- Email: [email protected]
Electrochemotherapy with Bleomycin in the Treatment of Squamous Cell Carcinoma of the Uropygial Gland in a Cockatiel (Nymphicus Hollandicus)
Accepted Manuscript
ELECTROCHEMOTHERAPY WITH BLEOMYCIN IN THE TREATMENT OF SQUAMOUS CELL CARCINOMA OF THE UROPYGIAL GLAND IN A COCKATIEL (NYMPHICUS HOLLANDICUS) Jozko Racnik , Tanja Svara , Marko Zadravec , Mitja Gombac , ˇ Maja Cemazar , Gregor Sersa , Nataˇsa Tozon PII: DOI: Reference:
S1557-5063(18)30103-4 https://doi.org/10.1053/j.jepm.2018.04.020 JEPM 50151
To appear in:
Journal of Exotic Pet Medicine
Please cite this article as: Jozko Racnik , Tanja Svara , Marko Zadravec , Mitja Gombac , ˇ Maja Cemazar , Gregor Sersa , Nataˇsa Tozon , ELECTROCHEMOTHERAPY WITH BLEOMYCIN IN THE TREATMENT OF SQUAMOUS CELL CARCINOMA OF THE UROPYGIAL GLAND IN A COCKATIEL (NYMPHICUS HOLLANDICUS), Journal of Exotic Pet Medicine (2018), doi: https://doi.org/10.1053/j.jepm.2018.04.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
ELECTROCHEMOTHERAPY WITH BLEOMYCIN IN THE TREATMENT OF SQUAMOUS CELL CARCINOMA OF THE UROPYGIAL GLAND IN A COCKATIEL (NYMPHICUS HOLLANDICUS)
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RUNNING TITLE: ELECTROCHEMOTHERAPY IN A COCKATIEL Jozko Racnik1, Tanja Svara2, Marko Zadravec1, Mitja Gombac2, Maja Čemazar3, Gregor Sersa2 and Nataša Tozon4
1
Clinic for Birds, Small mammals and Reptiles, Institute of Poultry, Birds, Small mammals
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and Reptiles, Gerbiceva 60, Veterinary Faculty University of Ljubljana, Slovenia 2
Institute of Pathology, Forensic and Administrative Veterinary Medicine, Gerbiceva 60,
Veterinary Faculty University of Ljubljana, Slovenia 3
Institute of Oncology Ljubljana, Zaloska cesta 2, Ljubljana, Slovenia
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Small Animal Clinic, Gerbiceva 60, Veterinary Faculty, University of Ljubljana, Slovenia
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Corresponding author: Dr Jozko Racnik phone +3864779251; fax +3864779339; e mail -
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[email protected]
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Abstract A 12-year-old spayed female cockatiel (Nymphicus hollandicus) was presented with a squamous cell carcinoma involving the uropygial gland. Electrochemotherapy (ECT) as a minimally invasive treatment option was applied. The patient was anesthetized, a chemotherapeutic agent was injected intratumorally, and electrical pulses were delivered using plate and needle
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electrodes. In the first session of ECT cisplatin was used, and no change in tumor volume was observed during the following month. In a further two treatments 1 month apart, bleomycin was used. The tumor responded and the mass became necrotic and eventually disappeared. Unfortunately, the tumorous mass reappeared after 3 months. Therefore, a fourth ECT using bleomycin was performed and the tumor completely regressed. No tumor growth was seen in 18 months after the last therapy. This report presents the clinical cell carcinoma in a cockatiel.
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significance of ECT using cisplatin and bleomycin in treatment of uropygial gland squamous
Keywords: electrochemotherapy, cisplatin, bleomycin, squamous cell carcinoma, uropygial
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gland, cockatiel (Nymphicus hollandicus)
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Case report A 12-year-old spayed female cockatiel was presented with a mass located on the dorsal tail region. On clinical examination, the bird was in good condition, with a body weight of 109 g, but a rapidly growing and ulcerating soft tissue mass measuring approximately 9 × 7 × 4 mm (Figure 1) involving the uropygial gland apparently caused
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some level of discomfort to the bird. The owner decided on further diagnostic tests, and a complete blood count, serum biochemical analysis, total body radiographs, and histopathological examination of tissue mass biopsy were performed. The results of the complete blood count revealed leukopenia (2.55 × 109/l; reference1 5–11 × 109/l) with heteropenia (11%; reference1 46–72%) and lymphocytosis (88%; reference1 26–60%), suggesting a chronic infectious or inflammatory condition.1 The results of the serum
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biochemistry panel were within reference levels. No evidence of distant metastases was detected on radiographs; only a calcified round lesion or mass in the caudal part of the celomic cavity was seen, probably connected with a history of reproductive disease managed by salpingohysterectomy 4 years earlier. Incisional biopsy under general anesthesia was performed and histopathological examination revealed a poorly demarcated, infiltrative
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tumor composed of islands of neoplastic squamous epithelium. Neoplastic epithelial cells showed marked anisocytosis and had a moderate to small amount of eosinophilic cytoplasm.
