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Purpose: To assess the clinical and biological activity of the association of bevacizumab and fotemustine as first-line treatment in advanced melanoma patients.Experimental Design: Previously untreated, metastatic melanoma patients (n = 20) received bevacizumab (at 15 mg/kg every 3 weeks) and fotemustine (100 mg/m2 by intravenous administration on days 1, 8, and 15, repeated after 4 weeks) in a multicenter, single-arm, open-label, phase II study. Primary endpoint was the best overall response rate; other endpoints were toxicity, time to progression (TTP), and overall survival (OS). Serum cytokines, angiogenesis, and lymphangiogenesis factors were monitored by multiplex arrays and by in vitro angiogenesis assays. Effects of fotemustine on melanoma cells, in vitro, on vascular endothelial growth factor (VEGF)-C release and apoptosis were assessed by ELISA and flow cytometry, respectively.Results: One complete response, 2 partial responses (PR), and 10 patients with stable disease were observed. TTP and OS were 8.3 and 20.5 months, respectively. Fourteen patients experienced adverse events of toxicity grade 3–4. Serum VEGF-A levels in evaluated patients (n = 15) and overall serum proangiogenic activity were significantly inhibited. A significant reduction in VEGF-C levels was found in several post-versus pretherapy serum samples. In vitro, fotemustine inhibited VEGF-C release by melanoma cells without inducing significant cell death. Serum levels of interleukin (IL)-10 and IL-12p70 showed the highest levels in sera of PR patients, compared with patients with stable or progressive disease whereas IL-23 showed the opposite pattern.Conclusions: The combination of bevacizumab plus fotemustine has clinical activity in advanced melanoma and promotes systemic modulation of angiogenesis and lymphangiogenesis factors. Clin Cancer Res; 16(23); 5862–72. ©2010 AACR.

Supplementary Figures S1-S3 and Supplementary Table S1.

Background Outcomes of patients with metastatic melanoma discontinuing BRAF‐targeted therapy for cumulative toxicity after sustained response are unknown. Materials and Methods This retrospective case series analysis conducted at a single Cancer Center in Italy included patients with BRAF mutated metastatic melanoma treated with a BRAF inhibitor as a single agent or in combination with a MEK inhibitor between June 1, 2011 and January 1, 2020 and interrupted treatment due to cumulative toxicity after achieving complete response (CR) or long‐lasting partial response (PR; i.e. >12 months). Results We included 24 patients with a median treatment duration of 59.4 months (95% confidence interval [CI], 55.4–63.4; range, 12–88). CR and PR were achieved in 71% and 29% of patients, respectively. At a median follow‐up after treatment discontinuation of 37.8 months (95% CI, 33.7–41.9), the 12‐month progression‐free survival after discontinuation (dPFS) rate was 70.8% (95% CI 54.8–91.6) and 24‐month dPFS rate was 58.3% (95% CI, 41.6–81.8). Baseline patient and tumor characteristics as well as treatment duration and best response did not significantly impact on dPFS. Patients with CR and negative circulating tumor DNA (ctDNA) at time of discontinuation had a significantly improved dPFS compared with patients with either radiological residual disease or ctDNA positivity (p = .007). No patient in CR with undetectable ctDNA experienced progression. Conclusion The risk of progression is high even in patients with sustained sensitivity to BRAF/MEK inhibitors. Integration of liquid biopsy in clinical trials investigating the optimal management of patients with sustained sensitivity to BRAF/MEK inhibitors is warranted. Implications for Practice Outcomes of patients with metastatic melanoma discontinuing BRAF‐targeted therapy for cumulative toxicity are unknown. This study analyzed patients with sustained responses (median treatment duration 59.4 months). Twelve‐ and 24‐month progression‐free survival following discontinuation were 70.8% and 58.3%, respectively. Complete response and negative circulating tumor DNA at time of discontinuation are promising prognostic biomarkers in this setting., This article reports clinical outcomes of patients with metastatic melanoma treated with BRAF/MEK inhibitors who interrupted the treatment after achieving a long‐lasting objective response, exploring the role of circulating tumor DNA assessed at time of discontinuation as a biomarker for post‐discontinuation progression‐free survival.

Since a non-negligible fraction of patients with metastatic melanoma does not experience long-term disease control, even with immunotherapy and targeted therapy, new biomarkers for patient stratification and treatment tailoring are needed in this setting. We investigated the association of a novel immune-inflammatory blood-based biomarker, the Pan-Immune-Inflammation Value (PIV), with clinical outcomes of patients with metastatic melanoma receiving first-line therapy. We retrospectively included patients treated at the Fondazione IRCCS Istituto Nazionale dei Tumori of Milan and having an available baseline complete blood cell count (CBC). PIV was calculated as: [neutrophil count (103/mm3) × platelet count (103/mm3) × monocyte count (103/mm3)]/lymphocyte count (103/mm3). A total of 228 patients were included: 119 (52%) had been treated with immunotherapy and 109 (48%) with targeted therapy. PIV was significantly higher in patients with ECOG PS ≥ 1, high disease burden, synchronous metastases, and elevated baseline LDH level. High baseline PIV was independently associated with poor overall survival (adjusted hazard ratio [HR]: 2.06; 95% confidence interval [CI]: 1.30–3.29; adjusted P = 0.002) and progression-free survival (adjusted HR 1.56; 95% CI 1.01–2.41; adjusted P = 0.044). High PIV was also associated with primary resistance to both immunotherapy (odds ratio [OR]: 3.98; 95% CI 1.45–12.32; P = 0.005) and targeted therapy (OR: 8.42; 95% CI 2.50–34.5; P < 0.001). PIV showed a promising discrimination ability in terms of AIC and c-index when compared with other CBC-based biomarkers. PIV may guide the treatment decision process and the development of novel first-line treatment strategies in melanoma, but warrants further study and validation.

Malignant melanoma is a malignant tumor with a poor prognosis. From 2010 the discovery of mutation of BRAF and the development of drugs against this target significantly improved the survival of these patients. The best results were achieved with the double inhibition of the MAPK pathway. There are 3 approved targeted therapies: vemurafenib and cobimetinib, dabrafenib and trametinib, encorafenib and binimetinib. They have similar efficacy, but different toxicity profiles. In this article, is critically review the case of a patient with metastatic melanoma treated with the combination encorafenib and binimetinib.

