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188 Background: Systemic anti-PD1 treatment is major advantage for metastatic melanoma patients over the last five years. Nevertheless up to the moment there are no solid predictive factors discovered. On the other hand immune related adverse events develop quite frequent and sometimes could be dangerous. Methods: Patient with advanced cutaneous melanoma with known BRAF status and at least one superficial lesion (suitable for injection) are eligible. One of two commercially available anti-PD1 drugs (nivolumab or pembrolizumab) is administered intralesionaly to patients every 2 weeks with total daily dose not more than 50 mg (5 ml) using a scale-base approach depending on lesions size. Clinical and immunological outcomes assessed every 12 weeks. For assessing lymphoid infiltrate tumor biopsy performed before treatment and on week 12. To detect a systemic objective response rate of 50% with sensitivity of 80% and error type alfa 5% at least 25 patients to be included. Interim analysis was preplanned when 3 (25%) of events occurred. Results: Among 7 pts OR was detected in 4 (54.7%), PD in 3 (42.9%). Bystander effect was seen in 2 of 4 patients (50%). Among 3 non-responders 2 failed to salvage systemic anti-PD1 treatment and one is now on anti-CTLA4. The most common adverse event was injection site pain, which was self-limiting. Baseline immune phenotype of tumor infiltrating lymphocytes (TILs) was available for 5 of 7 pts included. Baseline percent of TILs, CD3+CD8+ and CD8+CD279+ was higher in responders vs non-responders (3.6±4.08% vs 0.8±0, NS, 75.1±20.6% vs 54.8±1.13, NS; 36.75±18.7% vs 42.8±13.1, NS respectively ). Baseline difference in CD11b+CD28- between responders (7.06±7.768) and non-responders (25.0 ±0.00) was statistically significant. Conclusions: these results demonstrates feasibility, tolerability and potential clinical efficacy of intralesional anti-PD1 in melanoma patients. Further study are needed to determine if unsuccessful intralesional administration could predict failure to systemic treatment as well as TILs phenotype could predict intralesional or systemic anti-PD1 clincal benefit Clinical trial information: MEL004IL.

Introduction. The increasing clinical use of cellular technologies suggests the possibility of long-term storage of the cellular product while maintaining its viability and basic properties. The procedures of cell’s cryopreservation used in laboratory as well in clinical practice differ a lot. Each method includes two tasks to solve: what is the optimal freezing medium to use and what cryopreservation procedure to prefer. In this paper, we present the method utilized in our center for bone marrow cell cryopreservation. The freezing was carried out in nitrogen vapor after adding the medium containing 95 % dextran [average mw 50 000–70 000] and 5 % dimethylsulfoxid.Purpose. To show that the proposed method of cryopreservation of dendritic cells is highly effective, simple, reproducible and most convenient for clinical use.Materials and methods. Viability, expression of surface antigens and stimulating activity towards allogeneic T lymphocytes of cryopreserved mature dendritic cells cultured from peripheral blood monocytes were evaluated.Results. The first cryopreservation resulted in the death of a small amount of cells. The second freezing procedure increased the proportion of dead cells. Meanwhile, the difference in the expression of the surface antigens in fresh, cryopreserved and re-cryopreserved dendritic cells was not statistically significant. The level of stimulating activity of fresh and cryopreserved dendritic cells did not significantly differ. Conversely the proliferation of allogeneic T lymphocytes was decreased after stimulation with re-cryopreserved dendritic cells.Conclusion. The presented method of cryopreservation allows to preserve the viability and basic functions of dendritic cells. After thawing dendritic vaccine could be administered to patients after being diluted in an isotonic saline without washing, which makes this method the most convenient for clinical use.

Background. We previously found that a decrease in the number of NKT cells and activated CD 25+ peripheral blood lymphocytes (PBLs) before neoadjuvant chemotherapy was associated with an increased likelihood of disease progression in patients with locally advanced triple-negative breast cancer (TN BC).The purpose of this study was to determine the relationship between the initial number of NKT-and CD 25+ PBLs and relapsefree survival (RFS)/overall survival (OS ) in patients with TN BC who received neoadjuvant chemotherapy with cisplatin and paclitaxel followed by surgery.Material and Methods. The study included patients with stage II and III TN BC. The follow-up time was 36 and 66.9 months. Immediately before chemotherapy, the percentage of CD 3+CD 16+CD 56+ (NKT) -, CD 25+- and CD 8+ PBLs was determined by flow cytometry. Statistical analysis of the data was carried out using the Statistics 7 software package. The Kaplan-Meier method was used to determine the relationship between immunological parameters and RFS/ OS .Results. The decreased level of NKT cells before treatment was associated with a decrease in the 3-year RFS [Me: 20.1 (0.533 and 39.7) months] compared to that observed in patients with higher percentage of these cells than in the control (Me was not achieved). There were no statistically significant differences in the 3-year OS between the groups. The initially reduced number of CD 25+ lymphocytes in comparison with the control was associated with decreased rates of both RFS and OS . The difference in DFS and OS was more significant between the groups of patients who simultaneously had an increased initial number of both NKT and CD 25+ cells and patients in whom both cell populations were below normal levels.Conclusion. The initial (prior to chemotherapy) number of NKT and activated CD 25+ PBLs can apparently be a predictive factor in TN BC patients, who received neoadjuvant chemotherapy with cisplatin and paclitaxel.