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The vesicular nuclei varied from round to oval, with marked anisokaryosis and one large nucleolus. Mitoses were numerous, 138 per 10 HPF. Keratinization was found in some
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neoplastic foci. The histopathological findings were consistent with SCC (Figure 2). Treatment possibilities, including surgery, were discussed with the owner, and in line with
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the owner’s decision ECT was selected as a minimally invasive treatment option.2 Altogether, four ECT sessions were performed. Generally, for the ECT the patient
was anesthetized using isoflurane, a chemotherapeutic agent was injected intratumorally
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using a 29 gauge needle (Figure 3), and after 1 to 2 minutes eight electrical pulses of 100 µs duration with an amplitude to electrode distance ratio 1300 V/cm and frequency of 5 kHz were delivered using plate electrodes (first session; Figure 4) and needle electrodes (next three sessions). Good contact between the electrodes and tumor mass was achieved by application of a conductive gel to the treatment area. Electrical pulses were generated by an electrical pulse generator (Cliniporator,TM IGEA srl, Carpi [MO], Italy). The entire tumor was exposed to electrical pulses by moving the electrodes through entire volume of the mass 3
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and surrounding skin. After the application of electrical pulses, the tumor became pale, and the bleeding produced by intratumoral injections with a syringe needle stopped shortly after. The duration of each ECT cycle was approximately 15 minutes, and in all cases the recovery from anesthesia was uneventful. During ECT, some muscle contractions of the patient were observed after application of electrical pulses. The contractions were mild, disappearing after
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the end of each electrical pulse sequence. After treatment, the patient was examined at regular intervals to evaluate the treatment successes. In the meantime, the owner observed the treated area and reported the progress. At each visit, the treated tumor was examined and photographed. Meloxicam (Loxicom; Norbrook Laboratories) in a dose 0.5 mg/kg/day was prescribed for 14 days consecutive orally after every cycle of ECT to reduce pain and inflammation. No local or/and systemic side effects were noted on our patient’s condition
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for the entire period during and after ECT.
In the first ECT session, 1.0 mg/kg BW of cisplatin (Cisplatyl; Aventis) dissolved in distilled water at a concentration of 2 mg/ml was used. The tumor size and morphology remained the same in following month after treatment. Because no improvement was observed, in a further second and third ECT 1 month apart 0.5 mg/kg ~ 0.02 ml of
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bleomycin (3 mg/ml Blenoxane; Bristol-Myers) was used, respectively. The tumor responded well, became necrotic, and a partial response of more than a 90% reduction in the tumor size
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was observed in a 4-week period after the second ECT (Figure 5), when the third ECT session was performed. After the third ECT, the remaining small part of the tumor
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responded and eventually visually totally regressed. Recurrence of the tumor mass was noted 3 months after the third treatment. Thus, a fourth ECT using 1.0 mg/kg BW of bleomycin
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was performed and the tumor totally regressed (Figure 6); no tumor growth was seen until the death of the patient, 18 months after last ECT. The patient died 1 day after explorative celiotomy investigating and treating reproductive disease unrelated to oncological disease,
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and no evidence of distant metastasis was noted during the time of explorative surgery and on whole-body radiographs made before the procedure. In line with the owner’s request, no post mortem examinations were performed.
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Discussion ECT is a new local tumor treatment possibility in veterinary oncology that combines application of the chemotherapeutic agent cisplatin or bleomycin with short intense directcurrent electrical pulse to the tumorous mass.3-11 Electrical pulses produce a nonselective increase in plasma membrane permeability, and facilitate and increase uptake of the
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chemotherapeutic drugs, which leads to much higher tumor treatment effectiveness.3,12 ECT with cisplatin or bleomycin was already tested and proved effective in tumors of various histology in dogs,4,5 cats,5,6 horses,7 pet rats,8 some reptiles9,10 and ferrets11.
Surgical excision is still the primary therapy of choice in the case of uropygial tumors, including SCC; however these tumors tend to be very invasive and vascular, and so surgery alone is usually not enough to achieve a cure.13,14,15 Radiation therapy using strontium-90 has
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shown good results in uropygeal gland tumor treatment; however, radioactive materials need special handling and safety protocols, and are restricted to persons that possess special licences.15 Other therapeutic possibilities (e.g., photodynamic therapy and systemic or intratumoral chemotherapy) also showed limited success in the treatment of SCC in various forms in birds.16
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Intratumoral injection of a chemotherapeutic agent such as cisplatin or carboplatin was commonly used in birds as simple local chemotherapy of accessible solid tumors
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without risking systemic side effects and also as adjuvant therapy to other treatment modalities, including surgery and radiotherapy.16,17 However, intratumoral applications of
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aqueous solutions of chemotherapeutic agents usually have only marginal success, possibly because of rapid clearance of the drug from the tumor mass after injection.17 In some cases, sesame oil emulsion of cisplatin can improve therapeutic results with a prolonged-exposure
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chemotherapeutic agent with tumor cells7,16,17, nonetheless, the treatment outcome is usually unpredictable.