The genetic landscape of melanoma resistance to targeted therapy with small molecules inhibiting BRAF and MEK kinases is still largely undefined. In this study, we portrayed in detail the somatic alterations of resistant melanoma and explored the associated biological processes and their integration with transcriptional profiles. By targeted next-generation sequencing and whole-exome sequencing analyses, a list of 101 genes showing imbalance in metastatic tumors from patients with a complete/durable response or disease progression during therapy with vemurafenib or with dabrafenib and trametinib was defined. Classification of altered genes in functional categories indicated that the mutational pattern of both resistant tumors and melanoma cell lines was enriched in gene families involved in oncogenic signaling pathways and in DNA repair. Integration of genomic and transcriptomic features showed that the enrichment of mutations in gene sets associated with anabolic processes, chromatin alterations, and IFN-α response determined a significant positive modulation of the same gene signatures at the transcriptional level. In particular, MTORC1 signaling was enriched in tumors from poorly responsive patients and in resistant tumors excised from treated patients. Results indicate that genetic patterns are associated with melanoma resistance to targeted therapy and disclose the underlying key molecular pathways to define drug combinations for improved personalized therapies.

The MITF-E318K variant has been implicated in genetic predisposition to cutaneous melanoma. We addressed the occurrence of MITF-E318K and its association with germline status of CDKN2A and MC1R genes in a hospital-based series of 248 melanoma patients including cohorts of multiple, familial, pediatric, sporadic and melanoma associated with other tumors. Seven MITF-E318K carriers were identified, spanning every group except the pediatric patients. Three carriers showed mutated CDKN2A, five displayed MC1R variants, while the sporadic carrier revealed no variants. Germline/tumor whole exome sequencing for this carrier revealed germline variants of unknown significance in ATM and FANCI genes and, in four BRAF-V600E metastases, somatic loss of the MITF wild-type allele, amplification of MITF-E318K and deletion of a 9p21.3 chromosomal region including CDKN2A and MTAP. In silico analysis of tumors from MITF-E318K melanoma carriers in the TCGA Pan-Cancer-Atlas dataset confirmed the association with BRAF mutation and 9p21.3 deletion revealing a common genetic pattern. MTAP was the gene deleted at homozygous level in the highest number of patients. These results support the utility of both germline and tumor genome analysis to define tumor groups providing enhanced information for clinical strategies and highlight the importance of melanoma prevention programs for MITF-E318K patients.

Immune-checkpoint inhibitors (ICIs) exposed the oncology community to novel immune-related adverse events (irAEs). Here, we report on a retrospective analysis of patients with melanoma who developed an ICI-related, unilateral, acute and peripheral facial nerve paralysis (Bell's palsy).We retrospectively reviewed all the cases of ICI-related Bell's palsy in patients with melanoma treated at our institution from January 2015 to January 2020. A total of five cases of ICI-related Bell's palsy were identified. Median age was 63 years. Median time-to-onset of Bell's palsy from ICIs initiation was 15 weeks. Four patients were treated with prednisone alone, whereas one patient was treated with prednisone plus valaciclovir. All the patients completely recovered from Bell's palsy without neurological sequelae. In melanoma patients treated with ICIs, Bell's palsy is a rare, neurologic irAE with a favorable outcome following administration of oral corticosteroids.

Pembrolizumab is an anti-programmed cell death-1 monoclonal antibody, currently representing the first-line treatment for advanced melanoma. Apart from registration trials, there is a paucity of data on its effectiveness and safety in a real-world setting. We retrospectively analyzed patients with metastatic melanoma treated at our institution in the context of an Expanded Access Program. Survival outcomes were assessed using Kaplan-Meier and Cox hazard models. Overall, 42 melanoma patients were treated. BRAF status was wild type in 30 (71%) patients and mutated in 12 (29%). Twelve (29%) patients received pembrolizumab as the II line treatment, the other 30 (71%) as at least III line treatment. One (2%) patient experienced complete response, six (14%) partial response, and seven (17%) stable disease; 39 (93%) patients had disease progression. Median progression-free survival and overall survival were 2.4 (range: 0.2-46.5) and 5.5 months (range: 0.2-47.1), respectively. Results of the multivariate analysis showed that performance status [hazard ratio (HR): 7.10; 95% confidence interval (CI): 2.73-18.4; P

Therapeutic chances for metastatic melanoma have consistently changed over the last years with the advent of antibodies targeting the programmed cell death protein-1 (PD-1). Onset of immune-related adverse events (irAEs) during treatment can be a source of concern, and the association with survival outcome is yet to be defined. Data of consecutive patients treated with anti-PD1 (nivolumab or pembrolizumab) for metastatic melanoma between July 2013 and January 2018 were retrospectively reviewed. Baseline factors, together with onset of irAEs and vitiligo during treatment, were evaluated to identify predictors of progression-free (PFS) and overall (OS) survival. PFS and OS were assessed using Kaplan–Meier and Cox models. Overall, 173 patients were included in the present analysis, and 102 patients (59%) experienced irAEs. Disease control rate was 51%. Median (interquartile range) PFS and OS were 4.9 (2.6–13.3) and 8.6 (3.5–18.3) months, respectively. At multivariate analysis, irAEs occurrence was independently associated with improved PFS [HR 0.47 (95% CI 0.26, 0.86); p = 0.016], and correlated with better OS [HR 0.39 (95% CI 0.18, 0.81); p = 0.007]. Among various irAEs, the occurrence of vitiligo was associated with a trend toward a non-significant improved OS in comparison with other irAEs (p = 0.061). Median OS was undefined for patients experiencing vitiligo vs. 21.9 months for patients with other irAEs vs. 9.7 months for patients who had no irAEs (p = 0.003). Our study underlines the association between irAEs and survival outcomes from anti-PD1 therapy. Careful management of treatment-related toxicity can lead to achieve maximum clinical benefit from this therapy.

// Riccardo Marconcini 1 , Francesco Spagnolo 2 , Luigia Stefania Stucci 3 , Simone Ribero 4 , Elena Marra 4 , Francesco De Rosa 5 , Virginia Picasso 2 , Lorenza Di Guardo 6 , Carolina Cimminiello 6 , Stefano Cavalieri 6 , Laura Orgiano 7 , Enrica Tanda 2 , Laura Spano 2 , Alfredo Falcone 1 and Paola Queirolo 2 for the Italian Melanoma Intergroup (IMI) 1 Unit of Medical Oncology 2, Azienda Ospedaliera-Universitaria Pisana, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Italy 2 Department of Medical Oncology, IRCCS AOU San Martino-Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 3 Medical Oncology Unit, Department of Biomedical Sciences and Clinical Oncology, University of Bari, Bari, Italy 4 Dermatologic Clinic, Department of Medical Sciences, University of Turin, Turin, Italy 5 Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, IRST IRCCS, Meldola, Italy 6 Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 7 AOU Cagliari, Department of Medical Oncology, University of Cagliari, Cagliari, Italy Correspondence to: Riccardo Marconcini, email: marconcini.riccardo@gmail.com Keywords: melanoma; immunotherapy; ipilimumab; pembrolizumab; nivolumab Received: April 23, 2017 Accepted: November 03, 2017 Published: January 03, 2018 ABSTRACT Metastatic melanoma was the first malignancy in which immune checkpoint inhibitors demonstrated their successful efficacy. Currently, the knowledge on the interaction between the immune system and malignant disease is steadily increasing and new drugs and therapeutic strategies are overlooking in the clinical scenario. To provide a comprehensive overview of immune modulating drugs currently available in the treatment of melanoma as well as to discuss of possible future strategies in the metastatic melanoma setting, the present review aims at analyzing controversial aspects about the optimal immunomodulating treatment sequences, the search for biomarkers of efficacy of immunocheckpoint inhibitors, and innovative combinations of drugs currently under investigation.

