Evaluation of the safety and feasibility of electrochemotherapy with intravenous bleomycin as local treatment of bladder cancer in dogs

doi:10.21203/rs.3.rs-2658010/v1

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Evaluation of the safety and feasibility of electrochemotherapy with intravenous bleomycin as local treatment of bladder cancer in dogs

https://doi.org/10.21203/rs.3.rs-2658010/v1

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published 29 Nov, 2023

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Local treatment of canine urothelial carcinoma (UC) of the bladder is a challenge. More than 90% of cases arise as muscle-invasive tumors, with more than 50% developing on bladder sites with a difficult surgical approach often requiring radical procedures. This study aimed to evaluate the safety and feasibility of electrochemotherapy (ECT) with intravenous bleomycin (BLM) as selective local therapy for bladder UC. This prospective study included 21 dogs with spontaneous bladder UC. Neoplastic infiltration in the serosa layer was considered the main exclusion criterion. No patient died during ECT or in the immediate postoperative period, and no patient presented with suture dehiscence. Most dogs (19/21) developed mild adverse effects, whereas two dogs developed ureteral stenosis. Complete response (CR) was achieved in 62% of the dogs (13/21), while partial response (PR) was achieved in 24% (5/21). The mean survival and disease-free survival times were 420 and 405 days, respectively. Overall survival was significantly better in the patients who achieved CR. ECT was well-tolerated in dogs with UC, demonstrating its safety and feasibility. These data pave the way for new studies aimed at evaluating the effectiveness of ECT in canine bladder UC as a translational model for human disease.

Biological sciences/Cancer/Urological cancer

Health sciences/Oncology/Cancer/Cancer therapy

Health sciences/Medical research/Translational research

Health sciences/Oncology

Urothelial carcinoma (UC) is considered the most commonly diagnosed malignant bladder neoplasm, accounting for approximately 1.5-2% of all naturally occurring cancers, both in humans and dogs [1]. According to the World Health Organization staging system for canine bladder tumors, tumors are classified according to their degree of invasion into the bladder wall (2) (Table 1). In humans, the staging system is similar, but muscle-invasive disease is classified as T3 or higher [2]. More than 90% of affected dogs present with the muscle-invasive form of the disease (InvUC), which is most commonly located in the trigone of the bladder, extending down to the urethra in more than 50% of cases [3, 5]. Therefore, the surgical approach for these tumors is a great challenge, as partial cystectomy is possible in only a few cases, mainly when the trigone is not involved [6, 7, 8].

Table 1

TNM Clinical staging system for canine bladder cancer.
T - Primary Tumor
Tis	Carcinoma in situ
T0	No evidence of a primary tumor
T1	Superficial papillary tumor
T2	Tumor invading the bladder wall, with induration
T3	Tumor invading neighboring organs (prostate, uterus, vagina ad pelvic canal)
N - Regional Lymph Node (Internal and External Iliac Lymph Node)
N0	No regional lymph node involvement
N1	Regional lymph node involved
N2	Regional lymph node and juxtaregional lymph node involved
M - Distant Metastases
M0	No evidence of metastasis
M1	Distant metastasis present

Total cystectomy is not usually performed in dogs because patients are commonly in advanced stages of disease and because of the higher morbidity and mortality associated with the procedure [4, 9, 10, 11, 12]. It is important to highlight that even in the best scenarios when partial cystectomy is possible, the tumor recurrence rate is still high, ranging from 66 to 100% [5, 6, 8, 13, 14].

Radiation therapy is not commonly used for bladder cancer in dogs mainly because of its side effects, as reported in previous studies [15, 16, 17]. However, newer radiation protocols have shown better tolerability, encouraging further investigations [18, 19, 20].

Currently, medical therapy is the mainstay for InvUC in canines. The combination of chemotherapy with non-steroidal anti-inflammatory drugs (NSAIDs) improves clinical signs and provides disease control in 80% of dogs. However, it is not curative, and the tumor response is temporary [7, 21].

In contrast, in humans, the superficial non-invasive form of bladder UC comprises approximately 75% of cases, with no predilection for specific anatomic sites of the bladder [1, 22]. These features allow for a more feasible surgical approach, typically transurethral resection of the tumor. Despite this better scenario, recurrence after surgery is also a problem, reaching rates of up to 75% at five years, progressing to InvUC [22, 23]. As in dogs, human InvUC also possesses potentially lethal behavior, requires aggressive treatment, and only 5% of patients with metastatic disease remain alive at five years [24, 25].

