L.M.G. Silveira; C.H.M. Brunner; F.M. Cunha; F. Futema; B.B.K. Martins; F.F. Calderaro; D. Kozlowski
College of Veterinary Medicine, Universidade Paulista (UNIP), São Paulo, Brazil
Electrochemotherapy consists of a protocol which combines the use of antineoplastic agents with localized application of electric pulses to improve the intracellular concentration of the mentioned drugs, increasing, thus, its cytotoxic action (Entin et al. 2003, Miklavcic et al. 2005, Giardino et al. 2006, Sersa et al. 2006). By definition, the term electroporation is a physical method based on the employment of short high voltage electric pulses, regionally applied, which induce the development of nonselective, temporary and reversible pores in cellular membrane. This technique presents the intrinsic property of improving the transport of chemical substances, nucleic acids, immunoglobulins and plasmids into cell interior (Cirial & López 2001, Lebar et al. 2002, Pucihar et al. 2002, Chen et al. 2006, Sersa et al. 2008). Multiple antineoplastic agents, when simultaneously administered with electroporation, exhibit an increased therapeutic response in lower doses. Amongst the backward-cited agents, bleomycin has been remarkably noticed for its amplifying cytotoxic effect when combined with application of local electric pulses. Bleomycin is an antibiotic agent which presents antineoplastic properties and, because it is a hydrophilic molecule, shows a restricted transport through the cellular membrane. However, when it is administered intralesionally or intravenously and associated to electroporation, its cytotoxicity increases over a thousand times (Heller et al. 1998, Gothelf et al. 2003, Aminkov & Manov 2004, Sersa et al. 2006). Electrochemotherapy is a therapeutic modality which presents several indications in both human and veterinary clinical oncology. Previous studies revealed a satisfactory response to such procedure, characterized by high rates of partial or total tumor regression, low percentages or increased time interval for occurrence of tumoral recidivation or metastasis as well as inexpressive side effects (Heller et al. 1998, Mir et al. 1998, Lebar et al. 2002, Spugnini et al. 2006, Larkin et al. 2007). The purpose of this study was to evaluate the applicability, effectiveness and safety of electrochemotherapy in canine neoplasms, aiming the characterization of benefits intrinsic to the protocol.
Materials and Methods
Thirty-four domestic dogs presenting solitary tegumentary or mucosal neoplasms were admitted at the Surgery Service of the Veterinary Hospital from Universidade Paulista, San Paulo, SP, Brazil. The study was integrally led following the animal experimentation guidelines established by the Ethical Committee from Universidade Paulista (UNIP).
All the patients were evaluated by physical examination and pertinent complementary exams such as hematological, biochemical, thoracic radiological and abdominal ultrasonographically aiming to investigate coexistent infirmities and/or intracavitary neoplasia. Every lesion was submitted to a micromorphological analysis. The samples were obtained by incisional biopsy, immediately fixed in 10% tamponated formalin solution. After being registered and identified, such samples were submitted to a histological study so that information referring to neoplastic type, tissue origin and biological behavior could be achieved. All the animals were submitted to general anesthesia before the performance of the related procedures (biopsy and therapy).
The electrochemotherapy protocol was standardized using intralesional bleomycin sulfate at a dose of 1U/cm3 of tumoral area. The tumor volume was calculated with the aid of a pachymeter, in accordance with the equation height x width x depth x π/6.
Three minutes after intralesional administration of the antineoplastic agent, electric pulses were applied all over the neoplastic extension. Electroporation was performed using an electrode composed of 8 inbox needles, parallel disposed and 0.7 cm equidistant from one another. Electric pulses with a 1000V voltage, in unipolar square wave and 100 microseconds duration, totalizing 8 cycles, were regionally applied. Every procedure was repeated and sequentially performed once a month until the complete neoplastic remission.
Dogs were reevaluated seven, 14, 21 and 30 days after the protocol performance. Once observed the complete tumoral remission, patients were monitored every two months for a 12-month-period.