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In contrast, electroporation of cells during intratumoral chemotherapy can drastically
improve the therapeutic outcome. Electroporation increases the cytotoxicity of chemotherapeutic agents (bleomycin up to several thousand-fold and cisplatin up to 70 to 80-fold) and induces a transient but reversible reduction of blood flow, inducing drug entrapment in the tissue for several hours.2,3,18 Because cisplatin with ECT is described in dogs,4,5 horses,7 and pet rats,8 and is also commonly used in avian oncology, systemically and intratumoraly,14,16,17 it was a first 5
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chemotherapeutic option in our case. No improvement was seen in the following month after treatment with no local or/and systemic side effects noted in our patient. In birds, the pharmacology of cisplatin was studied in sulphur-crested cockatoos (Cacatua galerita) and gastrointestinal side effects were noted.16 In mammals, cisplatin is nephrotoxic and myelosupressive,16 and it is very likely this is also the case in birds. Fluid therapy following
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systemic chemotherapy helps lower the risk of kidney problems, and systemic antibiotic therapy can prevent secondary infections.
In the two further ECT sessions, administered 1 month apart, bleomycin was used. Intratumoral administration of bleomycin with ECT is described in dogs,4 cats,6 horses,7 some reptiles9,10 and ferrets11. Bleomycin is an antitumor antibiotic generally administered intravenously,3,9,19 is not efficiently transported through the cellular membrane under normal
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conditions, and is less toxic to tumor cells when used locally. Moreover, a hydrophilic drug like bleomycin can only be efficiently transported through the cellular membrane under conditions produced by electroporation to become more toxic to tumor cells.3,9 In our case, the tumor responded well, and after three ECT sessions using bleomycin the tumor regressed and no tumor growth was seen for 18 months after the last ECT.
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In general, there are no literature data about pharmacology including toxic side effects of bleomycin in birds; however, in other animals, bleomycin can produce side effects
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such as increased body temperature, anorexia, vomiting, allergic reaction, alopecia, rashes, stomatitis, pneumonitis, and pulmonary fibrosis.6,19 In our case, three subsequent
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intratumoral applications of bleomycin in doses of 0.5, 0.5, and 1.0 mg/kg BW, 1 and 3 months apart, respectively, did not induce any clinically evident systemic side effects in our
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patient; however, only mild local tumor necrosis was seen in the week following every treatment without a notable effect on the patient’s general condition. In dogs, partial transient necrosis with superficial scabbing of perianal tumors was noticed a week after ECT
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with cisplatin and/or bleomycin.4 In many cases of uropygial neoplasia, total surgical removal or partial surgical
debulking of the tumorous mass before adjuvant therapy (e.g., local chemotherapy or radiation therapy with strontium-90) is still reasonable, but surgery in small birds like a cockatiel can be challenging and a massive tissue lesion could facilitate fatal blood loss. In that context, the so-called “hemostatic” side effect of electroporation could be very useful in the case of a hemorrhage after biopsy or invasive tumor surgery in birds. Application of 6
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electrical pulses during adjuvant ECT to bleeding tumor tissue induces a transient but reversible reduction of blood flow, preventing bleeding from the tumor. This effect was also seen in our case: the tumor became pale, and the bleeding produced by intratumoral injections with a syringe needle stopped shortly after the application of electrical pulses. Moreover, this vascular disrupting action also contributes to the antitumoral effectiveness of
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ECT.2,12,18 Last, and possibly most importantly, there were no local or/and systemic side effects noted in our patient through the entire period during and after ECT. During ECT, some muscle contractions of the patient were observed after the application of each electrical pulse sequence. The contractions were mild, disappearing after the end of each run of electrical pulses. In human medicine, the main disadvantage of ECT is the pain produced
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because of electrical pulses. Pain during ECT can be avoided using sedation and/or general anaesthesia.4-11 Gaseous anesthesia with isoflurane was used in our case for preventing pain and facilitating proper position of the patient for tumor manipulation, application of chemotherapeutic agents, and positioning of electrodes. Meloxicam, a nonsteroidal antiinflammatory analgesic, was prescribed to our patient during and after ECT to reduce pain
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and inflammation, and possibly negatively affect tumor growth. Meloxicam has activity as a cyclooxygenase 2 (COX 2) inhibitor and its inhibition is believed to have a negative effect on
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the progression and development of tumor cells. Related nonsteroidal anti-inflammatory agents with anti-tumor therapeutic properties through COX 2 inhibition such as piroxicam
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are commonly used in the treatment of SCC in dogs.16,17,19,20 The drawback of our study is that it presents the ECT treatment effectiveness on
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only one patient. For broader acceptance of ECT in treatment of tumours in birds, larger group of patients and higher number and types of tumours is needed. However, this study as the first provides evidence of safety and effectiveness of ECT using bleomycin in this animal
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species.
In conclusion, ECT with bleomycin proved to be safe and effective against SCC of
the uropygial gland in a cockatiel and is very likely a good alternative to other treatment options currently used. Because of the good results, no side effects, low cost, and relatively easy procedure, ECT could represent a single and/or adjuvant treatment option for selected oncology cases in birds and also multiple and/or chronic palliative therapy.