Therefore, preclinical animal studies have been conducted to investigate promising therapies for human bladder UC. Experimental mouse models have been used for many years in bladder cancer research. However, they lack key features of the disease in humans and cannot accurately predict therapeutic outcomes [4, 26, 27]. Advances in translational research have proven that dogs with naturally occurring InvUC represent a highly relevant animal model for studying this invasive disease in humans. These studies have identified notable similarities between dogs and humans with InvUC, including etiological factors, biological behavior, molecular features, and chemotherapeutic responses [1, 4, 28].

Considerable efforts are being made to develop less invasive and effective therapeutic approaches for the local treatment of human and canine bladder cancer as well as for the prevention of recurrence. Electrochemotherapy (ECT) is a local technique that combines the injection of antineoplastic drugs followed by the application of electrical pulses at the tumor site and margins, aiming to increase drug uptake by electropermeabilized cells and maximize its cytotoxic effect [29]. The efficacy of ECT has already been proven in different types of cancers in humans and canines, including melanomas, carcinomas, mast cell tumors, and sarcomas [30, 31]. Some studies have evaluated the efficacy of ECT in vitro and in vivo in cultured human bladder cancer cells and in rats with induced UC [32, 33, 34, 35]. Recently, ECT treatment of dogs with naturally occurring bladder UC was first reported in three canine patients by Rangel et al. (2018) [36] with promising results.

This study aimed to evaluate the safety and viability of ECT as a local treatment for canine bladder UC. Second, this work also evaluated the local antitumor effect of the therapy and paves the way for future clinical trials in human medicine.

Patient data

The demographics of the treated patients and general data regarding tumor features and treatment outcomes for each patient are described in Tables 2 and 3, respectively.

Table 2

Patients’ demographics.
Characteristics of 21 patients	Mean (range) (%)
Age (years)	11.4 (8–16)
Sex ratio (F:M)	13:8
Body weight (kg)	8.69 (2.9–27)
Breeds
Shih-tzu Poodle Lhasa Apso Maltese Dachshund Pug Schnauzer Yorkshire Labrador Retriever French Bulldog Crossbreed	4 (19%) 4 (19%) 3 (14.2%) 2 (9.5%) 2 (9.5%) 1 (4.7%) 1 (4.7%) 1 (4.7%) 1 (4.7%) 1 (4.7%) 1 (4.7%)

Table 3. General patient information, tumor features and treatment outcome. ECT: Electrochemotherapy; VCOG-CTCAE: Veterinary Cooperative Oncology Group – Common Terminology Criteria for Adverse Events; PR: Partial response; CR: Complete response; UR: Unassessed response; ST: Survival time; DFS: Disease-free survival. *Days; **Inside the bladder.

Patient n^o	Tumor location**	TNM	N^o of ECT sessions	Final local response		Local relapse	VCOG-CTCAE grade	DFS*	ST*
1	Cranial-dorsal	T₁N₀M₀	1	PR	-		1	-	517
2	Trigone	T₂N₀M₀	3	CR	Yes		1	790	866
3	Apex	T₂N₀M₀	1	CR	No		1	30	30
4	Cranial-dorsal	T₂N₀M₀	1	CR	No		1	147	147
5	Trigone	T₂N₀M₀	3	CR	No		1	981	1021
6	Apex	T₂N₀M₀	1	CR	Yes		1	353	740
7	Trigone	T₂N₀M₀	2	CR	No		1	199	199
8	Trigone	T₂N₀M₀	1	CR	No		1	166	166
9	Trigone	T₂N₀M₀	2	CR	Yes		1	257	595
10	Trigone	T₂N₀M₀	1	CR	No		1	978	978
11	Trigone	T₂N₀M₀	2	CR	Yes		1	244	1015
12	Trigone	T₂N₀M₀	1	UR	-		1	-	20
13	Trigone	T₂N₀M₀	1	UR	-		1	-	7
14	Trigone	T₂N₀M₀	2	PR	-		1	-	283
15	Trigone	T₁N₀M₀	1	CR	No		1	495	515
16	Trigone	T₂N₀M₀	2	PR	-		1	-	284
17	Trigone	T₂N₀M₀	2	PR	-		3	-	168
18	Trigone	T₂N₀M₀	1	PR	-		1	-	155
19	Trigone	T₂N₀M₀	1	RC	No		1	499	499
20	Cranial-dorsal	T₂N₀M₀	3	RC	Yes		3	130	571
21	Trigone	T₂N₀M₀	1	UR	-		1	-	34

Twelve of the twenty-one patients (57.1%) underwent one ECT session, six patients (28.5%) underwent two sessions, and three patients (14.2%) underwent three sessions.