Background Serous chorioretinopathy has been associated with MEK inhibitors, including cobimetinib. We describe the clinical features of serous retinopathy observed with cobimetinib in patients with BRAFV600-mutated melanoma treated in the Phase III coBRIM study. Methods In the coBRIM study, 493 patients were treated in two randomly assigned treatment groups: cobimetinib and vemurafenib (n = 247) or vemurafenib (n = 246). All patients underwent prospective ophthalmic examinations at screening, at regular intervals during the study, and whenever ocular symptoms developed. Patients with serous retinopathy were identified in the study database using a group of relevant and synonymous adverse event terms. Results Eighty-six serous retinopathy events were reported in 70 patients (79 events in 63 cobimetinib and vemurafenib-treated patients vs seven events in seven vemurafenib-treated patients). Most patients with serous retinopathy identified by ophthalmic examination had no symptoms or had mild symptoms, among them reduced visual acuity, blurred vision, dyschromatopsia, and photophobia. Serous retinopathy usually occurred early during cobimetinib and vemurafenib treatment; median time to onset was 1.0 month. Most events were managed by observation and continuation of cobimetinib without dose modification and resolved or were resolving by the data cutoff date (19 Sept 2014). Conclusions Cobimetinib treatment was associated with serous retinopathy in patients with BRAFV600-mutated melanoma. Retinopathy was generally asymptomatic or mild. Periodic ophthalmologic evaluations at regular intervals and at the manifestation of any visual disturbance are recommended to facilitate early detection and resolution of serous retinopathy while patients are taking cobimetinib. Trial Registration Clinicaltrials.gov (NCT01689519). First received: September 18, 2012

BackgroundObesity is a risk factor for malignancy; however, its prognostic role in patients with metastatic melanoma is controversial. We aim to investigate the prognostic role of body mass index (BMI) in patients with metastatic melanoma receiving mitogen-activated pathway kinase inhibitors (MAPKi), immune checkpoint inhibitors (ICIs) alone or their sequence.MethodsData on patients with metastatic melanoma receiving ≥1 line of systemic treatment were retrieved from prospectively collected databases. Progression-free survival (PFS) and overall survival (OS) were analyzed by means of multivariable stratified Cox regression models; disease control rate (DCR) was analyzed by multivariable stratified logistic regression models. Subgroup analyzes according to the type of treatments received, and in BRAF-mutated patients were pre-planned. All multivariable models included BMI, age, gender, American Joint Committee on Cancer stage, performance status, lactate dehydrogenase and treatment sequencing strategy as covariates.ResultsBetween November 2010 and November 2018, 688 patients from three Italian and two Polish centers were enrolled. 379 (57%) patients had M1c/d disease, 273 (41%) were female and the mean BMI was 27.1 (SD=4.9). Considering first-line treatment, 446 patients (66.8%) received ICIs and 222 MAPKi. No impact of BMI on OS was detected either considering the first line of ICIs, or ICIs sequencing (HR=1.02, 95% CI: 0.99 to 1.05, p=0.202, and HR=1.02, 95% CI: 0.99 to 1.04, p=0.237, respectively). A late effect of BMI on OS was found in patients treated with MAPKi: for five units increment, a 51% of risk reduction at 18 months and a 76% of risk reduction at 30 months were observed. No significant effect of BMI on PFS and DCR was found in any of the subgroup analyzes.ConclusionIn patients with metastatic melanoma receiving ICIs, there is no impact of BMI on DCR, PFS and OS. The late prognostic effect of BMI in patients treated with MAPKi should be considered hypothesis generating and needs to be further investigated.

Background: Prognosis of patients with metastatic melanoma has improved due to the advent of antibodies targeting the programmed cell death protein-1 (PD-1). However, therapeutic outcomes from anti-PD-1 therapy widely differ among patients. Biomarkers for outcome are needed as these may influence patient selection and treatment decision. Methods: Data of patients with metastatic melanoma treated with anti-PD-1 were retrospectively reviewed. Baseline biochemical (serum lactate dehydrogenase [LDH] levels, complete blood count) and clinical characteristics were evaluated to identify predictors of progression-free survival (PFS) and overall survival (OS). PFS and OS were assessed using Kaplan-Meier and Cox models. The comparison of predictive power of independent predictors for response to anti-PD-1 was evaluated by receiver operating characteristic (ROC) curves. Results: Overall, 173 patients were included. Low metastases burden, normal baseline LDH levels, and high relative lymphocyte count (RLC) were associated with favorable outcomes ( p < 0.01). According to ROC curves, RLC >17.5% improved survival outcomes. PFS was 3.7 and 15.8 months for patients with RLC 17.5%, respectively ( p = 0.004); OS was 5.0 and 33.6 months for patients with RLC 17.5%, respectively ( p < 0.001). Stratification of patients according to these variables showed that survival outcomes strongly differ in patients with 3 of 3 compared to those with 2, 1, and none of these 3 factors present ( p < 0.001). Conclusions: Metastases burden, LDH levels, and RLC are independent baseline characteristics associated with outcome in patients with melanoma receiving anti-PD-1. Further investigations are needed to clarify if evaluation of these parameters can translate into clinical strategy and apply to patient selection.

