Evaluation of electrochemotherapy with bleomycin as an adjuvant treatment to surgical resection of feline application sarcoma: retrospective study from 2017 to 2023ABSTRACT
Feline application sarcoma (FAS) is a mesenchymal malignancy associated with chronic inflammation caused by subcutaneous or intramuscular applications. Surgical resection is the most recommended treatment, but high local recurrence demands multimodal therapies to improve clinical results. Among them, electrochemotherapy (ECT) stands out. This retrospective study evaluated the efficacy of ECT with bleomycin as adjuvant therapy to surgical resection in 40 cats with APS, divided into animals undergoing surgery alone (CG, n=20) and animals undergoing surgery associated with ECT (GE, n=20). Mixed breed females, with a mean age of 10.45 years, were the most affected and fibrosarcoma was the predominant histological type. ECT significantly reduced tumor recurrences (85% in CG and 35% in EG) and increased the chance of tumor control by 10.52 times. With minimal side effects, ECT has demonstrated to be a promising therapy for the management of APS in veterinary practice.
Keywords:
Electroporation; Fibrosarcoma; Cat; Tumor; Vaccine
RESUMO
O sarcoma de aplicação felino (SAF) é uma neoplasia maligna mesenquimal associada a inflamações crônicas por aplicações subcutâneas ou intramusculares. A ressecção cirúrgica é o tratamento mais indicado, porém a alta recorrência local demanda terapias multimodais para melhorar os resultados clínicos. Entre elas, destaca-se a eletroquimioterapia (EQT). Este estudo retrospectivo avaliou a eficácia da EQT com bleomicina como terapia adjuvante à ressecção cirúrgica em 40 gatos com SAF, divididos em animais submetidos somente à cirurgia (GC, n=20) e animais submetidos à cirurgia associada à EQT (GE, n=20). Fêmeas sem raça definida, com idade média de 10,45 anos, foram as mais acometidas e o fibrossarcoma foi o tipo histológico predominante. A EQT reduziu significativamente as recidivas tumorais (85% no GC e 35% no GE) e aumentou em 10,52 vezes a chance de controle tumoral. Com efeitos colaterais mínimos, a EQT demonstrou ser uma terapia promissora para o manejo do SAF na prática veterinária.
Palavras-chave:
eletroporação; fibrossarcoma; gato; tumor; vacina
INTRODUCTION
Feline application sarcoma (FAS) is a mesenchymal neoplasm associated with chronic inflammatory processes caused by subcutaneous or intramuscular applications of substances such as vaccines, antibiotics, corticosteroids, nonsteroidal anti-inflammatory drugs, chemotherapeutics and surgical threads (Kass et al., 1993; Bloch et al., 2019). Several histological types are described, with fibrosarcoma being the most reported (Carneiro et al., 2018) and the anatomical sites of highest incidence are associated with the most common regions of application such as femoral muscles, paralumbar region, flanks, dorsolateral interscapular and cervical thorax (Shaw et al., 2009; Hartmann et al., 2015). Metastases are observed in 10 to 24% of cases, mainly affecting the lungs (Ladlow, 2013). The treatment of choice is surgical resection with wide safety margins due to the infiltrative behavior of the tumor. However, recurrence rates range from 50 to 86% in periods of 4 to 6 months, making the use of adjuvant therapies necessary (Hershey et al., 2000; Spugnini et al., 2020). Chemotherapy and radiotherapy are widely used but have limitations due to toxicity and variable efficacy. On the other hand, electrochemotherapy (ECT) has been standing out as a promising approach. This technique combines electrical pulses and antineoplastic agents, promoting greater efficacy in tumor control and presenting low side effects (Spugnini et al., 2008; Carmo et al., 2020). The objective of this study was to evaluate the efficacy of ECT with bleomycin as adjuvant therapy to surgical resection in animals with APS, through a retrospective study, using as criteria the disease-free time and tumor recurrence rates in the animals, after the procedures were performed.