Overall, the interval between sessions ranged from 33 to 70 days and was individualized for each patient, depending on the treatment response. The mean interval between the first and second sessions was 52 days and that between the second and third sessions was 45 days.

Safety evaluation

None of the patients died during the surgical procedure, ECT treatment, or in the immediate postoperative period. Furthermore, none of the patients had suture dehiscence in the bladder or uroperitoneum. Nineteen dogs (90.4%) presented with mild adverse effects during the postoperative period, including pollakiuria, stranguria, dysuria, and transitory urinary incontinence. The degree of toxicity for these patients was classified as grade 1 according to the “Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events” (VCOG-CTCAE v2) [37].

Two patients had more serious complications (VCOG = 3), developing ureteral stenosis 69 and 73 days after the procedure. These dogs underwent a surgical procedure for the placement of a temporary “double-J” ureteral stent which was removed after recovery of ureteral patency.

Quality of life

According to the quality of life questionnaire filled by the owners and the oncologist’s clinical evaluations, all 19 patients who developed mild adverse events had no relevant negative impact on their quality of life related to ECT treatment.

As expected, the two dogs that developed ureteral stenosis manifested more serious adverse events, including a higher level of pain and changes in behavior. However, this negative impact on the quality of life was transitory, lasting until surgical treatment. The procedures were well tolerated by the dogs, who completely recovered their previous quality of life according to subsequent assessments.

Cause of death

Two patients died of disease progression. Of these, one developed metastatic disease to the lungs, and the other had local progression of the tumor, which occurred 517 days after the first and only ECT treatment was performed. The remaining 19 patients died of causes unrelated to the disease or ECT.

Local response

Sixty-two percent of the patients (13/21) achieved complete response (CR), and twenty-four percent (5/21) had partial response (PR). An objective response rate (ORR) of 86% was achieved. The remaining three cases were not evaluated, as the minimum time required to apply the RECIST criteria was not met.

The local response was not associated with sex (Fisher’s exact test, p = 0.4) or tumor location (Fisher’s exact test, p = 0.4).

Among the patients who achieved CR, most achieved it in one session (77%). Among the patients who required two sessions (15%), a median of 56 days (range 22–71) passed between the two sessions. For the patients who required three sessions (8%), a median of 43 days (range 33–60) passed after the second session.

Microscopic neoplastic foci were identified by additional surgery in five patients who had CR, following which they underwent a new ECT treatment. However, the owners of the remaining eight dogs with CR refused any additional surgery.

Of the five patients who achieved PR, no additional ECT sessions were performed because of the owner's refusal. Only one dog showed local disease progression.

The overall duration of response was on average 371 days (range, 30–981 days). Tumor recurrence occurred in five of the dogs (38.4%) in which CR was achieved, and the average time to its development was 354 days (range, 170–790 days) (Table 3).

At the end of the study, two dogs remained alive, one with CR and the other with tumor recurrence. Among the 19 patients who died and had their local response evaluated, six (31.5%) died without tumor evidence (CR), 10 (52.6%) died with macroscopic tumor evidence, and three 3 (15.7%) died before the period of response evaluation (Table 3).

Survival data

Disease-free survival time was on average 405 days (median 257, ranging, 30–981 days). DFS was not associated with sex (p = 0.2839), tumor location (p = 0.2552), or number of sessions (p = 0.1711).

The average overall survival of the patients was 420 days (median 284, ranging, 7–1021 days). Sex (p = 0.0355), number of sessions (p = 0.0622; Fig. 4C), and tumor location (p = 0.5590) were not associated with survival time. However, obtaining a complete response was associated with longer survival times (log-rank test, p = 0.0035; Fig. 4D), as expected.