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References 1. Doneley B: Appendix 2: Reference intervals for commonly kept companion birds, in Doneley B (ed): Avian Medicine and Surgery in Practice. (ed 2). London, CRC Press, p 447, 2016
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2. Sersa G, Cemazar M, Miklavcic D, Rudolf Z: Electrochemoterapy of tumours. Radiol Oncol 40:163-174, 2006
3. Cemazar M, Tamzali Y, Sersa G, Tozon N, Mir LM, Miklavcic D, et al: Electrochemotherapy in veterinary oncology. J Vet Intern Med 22:826-831, 2008
4. Tozon N, Kodre V, Sersa G, Cemazar M: Effective treatment of perianal tumors in dogs with electrochemotherapy. Anticancer Res 25:839-846, 2005
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5. Tozon N, Sersa G, Cemazar M: Electrochemotherapy: potentiation of local antitumor effectiveness of cisplatin in dogs and cats. Anticancer Res 21:2483-2486, 2001
6. Tozon N, Pavlin D, Sersa G, Dolinsek T, Cemazar M: Electrochemotherapy with intravenous bleomycin injection: an observational study in superficial squamous cell
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carcinoma in cats. J Feline Med Surg 16(4):291-299, 2014
7. Tozon N, Kramaric P, Kadunc Kos P, Sersa G, Cemazar M: Electrochemotherapy
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as a single or adjuvant treatment to surgery of cutaneous sarcoid tumours in horses: a 31-case retrospective study. Vet Rec 179:627, 2016 doi: 10.1136/vr.103867
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8. Lanza A, Pettorali M, Baldi A, Spugnini EP: Surgery and electrochemotherapy treatment of incompletely excised mammary carcinoma in two male pet rats (Rattus
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norvegicus). J Vet Med Sci 79(3):623-625, 2017 9. Bruner CHM, Dutra G, Silva CB, Silviera GML Martins MFM: Electrochemotherapy for the treatment of fibropapillomas in Chelonia mydas. J Zoo Wildl Med 45(2):213-
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218, 2014
10. Lanza A, Baldi A, Spugnini EP: Surgery and electrochemotherapy for the treatment of cutaneous squamous cell carcinoma in a yellow-bellied slider (Trachemys scripta scripta). J Am Vet Med Assoc 246(14):455-457, 2015
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11. Racnik J, Svara T, Zadravec M, Gombac M, Cemazar M, Sersa G, Tozon N: Electrochemoterapy with bleomycin in different types of cutaneous tumours in a ferret (Mustela putorius furo). Radiol Oncol 52(1):98-104, 2018 12. Yarmush ML, Golberg A, Serša G, Kotnik T, Miklavčič D: Electroporation-based
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technologies for medicine: Principles, applications, and challenges. Ann Rev Biomed Eng 16:295-320, 2014
13. Latimer KS: Oncology, in Ritchie BW, Harrison GJ, Harrison LR (eds): Avian Medicine: Principles and Application. Delray Beach, FL, HBD International, Inc, pp 640-672, 1999
14. Robat CS, Ammersbach M, Mans C: Avian oncology. Diseases, diagnostic, and
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therapeutics. Vet Clin North Am Exot Anim Pract 20:57-86, 2017
15. Nemetz L, Broome M: Strontium-90 therapy for uropygial neoplasia. Paper presented at: Proc Annu Conf Assoc Avian Vet 25th Annual Conference. New Orleans, 2004
16. Zehnder A, Graham J, Reavill DR, McLaughlin A: neoplastic diseases in avian
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species, in Speer BL (ed): Current Therapy in Avian Medicine and Surgery. St. Louis, MO, Elsevier, pp 107-141, 2016
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17. Graham JE, Kent MS, Theon A: Current therapies in exotic animal oncology. Vet Clin North Am Exot Anim Pract 7:757-781, 2004
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18. Sersa G, Miklavcic D, Cemazar M, Rudolf Z, Pucinar G, Snoj M: Electrochemotherapy in treatment of tumours. Eur J Surg Oncol 34(2):232-40, 2008
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19. Harrison TM, Kitchell BE: Principles and applications of medical oncology in exotic animals. Vet Clin North Am Exot Anim Pract 20:209-234, 2017
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20. Sander SJ, Hope KL, McNeill CJ, Roberts JF, Boedeker NC, Murray SZ: Metronomic chemotherapy for myxosarcoma treatment in a kori bustard (Ardeotis kori). J Avian Med Surg 29(3):210-215, 2015
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Figures (capitations)
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Figure 1. Macroscopic presentation of squamous cell carcinoma of the uropygial gland.
Figure 2. Histological presentation of squamous cell carcinoma. H&E stain, ×200.
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Figure 3. Intralesional injection of bleomycin into the tumor.
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Figure 4. Electrical pulses were delivered to the tumor using plate electrodes.
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Figure 5. Partial response of the tumor 4 weeks after the second ECT using bleomycin.
Figure 6. Complete response of the tumor 8 months after the last ECT using bleomycin.
