In the last 10 years, the treatment of metastatic cutaneous melanoma has changed dramatically. The new systemic treatment with immunotherapy has led to a dramatic improvement in quality of life and overall survival. Systemic treatment means that the patient receives the drug as an infusion into a vein. Unfortunately, investigators know that immunotherapy is not equally successful in all patients. Recent studies have shown that the success of the treatment is not only influenced by the cellular composition of the metastasis, but also by its surroundings. This is called tumor microenvironment. Depending on the differences in the composition of this microenvironment, some metastases can be described as immunologically hot and others as immunologically cold. Immunologically hot metastases respond better to immunotherapy than immunologically cold metastases.

Studies have shown that with some interventions can change the tumor microenvironment from being immune-cold to being immune-hot. Electrochemotherapy is one of the interventions that might improve the efficacy of immunotherapy in cutaneous melanoma. Electrochemotherapy is an established method for the local treatment of tumors, in which only a certain tumor is treated with special electrodes, to which a weak electric current is applied. Investigators hypothesize that electrochemotherapy stimulates the body's own immune response and enables more effective treatment. Since immunotherapy also stimulates the body's own immune response to cutaneous melanoma cells, the interaction of the two drugs could be even more successful. Recent research results support this assumption.

The primary objective is to evaluate the changes in the tumor microenvironment of cutaneous and subcutaneous melanoma metastases induced by electrochemotherapy, based on the histologic analysis of treated and untreated metastases before and after treatment. The secondary aim is to determine whether the changes in the tumor microenvironment differ depending on the chemotherapeutic agent used.

The results will help Investigators better understand the synergistic effects of electrochemotherapy and immunotherapy on cutaneous melanoma metastases. The combination of systemic immunotherapy and electrochemotherapy could become an important treatment method for patients with metastatic melanoma.

The study is prospective. The primary objective is to evaluate the changes in the tumor microenvironment of cutaneous and subcutaneous melanoma metastases induced by electrochemotherapy (ECT), based on the histologic analysis of treated and untreated metastases before and after treatment. The secondary aim is to determine whether the changes in the tumor microenvironment differ depending on the chemotherapeutic agent used.

In the study 10-15 patients will be enrolled and devided in two arms, ECT with bleomycin and ECT with cysplatin.

ECT will be offered to patients with cuteaneous melanoma and at least 5 in-transit or distant cutaneous and/or subcutaneous melanoma metastases regardless of previous treatments. The decision will be made in a multidisciplinary tumor board. The choice of chemotherapeuthic drug will depend on the size and number of lesions to be treated. Inclusion in the study has no influence on the decision regarding the timing of treatment with immunotherapy. Treatment with immunotherapy will later be included as a factor in the statistical analysis.

ECT will be performed according to the standard operating procedures for the treatment of cutaneous and subcutaneous tumors with ECT. ECT will be performed within 8 - 28 minutes after intravenous bolus administration of bleomycin (15.000 IU/m2 BSA) or directly after the intratumorally administration of cysplatin (0,5-2 mg/cm3 tumor). CliniporatorTM (IGEA S.P.A., Carpi, Italy) will be used to apply the pulses (8 pulses, 1300 V/cm, 100 μs, 5 kHz). Triggering of the electrical pulses will be synchronized with ECG signals, through the ECG triggering device AccuSync to avoid delivery of pulses in vulnerable period of the heart. The type of electrode used will be selected according to the size and location of the tumors.

One cutaneous/subcutaneous metastasis will be excised before ECT. One treated cutaneous/subcutaneous metastasis will be excised 2-4 and 9-13 days after the procedure. An untreated cutaneous/subcutaneous metastasis will be excised on day 9-13. The excisions will be performed under local anesthesia. All patients will be enrolled in the study after the procedures and the study have been explained to them in detail and they have signed an informed consent form. A venous blood sample will be taken at the same time points (before ECT, 2-4 days and 9-13 days after ECT).

Histological examination, assessment of the degree of regression and the presence of tumor infiltrating lymphocytes (TIL) will be performed according to standardized procedures on 2-3 μm thick tissue sections, previously fixed in formalin and embedded in paraffin (FFPE), stained with the hematoxylin-eosin (HE) staining method.

Immunohistochemical characterization of the tumor microenvironment will be performed on 2-4 μm thick tissue sections pre-fixed in formalin and embedded in paraffin. Investigators will use commercially available primary monoclonal antibodies to define the tumor inflammatory infiltrate, stroma and vasculature. We will use the following antibodies: CD3, CD4, CD8, CD56, CD163, FoxP3, ERG, PGM1, CD274 (PD-L1). The choice of antibodies used and the method of pathohistologic analysis may change depending on the results. Specific binding of primary antibodies will be visualized using the recommended three-step detection system OptiView DAB IHC Detection Kit (Cat. No. 760-700; manufactured by Ventana ROCHE inc., Tucson, AZ, USA) according to the manufacturer's instructions. The analysis will be performed by two independent pathologists.

Investigators will also collect the information about previous treatments for cutaneous melanoma and photographic documentation of the effectiveness of ECT treatment. Patients will also fill out internationally recognized, validated quality of life questionnaires (EORTC QLQ-C 30 and EQ-5D-5L) at entollment, after ECT, 3 months, 6 months and 12 months after ECT and then once a year during the follow-up period.

Inclusion Criteria:

• more than 4 cytologically and/or histologically confirmed intransit or distant cutaneous/subcutaneous cutaneous melanoma metastases
• ECT should be proposed as a treatment in the multidisciplinary tumor board
• cutaneous/subcutaneous melanoma metastases, that can be excised under local anesthesia with primary wound closure, minimal morbidity of the procedure (risk of complications < 5%) and nocosmetic or functional consequences of the procedure
• stage IIIB, IIIC or IV of the disease
• age over 18 years
• performance status World Health Organization more than 2
• patients must give informed consent

Exclusion Criteria:

• age less than 18 years
• polimorbidity
• performance status World Health Organization more than 2
• high risk for intervention under general anesthesia;
• wound closure would require coverage with a skin graft or local flap;
• undesirable cosmetic or functional consequences would be expected (face, extensor side of joints)
• patients incapable of understanding the aim of the study or disagree with the entering into the clinical study