European e-Delphi process to define expert consensus on electrochemotherapy treatment indications, procedural aspects, and quality indicators in melanoma.

BACKGROUND: Skin metastases are an important co-morbidity in melanoma. Despite broad adoption, electrochemotherapy implementation is hindered by a lack of treatment indications, uncertainty regarding procedural aspects, and the absence of quality indicators. An expert consensus may harmonize the approach among centres and facilitate comparison with other therapies. METHODS: An interdisciplinary panel was recruited for a three-round e-Delphi survey. A literature-based 113-item questionnaire was proposed to 160 professionals from 53 European centres. Participants rated each item for relevance and degree of agreement on a five-point Likert scale, and received anonymous controlled feedback to allow revision. The items that reached concordant agreement in two successive iterations were included in the final consensus list. In the third round, quality indicator benchmarks were defined using a real-time Delphi method. RESULTS: The initial working group included 122 respondents, of whom 100 (82 per cent) completed the first round, thus qualifying for inclusion in the expert panel (49 surgeons, 29 dermatologists, 15 medical oncologists, three radiotherapists, two nurse specialists, two clinician scientists). The completion rate was 97 per cent (97 of 100) and 93 per cent (90 of 97) in the second and third rounds respectively. The final consensus list included 54 statements with benchmarks (treatment indications, (37); procedural aspects, (1); quality indicators, (16)). CONCLUSION: An expert panel achieved consensus on the use of electrochemotherapy in melanoma, with a core set of statements providing general direction to electrochemotherapy users to refine indications, align clinical practices, and promote quality assurance programmes and local audits. The residual controversial topics set future research priorities to improve patient care.

Electrochemotherapy is an effective locoregional therapy for skin metastases from melanoma, a problem faced by almost half of patients with metastatic disease. The lack of comparative studies and the heterogeneity of its clinical application among centres make it challenging to support consistent, evidence-based recommendations. To address this unmet need, a three-round online survey was conducted to establish a consensus on treatment indications, standard operating procedures, and quality indicators. In the survey, a panel of 100 European melanoma experts agreed on 56 statements that can be used to improve patient selection, homogenize treatment application, and monitor outcomes.

Tolerogenic IDO1+CD83- Langerhans Cells in Sentinel Lymph Nodes of Patients with Melanoma.

Langerhans cells (LCs) are crucial regulators of anti-cancer immune responses. Cancer, however, can alter DCs functions leading to tolerance. The enzyme indoleamine 2,3-dioxygenase (IDO1) plays a crucial role in this process. In sentinel lymph nodes (SLNs) of patients with melanoma, LCs show phenotypical and functional alterations favoring tolerance. Herein we aimed to investigate IDO1 expression in SLN LCs from patients with melanoma. We showed by immunofluorescence analysis that a portion of Langerin+ LCs, located in the SLN T cell-rich area, displayed the typical dendritic morphology and expressed IDO1. There was no significant difference in the expression of IDO between SLN with or without metastases. Double IDO1/CD83 staining identified four LCs subsets: real mature IDO1−CD83+ LCs; real immature IDO1−CD83− LCs; tolerogenic mature IDO1+CD83+ LCs; tolerogenic immature IDO1+CD83− LCs. The latter subset was significantly increased in metastatic SLNs as compared to negative ones (p < 0.05), and in SLN LCs of patients with mitotic rate (MR) > 1 in primary melanoma, as compared to MR ≤ 1 (p < 0.05). Finally, immature SLN LCs, after in vitro stimulation by inflammatory cytokines, acquired a maturation profile by CD83 up-regulation. These results provide new input for immunotherapeutic approaches targeting in vivo LC of patients with melanoma.

High Antigen Processing Machinery component expression in Langerhans cells from melanoma patients' sentinel lymph nodes.