OBJECTIVE Prognosis of patients with metastatic melanoma has dramatically improved over recent years because of the advent of antibodies targeting programmed cell death protein-1 (PD1). However, the response rate is ~40% and baseline biomarkers for the outcome are yet to be identified. Here, we aimed to determine whether artificial intelligence might be useful in weighting the importance of baseline variables in predicting response to anti-PD1. METHODS This is a retrospective study evaluating 173 patients receiving anti-PD1 for melanoma. Using an artificial neuronal network analysis, the importance of different variables was estimated and used in predicting response rate and overall survival. RESULTS After a mean follow-up of 12.8 (±11.9) months, disease control rate was 51%. Using artificial neuronal network, we observed that 3 factors predicted response to anti-PD1: neutrophil-to-lymphocyte ratio (NLR) (importance: 0.195), presence of ≥3 metastatic sites (importance: 0.156), and baseline lactate dehydrogenase (LDH) > upper limit of normal (importance: 0.154). Looking at connections between different covariates and overall survival, the most important variables influencing survival were: presence of ≥3 metastatic sites (importance: 0.202), age (importance: 0.189), NLR (importance: 0.164), site of primary melanoma (cutaneous vs. noncutaneous) (importance: 0.112), and LDH > upper limit of normal (importance: 0.108). CONCLUSIONS NLR, presence of ≥3 metastatic sites, LDH levels, age, and site of primary melanoma are important baseline factors influencing response and survival. Further studies are warranted to estimate a model to drive the choice to administered anti-PD1 treatments in patients with melanoma.

10053 Background: In pts with metastatic melanoma bearing BRAF V600E/K mutations BRAF V600±MEK inhibitors are administered until PD/unacceptable toxicity. In patients achieving durable responses, outcomes following discontinuation for reasons other than PD are largely unknown. Methods: We identified all patients who interrupted BRAF±MEK inhibitors for reasons other than PD after complete (CR) or partial response (PR) from a clinical dataset of patients with BRAF mutated metastatic/unresectable melanoma treated with targeted therapy at a single Institution. Results: We included 24 pts. Fifteen (62.5%) and 9 (37.5%) pts were treated respectively with BRAF inhibitor monotherapy and BRAF+MEK inhibitor combination. All pts had normal baseline LDH and ECOG PS0, 2 (8%) pts had brain metastases and 15 (62.5%) had multi-organ metastatic involvement. Dose reduction was required for 12 (50%) pts. Median treatment duration was 59 (12-88) months. Causes of discontinuation were unacceptable toxicity (19 pts-79%) and consent withdrawal (5 pts-21%). At the time of discontinuation, 17 (71%) and 7 (29%) pts had achieved respectively CR and PR. At a median follow up of 31 (8-59) months after treatment discontinuation, 9 (37.5%) pts had experienced PD. Median time to PD after treatment discontinuation was 9 (3-16) months. At time of PD, 2 (22%) pts displayed involvement of new organ sites. Risk of PD following discontinuation was respectively 31% and 45% at 12 and 24 months. Neither baseline characteristics nor treatment duration and time to best response influenced risk of PD; we found a non-significant trend towards higher risk of relapse for patients interrupting treatment with residual disease compared to those who interrupted treatment after achieving CR [HR 3.3; 95%CI (0.8–14.1); log-rank p = 0.081]. After PD, 6 pts received BRAF+MEK inhibitors with a response rate of 100% and 3/6 pts achieving CR. Conclusions: In a subset of patients with favorable prognostic characteristics and retained sensitivity to BRAF±MEK inhibitors, treatment discontinuation was associated with relevant risk of relapse with about one third of pts experiencing PD within one year. Biomarker studies are needed to identify pts who might safely discontinue therapy due to sustained toxicity, especially after achieving CR.

Objective immunotherapy with immune checkpoint inhibitors has become one of the standard therapeutic modalities for patients with advanced melanoma. Melanoma of the female lower genital tract is a rare and aggressive disease, with poor long-term clinical outcomes. To date, no study evaluated the role of immunotherapy in metastatic melanoma of the lower genital tract. Methods Data of women with metastatic melanoma of the lower genital tract were prospectively collected. Survival outcomes over time was assessed using Kaplan-Meier model. Results Seven cases of metastatic melanoma of the lower genital tract (vulva [n=2], vagina [n=4], and uterine cervix [n=1]) treated with immune checkpoint inhibitors are reviewed. Two patients had metastatic disease at diagnosis, while 5 patients developed metastatic disease at a mean (standard deviation) time of 9.9 (±3.0) months from primary diagnosis. Four patients received an anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA4) (ipilimumab) and 3 received an anti-programmed cell death 1 (PD-1) (pembrolizumab [n=2], nivolumab [n=1]) therapy. The response rate to immunotherapy was 28.5%. Patients receiving an anti-PD-1 experienced a better progression-free survival than patients treated with anti-CTLA4 (p=0.01, log-rank test). Although not reaching statistical significance, overall survival was better in patients having an anti-PD-1 therapy in comparison to anti-CTLA4 (p=0.15, log-rank test). Conclusion Results from our series confirm the poor prognosis of women with metastatic melanoma of the lower genital tract, thus supporting the need of exploring new treatment modalities. Further studies are warranted to improve knowledge on the role of immunotherapy in metastatic melanoma of the lower genital tract.

Background: Up to 40% of patients with metastatic melanoma (MM) develop brain metastases. Radiotherapy (RT) may potentiate the effects of immunotherapy (IO), even on distant sites (abscopal effect). Material and Methods: We retrospectively analyzed all our MM patients treated with IO within 6 months before/after brain RT between 2012 and 2016. Progression-free (PFS) and overall survival (OS) were estimated with the Kaplan-Meier method and compared with those of controls treated with IO during the same period. Results: Thirty-six cases and 25 controls were identified. Among cases, 23 patients received an anti-CTLA4 agent and 13 an anti-PD1 agent. Eighteen cases were treated with stereotactic RT and 18 with whole-brain RT. Median PFS from the beginning of RT was 4 months in first-line and 2 months in second-line treatment. A third of the cases progressed at first evaluation after RT. Median OS from the beginning of RT was 7 months in first-line and 4 months in second-line treatment. Median PFS and OS of each treatment line showed a trend towards inferiority compared with those of controls. Conclusion: Synergism between RT and IO was not observed in our case series. No cases of abscopal effect were seen, and most patients underwent early systemic progression after RT.

The role of vitamin D in melanoma is still controversial. Although several Authors described a correlation between vitamin D deficiency and poor survival in metastatic melanoma patients, clinical trials exploring the effects of vitamin D supplementation in this clinical setting were mostly inconclusive. However, recent evidence suggests that vitamin D exerts both anti-proliferative effects on tumor cells and immune-modulating activities, that have been widely explored in auto-immune disorders. On the one hand, vitamin D has been shown to inhibit T-helper17 lymphocytes, notoriously involved in the pathogenesis of immune-related adverse events (iAEs) which complicate immune-checkpoint inhibitor (ICI) treatment. On the other hand, vitamin D up-regulates PDL-1 expression on both epithelial and immune cells, suggesting a synergic effect in combination with ICIs, for which further investigation is needed.