Histological analysis was performed in every lesion included in the study (n = 34) (100%) and revealed seven conjunctival malignant melanomas, two oral malignant melanomas and one interdigital malignant melanoma. Besides, seven spinocellular carcinomas, five hepatoid adenocarcinomas, three hepatoid adenomas, three sebaceous adenocarcinomas, two palpebral melanocytomas, one basal cell carcinoma, one single tegumentary plasmocytoma, one palpebral epithelioma and one sebaceous adenoma were verified.
Except for one case of interdigital melanoma which metastasized to ipsilateral popliteal lymph node, there were no physical, laboratorial, radiological or ultrasonographic evidences of adjacent and/or distant metastasis in these patients.
There were no complications and/or side effects decurrently from intralesional administration of bleomycin sulfate or electroporation. Involuntary muscular spasms were observed only in a few cases during application of electric pulses, which discontinued soon afterwards. No thermal or hemorrhagic tecidual lesions were evidenced due to electrode contact. The number of electrochemotherapy sessions necessary to integral neoplastic remission varied from one to three, which showed to be influenced by neoplastic type and volume of each lesion. Great part of the dogs (n = 30) (88.3%) presented complete neoplastic remission. Only four neoformations (11.7%) presented unsatisfactory response after two protocol sessions, those represented by three spinocellular carcinomas and one sebaceous adenocarcinoma, which were later submitted to surgical excision.
Seven days after electrochemotherapy, a discreet reduction of the neoplastic volume was evidenced and in some cases, neoplasm presented eroded and covered by hematic crusts. By the 14th day it was observed partial remission of neoformations and persistence of eroded areas, still covered by the same type of crust. By the 21st day of observation, there was a marked neoplastic regression, sometimes followed by subtle tegumentary injuries.
Macroscopic reevaluation performed 30 days after therapy showed integral tumoral remission in nine patients (30%) submitted to a single protocol session. Neoplasms which exhibited incomplete regression after one session (n = 21) (70%) were subsequently submitted to new procedure sessions, once a month, until total remission could be observed. All patients were reevaluated every two months through a detailed physical examination and pertinent complementary exams for a 12-month-period, showing no clinical, laboratorial, radiological or ultrasonographic manifestations of neoplastic recidivating, adjacent and/or distant metastasis within this period.
Discussion and Conclusions
Nowadays, several protocols, sometimes multidisciplinary, are available for oncology therapy. However, such procedures exhibit expressive differences concerning effectiveness, time of treatment, patients' recovery, safety and cost. The method that combines the employment of chemotherapy agents to application of electric pulses know as electrochemotherapy was characterized as an applicable, effective and safe modality of oncological therapy according to the assembled bibliography (Mir et al. 1998, Cirial & López 2001, Aminkov & Manov 2004, Spugnini et al. 2006). Intralesional administration of bleomycin sulfate revealed effectiveness. Nevertheless, intratumoral application of the agent was difficult to be performed in eroded or ulcerated neoplasms, suggesting refractivity to the protocol in four studied cases. Complications and/or side effects decurrently from this administration via were not observed, as related in previous assembled papers (Heller et al. 1998, Mir et al. 1998, Gothelf et al. 2003, Aminkov & Manov 2004). During application of electric pulses, involuntary regional muscular spasms were observed, which ceased right after the ending of the procedure. Such event was noticed in only a small number of cases in a transitional way causing minor discomfort to the patient, depending on the topography of the neoplasm (Heller et al. 1998, Gothelf et al. 2003, Miklavcic et al. 2005). Thermal or hemorrhagic lesions due to electroporation were not observed in animals from the considered sampling. Such phenomenon are directly related to amplitude, duration, frequency and number of the electric pulse (Lebar et al. 2002, Giardino et al. 2006, Sersa et al. 2006, Sersa et al. 2008).
Pharmacological group and dose of the employed chemotherapy agent, nature of electrodes as well as intensity, number and type of wave of the electric pulses differ from the consulted studies (Lebar et al. 2002, Pucihar et al. 2002, Entin et al. 2003, Aminkov & Manov 2004; Chen et al. 2006; Sersa et al. 2006, Larkin et al. 2007). However, bleomycin sulfate, employed at a dose of 1U/cm3, combined with application of electric 1000V pulses during 100 microseconds, in unipolar square wave, totalizing eight pulses, demonstrated to be effective for remission of the considered neoformations.