Langerhans cells (LCs) from melanoma patients sentinel lymph nodes (SLN) are poor T cell activators mostly due to an immature immunophenotype. However Antigen Presenting Machinery (APM) role is unknown. We investigated HLA-class I APM components (Delta, LMP-7/10, TAP-1, Calnexin, Tapasin, ß2-microglobulin and HLA-A,B,C) in LCs from healthy donors skin and melanoma patients SLN. APM component levels were low in immature epidermal LCs and significantly increased after maturation (p<0.05); their levels were significantly high in SLN LCs (p<0.01). APM component expression correlated with melanoma Breslow's thickness and SLN metastases: HLA-A,B,C level was significantly lower in SLN LCs from thick lesions patients compared with those from thin/intermediate lesions (p<0.05); ß2-microglobulin level was significantly higher in positive SLN LCs compared to negative ones (p<0.05). Functionally, SLN LCs did not phagocytose exogenous antigens. These findings extend LCs knowledge indicating that they are not fully impaired by melanoma, contributing to design new LCs-based therapeutic approaches.

A rare case of anal porocarcinoma treated by electrochemotherapy.

We report the case of an old woman with an eccrine porocarcinoma unusually localized in the perianal area treated by electrochemotherapy, a new technique, emerging as a very effective local treatment of different skin metastases and selected primary tumors. Electrochemotherapy was performed taking into account patient wishes and refusal of demolitive surgery. The electrochemotherapy treatment was well tolerated by the patient, it gave an excellent clinical response and a complete clinical regression with no sphincter dysfunction and signs of relapse observed during follow-up. The case is of particular interest for the exceptional localization of porocarcinoma for the first time treated by electrochemotherapy in this area. Electrochemotherapy could be considered as an alternative option for selected cases of cutaneous tumors.

Langerhans Cells in Sentinel Lymph Nodes from Melanoma Patients.

BACKGROUND: Langerhans cells (LCs) are professional Dendritic Cells (DCs) involved in immunoregulatory functions. At the skin level, LCs are immature. In response to tissue injuries, they migrate to regional Lymph Nodes (LNs), reaching a full maturation state. Then, they become effective antigen-presenting cells (APCs) that induce anti-cancer responses. Notably, melanoma patients present several DC alterations in the Sentinel Lymph Node (SLN), where primary antitumoral immunity is generated. LCs are the most represented DCs subset in melanoma SLNs and are expected to play a key role in the anti-melanoma response. With this paper, we aim to review the current knowledge and future perspectives regarding LCs and melanoma. METHODS: A systematic review was carried out according to the PRISMA statement using the PubMed (MEDLINE) library from January 2004 to January 2024, searching for original studies discussing LC in melanoma. RESULTS: The final synthesis included 15 articles. Several papers revealed significant LCs-melanoma interactions. CONCLUSIONS: Melanoma immune escape mechanisms include SLN LC alterations, favoring LN metastasis arrival/homing and melanoma proliferation. The SLN LCs of melanoma patients are defective but not irreversibly, and their function may be restored by appropriate stimuli. Thus, LCs represent a promising target for future immunotherapeutic strategies and cancer vaccines.

Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.

The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDH(high)), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH(low) population. A good correlation between the number of ALDH(high) cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDH(high) melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH(high) melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment.

The importance of mitosis ≥2 in selecting patients with T1 cutaneous melanomas for sentinel lymph node biopsy.

