Comparison of Nucleosome, Ferritin and LDH Levels in Blood with Clinical Response before and after Electrochemotherapy Combined with IL-12 Gene Electrotransfer for the Treatment of Mast Cell Tumours in Dogs.

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.

Treatment of spontaneous canine mast cell tumors by electrochemotherapy combined with IL-12 gene electrotransfer: Comparison of intratumoral and peritumoral application of IL-12.

The combined treatment of electrochemotherapy (ECT) and interleukin-12 (IL-12) gene electrotransfer (GET) has already been used in clinical studies in dogs to treat various histological types of spontaneous tumors. The results of these studies show that the treatment is safe and effective. However, in these clinical studies, the routes of administration of IL-12 GET were either intratumoral (i.t.) or peritumoral (peri.t.). Therefore, the objective of this clinical trial was to compare the two IL-12 GET routes of administration in combination with ECT and their contribution to the enhanced ECT response. Seventy-seven dogs with spontaneous mast cell tumors (MCTs) were divided into three groups: one treated with a combination of ECT + GET peri. t. (29 dogs), the second with the combination of ECT + GET i.t. (30 dogs), and the third with ECT alone (18 dogs). In addition, immunohistochemical studies of tumor samples before treatment and flow cytometry of peripheral blood mononuclear cells (PBMCs) before and after treatment were performed to determine any immunological aspects of the treatment. The results showed that local tumor control was significantly better in the ECT + GET i.t. group (p < 0.050) than in the ECT + GET peri.t. or ECT groups. In addition, disease-free interval (DFI) and progression-free survival (PFS) were significantly longer in the ECT + GET i.t. group than in the other two groups (p < 0.050). The data on local tumor response, DFI, and PFS were consistent with immunological tests, as we detected an increased percentage of antitumor immune cells in the blood after treatment in the ECT + GET i.t. group, which also indicated the induction of a systemic immune response. In addition, we did not observe any unwanted severe or long-lasting side effects. Finally, due to the more pronounced local response after ECT + GET i.t., we suggest that treatment response assessment should be performed at least two months after treatment, which meets the iRECIST criteria.

Metronomic Chemotherapy for Palliative Treatment of Malignant Oral Tumors in Dogs.

The aim of this study was to evaluate the efficacy of metronomic chemotherapy in the palliative treatment of various malignant oral tumors in dogs. Our focus was to determine the effect of treatment on local disease control and to assess the tolerability and safety of the treatment in dogs with various oral malignancies. Metronomic chemotherapy with cyclophosphamide was used to treat 12 dogs and was combined with non-steroidal anti-inflammatory drugs in 6/12 (50%) of dogs. A clinical benefit was observed in 6/12 (50%) patients 1 month and in 4/12 (33%) 3 months after treatment initiation. The median survival time of the dogs was 155 days (range 21-529 days). At the end of the observation period, the disease had progressed in 10/12 (83.3%) of the patients. Sterile hemorrhagic cystitis was the most commonly reported side effect of treatment, occurring in 4/12 (33.3%) dogs. The results of our study suggest that metronomic chemotherapy with cyclophosphamide can be, in a subset of dogs, beneficial in the palliation of malignant oral tumors.

Results of Dynamic Contrast-Enhanced Ultrasound Correlate With Treatment Outcome in Canine Neoplasia Treated With Electrochemotherapy and Interleukin-12 Plasmid Electrotransfer.

Electrochemotherapy (ECT) and/or gene electrotransfer of plasmid DNA encoding interleukin-12 (GET pIL-12) are effective treatments for canine cutaneous, subcutaneous, and maxillofacial tumors. Despite the clinical efficacy of the combined treatments of ECT and GET, data on parameters that might predict the outcome of the treatments are still lacking. This study aimed to investigate whether dynamic contrast-enhanced ultrasound (DCE-US) results of subcutaneous tumors differ between tumors with complete response (CR) and tumors without complete response (non-CR) in dogs treated with ECT and GET pIL-12. Eight dogs with a total of 12 tumor nodules treated with ECT and GET pIL-12 were included. DCE-US examinations were performed in all animals before and immediately after therapy as well as 8 h and 1, 3, and 7 days later. Clinical follow-up examinations were performed 7 and 14 days, 1 and 6 months, and 1 year after treatment. Numerous significant differences in DCE-US parameters were noted between tumors with CR and non-CR tumors; perfusion and perfusion heterogeneity were lower in CR tumors than in non-CR tumors. Therefore, studies with larger numbers of patients are needed to investigate whether DCE-US results can be used to predict treatment outcomes and to make effective decisions about the need for repeated therapy or different treatment combinations in individual patients.

Electroporation-Based Treatments in Small Animal Veterinary Oral and Maxillofacial Oncology.

Electroporation is a method of inducing an increase in permeability of the cell membrane through the application of an electric field and can be used as a delivery method for introducing molecules of interest (e.g., chemotherapeutics or plasmid DNA) into cells. Electroporation-based treatments (i.e., electrochemotherapy, gene electrotransfer, and their combinations) have been shown to be safe and effective in veterinary oncology, but they are currently mostly recommended for the treatment of those solid tumors for which clients have declined surgery and/or radiotherapy. Published data show that electroporation-based treatments are also safe, simple, fast and cost-effective treatment alternatives for selected oral and maxillofacial tumors, especially small squamous cell carcinoma and malignant melanoma tumors not involving the bone in dogs. In these patients, a good local response to treatment is expected to result in increased survival time with good quality of life. Despite emerging evidence of the clinical efficacy of electroporation-based treatments for oral and maxillofacial tumors, further investigation is needed to optimize treatment protocols, improve clinical data reporting and better understand the mechanisms of patients' response to the treatment.

