The aim of this study was to evaluate the owners' perception of health-related quality of life (HRQoL) of dogs after treatment with electrochemotherapy (ECT) alone or combined with interleukin-12 gene electrotransfer (IL-12 GET) and/or surgery. The owners of 44 dogs with histologically different tumours were offered the >> Cancer Treatment Form << at least one month after treatment. The owners assessed their dogs' quality of life (QoL) after treatment as good (mean 7.4) (from 1-very poor to 10-excellent) and the general health compared with the initial diagnosis of cancer as improving (mean 3.9) (from 1-worse to 5-better). The assessment of the current QoL was better within the group of dogs treated with non-invasive treatment (ECT and/or IL-12 GET only), compared with those that received invasive treatment, where, in addition to ECT and/or IL-12 GET, surgery was performed (p < .05). The owners of dogs that achieved an objective response (OR) to the treatment assessed the QoL as significantly better compared with those whose dogs did not respond to the treatment (p < .05). The majority of the owners (86.4%) would opt for the therapy again, regardless of the financial costs. In conclusion, the results of this study demonstrate that the majority of the owners of dogs assessed their dogs' QoL as good and felt that it improved after the treatment, especially in dogs, treated with non-invasive treatment and in those that responded to the treatment.This supports further use of ECT and IL-12 GET as suitable methods for the treatment of selected tumours in veterinary medicine.

Addition of 2-(ethylamino)acetonitrile group to nitroxoline results in significantly improved anti-tumor activity in vitro and in vivo Addition of 2-(ethylamino)acetonitrile group to nitroxoline results in significantly improved anti-tumor activity in vitro and in vivo

Gene immunotherapy has become an important approach in the treatment of cancer. One example is the introduction of genes encoding immunostimulatory cytokines, such as interleukin 2 and interleukin 12, which stimulate immune cells in tumours. The aim of our study was to determine the effects of gene electrotransfer of plasmids encoding interleukin 2 and interleukin 12 individually and in combination in the CT26 murine colon carcinoma cell line in mice. In the in vitro experiment, the pulse protocol that resulted in the highest expression of IL-2 and IL-12 mRNA and proteins was used for the in vivo part. In vivo, tumour growth delay and also complete response were observed in the group treated with the plasmid combination. Compared to the control group, the highest levels of various immunostimulatory cytokines and increased immune infiltration were observed in the combination group. Long-term anti-tumour immunity was observed in the combination group after tumour re-challenge. In conclusion, our combination therapy efficiently eradicated CT26 colon carcinoma in mice and also generated strong anti-tumour immune memory.

In the treatment of cancer, immunomodulatory approaches are developed to support the organism in fighting cancer or to enhance the immunomodulatory effects of local ablative techniques. To this end, we conducted an interventional, open-label, single-arm Phase I trial to evaluate the safety and tolerability of intratumoral phIL12 plasmid DNA gene electrotransfer as primary objectives. The study was dose-escalating with 3 consecutive cohorts of 3 patients per phIL12 dose level (0.5 mg/ml, 1 mg/ml or 2 mg/ml) according to a matched 3 + 3 design. Recruitment of patients was staggered. The waiting period was 30 days after treatment of the previous patient, based on the expected duration of acute and subacute toxicity. The results of this phase I clinical trial in basal cell carcinoma demonstrated the feasibility and safety of the phIL12 plasmid by gene electrotransfer. We were able to demonstrate that phIL12 gene electrotransfer induced local IL-12 production, which was accompanied with IFN-γ expression. Triggering of the immune response was demonstrated by increased infiltration of immune cells and some antitumor effect. Based on these data, we would recommend the use of a concentration of 2 mg/ml of the plasmid in future trials. The trial lays the foundation for future Phase II clinical trials in which phIL12 gene electrotransfer is used in combination with local tumor-ablative approaches, such as electrochemotherapy or radiotherapy.

Gene electrotransfer (GET) of plasmids encoding interleukin 12 (IL-12) has already been used for the treatment of various types of tumors in human oncology and as an adjuvant in DNA vaccines. In recent years, we have developed a plasmid encoding human IL-12 (phIL12) that is currently in a phase I clinical study. The aim was to confirm the results of a non-clinical study in mice on pharmacokinetic characteristics and safety in a porcine model that better resembled human skin. The GET of phIL12 in the skin was performed on nine pigs using different concentrations of plasmid phIL12 and invasive (needle) or noninvasive (plate) types of electrodes. The results of our study demonstrate that the GET of phIL-12 with needle electrodes induced the highest expression of IL-12 at the protein level on day 7 after the procedure. The plasmid was distributed to all tested organs; however, its amount decreased over time and was at a minimum 28 days after GET. Based on plasmid copy number and expression results, together with blood analysis, we showed that IL-12 GET is safe in a porcine animal model. Furthermore, we demonstrated that pigs are a valuable model for human gene therapy safety studies.

Electrochemotherapy (ECT) in combination with the gene electrotransfer of interleukin 12 (IL-12 GET) has been successfully used in veterinary medicine for the treatment of mast cell tumours (MCT), but the biomarkers that could predict response to this treatment have not yet been investigated. The aim of this study was to determine the plasma nucleosome and serum ferritin concentrations, as well as the lactate dehydrogenase (LDH) activity, in the serum of treated patients before and one and six months after treatment to evaluate their utility as potential biomarkers that could predict response to the combined treatment. The study was conducted in 48 patients with a total of 86 MCTs that we treated with the combined treatment. The blood samples used for analysing the potential predictive biomarkers were taken before treatment and one and six months after treatment, when the response to treatment was also assessed. The Nu. Q^® Vet Cancer Test, the Canine Ferritin ELISA Kit, and the RX Daytona+ automated biochemical analyser were used to analyse the blood samples. The results showed that the plasma nucleosome concentration (before treatment (BT): 32.84 ng/mL (median); one month after treatment (1 M AT): 58.89 ng/mL (median); p = 0.010) and serum LDH activity (BT: 59.75 U/L (median); 1 M AT: 107.5 U/L (median); p = 0.012) increased significantly one month after treatment and that the increase correlated significantly with the presence of a more pronounced local reaction (necrosis, swelling, etc.) at that time point for both markers (nucleosome: BT (necrosis): 21.61 ng/mL (median); 1 M AT (necrosis): 69.92 ng/mL (median), p = 0.030; LDH: BT (necrosis): 54.75 U/L (median); 1 M AT (necrosis): 100.3 U/L (median), p = 0.048). Therefore, both the plasma nucleosome concentration and serum LDH activity could serve as early indicators of the effect of the treatment. In this context, the serum ferritin concentration showed no significant predictive potential for treatment response (p > 0.999 for all comparisons). In conclusion, this study provides some new and important observations on the use of predictive biomarkers in veterinary oncology. Furthermore, it emphasises the need for the continued identification and validation of potential predictive biomarkers in dogs with MCT and other malignancies undergoing ECT treatment in combination with IL-12 GET to ultimately improve treatment outcomes.