ETHICAL ASPECTS
This research was submitted to the Ethics Committee on Animal Use of the Universidade Paulista (UNIP) and approved under protocol number 1902181122. Medical records of 40 felines diagnosed with FAS were analyzed between August 2017 and August 2023 at veterinary hospitals in the city of São Paulo (SP, Brazil). Data were collected on breed, age, sex, histopathological reports, tumor grading, anatomical location, tumor diameter, and whether the case was primary or recurrent neoplasia.
METHODS
The animals were divided into two groups based on treatment - the Surgery Group (control group, CG), submitted only to surgical resection, and the Electrochemotherapy Group (EG), submitted to surgical resection associated with electrochemotherapy (ECT) with bleomycin.
The surgeries were performed by different professionals with more than 3 years of experience, following margins of 3-5cm and removal of adjacent planes, with amputations when necessary. ECT was conducted by an oncologist with more than 12 years of experience, using bleomycin (15U/m², IV) and electrical pulses (ELECTROvet EZ®, 1200V/cm, 1 Hz, 8 pulses of 100µs) during surgery.
Felines with sarcomas confirmed by histopathology in sites compatible with applications and undergoing surgery alone or surgery associated with ECT were included. Animals of other species, with morbidities other than sarcoma, diagnosis only by cytology, preoperative metastases, incomplete medical records or deaths from unrelated causes were excluded.
The histopathological reports assessed tumor type, grade, and surgical margins, which were classified as free or compromised. Disease-free time was calculated from therapy to recurrence, with a minimum follow-up of 280 days for animals without recurrences. Survival time was not assessed due to several influences that may alter the results, such as age of the animals, diseases developed throughout life, and difficulty in monitoring them for long periods.
The data were tabulated in Excel and analyzed statistically. For quantitative variables, normality was assessed using the Shapiro-Wilk test and homogeneity of variances was assessed using the Levene test, with the support of quantile-quantile graphs (QQ-Plots) to visualize the data distribution. Statistical analysis involved tests comparing means (t-test for independent samples) or medians (Mann-Whitney test), depending on the normality of the variable. Qualitative variables were analyzed using contingency tables, comparing the frequency of events between groups. The chi-square test of independence was applied when the expected frequencies were greater than five; otherwise, Fisher's exact test was used. The significance level adopted was α=0.05. In addition to p-values, effect size measures were reported when applicable, such as Cohen's d for comparison of means and Cohen's w for the chi-square test, following classical interpretation criteria. All analyses were conducted in Excel for Windows®.
RESULTS AND DISCUSSION
In this study, the average age of the affected animals was 10.45 years, compatible with the average described by other authors, ranging from 9 to 11 years and, although previous studies indicated that there was no sexual predisposition (Hershey et al., 2000; Ferrari et al., 2015; Müller; Kessler, 2018; Dobromylsky et al., 2020), we found a higher prevalence of affected females, totaling 62.5% (25/40). There was no evident racial predisposition, since 85% (34/40) of the animals were of no defined breed (SRD), corroborating the findings described by some authors (Dobromylsky et al., 2020). The highest incidence of neoplasia was in the right flanks (25%; 10/40) and left flanks (20%; 8/40), interscapular region (10%; 4/40), right pelvic limb (10%; 4/40), left pelvic limb (7.5%; 3/40), dorsal thorax (7.5%; 3/40), right lateral thorax (7.5%; 3/40), left lateral thorax (5%; 2/40), right thoracic limb (5%; 2/40) and ventral thorax (2.5%; 1/40), making it clear that many veterinarians still do not follow the recommendations established by the Vaccine-Associated Feline Sarcoma Task Force (VAFSTF) in 1996, to standardize the sites of vaccine application in felines, delimiting those that present the greatest potential for sarcoma development and offering surgical alternatives with greater safety margins, such as amputations (Morrison and Starr, 2001).
The median area of the tumors was evaluated, being 27.5cm² (with an interquartile range of 40.69) in the CG, and 27.63cm² (with an interquartile range of 45.48) in the EG. There was no significant difference in the median area of the tumors of both groups, thus, the size of the formations did not influence the recurrence results in this study. The predominant histological type found was fibrosarcoma, representing 50% (20/40) of the histopathological reports, corroborating the findings of other authors (Kliczkouska et al., 2015; Carneiro et al., 2018).