The 8th Edition of the American Joint Committee on Cancer (AJCC) Staging Manual removed the mitotic rate (MR) as a staging criterion for T1 melanomas, thus leading to a debate on sentinel lymph node biopsy (SLNB) in thin melanomas. This study investigates whether MR plays a role in selecting patients with T1 melanoma for SLNB. We analyzed clinical and histological data from the Florence Melanoma & Skin Cancer Unit database for 313 patients with a single thin melanoma who had undergone SLNB. We determined sentinel lymph node (SLN) positivity percentages in T1 melanomas according to the AJCC 8th Edition focusing on MR. Of the 313 T1 patients, 108 had MR = 0, 127 had MR = 1 and 78 had MR ≥2. The overall SLN positivity rate was 8.6%, (5.6% with MR = 0, 6.3% with MR = 1 and 16.7% with MR ≥2). The SLNB positivity rate in T1b melanomas was 12.1%, (8.5% with MR = 0, 5.7% with MR = 1 and 24.4% with MR ≥2), whereas in T1a melanomas it was 5.8%, (3.3% with MR = 0, 6.8% with MR = 1 and 8.1% with MR ≥2). In a logistic regression analysis, MR ≥2 had an odds ratio of almost three in comparison with MR = 0/1 also adjusting for thickness. Thus, MR ≥2 significantly predicted SLN metastases in T1 melanomas. Of those patients with positive SLN, 37% were classified as T1a according to the AJCC 8th edition. These findings underline the importance of MR ≥2 in selecting patients with T1 cutaneous melanomas for SLNB.

"Saddle" tailored upper eyelid island myocutaneous flap to repair full-thickness lower eyelid defects after melanoma excision.

PURPOSE: To perform an early melanoma diagnosis and to repair the full-thickness lower eyelid defect with an island upper eyelid myocutaneous flap tailored into a new shape. METHODS: Two patients with pigmented lesion involving skin and tarsus of the lower eyelid were reported. Histologic examination, performed after diagnostic punch biopsy, confirmed the diagnosis of in situ melanomas in both cases. A full-thickness excision was done and a single pedicle island myocutaneous flap from the upper eyelid was performed. The flap was designed in a blepharoplastic manner and tunnelized to reach the lower eyelid defect. The flap was tailored into a "saddle" shape, doubled, and folded to restore both the internal and external eyelid walls in a single-stage procedure. RESULTS: Good functional and aesthetic results were obtained with no complications. Interestingly enough, the tissue of the internal layer lost the features of skin epithelium due to metaplasia processes and appeared similar to the conjunctiva. After 3 years, no sign of melanoma recurrence was noted. CONCLUSIONS: Early diagnosis was performed in both reported lower eyelid melanoma cases. For the reconstruction, a modified upper eyelid island myocutaneous flap tailored into a "saddle" shape was used, which had the advantages of being a single-stage procedure and avoiding mucosa grafts. The technique could also be used to repair full-thickness lower eyelid defects from other causes.

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha.

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.

Efficacy of Electrochemotherapy in the Treatment of Cutaneous Melanoma Metastases and Rare Non-melanoma Skin Cancer.

BACKGROUND: We aimed to verify the clinical efficacy and safety of the electrochemotherapy in melanoma metastases and in cases of rare non-melanoma tumors that were difficult to treat for the specific anatomical site or for patient comorbidities. PATIENTS AND METHODS: We treated 68 patients (699 cutaneous nodules), 44 patients with metastatic melanomas and 24 patients with non-melanoma tumors, at the Melanoma & Skin Cancer Unit, Florence, Italy. RESULTS: We obtained an objective response of 89.7% (88.6% in melanomas and 91.7% in non-melanoma tumors), complete response 54.4% and partial response 35.3%. CONCLUSION: This study showed that electrochemotherapy is effective in the treatment of melanoma metastases and in rare types of non-melanoma tumors. In particular, we successfully treated rare tumors as angiosarcoma, pleomorphic sarcoma, myxofibrosarcoma, sarcoma di Kaposi, porocarcinoma, sebaceous carcinoma, Merkel cell carcinoma, malignant blue nevus, undifferentiated epitheliomorphic cell neoplasia and metastases from thyroid carcinoma. No serious adverse events were observed.

Correction to: Thick melanoma in Tuscany.

This corrects the article DOI: 10.23736/S0392-0488.17.05584-5.

Enhancing the prognostic role of melanoma sentinel lymph nodes through microscopic tumour burden characterization: clinical usefulness in patients who do not undergo complete lymph node dissection.