The accrual of myeloid-derived suppressor cells (MDSCs) represents a major obstacle to effective immunotherapy in cancer patients, but the mechanisms underlying this process in the human setting remain elusive. Here, we describe a set of microRNAs (miR-146a, miR-155, miR-125b, miR-100, let-7e, miR-125a, miR-146b, miR-99b) that are associated with MDSCs and resistance to treatment with immune checkpoint inhibitors in melanoma patients. The miRs were identified by transcriptional analyses as being responsible for the conversion of monocytes into MDSCs (CD14(+)HLA-DR(neg) cells) mediated by melanoma extracellular vesicles (EVs) and were shown to recreate MDSC features upon transfection. In melanoma patients, these miRs were increased in circulating CD14(+) monocytes, plasma, and tumor samples, where they correlated with the myeloid cell infiltrate. In plasma, their baseline levels clustered with the clinical efficacy of CTLA-4 or programmed cell death protein 1 (PD-1) blockade. Hence, MDSC-related miRs represent an indicator of MDSC activity in cancer patients and a potential blood marker of a poor immunotherapy outcome.

The most frequently reported skin tumours during treatment with targeted therapies for BRAF (B type Rapidly Accelerated Fibrosarcoma kinase) mutated metastatic melanoma are squamous cell carcinomas (SCCs). Basal cell carcinomas (BCCs) have been described in such setting, but no cases of multiple and recurring tumours have been reported so far. A patient with a history of chronic sun exposure and more than 10 BCCs removed since 1998 started treatment with vemurafenib for BRAF mutated metastatic melanoma. Therapy was complicated by sporadic episodes of atrial fibrillation and by the development of recurrent, multiple and diffuse BCCs. So, vemurafenib was discontinued and dabrafenib and trametinib were started. Since then, only four BCCs occurred in the patient. Histopathological re-examination showed that most BCCs occurred under vemurafenib presented with squamous features. Such characteristic was significantly less evident before therapy start and in lesions removed under treatment with dabrafenib and trametinib. BRAF inhibition (BRAFi) without MEK inhibition induces mitogen activated kinases overactivation, with consequent skin toxicity and acquired drug resistance. The BCCs removed from our patient showed squamous features, more evident during vemurafenib monotherapy. Both the switch from vemurafenib to dabrafenib and the addition of MEK inhibitor (MEKi) might have reduced the incidence of BCCs and their squamous differentiation.

Patients with melanoma brain metastases have a poor prognosis and historically have been excluded from clinical trials. The Expanded Access Program (EAP) provided an opportunity to evaluate the feasibility of ipilimumab (3 mg/kg every 3 weeks for four doses) in patients with stage 3 (unresectable) or 4 melanoma and asymptomatic brain metastases, who had failed or did not tolerate previous treatments and had no other therapeutic option available. Tumor assessments were conducted at baseline and week 12 using immune-related response criteria and patients were monitored for adverse events (AEs). Of 855 patients participating in the EAP in Italy, 146 had asymptomatic brain metastases. With a median follow-up of 4 months, the global disease control rate was 27 %, including 4 patients with a complete response and 13 with a partial response. Median progression-free survival and overall survival were 2.8 and 4.3 months, respectively and approximately one-fifth of patients were alive 1 year after starting ipilimumab. In total, 29 % of patients reported a treatment-related AE of any grade, which were grade 3/4 in 6 % of patients. AEs were generally reversible with treatment as per protocol-specific guidelines. Ipilimumab shows durable benefits in some patients with advanced melanoma metastatic to the brain, with safety results consistent with those previously reported in clinical trials.

Objective The incidence of brain metastases from melanoma is increasing. Several effective treatment options are now available but what can be considered the optimal therapeutic strategy is not yet defined. We evaluated the outcome of patients with brain metastatic melanoma in terms of local control rate, brain distant progression, and overall survival. Methods The present retrospective study includes only patients with limited brain metastases (≤4) who underwent surgery plus stereotactic radiosurgery (SRS), or SRS alone. Surgical resection was performed in patients with good Karnofsky performance score, single large brain lesions, controlled extracranial disease, and SRS alone in all other cases. Supramargical resection was performed in all patients. The prescribed radiotherapy doses were 24 Gy/1 fraction and 30 Gy in 3–5 fractions for lesions >2.5 cm. Clinical outcome was evaluated by brain magnetic resonance imaging performed 2 months after radiotherapy and then every 3 months. Results From April 2011 to October 2015, 53 patients were treated. The median age was 54 years (range, 29–82 years). Most of patients had 1–2 brain metastases (86.8%). Twelve patients (22.6%) underwent surgical resection followed by SRS on the tumor bed, and 41 (77.4%) received SRS alone. The median follow-up time was 20.9 months (range, 5.7–61.3 months). The median, 1-, 2-, 3-year overall survival were 11.8 months, 47.2%, 28%, and 21.8%, respectively. Factors recorded as influencing survival were the number of brain metastases, the melanoma-specific graded prognostic assessment score, and BRAF mutated status. Conclusions Our data identifier a subset of patients with a more favorable outcome who could take advantage of a more aggressive local approach followed by targeted therapy.

Purpose Combination therapy with dabrafenib and trametinib is safer and more effective than BRAF inhibitor-based monotherapy for metastatic melanoma. Methods We retrospectively analyzed BRAF-mutated metastatic melanoma patients treated at our institution with daily oral dabrafenib 300 mg and trametinib 2 mg from November 2013 to April 2016. This clinical record included both untreated and previously treated stage IV melanomas. Physical examination and laboratory examinations were performed monthly and disease re-evaluations were performed every 3 months. Results A total of 48 patients (24 male, 24 female) with BRAF-mutated metastatic melanoma received dabrafenib and trametinib; median age was 48 years (range 23-75). Median follow-up was 362.5 days (range 72-879). Best overall response rate consisted of 6.2% (3 patients) complete response, 64.6% (31) partial response, and 25% ( 12 ) stable disease; median time to best response was 11 weeks (range 5.7-125.5). Progression of disease was seen in 19 patients (39.6%), with median time to progression (TTP) of 26 weeks (range 8-54). A total of 15 patients (31.2%) died due to progression of disease. Median progression-free survival and median overall survival were not reached. To date, 30 patients (62.5%) are still under treatment. A total of 27 (56.2%) patients had at least one adverse event (AE); grade 3-4 AEs were seen in 4 cases (8.3%). The main toxicities were fever (25%), skin rash (14.6%), arthralgias (10.4%), and aspartate aminotransferase/alanine aminotransferase increase (8.3%). Treatment dose was reduced in 7 subjects (14.6%), with only one case of discontinuation due to AE. Conclusions Our data, using combined targeted therapy, are in line with the scientific literature in terms of both safety and effectiveness in a real-life setting.