FASs present higher histological grades, due to their higher rate of anaplasia, mitosis and areas of necrosis, when compared to sarcomas that do not have an application origin, thus resulting in a more aggressive biological behavior (Ladlow, 2013; Almeida et al., 2021). These data are compatible with those identified in this investigation, since, in the CG, 55% (11/20) were classified as grade II and 25% as grade III, and, in the EG, they were, respectively, 40% (8/20) and 40% (8/20). Statistically, the proportion of grades was equal between the groups, therefore, the grade had no impact on the recurrence results of this study. Surgical en bloc resection of the neoplasm is still the main form of treatment for APS, removing the entire pseudocapsule, with lateral margins of 3 to 5 cm, the adjacent deep planes and even amputation of the affected limb (Ogilvie and Moore, 2002). However, it is known that, even respecting all these criteria of oncological surgery, the recurrences of APS when treated only with surgical removal vary between 50 and 86%, with an interval of 4 to 6 months, and may be higher or lower, depending on the location of the tumor, histological grading and experience of the surgeon (Hershey et al., 2000; Spugnini et al., 2008, 2011). Such data corroborates the findings in this study, in which the GC presented a recurrence of 85% (17/20), with a median time of 88 days (approximately 3 months). Thus, multimodal therapy is currently recommended, associating surgical resection with other adjuvant therapies, whose objectives are to minimize recurrence rates or increase the disease-free interval and, consequently, animal survival (Ferreira et al., 2016; Almeida et al., 2021). Some investigations have shown that ECT, when associated with surgical resections, presents a good response in tumor control, especially in tumors of mesenchymal origin, which have high numbers of recurrences and are difficult to control (Spugnini et al., 2016; Carmo et al., 2020). Although in this study, when we compared the two groups regarding the disease-free interval, we found that the time variable did not behave as expected for a normal distribution, showing some discrepancy. Thus, a median comparison test was chosen. The median in the CG was 88 days and, in the EG, it was 133 days (with interquartile ranges of 125 and 236, respectively). Based on the test conducted, we cannot infer that there was a difference between the medians of the two groups or, in other words, given the limitations of the study, the sample size in question and the dispersion of the elements within the groups, the time to relapse was not influenced by the treatment (Fig. 1), thus not corroborating the data found by other authors (Spugnini et al., 2008, 2011).
Box-plot of the variation in recurrence according to the group. There was no significant difference in the median time to recurrence in the 2 groups. Control group (CG, n=20), cats diagnosed with feline application sarcoma (FAS) and submitted only to surgical resection; Electrochemotherapy group (EG, n=20), cats diagnosed with feline application sarcoma and submitted to surgical resection associated with electrochemotherapy (ECT) with bleomycin. Mann-Whitney test.
However, considering that tumor recurrence in the EG was 35% (7/20), a considerably lower number when compared to the animals in the CG, whose recurrence was 85% (17/20), the proportion of animals with recurrence could be considered different between the groups (Fig. 2). Thus, according to the data analyzed, statistically, an animal treated with ECT had a 10.52 times greater chance of not presenting recurrence compared to an animal treated only with surgery.
Spugnini et al. (2020) observed a decrease in the efficacy of the technique in animals that underwent other surgeries before choosing the combination of ECT as adjuvant therapy. This result was similar in our study, since, in the EG, of the 35% (7/20) of the animals that presented tumor recurrence, 57.14% (4/7) had a history of AFS, that is, they had already been treated only with surgical excision before being submitted to ECT for this study, thus suggesting that, when associated as soon as possible, it presents better results in tumor control. The survival time of the animals was not monitored, since factors such as the age of the animals can influence the results. However, a minimum period of 280 days (approximately 9 months) was established for animals that did not present recurrences in both groups.