Urothelial carcinoma (UC) is the most commonly diagnosed malignant bladder neoplasm in dogs and affects the trigone region in more than half of the cases. Obtaining adequate local control of the disease is one of the greatest challenges owing to surgical limitations and a high rate of tumor recurrence. Total cystectomy can be considered when complete tumor resection is not possible, and often when the trigone is involved. However, this surgical approach is not commonly performed because of the high rates of postsurgical complications, low acceptance by owners, and compromised quality of life in most patients. Unfortunately, even when tumors arise away from the trigonal area and partial cystectomy is feasible, local recurrence rates remain considerable.

ECT is an effective therapeutic modality for tumors with different histologies [31, 38]. This technique stands out for its high efficacy, low cost, and selectivity towards neoplastic tissues. The latter feature allows for the conservation of important anatomical structures, especially when tumors develop in sites with a difficult surgical approach. Recently, ECT has demonstrated significant efficacy in the treatment of canine carcinomas such as squamous cell, anal sac, and nasal carcinomas [38, 39, 40, 41]. ECT has also been shown to be safe and effective for deep-seated tumors in humans, such as pancreatic and hepatocellular carcinomas [42, 43].

To date, two preliminary studies have reported the application of ECT in canine bladder UC; however, both studies used a small sample of dogs [36, 44]. Considering the anatomical characteristics of the urinary bladder as well as the possible side effects associated with ECT, the present study evaluated the safety and feasibility of this technique for the treatment of bladder UC in 21 dogs. Additionally, the local ECT antitumor effect was preliminarily assessed.

Demographic data regarding sex predisposition, age at diagnosis, and reproductive status are in agreement with previous studies [1, 7, 21]. Shih Tzu and Poodle (38%) were the most affected breeds in this study, with no occurrence of the most known predisposed breeds. This fact may be related to differences in racial distribution between the canine population in Brazil, the United States, and Europe.

Most dogs (90.4%) had T2 tumors, while 9.6% had T1 tumors, corroborating the data found in the literature, which states that T2 tumors account for approximately 68–78% of canine bladder neoplasms [1, 21].

Intravenous bleomycin (BLM) was selected as the antineoplastic drug for ECT in this study. BLM possesses a selective mechanism of cell death, and its systemic administration allows for a more homogenous distribution through tissues. In addition, electropermeabilization promotes BLM intracellular uptake by a factor of 700-fold [29, 45, 46]. Once inside the nucleus, BLM leads to single- and double-strand breaks in the DNA. This damage accumulates as the cells proliferate, and after a threshold is reached, cell death occurs via a process called mitotic cell death. This mechanism confers BLM with unique selectivity towards rapidly dividing cancer cells. In turn, cells from healthy tissues (such as those from tumor margins) are quiescent and slow or non-dividing, and remain viable even if electropermeabilized [29, 46, 47].

Thus, the process of cell death and consequent tissue necrosis caused by ECT occurs in any tissue where cancer cells infiltrate. Consequently, ECT application in bladder tumors that infiltrate the serosa would entail significant risks of bladder rupture and uroperitoneum due to necrosis, as the serosa is the last layer of the bladder lining. Therefore, the non-involvement of serosa was a crucial inclusion criterion in this study.

None of the patients in this study died during the procedure or in the immediate postoperative period, and there were no occurrences of bladder rupture, uroperitoneum, or suture dehiscence. Similarly, Rangel et al. (2018) [36] performed ECT with intravenous BLM in two dogs with bladder UC and adopted the non-involvement of the serosa as a safety criterion. None of their patients developed any of these complications.

The vast majority of patients in this study developed mild adverse effects (90%; VCOG = 1), which had already manifested at the first presentation. However, transient urinary incontinence (lasting 7–86 days) was the only adverse effect not previously reported. Likewise, Rangel et al. (2018) [36] also reported transient urinary incontinence in patients treated for 56 and 76 days. The development of this condition may be due to transient inflammation and edema in the bladder mucosa, which are expected to be caused by ECT, leading to nerve stunning and involuntary muscle spasms.

Two patients (9.5%) developed ureteral stenosis at 69 and 73 days after the second ECT session (VCOG = 3). They were treated surgically by placement of a “double-J” stent with good tolerability. Ureteral stenosis might be attributed to scarring fibrosis resulting from tumor death, which is an expected effect after ECT. However, as the amount of fibrotic tissue increases, the risk of stenosis increases.