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ELECTROCHEMOTHERAPY WITH BLEOMYCIN IN THE TREATMENT OF SQUAMOUS CELL CARCINOMA OF THE UROPYGIAL GLAND IN A COCKATIEL (NYMPHICUS HOLLANDICUS) Jozko Racnik , Tanja Svara , Marko Zadravec , Mitja Gombac , ˇ Maja Cemazar , Gregor Sersa , Nataˇsa Tozon PII: DOI: Reference:
S1557-5063(18)30103-4 https://doi.org/10.1053/j.jepm.2018.04.020 JEPM 50151
To appear in:
Journal of Exotic Pet Medicine
Please cite this article as: Jozko Racnik , Tanja Svara , Marko Zadravec , Mitja Gombac , ˇ Maja Cemazar , Gregor Sersa , Nataˇsa Tozon , ELECTROCHEMOTHERAPY WITH BLEOMYCIN IN THE TREATMENT OF SQUAMOUS CELL CARCINOMA OF THE UROPYGIAL GLAND IN A COCKATIEL (NYMPHICUS HOLLANDICUS), Journal of Exotic Pet Medicine (2018), doi: https://doi.org/10.1053/j.jepm.2018.04.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
ELECTROCHEMOTHERAPY WITH BLEOMYCIN IN THE TREATMENT OF SQUAMOUS CELL CARCINOMA OF THE UROPYGIAL GLAND IN A COCKATIEL (NYMPHICUS HOLLANDICUS)
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RUNNING TITLE: ELECTROCHEMOTHERAPY IN A COCKATIEL Jozko Racnik1, Tanja Svara2, Marko Zadravec1, Mitja Gombac2, Maja Čemazar3, Gregor Sersa2 and Nataša Tozon4
1
Clinic for Birds, Small mammals and Reptiles, Institute of Poultry, Birds, Small mammals
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and Reptiles, Gerbiceva 60, Veterinary Faculty University of Ljubljana, Slovenia 2
Institute of Pathology, Forensic and Administrative Veterinary Medicine, Gerbiceva 60,
Veterinary Faculty University of Ljubljana, Slovenia 3
Institute of Oncology Ljubljana, Zaloska cesta 2, Ljubljana, Slovenia
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Small Animal Clinic, Gerbiceva 60, Veterinary Faculty, University of Ljubljana, Slovenia
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Corresponding author: Dr Jozko Racnik phone +3864779251; fax +3864779339; e mail -
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[email protected]
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Abstract A 12-year-old spayed female cockatiel (Nymphicus hollandicus) was presented with a squamous cell carcinoma involving the uropygial gland. Electrochemotherapy (ECT) as a minimally invasive treatment option was applied. The patient was anesthetized, a chemotherapeutic agent was injected intratumorally, and electrical pulses were delivered using plate and needle
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electrodes. In the first session of ECT cisplatin was used, and no change in tumor volume was observed during the following month. In a further two treatments 1 month apart, bleomycin was used. The tumor responded and the mass became necrotic and eventually disappeared. Unfortunately, the tumorous mass reappeared after 3 months. Therefore, a fourth ECT using bleomycin was performed and the tumor completely regressed. No tumor growth was seen in 18 months after the last therapy. This report presents the clinical cell carcinoma in a cockatiel.
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significance of ECT using cisplatin and bleomycin in treatment of uropygial gland squamous
Keywords: electrochemotherapy, cisplatin, bleomycin, squamous cell carcinoma, uropygial
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gland, cockatiel (Nymphicus hollandicus)
2
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Case report A 12-year-old spayed female cockatiel was presented with a mass located on the dorsal tail region. On clinical examination, the bird was in good condition, with a body weight of 109 g, but a rapidly growing and ulcerating soft tissue mass measuring approximately 9 × 7 × 4 mm (Figure 1) involving the uropygial gland apparently caused
CR IP T
some level of discomfort to the bird. The owner decided on further diagnostic tests, and a complete blood count, serum biochemical analysis, total body radiographs, and histopathological examination of tissue mass biopsy were performed. The results of the complete blood count revealed leukopenia (2.55 × 109/l; reference1 5–11 × 109/l) with heteropenia (11%; reference1 46–72%) and lymphocytosis (88%; reference1 26–60%), suggesting a chronic infectious or inflammatory condition.1 The results of the serum
AN US
biochemistry panel were within reference levels. No evidence of distant metastases was detected on radiographs; only a calcified round lesion or mass in the caudal part of the celomic cavity was seen, probably connected with a history of reproductive disease managed by salpingohysterectomy 4 years earlier. Incisional biopsy under general anesthesia was performed and histopathological examination revealed a poorly demarcated, infiltrative
M
tumor composed of islands of neoplastic squamous epithelium. Neoplastic epithelial cells showed marked anisocytosis and had a moderate to small amount of eosinophilic cytoplasm.