Safety and Feasibility of Electrochemotherapy of the Pancreas in a Porcine Model.

OBJECTIVES: The use of thermal ablative therapies in the pancreatic tumors is limited because of the risk of the vessel injury and potential pancreatitis or fistula formation. Electrochemotherapy (ECT) is an ablative therapy with an established role in the treatment of cutaneous and liver tumors. This study was designed to evaluate the safety and feasibility of ECT of the pancreas in a porcine survival model. METHODS: In the first group, 4 animals underwent computed tomography (CT)-guided percutaneous ECT with bleomycin of the pancreatic tail. In the second group (4 animals), the intraoperative ECT with bleomycin of pancreatic tail and head was performed. Animals were followed for 7 days and then killed. Clinical parameters, CT imaging, laboratory, and histologic analysis were performed. RESULTS: All pigs survived the ECT procedure and none of them developed clinical signs of acute pancreatitis or related complications. There were no signs of acute pancreatitis or damage to the large vessels present in the follow-up CT scans. No significant change in laboratory parameters was obtained after procedure. CONCLUSIONS: This study shows that ECT with bleomycin is feasible and safe in the pancreatic parenchyma. Clinical studies are needed to evaluate the efficacy of ECT in pancreatic cancer.

Health-related quality of life in dogs treated with electrochemotherapy and/or interleukin-12 gene electrotransfer.

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 ¼Cancer 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.

Radiological findings of porcine liver after electrochemotherapy with bleomycin.

Background Radiologic findings after electrochemotherapy of large hepatic blood vessels and healthy hepatic parenchyma have not yet been described. Materials and methods We performed a prospective animal model study with regulatory approval, including nine grower pigs. In each animal, four ultrasound-guided electroporated regions were created; in three regions, electrodes were inserted into the lumen of large hepatic vessels. Two types of electrodes were tested; variable linear- and fixed hexagonal-geometry electrodes. Ultrasonographic examinations were performed immediately and up to 20 minutes after the procedure. Dynamic computed tomography was performed before and at 60 to 90 minutes and one week after the procedure. Results Radiologic examinations of the treated areas showed intact vessel walls and patency; no hemorrhage or thrombi were noted. Ultrasonographic findings were dynamic and evolved from hyperechogenic microbubbles along electrode tracks to hypoechogenicity of treated parenchyma, diffusion of hyperechogenic microbubbles, and hypoechogenicity fading. Contrast-enhanced ultrasound showed decreased perfusion of the treated area. Dynamic computed tomography at 60 to 90 minutes after the procedure showed hypoenhancing areas. The total hypoenhancing area was smaller after treatment with fixed hexagonal electrodes than after treatment with variable linear geometry electrodes. Conclusions Radiologic findings of porcine liver after electrochemotherapy with bleomycin did not show clinically significant damage to the liver, even if a hazardous treatment strategy, such as large vessel intraluminal electrode insertion, was employed, and thus further support safety and clinical use of electrochemotherapy for treatment of hepatic neoplasia.

Large Liver Blood Vessels and Bile Ducts Are Not Damaged by Electrochemotherapy with Bleomycin in Pigs.

The first clinical studies on the use of electrochemotherapy to treat liver tumours that were not amenable to surgery or thermal ablation techniques have recently been published. However, there is still a lack of data on the effects of electrochemotherapy on normal liver tissue. Therefore, we designed a translational animal model study to test whether electrochemotherapy with bleomycin causes clinically significant damage to normal liver tissue, with emphasis on large blood vessels and bile ducts. We performed electrochemotherapy with bleomycin or delivered electric pulses alone using a potentially risky treatment strategy in eight pigs. Two and seven days after treatment, livers were explanted, and histological analysis was performed. Blood samples were collected before treatment and again before euthanasia to evaluate blood biomarkers of liver function and systemic inflammatory response. We found no thrombosis or other clinically significant damage to large blood vessels and bile ducts in the liver. No clinical or laboratory findings suggested impaired liver function or systemic inflammatory response. Electrochemotherapy with bleomycin does not cause clinically significant damage to normal liver tissue. Our study provides further evidence that electrochemotherapy with bleomycin is safe for treatment of patients with tumours near large blood vessels in the liver.

A combination of electrochemotherapy, gene electrotransfer of plasmid encoding canine IL-12 and cytoreductive surgery in the treatment of canine oral malignant melanoma.

The aim of this study was to evaluate the safety and efficacy of the combination of electrochemotherapy (ECT) with bleomycin and gene electrotransfer (GET) of plasmid encoding canine interleukin 12 (IL-12) for the treatment of canine oral malignant melanoma (OMM). Our focus was to determine the effect of the treatment on achieving local tumor control and stimulation of an antitumor immune response. Nine dogs with histologically confirmed OMM stage I to III were included in a prospective, non-randomized study. The dogs were treated with a combination of cytoreductive surgery, ECT and IL-12 GET, which was repeated up to five times, depending on the clinical response to the treatment, evaluated according to the follow-up protocol (7, 14 and 28 days after, the last treatment). One month after treatment, the objective response (OR) rate was 67% (6/9). Median survival time (MST) was 6 months and, even though the disease progressed in 8/9 patients at the end of the observation period (2 to 22 months), four animals were euthanized due to tumor-unrelated reasons. In addition, we observed a decline in the percentage of regulatory T cells (Treg) in the peripheral blood in the course of the treatment, which could be attributed to a systemic antitumor response to IL-12 GET. The results of this study suggest that a combination of ECT and IL-12 GET may be beneficial for dogs with OMM, especially when other treatment approaches are not acceptable due to their invasiveness or cost.