In comparison, studies evaluating partial or total cystectomy in dogs with bladder UC have found more significant complication rates (range 43–70%), including suture dehiscence, ureteral obstruction, pyelonephritis, and uroperitoneum [6, 11, 14].

Studies evaluating partial cystectomy, with or without adjuvant chemotherapy, have reported death rates due to disease progression ranging from 45 to 91% [6, 8, 14]. In the present study, only two dogs (9.5%) died of UC. One of them died due to local progression of the disease 517 days after the first and only ECT session. The owners refused additional sessions or other treatment. However, the survival time was above the average for dogs treated with conventional therapies (surgery and/or chemotherapy) [6, 8, 48, 49].

The other patient who died because of UC progression developed metastatic disease in the lungs, which was identified on chest radiographs approximately 5 months after ECT. This dog was the only patient who developed distant metastasis until the end of the study, resulting in a distant metastasis rate of 4.7%. In contrast, according to a study by Knapp et al. (2000) [5], the rate of distant metastasis at death was 49% among 80 dogs with bladder UC. This discrepancy could be attributed to the earlier stages of the disease in the present study. Another hypothesis may be attributed to the potential efficacy of ECT in eliminating cancer cells at the primary tumor site. This good local control would decrease the probability of cancer progression to distant sites, which is facilitated when residual tumor remains in the patient’s bladder (as occurs during chemotherapy and after debulking surgery).

Regional metastasis to the lymph nodes was assessed through intraoperative cytological evaluation by fine needle aspiration of the regional iliac lymph nodes, even if apparently normal. Although histopathology is the gold standard for definitive diagnosis of lymph node metastasis, we elected cytological evaluation because it is a less time-consuming examination, either for sample collection or intraoperative evaluation, and it is associated with lower rates of intraoperative complications. It is important to note that all patients who had regional lymph node metastasis were also staged as T2 with serosa involvement or T3 during intraoperative evaluation, and consequently excluded from the study.

Local spread of tumor cells is also a concern in patients with bladder UC who undergo cystotomy. According to Higuchi et al. (2013) [50], 75% (18/24) of dogs with bladder UC developed metastatic foci in the abdominal wall after cystotomy. In the study by Marvel et al. (2017), 10% (4/37) of dogs that underwent partial cystectomy developed tumor spread in the abdominal wall [7]. In contrast, none of the patients in the present study developed this complication after cystotomy and ECT. These results could be attributed to the safety criteria carefully adopted during bladder manipulation and ECT application, minimizing the risk of tumor spread.

Regarding local response, out of the 21 dogs treated in this study, 13 (62%) achieved CR, 5 (24%) had PR, and 3 (14.3%) died before the minimum period for response evaluation because of reasons unrelated to the disease or ECT therapy. This overall response rate (86%) was greater than those reported in studies evaluating different chemotherapy protocols for dogs with bladder UC, ranging from 3–71%, in which partial responses were predominant [7].

The overall duration of response obtained was 370 days on average, which was higher than the 235 and 85 days obtained by Marvel et al. (2017) and Bradburry et al. (2021) [6, 8], respectively. The mean duration of response was also higher than that reported in several studies evaluating medical therapy, which ranged from 96 to 199 days [51, 52, 53, 54].

One of the greatest challenges in treating bladder UC is the high recurrence rate after partial cystectomy, which ranges from 66 to 100% even when the tumor is resected with complete histological margins [6, 8, 14]. This finding may be explained by the phenomenon called “field cancerization effect,” which hypothesizes that bladder urothelium is uniformly exposed to carcinogens through urine contact, making it more susceptible to the development of multiple microscopic foci of malignant transformation, ultimately leading to macroscopic UC [6, 8, 55].

In comparison, of the 13 dogs that achieved a CR in this study, five had local tumor recurrence, with a recurrence rate of 38.4%, which was considerably inferior to the aforementioned studies. The mean time to recurrence in these patients was 354 days, which is longer than that reported by Marvel et al. (2017) and Bradburry et al. (2021) [6, 8] (101 and 85 days, respectively). It is important to note that in these two studies, medical therapy (chemotherapy and/or NSAIDs) was initiated after surgery. However, in the present study, ECT was performed as the sole treatment for all 21 dogs, indicating efficient disease control even without medical therapy.