This study aimed to investigate the sentinel lymph node (SLN) tumour burden to predict the non-SLN positivity rate and the survival of melanoma patients to evaluate whether SLN microstaging could predict the prognosis, similar to what is currently performed by examining the lymph nodes excised by complete lymph node dissection. Of 1130 consecutive melanoma patients who underwent SLN biopsy, 226 were tumour-positive and 204 were included in this study. SLN metastases were classified on the basis of dimensional (Rotterdam) and topographic (Dewar) criteria either separately or combined. SLN metastases more than 1 mm in diameter had the highest non-SLN positivity rate (31%) compared with metastases 0.1-1 mm (10%) and less than 0.1 mm (4%). The non-SLN positivity rate was 45% for extensive metastases, 5% for subcapsular metastases and 23-29% for parenchymal, combined and multifocal classes, therefore suggesting a simplification of the parenchymal SLN metastases into only two classes: extensive and 'not extensive'. The dimension of the metastasis was correlated with a different non-SLN positivity rate only when the metastasis was in the parenchyma (20-36%) and not when it was in the subcapsular location (4-7%). Interestingly, the 5-year melanoma-specific survival (MSS) was 89% for patients with subcapsular less than 0.1 mm metastases and 45% for patients with nonsubcapsular more than 1 mm metastases (P=0.017). In the parenchyma, larger metastases (>1 mm) were related to a lower 5-year MSS (46%) than smaller (<1 mm) metastases (MSS 77%). SLN tumour burden characterization can be simplified and it can provide prognostic information on non-SLN positivity and survival, which is especially useful in patients who do not undergo complete lymph node dissection.

Thick melanoma in Tuscany.

BACKGROUND: The epidemiologic trends of cutaneous melanoma are similar in several countries with a Western-type lifestyle, where there is a progressively increasing incidence and a low but not decreasing mortality - even increasing in selected cases, especially in the older age groups. Also in Tuscany there is a steady rise in the incidence with prevalence of in situ and invasive thin melanomas, with also an increase of thick melanomas. It is necessary to reduce the frequency of thick melanomas to reduce specific mortality. The objective of the current survey has been to compare, in the Tuscany population, by a case-case study, thin and thick melanoma cases, trying to find out those personal and tumor characteristics which may help to customize preventive interventions. METHODS: The study included nine centers involved in the melanoma diagnosis. A consecutive series of incident invasive melanomas diagnosed in a period of about 18 months (July 2010 to December 2011) was collected and matched according in a ratio of one thick melanoma (cutoff thickness: 1 mm) every two thin melanomas. The investigators filled in a questionnaire on patients' self-reported sun exposure, way of melanoma detection, awareness and performance of self-skin examination, as well as propensity to prevention in general. RESULTS: The results of this survey confirm that older age and the lower education level are associated with a later detection. The habit of performing skin self-examination is crucial in the early diagnosis of thick melanoma. The results of this survey seem to suggest that population aged over 50 years, with few total and few atypical nevi, and limited sun exposure and burning are at higher risk of late diagnosis. It can be assumed that part of the population is not effectively reached by prevention campaigns because they do not recognize themselves as being at risk for skin cancers. CONCLUSIONS: In order to achieve a higher rate of early diagnosis of skin melanoma, a new strategy must be implemented. It could be useful to rethink educational campaigns - which seem to unintentionally leave out subjects more at risk for melanoma - and to renew the active involvement of the general practitioners.

Stem cell factor affects tumour progression markers in metastatic melanoma cells.

