Contrast-enhanced ultrasound for evaluation of tumor perfusion and outcome following treatment in a murine melanoma model.

Due to a lack of data on predictors of electroporation-based treatment outcomes, we investigated the potential predictive role of contrast-enhanced harmonic ultrasound (CEUS) in mice B16F10 melanoma treated by gene electrotransfer (GET) to silence melanoma cell adhesion molecule (MCAM) and radiotherapy, which has not been evaluated yet. CEUS evaluation was verified by tumor histological analysis. Mice bearing subcutaneous tumors were treated with GET to silence MCAM, irradiation or the combination of GET to silence MCAM and irradiation (combined treatment). CEUS of the tumors used to evaluate tumor perfusion was performed before and up to 10 days after the beginning of the experiment, and the CEUS results were compared with tumor growth and the number of blood vessels analyzed in the histological tumor sections. CEUS revealed a decrease in tumor perfusion in the combined therapy groups compared with the control groups and correlated with tumor histological analyses, which showed a decreased vascular density. In this study a trend of inverse correlation was observed between tumor perfusion and treatment efficacy. The greater the perfusion of the tumor, the shorter the expected doubling time. Furthermore, decreased perfusion showed a trend to correlate with higher antitumor efficacy. Thus, CEUS could be used to predict tumoral vascular density and treatment effectiveness.

Treatment of canine cognitive dysfunction with novel butyrylcholinesterase inhibitor.

Canine cognitive dysfunction (CCD) is common in aged dogs and has many similarities with Alzheimer's disease. Unfortunately, like Alzheimer's disease, CCD cannot be cured. In the present study, we treated dogs with CCD with our newly developed and characterized butyrylcholinesterase inhibitor (BChEi). Seventeen dogs were randomized into two groups (treated with BChEi and untreated) and followed for 6 months at regular check-ups. The dogs' cognitive status was determined by a Canine Dementia Scale (CADES) questionnaire and two cognitive tests. In dogs with moderate cognitive impairment, treatment caused significant improvement in the clinical rating of cognitive abilities and the performance-based tests of cognitive functioning when compared to the untreated group (p < 0.001). Dogs treated with BChEi showed markedly improved cognitive function with enhanced quality of life. No side effects were observed in the treated dogs with moderate cognitive impairment. According to the results of this preliminary study, there is an indication that novel BChEi may be a promising drug for the treatment of CCD in dogs and may be an interesting candidate for the treatment of Alzheimer's disease in humans. However, further clinical studies are needed to confirm this.

Tumor perfusion evaluation using dynamic contrast-enhanced ultrasound after electrochemotherapy and IL-12 plasmid electrotransfer in murine melanoma.

Electrochemotherapy with bleomycin (ECT BLM) is an effective antitumor treatment already used in clinical oncology. However, ECT alone is still considered a local antitumor therapy because it cannot induce systemic immunity. When combined with adjuvant gene electrotransfer of plasmid DNA encoding IL-12 (GET pIL-12), the combined therapy leads to a systemic effect on untreated tumors and distant metastases. Although the antitumor efficacy of both therapies alone or in combination has been demonstrated at both preclinical and clinical levels, data on the predictors of efficacy of the treatments are still lacking. Herein, we evaluated the results of dynamic contrast-enhanced ultrasound (DCE-US) as a predictive factor for ECT BLM and GET pIL-12 in murine melanoma. Melanoma B16F10 tumors grown in female C57Bl/6NCrl mice were treated with GET pIL-12 and ECT BLM. Immediately after therapy, 6 h and 1, 3, 7 and 10 days later, tumors were examined by DCE-US. Statistical analysis was performed to inspect the correlation between tumor doubling time (DT) and DCE-US measurements using semilinear regression models and Bland-Altman plots. Therapeutic groups in which DCE-US showed reduced tumor perfusion had longer tumor DTs. It was confirmed that the DCE-US parameter peak enhancement (PE), reflecting relative blood volume, had predictive value for the outcome of therapy: larger PE correlated with shorter DT. In addition, perfusion heterogeneity was also associated with outcome: tumors that had more heterogeneous perfusion had faster growth, i.e., shorter DTs. This study demonstrates that DCE-US can be used as a method to predict the efficacy of electroporation-based treatment.