One of the factors that may have contributed to the lower recurrence rate in this study might be attributed to the treatment of the entire bladder inner wall with ECT using intravenous BLM. This approach could lead to the destruction of possible microscopic neoplastic foci along the urothelium, minimizing the development of tumor recurrence and the risk of tumor implantation in other bladder regions. These effects are related to the selective mechanism of action of BLM, which leads to the preservation of the healthy urothelium [29, 46, 47].

Regarding survival data, studies evaluating partial cystectomy in bladder tumors away from the trigone have reported a median survival time (MST) varying from 348 to 498 days [8, 13, 14]. For most patients in these studies, adjuvant chemotherapy and/or NSAIDs were combined with surgery, making it difficult to determine whether this association resulted in an increase in survival time. In this context, it is important to highlight two main points in the present study:1) ECT was not combined with any other adjuvant therapy and 2) the majority of dogs (76.1%) had tumors located in the bladder trigone. Dogs presenting with tumors in this location naturally have a worse prognosis and shorter survival times because of the complex surgical approach [6, 21]. Despite this unfavorable scenario, the MST achieved by the dogs in this study was 420 days, which is similar or superior to those obtained in the aforementioned studies [8, 13, 14]. Furthermore, tumor location was not correlated with DFS and survival time.

The MST achieved was also superior to that obtained in studies that evaluated the use of chemotherapy (and/or NSAIDs) as a single treatment, with MST ranging from 108 to 338 days [48, 49, 51, 53, 54, 56, 57].

The time between ECT sessions varied in each case depending on the individual response, and usually ranged between 30 and 60 days. During the follow-up visits, it was noted that patients who underwent a new procedure at shorter time intervals (33–40 days) had a greater amount of necrotic tissue in the tumor region, which remained from the previous session. This fact can be explained by the mitotic cell death caused by BLM, which is a slower process that relies on the mitotic rate of the tumor cells and may take a longer time to achieve a response and tissue healing [46, 47]. Although there is still no consensus on when it is more appropriate to perform a subsequent ECT session, it is possible to determine the preferable interval between ECT sessions to range from 45 to 60 days.

Regarding the number of ECT sessions, the majority of treated dogs (57%) received only one session. Furthermore, among patients who achieved CR, 77% achieved CR in the first session. In addition, in the Kaplan-Meier analysis, the number of sessions did not influence patient survival (P > 0,01). However, care must be taken when interpreting these data, as there was a non-homogenous distribution of patients among the groups, which may constitute a bias in the analysis.

These results indicate that ECT offers good local control and antitumor effects regardless of the number of sessions. This favorable initial response is in agreement with different published studies that demonstrate the efficiency of ECT for different types of carcinomas [31, 39, 40]; despite being of different origins, they show similar morphology and response to ECT.

In conclusion, ECT is a safe and feasible therapy for canine patients with T1 and T2 bladder UC without serosal involvement. The therapy was well tolerated by the patients, providing significant local control of the disease along with a good quality of life. Regarding its antitumor efficiency, the results obtained in this study are promising; however, they are still preliminary. Further prospective studies should be conducted to evaluate the efficiency of ECT in bladder tumors, using a larger sample size of dogs and performing comparative analysis between different treatment groups (ECT vs. conventional therapy).

Limitations

Although the primary aim of this study was to evaluate the safety and viability of ECT in canine bladder UC, one of its limitations is the lack of a control group with appropriate statistical analysis comparing ECT with conventional treatments of bladder UC. The absence of a control group is due the fact that all dogs presented with bladder UC, that met the inclusion criteria, were treated with ECT.

In addition, it is important to highlight that the small sample size can represent a statistical bias when the survival of patients is compared according to the number of sessions and type of response.

Ethical aspects

This prospective clinical study was performed in accordance with the relevant guidelines and regulations of the Brazilian National Council for the Control of Animal Experimentation (CONCEA) and was approved by the Ethics Committee in the Use of Animals of the São Paulo State University “Júlio de Mesquita Filho” (UNESP), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Brazil (protocol no. 3848/20). Written informed consent to perform the treatment was obtained from all the dogs’ owners. All experiments methods were performed in accordance with relevant guidelines and regulations and complies with the ARRIVE guidelines.

Study design and patient selection

Twenty-one dogs with spontaneous bladder UC were enrolled in this prospective, longitudinal study. The dogs were presented to the Vet Câncer Oncologia e Patologia Animal (São Paulo, Brazil) between March 2016 and March 2021. ECT was offered as an alternative therapy when the owners refused other standard treatments such as surgery or medical therapy. The recommendations made by Campana et al. (2016) [58] for clinical studies of ECT were followed.

