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When cells or tissues are exposed to an electric field, structural changes can be induced in the cell membrane, allowing molecules to flow into the cells. This condition is useful for delivering cytotoxic drugs whose transport into cells is impeded, such as bleomycin and cisplatin. This is referred to as electrochemotherapy (ECT). When DNA or RNA molecules are transported into cells, the approach is called gene electrotransfer (GET). ECT is an effective local treatment for tumors, but it lacks the systemic component of treatment that is essential for fighting metastatic disease. Cancer progression and metastasis are closely related to an inadequate antitumor immune response. Therefore, the antitumor efficacy of interleukin 12 (IL -12), a cytokine that stimulates the innate and adaptive immune response, has been tested in preclinical studies using plasmid DNA encoding IL -12 delivered into the tumor by gene electrotransfer (IL -12 GET). In addition, the combined treatment of ECT and IL -12 GET has already been shown to be effective in the treatment of mouse tumors and has been used in clinical trials in companion dogs to treat various histological types of spontaneous tumors. The results of these studies showed that the treatment is safe and effective. However, in these clinical trials, IL -12 GET was administered by different routes, either intratumorally (i.t.) or peritumorally (peri.t.). Therefore, the aim of this study was to compare the two IL -12 GET routes of administration in combination with ECT and to determine their contribution to the enhanced response to ECT. In the clinical trial, 59 dogs with spontaneous mast cell tumors (MCT) were divided into two groups: one group was treated with the combination of ECT and GET peri.t. (29 dogs) and the other with the combination of ECT + GET i.t. (30 dogs). The efficacy of the treatment was compared with the group of 18 dogs treated with ECT alone. In addition, immunohistochemical studies of tumor samples before treatment and flow cytometric studies of PMBC before and after treatment were performed to determine immunological aspects of the treatment. The results of this study showed that local tumor control was significantly better in the ECT + GET i.t. group (p < 0.05) than in the ECT + GET peri.t. and ECT groups. In addition, the 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 < 0.05). The data on local tumor response, DFI and PFS were consistent with immunological studies, as we detected increased infiltration of antitumor immune cells after treatment in the ECT + GET i.t. group, also suggesting a systemic effect. Citation Format: Maja Cemazar, Ursa Lampreht Tratar, Nina Milevoj, Katarina Znidar, Katja Ursic Valentinuzzi, Andreja Brozic, Gregor Sersa, Natasa Tozon. Electrochemotherapy and IL-12 gene electrotransfer in treatment of mast cell tumors in companion dogs. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5122.

Flow cytometry is helpful in differentiating between B-cell lymphoma (BCL) and reactive lymphocytic proliferation (RLP) in FNA biopsies. However, the presence of inconclusive surface immunoglobulin light chains (sIg LC) poses a problem. We investigated the usefulness of additional tests, namely Bcl-2 expression and expression of cytoplasmic Ig LC (cIg LC), mainly on samples with inconclusive sIg LC. Both tests were performed on 232 FNA samples from lymph nodes. Bcl-2 alone was determined qualitatively and quantitatively on 315 samples. The quantitative test was correctly positive in 76% of cases and falsely negative in 24%. The correctly positive results of the qualitative test were 11% points lower. cIg LC correctly identified 65% of BCL with dual positive sIg LC, 36% of BCL with difficult to interpret sIg LC and only 7% of BCL with negative sIg LC. The best results in differentiating between BCL and RLP were obtained when all three tests were used together. In samples with inconclusive sIg LC and additional monoclonal or polyclonal populations the &kappa, &lambda, ratios did not differentiate between RLP and BCL. We propose that in case of inconclusive sIg LC Bcl-2 test is used first. The addition of cIg LC test is sensible only in cases with dual positive and difficult to interpret sIg LC.