ED
The vesicular nuclei varied from round to oval, with marked anisokaryosis and one large nucleolus. Mitoses were numerous, 138 per 10 HPF. Keratinization was found in some
PT
neoplastic foci. The histopathological findings were consistent with SCC (Figure 2). Treatment possibilities, including surgery, were discussed with the owner, and in line with
CE
the owner’s decision ECT was selected as a minimally invasive treatment option.2 Altogether, four ECT sessions were performed. Generally, for the ECT the patient
was anesthetized using isoflurane, a chemotherapeutic agent was injected intratumorally
AC
using a 29 gauge needle (Figure 3), and after 1 to 2 minutes eight electrical pulses of 100 µs duration with an amplitude to electrode distance ratio 1300 V/cm and frequency of 5 kHz were delivered using plate electrodes (first session; Figure 4) and needle electrodes (next three sessions). Good contact between the electrodes and tumor mass was achieved by application of a conductive gel to the treatment area. Electrical pulses were generated by an electrical pulse generator (Cliniporator,TM IGEA srl, Carpi [MO], Italy). The entire tumor was exposed to electrical pulses by moving the electrodes through entire volume of the mass 3
ACCEPTED MANUSCRIPT
and surrounding skin. After the application of electrical pulses, the tumor became pale, and the bleeding produced by intratumoral injections with a syringe needle stopped shortly after. The duration of each ECT cycle was approximately 15 minutes, and in all cases the recovery from anesthesia was uneventful. During ECT, some muscle contractions of the patient were observed after application of electrical pulses. The contractions were mild, disappearing after
CR IP T
the end of each electrical pulse sequence. After treatment, the patient was examined at regular intervals to evaluate the treatment successes. In the meantime, the owner observed the treated area and reported the progress. At each visit, the treated tumor was examined and photographed. Meloxicam (Loxicom; Norbrook Laboratories) in a dose 0.5 mg/kg/day was prescribed for 14 days consecutive orally after every cycle of ECT to reduce pain and inflammation. No local or/and systemic side effects were noted on our patient’s condition
AN US
for the entire period during and after ECT.
In the first ECT session, 1.0 mg/kg BW of cisplatin (Cisplatyl; Aventis) dissolved in distilled water at a concentration of 2 mg/ml was used. The tumor size and morphology remained the same in following month after treatment. Because no improvement was observed, in a further second and third ECT 1 month apart 0.5 mg/kg ~ 0.02 ml of
M
bleomycin (3 mg/ml Blenoxane; Bristol-Myers) was used, respectively. The tumor responded well, became necrotic, and a partial response of more than a 90% reduction in the tumor size
ED
was observed in a 4-week period after the second ECT (Figure 5), when the third ECT session was performed. After the third ECT, the remaining small part of the tumor
PT
responded and eventually visually totally regressed. Recurrence of the tumor mass was noted 3 months after the third treatment. Thus, a fourth ECT using 1.0 mg/kg BW of bleomycin
CE
was performed and the tumor totally regressed (Figure 6); no tumor growth was seen until the death of the patient, 18 months after last ECT. The patient died 1 day after explorative celiotomy investigating and treating reproductive disease unrelated to oncological disease,
AC
and no evidence of distant metastasis was noted during the time of explorative surgery and on whole-body radiographs made before the procedure. In line with the owner’s request, no post mortem examinations were performed.
4
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Discussion ECT is a new local tumor treatment possibility in veterinary oncology that combines application of the chemotherapeutic agent cisplatin or bleomycin with short intense directcurrent electrical pulse to the tumorous mass.3-11 Electrical pulses produce a nonselective increase in plasma membrane permeability, and facilitate and increase uptake of the
CR IP T
chemotherapeutic drugs, which leads to much higher tumor treatment effectiveness.3,12 ECT with cisplatin or bleomycin was already tested and proved effective in tumors of various histology in dogs,4,5 cats,5,6 horses,7 pet rats,8 some reptiles9,10 and ferrets11.
Surgical excision is still the primary therapy of choice in the case of uropygial tumors, including SCC; however these tumors tend to be very invasive and vascular, and so surgery alone is usually not enough to achieve a cure.13,14,15 Radiation therapy using strontium-90 has
AN US
shown good results in uropygeal gland tumor treatment; however, radioactive materials need special handling and safety protocols, and are restricted to persons that possess special licences.15 Other therapeutic possibilities (e.g., photodynamic therapy and systemic or intratumoral chemotherapy) also showed limited success in the treatment of SCC in various forms in birds.16
M
Intratumoral injection of a chemotherapeutic agent such as cisplatin or carboplatin was commonly used in birds as simple local chemotherapy of accessible solid tumors
ED
without risking systemic side effects and also as adjuvant therapy to other treatment modalities, including surgery and radiotherapy.16,17 However, intratumoral applications of
PT
aqueous solutions of chemotherapeutic agents usually have only marginal success, possibly because of rapid clearance of the drug from the tumor mass after injection.17 In some cases, sesame oil emulsion of cisplatin can improve therapeutic results with a prolonged-exposure
CE
chemotherapeutic agent with tumor cells7,16,17, nonetheless, the treatment outcome is usually unpredictable.