Before selection, the patients underwent physical examination, complete blood count, serum biochemical analysis (creatinine, BUN, alkaline phosphatase, and ALT), and electrocardiogram. Three-view thoracic radiography and abdominal ultrasonography were also performed for metastasis screening and bladder tumor evaluation. Dogs were classified according to the TNM clinical staging system defined by the World Health Organization for canine bladder tumors [2] (Table 1).

Dogs eligible for inclusion in the study had to meet the following criteria:1) histopathological diagnosis of bladder UC; 2) T1 or T2 stage (without bladder serosa layer involvement); 3) absence of urethra and/or ureter involvement; 4) absence of regional and distant metastatic disease; and 5) choice of the dogs’ owners to pursue ECT treatment after presenting all treatment options. Dogs that did not meet these criteria were excluded from the study.

Surgical procedure

All procedures were performed under general anesthesia. For induction, propofol (Lipuro® 10 mg/ml; Braun, Melsungen, Germany) was administered intravenously at a dose of 5 mg/kg, followed by endotracheal intubation. Isoflurane (Genérico, Biochimico Laboratory, Itatiaia, Rio de Janeiro, Brazil) was used to maintain anesthesia.

Laparotomy was performed using the ventral midline approach, followed by examination of the regional iliac lymph nodes, which were aspirated for intraoperative cytological evaluation (even if macroscopically normal) for staging purposes. Subsequently, the bladder was carefully isolated from the abdominal cavity, and cystotomy was performed. Following tumor visualization (Fig. 1A), incisional biopsies were performed and an intraoperative histopathological evaluation was performed.

During cystotomy, to minimize tumor cell spread and subsequent implantation in neighboring organs, the following criteria were met:1) careful manipulation of the bladder; 2) exchange of surgical instruments and gloves after contact with the tumor; and 3) in case of bladder eversion, contact of the inner bladder wall with any other intra-abdominal structure was avoided.

Intraoperative histopathological evaluation

Intraoperative histopathological evaluation was performed using frozen sections. This technique comprised the steps of macroscopic analysis of the samples, cleavage, freezing with compressed CO₂ and obtaining sections approximately 5 µm in thickness in a portable freezing microtome (Leica). The sections were then stained with toluidine blue and analyzed under a light microscope (Nikon YS2).

Intraoperative evaluation allowed the assessment of regional lymph node involvement, tumor diagnosis, degree of tumor infiltration on the bladder wall, and consequently, the definitive inclusion of the patients (Fig. 2).

Electrochemotherapy protocol

In patients who met the inclusion criteria, ECT was initiated by intravenous injection of BLM (Bleovet®) at a dose of 15000 UI/m2, after dilution in 5 mL of saline solution. Eight minutes later, electrical pulses were applied over the tumor and the entire inner bladder wall (Fig. 1B). Pulses were triggered whenever the internal wall of the bladder was attached to the electrode needles, to avoid cancer cell implantation at other bladder sites. The pulses were applied within a period of 40 min from drug injection, as recommended by Tellado, Mir and Maglietti (2022) [59] in the Veterinary Guidelines for Electrochemotherapy of Superficial Tumors.

Each train of pulses consisted of eight square bipolar pulses, 1,000 V/cm, 100 µs long, at 5 kHz frequency, delivered by VETCP 125® (IMPLASTIC, São Paulo, Brazil). The pulses were delivered using a 6-needle electrode, which consisted of two sequences of three needles, with 5 mm distance between contralateral needles and a 3 mm distance between ipsilateral needles.

To monitor for immediate adverse effects, urinary output, and postsurgical pain, the patients were hospitalized with a urethral catheter for at least 48 hours. Anti-inflammatory and analgesics used during hospitalization included tramadol hydrochloride (4 mg/kg/BID) and meloxicam (0,1 mg/kg/SID), both subcutaneously. Patients were discharged with domiciliary prescription of tramadol hydrochloride (4 mg/kg/VO/BID) and piroxicam (0.3 mg/kg/VO/SID) for 3 more days.