Cancer is a common disease of client-owned dogs, with the majority of solid tumors located in the skin or oral cavity. One of the local ablative therapy is electrochemotherapy, where cytotoxic drugs, bleomycin or cisplatin, are introduced into the cells by application of electric pulses (electroporation). In veterinary oncology, electrochemotherapy proved to be an effective and safe local treatment of tumors in horses, cats, dogs, and exotic animals, where success is comparable to standard surgical therapy. The drawback of electrochemotherapy is that, despite its high local effectiveness, it does not have systemic antitumor effects. In preclinical and some clinical studies, gene electrotransfer (GET) of plasmid encoding IL-12 was effective local therapy eliciting also systemic immune response. By means of electroporation, the plasmid DNA is introduced into the cells, resulting in IL-12 expression. The aim of our study was to evaluate a combination of electrochemotherapy and GET of plasmid encoding canine IL-12 for the treatment of skin and oral tumors in dogs. From June 2015 till October 2018, 51 dogs were enrolled in the clinical study: 38 dogs with 68 skin tumors and 13 dogs with oral tumors. Under general anesthesia, electrochemotherapy and gene therapy with IL-12 were performed. For electrochemotherapy cytotoxic drugs were first administered: bleomycin intravenously or intratumorally or cisplatin intratumorally. Standard electrochemotherapy pulses (8 electric pulses, 100 μs, 1300 V/cm, 5 kHz) were applied 10 minutes after intravenous bleomycin administration and immediately after intratumoral bleomycin or cisplatin administration. After electrochemotherapy, the plasmid pCMVcaIL-12 was applied peritumorally at two sites of the tumor and immediately after application of plasmid DNA the electric pulses were applied, using noninvasive electrodes with 7 pins arranged in hexagonal arrays with central pin (24 pulses, 60 V, 150 ms, 0.40/1.39 Hz). The results of our study showed that the combination of electrochemotherapy and gene therapy of plasmid IL-12 is the most effective in treatment of skin tumors, especially in the case of mast cell tumors, with ~90% of local tumor control rate. Moreover, in the case of skin tumors we demonstrated an effect in distant untreated tumors in 5 dogs, which confirms the systemic (abscopal) effect of the therapy. In the treatment of oral tumors, we achieved 54% of objective local tumor response 1 month after treatment. Although progressive disease occurred in 11/13 patients at the end of observation period, a mean survival time of 7 months was obtained, which is comparable with standard surgical therapy, but without cosmetic or functional shortcomings that occur after surgical treatment. To conclude, the results of our study showed significant antitumor response of combined therapy in the case of skin tumors. Also, for dogs with oral tumors combined therapy represents a valuable treatment option, especially when other treatment approaches are not applicable or acceptable. Citation Format: Maja Cemazar, Ursa Lampreht Tratar, Nina Milevoj, Andreja Brozic, Ana Nemec, Gregor Sersa, Natasa Tozon. Immuno gene therapy with IL-12 combined with electrochemotherapy for the treatment of skin and oral tumors in client-owned dogs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B33.

A gene electrotransfer (GET) of interleukin 12 (IL-12) had already given good results when treating tumors in human and veterinary clinical trials. So far, plasmids used in veterinary clinical studies encoded a human or a feline IL-12 and an ampicillin resistance gene, which is not recommended by the regulatory agencies to be used in clinical trials. Therefore, the aim of the current study was to construct the plasmid encoding a canine IL-12 with kanamycin antibiotic resistance gene that could be used in veterinary clinical oncology. The validation of the newly constructed plasmid was carried out on canine malignant melanoma cells, which have not been used in GET studies so far, and on human malignant melanoma cells. Canine and human malignant melanoma cell lines were transfected with plasmid encoding enhanced green fluorescence protein at different pulse parameter conditions to determine the transfection efficiency and cell survival. The IL-12 expression of the most suitable conditions for GET of the plasmid encoding canine IL-12 was determined at mRNA level by the qRT-PCR and at protein level with the ELISpot assay. The obtained results showed that the newly constructed plasmid encoding canine IL-12 had similar or even higher expression capacity than the plasmid encoding human IL-12. Therefore, it represents a promising therapeutic plasmid for further IL-12 gene therapy in clinical studies for spontaneous canine tumors. Additionally, it also meets the main regulatory agencies' (FDA and EMA) criteria.