Stem cell factor (SCF), next to various relevant biological effects exerted on many cell types, is able to keep melanocyte homeostasis through its receptor c-kit. Only a minority of metastatic melanoma cells (MMC) express c-kit receptor, but c-kit positive MMC move more slowly towards tumour progression and have a more natural tendency to undergo apoptosis. In our study c-kit positive MMC from human melanoma metastases and a c-kit positive human melanoma cell line-SK-MEL-28-showed a clear-cut reduction of cytokines normally up-regulated along melanoma progression after SCF stimulation. SCF was also able to maintain all MMC and SK-MEL-28 cells in a well differentiated status with an increase in organellogenesis and in particular of melanosomes in various degree of differentiation, but it did not induce apoptosis as observed in other in vitro models. The increase of melanosomes matched an increase of tyrosinase production. SCF did not modify the expression of NOS while it enhanced the expression of HLA-DR molecules on MMC membranes. Taken altogether these data stress the biological activity of SCF as a cytokine which is able to maintain MMC in a well differentiated status, and suggest a more in depth evaluation of possible effects of SCF on melanoma cells.

Down-Regulation of SOX2 Underlies the Inhibitory Effects of the Triphenylmethane Gentian Violet on Melanoma Cell Self-Renewal and Survival.

Human melanomas contain a population of tumor-initiating cells that are able to maintain the growth of the tumor. We previously showed that the embryonic transcription factor SOX2 is essential for self-renewal and tumorigenicity of human melanoma-initiating cells. However, targeting a transcription factor is still challenging. Gentian violet (GV) is a cationic triphenylmethane dye with potent antifungal and antibacterial activity. Recently, a combination therapy of imiquimod and GV has shown an inhibitory effect against melanoma metastases. Whether and how GV affects melanoma cells remains unknown. Here we show that GV represses melanoma stem cell self-renewal through inhibition of SOX2. Mechanistically, GV hinders EGFR activation and inhibits the signal transducer and activator of transcription-3 [(STAT3)/SOX2] axis. Importantly, we show that GV treatment decreases STAT3 phosphorylation at residue tyrosine 705, thus preventing the translocation of STAT3 into the nucleus and its binding to SOX2 promoter. In addition, GV affects melanoma cell growth by promoting mitochondrial apoptosis and G2 cell cycle arrest. This study shows that in melanoma, GV affects both the stem cell and the tumor bulk compartments, suggesting the potential use of GV in treating human melanoma alone or in combination with targeted therapy and/or immunotherapy.

Thin and thick primary cutaneous melanomas reveal distinct patterns of somatic copy number alterations.

Cutaneous melanoma is one of the most aggressive type of skin tumor. Early stage melanoma can be often cured by surgery; therefore current management guidelines dictate a different approach for thin (<1mm) versus thick (>4mm) melanomas. We have carried out whole-exome sequencing in 5 thin and 5 thick fresh-frozen primary cutaneous melanomas. Unsupervised hierarchical clustering analysis of somatic copy number alterations (SCNAs) identified two groups corresponding to thin and thick melanomas. The most striking difference between them was the much greater abundance of SCNAs in thick melanomas, whereas mutation frequency did not significantly change between the two groups. We found novel mutations and focal SCNAs in genes that are embryonic regulators of axon guidance, predominantly in thick melanomas. Analysis of publicly available microarray datasets provided further support for a potential role of Ephrin receptors in melanoma progression. In addition, we have identified a set of SCNAs, including amplification of BRAF and ofthe epigenetic modifier EZH2, that are specific for the group of thick melanomas that developed metastasis during the follow-up. Our data suggest that mutations occur early during melanoma development, whereas SCNAs might be involved in melanoma progression.

Skin cancer and immunosuppression.

All immunosuppressive treatments, either pharmacological or physical, have the potential to impair the skin immune system network of cells and cytokines, thus leading to an increased incidence of skin cancer. Since skin cancer in transplant recipients may show uncommon clinical features and have an unusually aggressive course, transplant patients should be strictly followed up by experienced dermatologists in order to diagnose and treat properly any skin cancer in an early phase. Importantly, due to the fact that sun exposure increases immunosuppression in the skin, patients should be clearly informed about the additional risk of sun exposure and the preventive measures to be taken.