Patient follow-up

Initial follow-up was performed 7, 14, 21, and 30 days after surgery. The treatment period was individualized for each dog and was determined as the period between the day of the first ECT and 30 days after the last ECT session. If no further sessions were performed, regular check-ups were scheduled every 60 days for the first 6 months and every 90 days until a year after the treatment, which is considered the observation period. Physical examination, complete blood count, biochemical analysis, and abdominal ultrasonography were performed during each visit. Chest radiographs were obtained every 3 months to monitor for lung metastasis.

Safety evaluation

The development of adverse events was assessed during ECT treatment, immediately after ECT treatment, during postsurgical hospitalization, and at each follow-up visit. These events were graded according to the VCOG-CTCAE v2 [37] toxicity scale as follows:1) mild, 2) moderate, 3) severe or medically significant, but not immediately life-threatening, 4) life-threatening consequences, or 5) death related to adverse events.

Quality of life

Quality of life was assessed before the first ECT procedure and at each visit using a questionnaire inspired by the Canine Owner-Reported Quality of Life, developed by Giuffrida et al. (2018) [60].

The questionnaire contained 17 items related to observable behaviors commonly perceived by owners and was organized into four proposed factors: vitality, companionship, pain, and mobility. Each item was arranged as a sentence that described a common behavior, followed by a numerical analog scale (0–7). The owner was asked to circle the number of days in the previous week the behavior occurred.

Additional information obtained by the oncologist and owner at each follow-up visit was documented in the dog’s medical records.

Response evaluation

According to the RECIST criteria [61], the local response was assessed at 30 days after each ECT session and the final local response was determined at the end of the treatment period, when no more ECT sessions were needed or the owner refused them.

The local response and need for additional sessions were determined by ultrasound assessment performed at each visit by the same radiologist (Fig. 3). The owners were instructed to offer water ad libitum and not allow their animals to urinate for one hour before ultrasound examination. During the examinations, the dogs were positioned in dorsal recumbency with full urinary bladder repletion, according to the recommendations of Fulkerson and Knapp (2015) [62] for bladder tumor imaging assessment.

New ECT sessions were performed when macroscopic evidence of the tumor was identified during ultrasound evaluation at least 30 days after the previous session. If a complete remission was identified, consent for an additional surgical procedure was requested from the owners, in order to confirm the absence of residual microscopic disease. If neoplastic foci were identified on intraoperative evaluation, a new ECT treatment was initiated.

Survival time was defined as the period between diagnosis and death, or the end of the study period. Disease-free survival was calculated only for patients who achieved CR. It was defined as the period between CR achievement and the day of local or distant relapse, death, or the end of the study. The duration of response was calculated for all patients who achieved CR, PR, or stable disease and was defined as the period between the final local response achievement until local or distant relapse/progression, death, or end of the study. Dogs who developed progressive disease within the treatment period were excluded from the study and were directed to other treatment modalities.

Statistical analysis

Statistical analyses were performed using MedCalc version 14.8.1. To analyze the influence of different factors on survival and disease-free survival, Kaplan-Meier curves were used, and significance was assessed using the log-rank test. Fisher’s exact test was performed to determine the influence of different factors on the achievement of the response. Statistical significance was set at p < 0.01.

Acnowledgements

The authors would like to thank for Coordination for the Improvement of Higher Education Personnel (CAPES) for funding the research and the owners of the dogs participating in this study.

Author contributions

M.M.M.R. conceived, designed and executed the experiment. M.M.M.R.; D.O.H.S.; A.B.D.N. conceptualized and supervised the project. K.D.O. executed pathological analysis. L.C.M.L. conducted data collection and analysis and prepared the paper. F.H.M. Statistical analyzed data. All authors interpreted the data, critically revised the manuscript for important intellectual contents and approved the final version.

Data availability

The data generated during this study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Correspondence and requests for materials should be addressed to L.C.M.L.

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No competing interests reported.

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Evaluation of the safety and feasibility of electrochemotherapy with intravenous bleomycin as local treatment of bladder cancer in dogs

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Abstract

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Introduction

Results

Patient data

Safety evaluation

Quality of life

Cause of death

Local response

Survival data

Discussion

Limitations

Methods

Ethical aspects

Study design and patient selection

Surgical procedure

Intraoperative histopathological evaluation

Electrochemotherapy protocol

Patient follow-up

Safety evaluation

Quality of life

Response evaluation

Statistical analysis

Declarations

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