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In contrast, electroporation of cells during intratumoral chemotherapy can drastically
improve the therapeutic outcome. Electroporation increases the cytotoxicity of chemotherapeutic agents (bleomycin up to several thousand-fold and cisplatin up to 70 to 80-fold) and induces a transient but reversible reduction of blood flow, inducing drug entrapment in the tissue for several hours.2,3,18 Because cisplatin with ECT is described in dogs,4,5 horses,7 and pet rats,8 and is also commonly used in avian oncology, systemically and intratumoraly,14,16,17 it was a first 5
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chemotherapeutic option in our case. No improvement was seen in the following month after treatment with no local or/and systemic side effects noted in our patient. In birds, the pharmacology of cisplatin was studied in sulphur-crested cockatoos (Cacatua galerita) and gastrointestinal side effects were noted.16 In mammals, cisplatin is nephrotoxic and myelosupressive,16 and it is very likely this is also the case in birds. Fluid therapy following
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systemic chemotherapy helps lower the risk of kidney problems, and systemic antibiotic therapy can prevent secondary infections.
In the two further ECT sessions, administered 1 month apart, bleomycin was used. Intratumoral administration of bleomycin with ECT is described in dogs,4 cats,6 horses,7 some reptiles9,10 and ferrets11. Bleomycin is an antitumor antibiotic generally administered intravenously,3,9,19 is not efficiently transported through the cellular membrane under normal
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conditions, and is less toxic to tumor cells when used locally. Moreover, a hydrophilic drug like bleomycin can only be efficiently transported through the cellular membrane under conditions produced by electroporation to become more toxic to tumor cells.3,9 In our case, the tumor responded well, and after three ECT sessions using bleomycin the tumor regressed and no tumor growth was seen for 18 months after the last ECT.
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In general, there are no literature data about pharmacology including toxic side effects of bleomycin in birds; however, in other animals, bleomycin can produce side effects
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such as increased body temperature, anorexia, vomiting, allergic reaction, alopecia, rashes, stomatitis, pneumonitis, and pulmonary fibrosis.6,19 In our case, three subsequent
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intratumoral applications of bleomycin in doses of 0.5, 0.5, and 1.0 mg/kg BW, 1 and 3 months apart, respectively, did not induce any clinically evident systemic side effects in our
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patient; however, only mild local tumor necrosis was seen in the week following every treatment without a notable effect on the patient’s general condition. In dogs, partial transient necrosis with superficial scabbing of perianal tumors was noticed a week after ECT
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with cisplatin and/or bleomycin.4 In many cases of uropygial neoplasia, total surgical removal or partial surgical
debulking of the tumorous mass before adjuvant therapy (e.g., local chemotherapy or radiation therapy with strontium-90) is still reasonable, but surgery in small birds like a cockatiel can be challenging and a massive tissue lesion could facilitate fatal blood loss. In that context, the so-called “hemostatic” side effect of electroporation could be very useful in the case of a hemorrhage after biopsy or invasive tumor surgery in birds. Application of 6
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electrical pulses during adjuvant ECT to bleeding tumor tissue induces a transient but reversible reduction of blood flow, preventing bleeding from the tumor. This effect was also seen in our case: the tumor became pale, and the bleeding produced by intratumoral injections with a syringe needle stopped shortly after the application of electrical pulses. Moreover, this vascular disrupting action also contributes to the antitumoral effectiveness of
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ECT.2,12,18 Last, and possibly most importantly, there were no local or/and systemic side effects noted in our patient through the entire period during and after ECT. During ECT, some muscle contractions of the patient were observed after the application of each electrical pulse sequence. The contractions were mild, disappearing after the end of each run of electrical pulses. In human medicine, the main disadvantage of ECT is the pain produced
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because of electrical pulses. Pain during ECT can be avoided using sedation and/or general anaesthesia.4-11 Gaseous anesthesia with isoflurane was used in our case for preventing pain and facilitating proper position of the patient for tumor manipulation, application of chemotherapeutic agents, and positioning of electrodes. Meloxicam, a nonsteroidal antiinflammatory analgesic, was prescribed to our patient during and after ECT to reduce pain
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and inflammation, and possibly negatively affect tumor growth. Meloxicam has activity as a cyclooxygenase 2 (COX 2) inhibitor and its inhibition is believed to have a negative effect on
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the progression and development of tumor cells. Related nonsteroidal anti-inflammatory agents with anti-tumor therapeutic properties through COX 2 inhibition such as piroxicam
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are commonly used in the treatment of SCC in dogs.16,17,19,20 The drawback of our study is that it presents the ECT treatment effectiveness on
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only one patient. For broader acceptance of ECT in treatment of tumours in birds, larger group of patients and higher number and types of tumours is needed. However, this study as the first provides evidence of safety and effectiveness of ECT using bleomycin in this animal
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species.
In conclusion, ECT with bleomycin proved to be safe and effective against SCC of
the uropygial gland in a cockatiel and is very likely a good alternative to other treatment options currently used. Because of the good results, no side effects, low cost, and relatively easy procedure, ECT could represent a single and/or adjuvant treatment option for selected oncology cases in birds and also multiple and/or chronic palliative therapy.