Human papillomavirus (HPV) is an important etiological factor in oropharyngeal squamous cell carcinoma (SCC). Compared to HPV-negative tumors, HPV-positive oropharyngeal SCC has shown a better response to nonsurgical treatments. In this study, we determined the dose-modifying factors for HPV-positive tumors with single-dose irradiation, with or without low radiosensitizing doses of cisplatin. In vitro, we determined an increased radiosensitivity of HPV-positive SCC, which might be a consequence of HPV-induced changes in the cell cycle regulation and DNA damage response, leading to increased cell death. Additionally, compared to HPV-negative tumors, 30% higher radiosensitivity of HPV-positive tumors was determined by tumor growth delay monitoring in immunodeficient mice in vivo. Concurrent cisplatin treatment had an additive effect in both HPV-negative and HPV-positive tumors, resulting in 20% better response in HPV-positive tumors than in HPV-negative tumors.

Background Flow cytometric immunophenotyping (FCI), is widely used in cytology for distinguishing between B-cell lymphoma (BCL) and reactive lymphocytic proliferations (RLP), mainly by identifying monotypic B-cell populations. Since this cannot always be determined by ratios of surface immunoglobulin light chains (sIg LCs) we wanted to assess if cytoplasmic immunoglobulin (cIg) LCs, Bcl-2 and polymerase chain reaction (PCR) based clonality analysis can improve accuracy of cytological diagnoses of BCL. Methods Our study included 98 fine needle aspiration biopsies from lymph nodes suspicious for BCL with inconclusive sIg LCs. In all cases PCR clonality analysis was performed in order to determine immunoglobulin heavy chain (IGH) gene and T-cell receptor (TRC) gene rearrangement. In selected cases expression of Bcl-2 and cIg LC were determined by FC. Results Thirty patients had lymphoma and 68 had reactive lymphocytic proliferations. Three patterns of sIg LCs staining were found: negative, dual positive and difficult to interpret. Percentage of lymphomas was highest in the dual positive group (75 %). Morphology coupled with cIg LCs determination and/or Bcl-2 expression was able to give a correct diagnosis in 83 % of cases. Molecular tests would have been misleading in 15 % of cases because 7/30 BCL were polyclonal and 8/68 RLP were monoclonal. Conclusions Determination of cIg LCs, Bcl-2 expression and PCR clonality analysis of B cells improved accuracy of cytological diagnoses in BCL with inconclusive sIg LC. However, clonality determined by PCR was misleading in some cases.

Endoglin is a transforming growth factor-β (TGF- β) co-receptor that participates in the activation of a signaling pathway that mediates endothelial cell proliferation and migration in angiogenic tumor vasculature. Therefore, silencing of endoglin expression is an attractive approach for antiangiogenic therapy of tumors. The aim of our study was to evaluate the therapeutic potential of small interfering RNA (siRNA) molecules against endoglin in vitro and in vivo. Therapeutic potential in vitro was assessed in human and murine endothelial cells (HMEC-1, 2H11) by determining endoglin expression level, cell proliferation and tube formation. In vivo, the therapeutic potential of siRNA molecules was evaluated in TS/A mammary adenocarcinoma growing in BALB/c mice. Results of our study showed that siRNA molecules against endoglin have a good antiangiogenic therapeutic potential in vitro, as expression of endoglin mRNA and protein levels in mouse and human microvascular endothelial cells after lipofection were efficiently reduced, which resulted in the inhibition of endothelial cell proliferation and tube formation. In vivo, silencing of endoglin with triple electrotransfer of siRNA molecules into TS/A mammary adenocarcinoma also significantly reduced the mRNA levels, number of tumor blood vessels and the growth of tumors. The obtained results demonstrate that silencing of endoglin is a promising antiangiogenic therapy of tumors that could not be used as single treatment, but as an adjunct to the established cytotoxic treatment approaches.