It is known that one of the most relevant factors for the prognosis of AFSs is the evaluation of surgical margins. Although free margins do not influence the survival time of animals, they are related to reducing the recurrence rate or increasing the disease-free interval (Müller; Kessler, 2018; Carmo et al., 2020; Liptak and Christensen, 2020). In this study, 50% (10/20) of the animals in the CG and 70% (14/20) in the EG had margins free of tumor cells upon histological examination. This percentage difference could be considered equal between the groups, that is, it was not because the EG had a higher percentage of tumors with free margins that the result of tumor recurrences was influenced, especially because among the animals that recurred, most of the tumors were predominantly free of tumor cells. Of the CG, 41.18% (7/17) had free margins, while in the EG, 57.14% (4/7). This relationship probably occurred because free margins were the most frequently found in the examinations, since the animals that underwent surgery were generally treated by surgeons who were familiar with the need for a wide surgical margin for these neoplasms. The metastatic potential of AFS is relatively low, observed in approximately 10 to 24% of cases, with the lungs being the most affected, but with reports of involvement in regional lymph nodes, skin and liver (Hershey et al., 2000; Ladlow, 2013). This case series was observed in our data, with only 12.5% (5/40) of the animals developing lung metastasis during the study.
Profile of the frequency of animals with or without relapses according to the group. Control group (CG, n=20), cats diagnosed with feline application sarcoma (FAS) and submitted only to surgical resection; Electrochemotherapy group (EG, n=20), cats diagnosed with feline application sarcoma and submitted to surgical resection associated with electrochemotherapy (ECT) with bleomycin. In other words, an animal subjected to ECT had a 10.52 times greater chance of not presenting relapses compared to an animal treated only with surgery. The chi-square test of independence was used to assess whether the proportion of animals with relapse differed between groups, since all expected frequencies were greater than five units. The result indicated a significant association between relapse and group (Χ²(1)=10.417, p=0.001, w=0.51).
CONCLUSION
The results obtained in this study showed that ECT with bleomycin, used as an adjuvant therapy to surgical resection, was effective in controlling feline application sarcoma (FAS). Compared to the group that underwent surgery alone, the group that was submitted to ECT had a significantly lower rate of tumor recurrence (35% versus 85%), with a significant increase in the probability of disease control. Additionally, the technique demonstrated low rates of side effects, standing out as a viable and promising alternative for veterinary oncology. These findings reinforce the potential of ECT as part of multimodal therapies in the management of neoplasias in felines, contributing to the reduction of recurrence rates and improving the quality of life of patients.
ACKNOWLEDGEMENTS
This research was funded by the Coordination of Improvement of Higher Education Personnel (CAPES, Brazil / PROSUP - Code 001).
REFERENCES
- ALMEIDA, I.O.; PIMENTEL, P.A.B.; SENA, B.V.; MERISIO, A.C.R.; HORTA, R.S. Do sarcoma de aplicação felino com associação de cirurgia, quimioterapia neoadjuvante e adjuvante e eletroquimioterapia - relato de dois casos. In: ONCOVET CONGRESSO ONLINE, 11., 2021. Anais... São Paulo: ABROVET, 2021. p.8.
- BLOCH, J.; ROGERS, K.; WALKER, M. et al. Treatment of feline injection-site sarcoma with surgery and iridium-192 brachytherapy: retrospective evaluation of 22 cats. J. Feline Med. Surg., v.22, p.313-321, 2019.
- CARMO, E.S.; SMIDERLE, L.M.; ARAUJO, M.F. et al. Sarcoma de aplicação em felinos: revisão bibliográfica. Rev. Pesqui. Extensão Fac. Serra Gaúcha, v.8, p.367-377, 2020.
- CARNEIRO, C.S.; QUEIROZ, G.F.; PINTO, A.C.B.C.F. et al. Feline injection site sarcoma: immunohistochemical characteristics. J. Feline Med. Surg., v.21, p.314-321, 2018.
- DOBROMYLSKY, M.J.; RICHARDS, V.; SMITH, K. Prognostic factors and proposed grading system for cutaneous and subcutaneous soft tissue sarcomas in cats, based on a retrospective study. J. Feline Med. Surg., v.23, p.168-174, 2020.