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References 1. Doneley B: Appendix 2: Reference intervals for commonly kept companion birds, in Doneley B (ed): Avian Medicine and Surgery in Practice. (ed 2). London, CRC Press, p 447, 2016
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2. Sersa G, Cemazar M, Miklavcic D, Rudolf Z: Electrochemoterapy of tumours. Radiol Oncol 40:163-174, 2006
3. Cemazar M, Tamzali Y, Sersa G, Tozon N, Mir LM, Miklavcic D, et al: Electrochemotherapy in veterinary oncology. J Vet Intern Med 22:826-831, 2008
4. Tozon N, Kodre V, Sersa G, Cemazar M: Effective treatment of perianal tumors in dogs with electrochemotherapy. Anticancer Res 25:839-846, 2005
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5. Tozon N, Sersa G, Cemazar M: Electrochemotherapy: potentiation of local antitumor effectiveness of cisplatin in dogs and cats. Anticancer Res 21:2483-2486, 2001
6. Tozon N, Pavlin D, Sersa G, Dolinsek T, Cemazar M: Electrochemotherapy with intravenous bleomycin injection: an observational study in superficial squamous cell
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carcinoma in cats. J Feline Med Surg 16(4):291-299, 2014
7. Tozon N, Kramaric P, Kadunc Kos P, Sersa G, Cemazar M: Electrochemotherapy
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as a single or adjuvant treatment to surgery of cutaneous sarcoid tumours in horses: a 31-case retrospective study. Vet Rec 179:627, 2016 doi: 10.1136/vr.103867
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8. Lanza A, Pettorali M, Baldi A, Spugnini EP: Surgery and electrochemotherapy treatment of incompletely excised mammary carcinoma in two male pet rats (Rattus
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norvegicus). J Vet Med Sci 79(3):623-625, 2017 9. Bruner CHM, Dutra G, Silva CB, Silviera GML Martins MFM: Electrochemotherapy for the treatment of fibropapillomas in Chelonia mydas. J Zoo Wildl Med 45(2):213-
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218, 2014
10. Lanza A, Baldi A, Spugnini EP: Surgery and electrochemotherapy for the treatment of cutaneous squamous cell carcinoma in a yellow-bellied slider (Trachemys scripta scripta). J Am Vet Med Assoc 246(14):455-457, 2015
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11. Racnik J, Svara T, Zadravec M, Gombac M, Cemazar M, Sersa G, Tozon N: Electrochemoterapy with bleomycin in different types of cutaneous tumours in a ferret (Mustela putorius furo). Radiol Oncol 52(1):98-104, 2018 12. Yarmush ML, Golberg A, Serša G, Kotnik T, Miklavčič D: Electroporation-based
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technologies for medicine: Principles, applications, and challenges. Ann Rev Biomed Eng 16:295-320, 2014
13. Latimer KS: Oncology, in Ritchie BW, Harrison GJ, Harrison LR (eds): Avian Medicine: Principles and Application. Delray Beach, FL, HBD International, Inc, pp 640-672, 1999
14. Robat CS, Ammersbach M, Mans C: Avian oncology. Diseases, diagnostic, and
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therapeutics. Vet Clin North Am Exot Anim Pract 20:57-86, 2017
15. Nemetz L, Broome M: Strontium-90 therapy for uropygial neoplasia. Paper presented at: Proc Annu Conf Assoc Avian Vet 25th Annual Conference. New Orleans, 2004
16. Zehnder A, Graham J, Reavill DR, McLaughlin A: neoplastic diseases in avian
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species, in Speer BL (ed): Current Therapy in Avian Medicine and Surgery. St. Louis, MO, Elsevier, pp 107-141, 2016
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17. Graham JE, Kent MS, Theon A: Current therapies in exotic animal oncology. Vet Clin North Am Exot Anim Pract 7:757-781, 2004
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18. Sersa G, Miklavcic D, Cemazar M, Rudolf Z, Pucinar G, Snoj M: Electrochemotherapy in treatment of tumours. Eur J Surg Oncol 34(2):232-40, 2008
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19. Harrison TM, Kitchell BE: Principles and applications of medical oncology in exotic animals. Vet Clin North Am Exot Anim Pract 20:209-234, 2017
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20. Sander SJ, Hope KL, McNeill CJ, Roberts JF, Boedeker NC, Murray SZ: Metronomic chemotherapy for myxosarcoma treatment in a kori bustard (Ardeotis kori). J Avian Med Surg 29(3):210-215, 2015
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Figures (capitations)
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Figure 1. Macroscopic presentation of squamous cell carcinoma of the uropygial gland.
Figure 2. Histological presentation of squamous cell carcinoma. H&E stain, ×200.
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Figure 3. Intralesional injection of bleomycin into the tumor.
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Figure 4. Electrical pulses were delivered to the tumor using plate electrodes.
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Figure 5. Partial response of the tumor 4 weeks after the second ECT using bleomycin.
Figure 6. Complete response of the tumor 8 months after the last ECT using bleomycin.
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