; HR 0.377 IC95% 0.186-0.762), median OS was 12.1, 4.0 respectively (p value 0.0016 HR 0.287 IC95% 0.132-0.622). For pt with NLR 0,7 medianPFS was 3.2, 2.0 month respectively (p value 0.002 HR 0.241 IC95% 0.0978-0.593), median OS was 7.63, 2.67 respectively (p value 0.0037 HR 0.251 IC95% 0.0986-0.0637) A score was counted for each pt considering the number of favorable basal factors present (ECOG PS 0, NLR Conclusions ECOG PS 0, NLR Legal entity responsible for the study Italian Melanoma Intergroup. Funding Has not received any funding. Disclosure R. Marconcini: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Novartis; Honoraria (self), Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: La Roche; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: MSD; Honoraria (self), Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy: Incyte; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Ipsen. All other authors have declared no conflicts of interest.

Cancer survival rates are lower in Eastern Europe. To describe, based on a single-centre database in northern Italy, clinical, histopathological, and prognostic features of melanoma in a migrant population from Eastern Europe. We retrospectively analysed data from 18,190 consecutive foreign patients who visited our institution, with 49 cases of melanoma from Eastern Europe. The control group was represented by 1,003 Italian melanoma patients diagnosed and followed at our centre during the same time period. Patients from Eastern Europe were mainly females with lower median age, without significant differences regarding primary melanoma site, relative to the control group. Diagnosiswas made at the place of birth in 30.6% and in our centre for the remainder. Median Breslow thickness was greater (p = 0.0178), and aggressive histotypes (p = 0.0017) and ulcerated melanomas (p = 0.002) were significantly over-represented, particularly when diagnosed in the patients’ native country. Disease was more advanced at diagnosis (p = 0.0001), regardless of the place of initial diagnosis (51% had a progressive disease within one year which rose to 80% if diagnosed before admission to our centre), and the percentage of patients who died within one year was significantly higher (p = 0.022), relative to the control group. Our study shows a poor prognosis for melanoma patients diagnosed in Eastern Europe. Moreover, for migrant populations moving from Eastern to Western European countries, financial difficulties, poor social integration, and language barriers, with consequent late access to healthcare facilities, may account for a worse prognosis.

Mycosis fungoides (MF) is characterized by a switch from indolent behaviour in the early stages to a worse clinical outcome in the advanced ones. Recently, various studies have investigated the role the microenvironment might play in such a switch. We have analysed the distribution of Langerhans cells, plasmacytoid dendritic cells and myeloid-derived suppressor cells in 46 MF cases in various stages, aiming to assess whether changes occur from early to advanced stage. We have investigated the number of langerin, CD303 and arginase-1 positive cells and their distribution at high power. Data were analysed using t test for continuous variables, χ 2 tests or Fisher’s exact test for categorical variables, as well as analysis of covariance. In comparing stages IA/B to IIB, we observed a significant decrease in Langerhans cells (p value 0.03) and a significant increase in CD303 and arginase-1 positive cells (p value

Background Multiple primary melanoma (MPM), in concert with a positive family history, is a predictor of cyclin-dependent kinase (CDK) inhibitor 2A ( CDKN2A ) germline mutations. A rule regarding the presence of either 2 or 3 or more cancer events (melanoma and pancreatic cancer) in low or high melanoma incidence populations, respectively, has been established to select patients for genetic referral. Objective We sought to determine the CDKN2A / CDK4 /microphthalmia-associated transcription factor mutation rate among Italian patients with MPM to appropriately direct genetic counseling regardless of family history. Methods In all, 587 patients with MPM and an equal number with single primary melanomas and control subjects were consecutively enrolled at the participating centers and tested for CDKN2A , CDK4 , and microphthalmia-associated transcription factor. Results CDKN2A germline mutations were found in 19% of patients with MPM versus 4.4% of patients with single primary melanoma. In familial MPM cases the mutation rate varied from 36.6% to 58.8%, whereas in sporadic MPM cases it varied from 8.2% to 17.6% in patients with 2 and 3 or more melanomas, respectively. The microphthalmia-associated transcription factor E318K mutation accounted for 3% of MPM cases altogether. Limitations The study was hospital based, not population based. Rare novel susceptibility genes were not tested. Conclusion Italian patients who developed 2 melanomas, even in situ, should be referred for genetic counseling even in the absence of family history.