- FERRARI, R.; GIANCAMILLO, M.; STEFANELLO, D. et al. Clinical and computed tomography tumour dimension assessments for planning wide excision of injection site sarcomas in cats: how strong is the agreement? Vet. Comp. Oncol., v.15, p.374-382, 2015.
- FERREIRA, M.G.P.A.; FILHO, N.P.R.; PASCOLI, A.L.C.R. et al. Sarcoma de aplicação em felinos: aspectos clínicos, diagnóstico e terapia. Investigação, v.15, p.29-36, 2016.
- HARTMANN, K.; DAY, M.J.; THIRY, E. et al. Feline injection-site sarcoma: ABCD guidelines on prevention and management. J. Feline Med. Surg., v.17, p.606-613, 2015.
- HERSHEY, A.E.; SORENMO, K.U.; HENDRICK, M.J. et al. Prognosis for presumed feline vaccine-associated sarcoma after excision: 61 cases (1986-1996). J. Am. Vet. Med. Assoc., v.216, p.58-61, 2000.
- KASS, P.H.; BARNES, W.G.; SPANGLER, W.L. et al. Epidemiologic evidence for a causal relationship between vaccination and fibrosarcoma tumorigenesis in cats. J. Am. Vet. Med. Assoc., v.203, p.396-405, 1993.
- KLICZKOWSKA, K.; JANKOWSKA, U.; JAGIELSKI, D. et al. Epidemiological and morphological analysis of feline injection site sarcomas. Pol. J. Vet. Sci., v.18, p.313-322, 2015.
- LADLOW, J. Injection site-associated sarcoma in the cat: treatment recommendations and results to date. J. Feline Med. Surg., v.15, p.409-418, 2013.
- LIPTAK, J.M.; CHRISTENSEN, N.I. Soft tissue sarcomas. In: VAIL, D.M.; THAMM, D.H.; LIPTAK, J.M. Withrow & MacEwen`s small animal clinical oncology. 6.ed. Saint Louis: Elsevier Saunders, 2020. p.404-431.
- MORRISON, W.B.; STARR, R.M; Vaccine-associated feline sarcoma task force. vaccine-associated feline sarcomas. J. Am. Vet. Med. Assoc., v.218, p.697-702, 2001.
- MÜLLER, N.; KESSLER, M. Curative-intent radical en bloc resection using a minimum of a 3 cm margin in feline injection-site sarcomas: a retrospective analysis of 131 cases. J. Feline Med. Surg., v.20, p.509-519, 2018.
- OGILVIE, G.K.; MOORE, A.S. Feline oncology: a comprehensive guide to compassionate care. New Jersey: Veterinary Learning Systems, 2002. 503p.
- SHAW, S.C.; KENT, M.S.; GORDON, I.K. et al. Temporal changes in characteristics of injection-site sarcomas in cats: 392 cases (1990-2006). J. Am. Vet. Med. Assoc., v.234, p.376-380, 2009.
- SPUGNINI, E.P.; CITRO, G.; D’AVINO, A.; BALDI, A. Potential role of electrochemotherapy for the treatment of soft tissue sarcoma: first insights from preclinical studies in animals. Int. J. Biochem. Cell Biol., v.40, p.159-163, 2008.
- SPUGNINI, E.P.; FAIS, S.; AZZARITO, T.; BALDI, A. Novel instruments for the implementation of electrochemotherapy protocols: From bench side to veterinary clinic. J. Cell. Physiol., v.232, p.490-495, 2016.
- SPUGNINI, E.P.; RENAUD, S.M.; BUGLIONI, S. et al. Electrochemotherapy with cisplatin enhances local control after surgical ablation of fibrosarcoma in cats: an approach to improve the therapeutic index of highly toxic chemotherapy drugs. J. Transl. Med., v.9, p.1-5, 2011.
- SPUGNINI, E.P.; VINCENZI, B.; CAROCCI, F. et al. Combination of bleomycin and cisplatin as adjuvant electrochemotherapy protocol for the treatment of incompletely excised feline injection-site sarcomas: a retrospective study. Open Vet. J., v.10, p.267-271, 2020.
Data-available-upon-request - the research data are available upon request.