In melanoma, the adaptative cell response to BRAF inhibitors includes altered patterns of cytokine production contributing to tumor progression and drug resistance. Among the factors produced by PLX4032-resistant melanoma cell lines, CCL2 was higher compared to the sensitive parental cell lines and increased upon drug treatment. CCL2 acted as an autocrine growth factor for melanoma cells, stimulating the proliferation and resistance to apoptosis. In patients, CCL2 is detected in melanoma cells in tumors and in plasma at levels that correlate with tumor burden and lactate dehydrogenase. Vemurafenib treatment increased the CCL2 levels in plasma, whereas the long-term clinical response was associated with low CCL2 levels. Increased CCL2 production was associated with miRNA deregulation in the resistant cells. miR-34a, miR-100 and miR-125b showed high expression in both resistant cells and in tumor biopsies that were obtained from treated patients, and they were involved in the control of cell proliferation and apoptosis. Inhibition of CCL2 and of the selected miRNAs restored both the cell apoptosis and the drug efficacy in resistant melanoma cells. Therefore, CCL2 and miRNAs are potential prognostic factors and attractive targets for counteracting treatment resistance in metastatic melanoma.

ABSTRACT Aim: We describe the ad interim analysis of the Italian cohort of the global safety study on vemurafenib in patients with metastatic melanoma. Patients & methods: A total of 385 patients received vemurafenib 960 mg twice daily. Results: In total, 330 patients (86%) reported adverse events; 16 serious adverse events were observed (three related to vemurafenib). The response rate was 30.4%. Median progression-free survival (PFS) and overall survival (OS) were 5.9 months and 16.3 months, respectively. In patients with brain metastasis (BM; n = 83), median PFS was 4.3 months and OS was 7.6 months. In patients without BM, PFS was 6.5 months and OS was not reached. Median PFS was 12.6 months in patients with M1a stage of disease, 9.6 months in those with M1b stage and 5.4 months in subjects with M1c stage. Conclusion: Vemurafenib appears safe and active in clinical practice, and seems particularly active in patients without BM and low tumor burden.

To investigate the role of extracranial stereotactic body radiation therapy (SBRT) in the management of oligometastatic melanoma.Patients affected by Stage IV melanoma, with less than three extracranial metastatic lesions, who received SBRT were included in this analysis. Acute and late toxicity, local control (LC), overall survival (OS) and progression-free survival (PFS) were analysed.31 patients were included in the study. 16 patients (51.6%) were treated for lung meta-stases, 8 patients for liver metastases (25.8%) and 7 (22.6%) for nodal metastases. 38 lesions were irradiated. With a median follow-up time of 13 months, 11 patients (35.4%) were still alive, in four cases (12.9%) with no evidence of disease. Median OS was 10.6 months, and OS at 6, 12 and 24 months was 77, 41 and 21% respectively. LC at 12 and 24 months was 96.6 and 82.8%. 23 patients (74.2%) developed distant metastases. Median PFS was 5.8 months, and PFS at 6, 12 and 24 months was 48.2, 18.5 and 13.9% respectively. Number of irradiated lesions showed a statistically significant correlation only with LC (p = 0.03). Response of the irradiated lesion was related to OS (p = 0.019). Local response showed also a borderline correlation with PFS (p = 0.07).SBRT for extracranial metastases from melanoma is feasible and well tolerated. Response of the irradiated lesions is predictive of OS. Advances in knowledge: SBRT for melanoma extracranial metastases is feasible and the response of the irradiated lesions is predictive of OS.

e21001 Background: Efficacy of Vb in the treatment of mm has been established. We aimed to analyze characteristics of pts achieving LTR to Vb in order to identify potential predictive factors of prolonged benefit. Methods: We collected information about pts affected by BRAF V600 mutated mm treated with Vb at Medical Oncology Unit of Istituto Nazionale dei Tumori, Milan, Italy, from 09/2010 to 08/2014. LTR was defined as clinical or radiological benefit longer than 14 months (mos). Biological data about circulating lymphocyte subpopulations and CCL2 levels were also determined. Results: 46 pts (26 men, 20 women) were identified. Median age was 58 years. ECOG performance status was 0 in 44 pts. Disease stage was M1a in 14 pts, M1c in 20 pts. 8 pts had high serum LDH levels and 5 brain metastases at baseline. 41 pts harbored BRAF V600E mutation, 5 V600K. Median follow up was 47 mos. 5 pts had received adjuvant interferon; 9 pts had been treated with up to 3 lines for metastatic disease (either chemotx or ipilimumab). According to RECIST 1.1, 22% of pts showed complete response (CR), 50% partial response and 22% stable disease. Notably, all CRs were maintained for more than 2 years. 26 pts progressed, mostly (38%) with brain metastases; 16 of them continued tx beyond progression. Median duration of response was 33 mos, being significantly longer in M1a than in M1c (44 versus 28 mos). Median PFS was 32.5 mos; median OS was not reached. All pts experienced at least 1 adverse event (AE); the AEs were moderate or severe in 22% of cases. 19 pts underwent dose reduction, but no discontinuation due to AEs was observed. After 6 mos of tx, pts with LTRs had less myeloid derived suppressor cells (CD11b+ CD14+) and more memory Th1 cells (CD8+ CXCR3+) if compared with pts not bearing LTRs. The same pts after 1 mo of tx showed lower plasma levels of CCL2, a marker of acquired resistance to Vb. Conclusions: Vb can induce LTRs in a subset of pts with mm also in presence of unfavorable prognostic factors, though M1a pts are more likely to obtain prolonged benefit. Toxicity is frequent, but manageable with limited dose reductions. Exploratory biological analyses suggest an immunomodulatory effect of Vb and the existence of circulating biomarkers of LTR.

Background: Elderly patients with metastatic melanoma have different disease characteristics and a poorer prognosis than younger patients. Data from clinical trials and expanded access programmes (EAPs) suggest ipilimumab confers a consistent survival benefit and has a similar safety profile across different age groups of patients with metastatic melanoma. Here we report the efficacy and safety of ipilimumab 3 mg/kg in elderly patients enrolled in an EAP in Italy. Methods: Patients aged > 70 years with pretreated melanoma received ipilimumab 3 mg/kg every 3 weeks for four doses through an EAP. Tumour response was evaluated at baseline and after completion of induction therapy using immune-related response criteria and patients were monitored throughout the treatment period for adverse events (AEs), including immune-related AEs. Results: The immune-related disease control rate among 188 evaluable patients was 38%, including four patients with an immune-related complete response, 24 with an immune-related partial response and 44 with immune-related stable disease. Median progression-free survival (PFS) was 4.0 months and the 1- and 2-year PFS rates were 21% and 12%, respectively. Median overall survival (OS) was 8.9 months; 1- and 2-year OS rates were 38% and 22%, respectively. The safety profile of ipilimumab was consistent with that observed in the general population of the Italian EAP and treatment-related AEs generally resolved within a median of 2 weeks with treatment as per protocol-specific guidelines. Conclusions: These results suggest ipilimumab is a feasible treatment option in elderly patients with metastatic melanoma. Ipilimumab treatment was generally well tolerated and resulted in clinical benefit and extended survival in elderly patients treated at centres in Italy.