Introduction The therapeutic landscape of metastatic melanoma drastically changed after the introduction of targeted therapies and immunotherapy, in particular immune checkpoints inhibitors (ICI). In recent years, positive effects on the immune system associated to radiotherapy (RT) were discovered, and radiation has been tested in combination with ICI in both pre-clinical and clinical studies (many of them still ongoing). We here summarize the rationale and the preliminary clinical results of this approach. Materials and methods In the first part of this review article, redacted with narrative non-systematic methodology, we describe the clinical results of immune checkpoints blockade in melanoma as well as the biological basis for the combination of ICI with RT; in the second part, we systematically review scientific publications reporting on the clinical results of the combination of ICI and RT for advanced melanoma. Results The biological and mechanistic rationale behind the combination of ICI and radiation is well supported by several preclinical findings. Retrospective observational series and few prospective trials support the potential synergistic effect between radiation and ICI for metastatic melanoma. Conclusion RT may potentiate anti-melanoma activity of ICI by enhancing response on both target and non-target lesions. Several prospective trials are ongoing with the aim of further exploring this combination in the clinical setting, hopefully confirming initial observations and opening a new therapeutic window for advanced melanoma patients.

The purpose of this study was to set up a prognostic model for the identification of survival predictors specific for melanoma patients treated with ipilimumab.The following prospectively collected data were utilised: patient and primary tumour characteristics, relapse-free-interval, site and number of metastases, previous therapies and level of serum biomarkers (lactic dehydrogenase (LDH), C-reactive protein, β2-microglobulin, vascular endothelial growth factor (VEGF), IL2, IL6, S-100, alkaline phosphatase (ALP), transaminases, leucocyte count, lymphocytes subpopulations). A multivariate prognostic model was developed using the Cox regression model fitted to the data of 113 consecutive metastatic patients treated with ipilimumab (3 mg/kg, q3w) at Veneto Institute of Oncology (IOV). External validation was obtained using the data of 69 and 34 patients treated at European Oncology Institute (IEO) and University of Torino (UT), respectively.Median survival was 8.3, 4.9 and 7.1 months from first ipilimumab administration at IOV, IEO and UT, respectively. Both higher baseline levels of LDH (Hazard Ratio [HR] v=1.36, 95% Confidence Interval [CI] 1.16-1.58, P.001) and neutrophils (HR=1.76, 95% CI 1.41-2.10, P.001) were associated with worse prognosis. Model performance was satisfactory both upon internal validation (Dxy=0.42) and external validation (Dxy=0.40). Serum LDH and neutrophil count discriminated patients who lived more (low neutrophils and low LDH) or less (high LDH or neutrophils) than 24 months.Serum LDH and neutrophil count were significant independent prognostic factors. This externally validated prognostic nomogram, could help clinicians to identify the patients who would benefit most from ipilimumab and consequently to improve resource allocation. These easily available biomarkers deserve further validation.

Background: No studies are available in the literature on the distribution of different melanoma features and risk factors in the Italian geographical areas. Objective: To identify the differences in clinical-pathological features of melanoma, the distribution of risk factors and sun exposure in various Italian macro-areas. Methods: Multicentric-observational study involving 1,472 melanoma cases (713 north, 345 centre, 414 south) from 26 referral centres belonging to the Italian Multidisciplinary Group for Melanoma. Results: Melanoma patients in northern regions are younger, with thinner melanoma, multiple primaries, lower-intermediate phototype and higher counts of naevi with respect to southern patients; detection of a primary was mostly connected with a physician examination, while relatives were more involved in the south. Northern patients reported a more frequent use of sunbeds and occurrence of sunburns before melanoma despite sunscreen use and a lower sun exposure during the central hours of the day. Conclusions: The understanding of differences in risk factors distribution could represent the basis for tailored prevention programmes.

Skin metastases represent a relatively frequent event in the natural history of melanoma, developing both in early and in late disease stages. Cutaneous or subcutaneous lesions arise in 15-20% of patients; almost 50% of subjects with metastatic disease have soft tissue metastases [1]. The treatment of these lesions is still a challenge: surgical excision and radiotherapy represent the standard treatments for isolated lesions, while melphalan and/or tumour necrosis factor limb perfusion is considered [...]

Summary Background The prognostic significance of regression in primary melanoma has been debated over the past few years. Once it was considered to be a negative prognostic factor, as it may have prevented proper melanoma thickness measurement, therefore affecting the staging of the tumours. For this reason, it was considered to be an indication for sentinel lymph node biopsy (SLNB) in melanoma

e20060 Background: Metastatic melanoma (MM) usually has a dismal prognosis and its treatment options have been deluding for long time: until recently, systemic therapies were limited, with poor res...