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.

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.

Solid type primary intraosseous squamous cell carcinoma in a cat.

BACKGROUND: Squamous cell carcinoma (SCC) is the most common nonodontogenic oral tumor in cats. In the jaw, it usually presents as an ulceroproliferative lesion associated with enlargement of the affected bone. CASE PRESENTATION: This report describes the case of a cat in which clinical and radiographic findings of a mandibular swelling were suggestive of an aggressive process, but the oral mucosa was unaffected. The results of histopathological and immunohistochemical examination of the samples obtained from the intraosseous lesion were consistent with SCC. The animal was euthanized 5 months after initial presentation as a result of the severe progression of the disease, and no other primary tumors were identified at necropsy. CONCLUSIONS: Based on the clinicopathological, microscopic, and immunohistochemical staining features, as well as the absence of a primary tumor at a distant site, we propose that the term, solid type primary intraosseous SCC (PIOSCC), be used to describe this neoplasia, as it shares similar features with human PIOSCC.

Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study.

BACKGROUND: The study was aimed to characterize tumor response after combined treatment employing electrochemotherapy with IL-12 gene electrotransfer in dogs with spontaneous mast cell tumors (MCT). MATERIALS AND METHODS: Eleven dogs with eleven MCTs were included in the study. Histological changes were investigated in biopsy specimens collected before the treatment (T0), and 4 (T1) and 8 weeks (T2) later. Cellular infiltrates were characterized immunohistochemically by using anti CD3, CD20, Foxp3 (Treg), CD68 and anti MHC-class II antibodies. Proliferation and anti-apoptotic activity of neoplastic cells were assessed using anti Ki-67 and Bcl-2 antibodies. Angiogenetic processes were investigated immunohistochemically by using anti Factor VIII and anti CD31 antibodies and micro vessel density quantification. RESULTS: Histopathological examination of samples at T0 confirmed the diagnosis and the presence of scanty infiltrates consisted mainly of T-lymphocytes and macrophages. At T1 and T2 neoplastic cells were drastically reduced in 7/11 cases, small clusters of neoplastic cells were detected in 3/11 cases and 1/11 cases neoplastic cells were still evident. Proliferation activity of neoplastic cells was significantly reduced at T1 and T2 and expression of anti-apoptotic protein at T1. Microvessel density was drastically reduced in all samples after treatment. The number of T-lymphocytes increased at T1, although not significant, while Treg were significant higher at T1 and macrophages at T2. CONCLUSIONS: The combined electrochemotherapy and IL-12 gene electrotransfer effectively induced a cellular response against neoplastic cells characterized mainly by the recruitment of T-lymphocytes and macrophages and a fibrotic proliferation with reduction of microvessels.

Electrochemotherapy with intravenous bleomycin injection: an observational study in superficial squamous cell carcinoma in cats.

The aim of this study was to evaluate the efficacy and safety of electrochemotherapy (ECT) with bleomycin for treatment of squamous cell carcinoma (SCC) in cats. Between March 2008 and October 2011, 11 cats with 17 superficial SCC nodules in different clinical stages (ranging from Tis to T4), located on nasal planum (6/11), pinnae (3/11) and both locations (2/11), were included in a prospective non-randomised study. Sixteen of 17 SCC nodules were treated with ECT (15/16 with single session and in one case with two sessions); one nodule was surgically removed. Altogether, complete response (CR) was achieved for 81.8% (9/11) cats and 87.5% (14/16) nodules, lasting from 2 months up to longer than 3 years. Only 2/9 cats in which CR was initially observed, had recurrence 2 and 8 months after the ECT procedure. In the remaining two cats with highly infiltrative spread into adjacent tissues, progression of the disease was observed, despite ECT, and both were euthanased 4 and 5 months after the procedure. ECT in cats was well tolerated and no evident local or systemic side effects were observed. The results of this study suggest that ECT is a highly effective and safe method of local tumour control of feline cutaneous SCCs. It should be considered as an alternative treatment option, especially when other treatment approaches are not acceptable by the owners, owing to their invasiveness, mutilation or high cost.