Background Mucosal melanoma is an extremely rare and aggressive malignancy that often remains undetected until it reaches an advanced stage, when effective treatment options are limited. The activity and safety of ipilimumab were assessed in an Expanded Access Programme (EAP) that included patients with metastatic, mucosal melanoma. Methods Ipilimumab was available upon physician request for patients aged ⩾16 years with stage III (unresectable) or IV skin, ocular or mucosal melanoma, who had failed or did not tolerate previous treatments and had no other therapeutic option available. Patients received ipilimumab 3 mg/kg every 3 weeks for four doses. Patients with stable disease or an objective response to ipilimumab were eligible for retreatment upon disease progression. Tumour assessments were conducted at baseline and week 12 using immune-related response criteria. Patients were monitored for adverse events (AEs), including immune-related AEs, within 3 to 4 days of each scheduled visit. Results Of 855 patients participating in the EAP in Italy, 71 (8%) had metastatic, mucosal melanoma. With a median follow-up of 21.8 months, the response rate was 12% and the immune-related disease control rate was 36%. Median progression-free survival and overall survival were 4.3 and 6.4 months, respectively. In total, 34% of patients reported treatment-related AEs of any grade, which were grade 3 or 4 in 9% of patients. AEs were generally manageable as per protocol-specific guidelines. Conclusion/interpretation Ipilimumab may be a feasible treatment option in pretreated patients with metastatic mucosal melanoma, and warrants further investigation in prospective clinical trials.

Background Ipilimumab improves survival in patients with advanced melanoma. The activity and safety of ipilimumab outside of a clinical trial was assessed in an expanded access programme (EAP). Methods Ipilimumab was available upon physician request for patients aged 16 or over with pretreated stage III (unresectable)/IV melanoma, for whom no other therapeutic option was available. Patients received ipilimumab 3 mg/kg every 3 weeks for four doses. Patients with stable disease or an objective response to ipilimumab were eligible for retreatment upon disease progression. Tumour assessments were conducted at baseline and week 12. Patients were monitored for adverse events (AEs) within 3 to 4 days of each scheduled visit. Results Of 855 patients participating in the EAP in Italy, 833 were evaluable for response. Of these, 13% had an objective immune response, and the immune-related disease control rate was 34%. Median progression-free survival and overall survival were 3.7 and 7.2 months, respectively. Efficacy was independent of BRAF and NRAS mutational status. Overall, 33% of patients reported an immune-related AE (irAE). The frequency of irAEs was not associated with response to ipilimumab. Conclusions Outside of a clinical trial setting, ipilimumab is a feasible treatment option in patients with pretreated metastatic melanoma, regardless of BRAF and NRAS mutational status. Data from this large cohort of patients support clinical trial evidence that ipilimumab can induce durable disease control and long-term survival in patients who have failed to respond to prior treatment.

e21009Background: Ipilimumab is an inhibitor of CTLA4 receptor of T lymphocytes that has been approved by FDA both as first and second line treatment for patients with metastatic melanoma. Despite ...

Purpose: To assess the clinical and biological activity of the association of bevacizumab and fotemustine as first-line treatment in advanced melanoma patients. Experimental Design: Previously untreated, metastatic melanoma patients (n = 20) received bevacizumab (at 15 mg/kg every 3 weeks) and fotemustine (100 mg/m2 by intravenous administration on days 1, 8, and 15, repeated after 4 weeks) in a multicenter, single-arm, open-label, phase II study. Primary endpoint was the best overall response rate; other endpoints were toxicity, time to progression (TTP), and overall survival (OS). Serum cytokines, angiogenesis, and lymphangiogenesis factors were monitored by multiplex arrays and by in vitro angiogenesis assays. Effects of fotemustine on melanoma cells, in vitro, on vascular endothelial growth factor (VEGF)-C release and apoptosis were assessed by ELISA and flow cytometry, respectively. Results: One complete response, 2 partial responses (PR), and 10 patients with stable disease were observed. TTP and OS were 8.3 and 20.5 months, respectively. Fourteen patients experienced adverse events of toxicity grade 3–4. Serum VEGF-A levels in evaluated patients (n = 15) and overall serum proangiogenic activity were significantly inhibited. A significant reduction in VEGF-C levels was found in several post-versus pretherapy serum samples. In vitro, fotemustine inhibited VEGF-C release by melanoma cells without inducing significant cell death. Serum levels of interleukin (IL)-10 and IL-12p70 showed the highest levels in sera of PR patients, compared with patients with stable or progressive disease whereas IL-23 showed the opposite pattern. Conclusions: The combination of bevacizumab plus fotemustine has clinical activity in advanced melanoma and promotes systemic modulation of angiogenesis and lymphangiogenesis factors. Clin Cancer Res; 16(23); 5862–72. ©2010 AACR.

9033 Background: Various ocular adverse events (AE) have been described with targeted agents. MEK inhibitors (MEKi) are associated with SR. We describe the clinical features of SR observed with COB...

Exosomes are endosome-derived nanovesicles involved in intercellular cross-talk through the transfer of proteins and genetic material. In cancer, exosomes can contribute to mould host micro- and macroenvironment, conditioning tumour immunity both at local and systemic level. We have been collecting evidence that these nanovesicles exert a broad array of detrimental effects on the immune system, ranging from apoptosis in activated antitumour T cells to impairment of monocyte differentiation into dendritic cells and induction of suppressive effectors. More recently, we observed that cells with phenotypic and functional properties overlapping with those displayed by myeloid-derived suppressor cells (MDSC) can be generated in vitro by culturing normal monocytes with exosomes released by melanoma cells (Exo-MDSC). Compared to untreated monocytes, Exo-MDSC show increased mRNA level and release of protumourigenic and immunosuppressive cyto/chemokines, down-modulate the expression of HLA-DR and TLRs, and acquire the ability to inhibit T cell proliferation. A defined gene-expression and miRNA signature, involving HIF1alfa, IL6 and several immune-related genes, has been identified in Exo-MDSC as triggered by exosomal TGFb, CCL2 and selected miRNA. Given their ability to efficiently recirculate in body fluids, tumour exosomes could spread systemically and directly condition myelopoiesis. This hypothesis is supported by the evidence that peripheral MDSC from melanoma patients share defined traits of Exo-MDSC gene-expression and miRNA signature, in addition with phenotypic and functional features of these cells. MDSC accumulate in peripheral blood of melanoma patients as an early event and in association with disease progression. High MDSC frequency is an acknowledged negative prognostic factor in several tumour histologies and predicts poor response to immunotherapy, including checkpoint blockade. The identification of exosomes as a potential pathway responsible for MDSC mobilisation and accrual paves the way to the development of novel immune-based therapeutic strategies and prognostic biomarkers in cancer patients. Supported by Italian Association For Cancer Research – AIRC Grant code 12162.