The studied protocol revealed to be applicable, feasible, effective and safe for antineoplastic therapy in dogs. There was no toxicity related to the employed agent. Nevertheless, anatomophysiological integrity of the involved tissue or organ was preserved.
1. Aminkov B, Manov V. 2004. Electrochemotherapy--a novel method of treatment of malignant tumors in dog. Bulg. J. Vet. Med., 7(4): 209-213.
2. Chen C, Smye SW, Robinsos MP, Evans JA. 2006. Membrane electroporation theories: a review. Med. Biol. Eng. Comput., 44(1-2): 5-14.
3. Cirial HC, López DS. 2001. La electroquimioterapia: una nueva alternativa terapéutica en la oncología.Rev. Cubana Oncol., 17(3): 188-194.
4. Entin I, Plotnikov A, Korenstein R, Keisari Y. 2003. Tumor growth retardation, cure, and induction of antitumor immunity in B16 melanoma-bearing mice by low electric field-enhanced chemotherapy. Clin. Cancer Res., 9(8): 3190-3197.
5. Giardino R, Fini M, Bonazzi V, Cardossi R, Nicolini A, Carpi A. 2006. Electrochemotherapy--a novel approach to the treatment of metastatic nodules on the skin and subcutaneous tissues. Biomed. Pharmacother., 60(8): 458-462.
6. Gothelf A, Mir LM, Gehl J. 2003. Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat. Rev., 29(5): 371-387.
7. Heller R, Jaroszeski MJ, Reintgen DS, Puleo CA, DeConti RC, Gilbert RA, Glass LF. 1998. Treatment of cutaneous and subcutaneous tumors with electrochemotherapy using intralesional bleomycin. Cancer, 83:148-157.
8. Larkin JO, Collins CG, Aarons S, Tangney M, Whelan M, O'Reily S, Breathnach O, Soden DM, O' Sullivan GC. 2007. Electrochemotherapy--aspects of preclinical development and early clinical experience. Ann. Surg., 245(3):469-479.
9. Lebar AM, Sersa G, Groselj A, Miklavcic D. 2002. Optimization of pulse parameters in vitro for in vivo electrochemotherapy. Anticancer Res., 22(3):1731-1736.
10. Miklavcic D, Pucihar G, Pavlovec M, Ribaric S, Mali M, Lebar AM, Petkovsek M, Nastran J, Kranjc S, Cemazar M, Sersa G. 2005. The effect of high frequency electric pulses on muscle contractions and antitumor efficiency in vivo for a potential use in clinical electrochemotherapy. Bioelectrochem., 65(2):121-128.
11. Mir LM, Glass LS, Sersa G, Teissié J, Domenge C, Miklavcic D, Jaroszeski MJ, Reintgen DS, Rudolf Z, Belehradek M, Gilbert R, Rols MP, Belehradek Jr. J, Bachaud JM, De Conti R, Stabuc B, Cemazar M, Coninx P, Heller R. 1998. Effective treatment of cutaneous and subcutaneous malignant tumors by electrochemotherapy. Br. J. Cancer, 77(12):2336-2342.
12. Pucihar G, Mir LM, Miklavcic D. 2002. The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy. Bioelectrochem., 57(2):167-172.
13. Sersa G, Cemazar M, Miklavcic D, Rudolf Z. 2006. Electrochemotherapy of tumors. Radiol. Oncol., 40(3):163-174.
14. Sersa G, Jarm T, Kotnik T, Coer A, Podkrajsek M, Sentjurc M, Miklavcic D, Kadivec M, Kranjc S, Secerov A, Cemazar M. 2008. Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma. Br. J. Cancer., 98(2):388-398.
15. Spugnini EP, Dragonetti E, Vicenzi B, Onori N, Citro G, Baldi A. 2006. Pulse-mediated chemotherapy enhances local control and survival in a spontaneous canine model primary mucosal melanoma. Melanoma Res., 16(1):23-27.