Surgical correction of labial fusion with vaginoplasty in a cat.

A spayed female domestic shorthair cat was first examined at the age of 16 months because of persistent licking of the perineal area. The cat had a grossly enlarged and oedematous vulva with pronounced superficial pyoderma of the perivulvar area, which responded favourably to systemic antibiotics, analgesics and local corticosteroids. A month after the initial examination, the cat was re-presented owing to pollakiuria, stranguria and dyschesia. The oedema of the vulva had disappeared and the vulvar labia were fused together; there was only a fistulous tract with a diameter of 1 mm present in the area of the vulva, and the cat strained to urinate through that opening. A contrast study revealed normal transit through the lower urinary tract, but labial adhesions resulted in the development of dilation cranially, where the vaginal vestibule was supposed to be. Vaginoplasty was subsequently performed, the cat recovered normally and, 10 months after the procedure, the lumen of the vaginostoma is preserved and the cat is urinating without difficulty.

IL-12 based gene therapy in veterinary medicine.

The use of large animals as an experimental model for novel treatment techniques has many advantages over the use of laboratory animals, so veterinary medicine is becoming an increasingly important translational bridge between preclinical studies and human medicine. The results of preclinical studies show that gene therapy with therapeutic gene encoding interleukin-12 (IL-12) displays pronounced antitumor effects in various tumor models. A number of different studies employing this therapeutic plasmid, delivered by either viral or non-viral methods, have also been undertaken in veterinary oncology. In cats, adenoviral delivery into soft tissue sarcomas has been employed. In horses, naked plasmid DNA has been delivered by direct intratumoral injection into nodules of metastatic melanoma. In dogs, various types of tumors have been treated with either local or systemic IL-12 electrogene therapy. The results of these studies show that IL-12 based gene therapy elicits a good antitumor effect on spontaneously occurring tumors in large animals, while being safe and well tolerated by the animals. Hopefully, such results will lead to further investigation of this therapy in veterinary medicine and successful translation into human clinical trials.

Electrogene therapy with interleukin-12 in canine mast cell tumors.

BACKGROUND: Mast cell tumors (MCT) are the most common malignant cutaneous tumors in dogs with extremely variable biological behaviour. Different treatment approaches can be used in canine cutaneous MCT, with surgical excision being the treatment of choice. In this study, electrogene therapy (EGT) as a new therapeutic approach to canine MCTs, was established. MATERIALS AND METHODS.: Eight dogs with a total of eleven cutaneous MCTs were treated with intratumoral EGT using DNA plasmid encoding human interleukin-12 (IL-12). The local response to the therapy was evaluated by repeated measurements of tumor size and histological examination of treated tumors. A possible systemic response was assessed by determination of IL-12 and interferon- γ (IFN-γ) in patients' sera. The occurence of side effects was monitored with weekly clinical examinations of treated animals and by performing basic bloodwork, consisting of the complete bloodcount and determination of selected biochemistry parameters. RESULTS: Intratumoral EGT with IL-12 elicits significant reduction of treated tumors' size, ranging from 13% to 83% (median 50%) of the initial tumor volume. Additionally, a change in the histological structure of treated nodules was seen. There was a reduction in number of malignant mast cells and inflammatory cell infiltration of treated tumors. Systemic release of IL-12 in four patients was detected, without any noticeable local or systemic side effects. CONCLUSIONS: These data suggest that intratumoral EGT with plasmid encoding IL-12 may be useful in the treatment of canine MCTs, exerting a local antitumor effect.

Controlled systemic release of interleukin-12 after gene electrotransfer to muscle for cancer gene therapy alone or in combination with ionizing radiation in murine sarcomas.