Melanoma immunotherapy based on immune checkpoint blockade, by targeting of CTLA-4 or PD-1, has markedly improved the treatment of advanced disease, as shown by several recent trials. Monotherapy with CTLA-4-specific antibody (Ipilimumab) prolongs overall survival, while PD-1-specific antibodies (Nivolumab and Lambrolizumab) induce durable tumor regression in a large fraction of patients. Moreover, a recent trial based on combination checkpoint blockade (Ipilimumab plus Nivolumab) has shown an objective response rate of 53%, with tumor reduction of 80% in responding patients, without escalation of toxic effects. In spite of these highly relevant results, clinical benefit by immunotherapy targeting CTLA-4 or PD-1 remains limited to a fraction of patients. This points to the existence of immune escape mechanisms, which, in some tumors, may prevent effective elimination of neoplastic cells, in spite of the reactivated T cell immunity. This hypothesis was tested in a melanoma patient, treated with Ipilimumab, and that we recently reported (J. Clin. Oncol. 29:e783-8, 2011) to have a strong T cell infiltrate and evidence of T cell maturation/activation only in a regressing right adrenal metastasis, but not in the progressing controlateral lesion. To this end, we carried out DASL whole genome gene expression analysis and extensive immunohistochemistry (IHC) on paraffin-embedded neoplastic tissues. The results indicated that, in comparison to the progressing metastasis, the regressing lesion had a distinct gene expression profile characterized by enhanced expression of several genes involved in T cell differentiation to cytolytic effector stage. Most importantly, the regressing lesion retained expression of HLA molecules on neoplastic cells. In contrast, the progressing lesion showed almost complete loss of HLA class I and Class II molecules by the tumor, as documented by IHC. Taken together, these results suggest that lack/loss of expression of HLA molecules, by tumor cells, may represent a highly effective tumor resistance mechanism to immune checkpoint blockade. Moreover, pre-therapy IHC on tumor samples may allow to test whether HLA antigen expression/loss is a predictive marker for response/resistance to therapy targeting CTLA-4 or PD-1. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A89. Citation Format: Andrea Anichini, Filippo G. de Braud, Roberta Montarini, Ilaria Bersani, Gabrina Tragni, Loris de Cecco, Silvana Canevari, Lorenza Di Guardo, Lorenzo Pilla, Michele Del Vecchio. Loss of HLA molecules as melanoma resistance mechanism in immune checkpoint blockade therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A89.

9092 Background: To select melanoma patients for treatment with BRAF inhibitors, the BRAF mutational status is determined in the most recent tumor biopsy. However, tumor specimens are not always available for the analysis, and relying on a single biopsy specimen can potentially exclude from treatment patients with heterogeneity among metastatic tumors due to polyclonality of BRAF mutation, which appears as a rather common condition. As an alternative approach we explored a blood-based mutation detection assay. Methods: We developed a method including enrichment for the BRAV600E variant by selective elimination of the wild type allele by TspRI digestion and BRAFV600E detection by TaqMan Mutation Detection Assay (BRAF_476_mu, Life Technologies). Sensitivity testing showed that BRAFV600E variant was detected starting from 6.25X10-5 ng of DNA, and specificity testing showed that the variant can be detected when diluted in 8X105 copies of wild-type alleles. Results: Mutational analysis performed by Sanger sequencing of exon15 in 114 melanoma biopsies showed that 4 (3%) harbored the c.1798_1799GT>AA (V600K) mutation, 1 (1%) the c.1799_1800TG>AA mutation (V600E), and 56 (49%) the most common c.1799T>A mutation (V600E), while by the novel method the latter mutation was detected in 9 additional specimens (8% increment) and confirmed by sequencing the PCR product after TspRI digestion. Pre-surgery plasma was available for 50/114 patients at advanced stages, 26/50 (52%) showing a mutated specimen, including 4 with a double nucleotide substitution (V600K and V600E). The matched plasma samples resulted mutated in 25/26 cases with mutated biopsy and in further 14/25 samples with biopsy resulting wild type; in contrast, plasma of 50 healthy controls tested negative. Taken together, these results indicate that V600E circulating variant was detectable in 39/50 (78%) plasma samples and in 22/50 (44%) tumor specimens. Conclusions: Detection of BRAF V600E variant in circulating free DNA may represent a more sensitive approach for patient selection and decision making process during the treatment with BRAF inhibitors.

Aims and background Carcinoids are infrequent neoplasms arising from neuroendocrine cells. Due to blurred symptoms and the presence of equivocal diagnostic findings, these tumors are sometimes misdiagnosed. Therefore, increased rates of false neuroendocrine tumors represent an emerging problem in clinical practice. Our aim is to alert clinicians on this matter by supplying them with useful warnings. Methods In the specialized neuroendocrine tumor study Center Centro di Riferimento per lo Studio e la Cura dei Carcinoidi e dei Tumori Neuroendocrini (Ce.Ri.Ca), some patients highly suspected to have a neuroendocrine tumor have been recognized as having false neuroendocrine tumors. The related clinical and instrumental findings leading to a previous wrong neuroendocrine tumor diagnosis are reported. Results From July 2006 to December 2008, 88 consecutive cases of neuroendocrine tumors (Nets) were referred at Ce.Ri.Ca. In the former group, 8 cases of false Nets were discovered while in the remaining 80 cases a correct Net diagnosis was carried out. Watchful differential diagnoses and skill appraisal of laboratory investigations resulted in: chronic atrophic gastritis with enterochromaffin-like cell hyperplasia (4 cases), estrogen-deprivation syndrome (1), hypochondriac disorder (1), metabolic syndrome (1), and sarcoidosis (1). Conclusions Neuroendocrine tumors are still relatively known clinical entities. To discriminate false neuroendocrine tumors from neuroendocrine tumors requires a good expertise and a large daily practice with the disease. Good knowledge and skillfulness in identifying biochemical alterations and false radiological positive results could avoid both patient inconvenience and very expensive workup. The importance of a multidisciplinary approach in specialized centers is emphasized.