BACKGROUND: The present study aimed to evaluate the antitumor effectiveness of systemic interleukin (IL)-12 gene therapy in murine sarcoma models, and to evaluate its interaction with the irradiation of tumors and metastases. To avoid toxic side-effects of IL-12 gene therapy, the objective was to achieve the controlled release of IL-12 after intramuscular gene electrotransfer. METHODS: Gene electrotransfer of the plasmid pORF-mIL12 was performed into the tibialis cranialis in A/J and C57BL/6 mice. Systemic release of the IL-12 was monitored in the serum of mice after carrying out two sets of intramuscular IL-12 gene electrotransfer of two different doses of plasmid DNA. The antitumor effectiveness of IL-12 gene electrotransfer alone or in combination with local tumor or lung irradiation with X-rays, was evaluated on subcutaneous SA-1 and LPB tumors, as well as on lung metastases. RESULTS: A synergistic antitumor effect of intramuscular gene electrotransfer combined with local tumor irradiation was observed as a result of the systemic distribution of IL-12. The gene electrotransfer resulted in up to 28% of complete responses of tumors. In combination with local tumor irradiation, the curability was increased by up to 100%. The same effect was observed for lung metastases, where a potentiating factor of 1.3-fold was determined. The amount of circulating IL-12 was controlled by the number of repeats of gene electrotransfer and by the amount of the injected plasmid. CONCLUSIONS: The present study demonstrates the feasibility of treatment by IL-12 gene electrotransfer combined with local tumor or lung metastases irradiation on sarcoma tumors for translation into the clinical setting.

Local and systemic antitumor effect of intratumoral and peritumoral IL-12 electrogene therapy on murine sarcoma.

Soft tissue sarcomas pose a challenge for successful treatment with conventional therapeutic methods, therefore newer therapeutic approaches are considered. In this study, we evaluated the antitumor effect of IL-12 electrogene therapy (EGT) on murine SA-1 fibrosarcoma. The therapeutic plasmid was injected either intratumorally into subcutaneous SA-1 nodules or intradermally into the peritumoral region. We achieved a remarkable local and systemic antitumor effect with both approaches after single plasmid DNA application, with significant intratumoral and systemic production of IL-12 and IFNgamma. Intratumoral IL-12 EGT resulted in over 90% complete response rate of the treated tumors with 60% of cured mice being resistant to challenge with SA-1 tumor cells. Peritumoral EGT resulted in a lower complete response rate (16%), with significant growth delay of remaining tumors. Both therapies also resulted in significant inhibition of growth of untreated tumors, growing simultaneously at a distant site. These data suggest that IL-12 EGT may be useful in the treatment of soft tissue sarcomas, exerting a local and systemic antitumor effect.

Gene electrotransfer into murine skeletal muscle: a systematic analysis of parameters for long-term gene expression.

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57Bl/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57Bl/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 microg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

Sequence and time dependence of transfection efficiency of electrically-assisted gene delivery to tumors in mice.

Electrically-assisted gene delivery is a non-viral gene delivery technique, using application of square wave electric pulses to facilitate uptake of plasmid DNA into the cells. Feasibility and effectiveness of this method in vivo was already demonstrated, elaborating on pulse parameters and plasmid construction. However, there were no studies performed on sequencing and timing of plasmid DNA injection into the tumors and application of electric pulses. For this purpose we measured luciferase expression in two tumor models (LPB fibrosarcoma, B16F1 melanoma) after electrically-assisted gene delivery at varying time intervals between the pCMV-Luc plasmid injection and electroporation. Expression of luciferase was determined by measurement of its activity using luminometer. The results demonstrated that pCMV-Luc plasmid has to be injected before the application of electric pulses, since no measurable expression was detected in the tumors when pCMV-Luc plasmid was injected after electroporation of tumors. In both tumor models the highest transfection efficiency was obtained when pCMV-Luc plasmid was injected not less than 5 minutes but also not more than 30 minutes before the application of electric pulses. The results also demonstrated variability in the transfection efficiency depending on the tumor model. High expression was obtained in B16F1 tumor model (approximately 5500 pg luc/mg tumor) and lower in LPB fibrosarcoma (approximately 200 pg luc/mg tumor). In conclusion, our results demonstrate that regardless of the susceptibility of the tumors to electrically-assisted gene delivery, the best timing for pCMV-Luc plasmid is between 30 to 5 minutes prior to the application of electric pulses to the tumors.