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

Local treatment of canine urothelial carcinoma (UC) of the bladder is a challenge. More than 90% of the cases invade the muscular layer, more than 50% develop on bladder sites with a difficult surgical approach and often requiring radical surgical procedures. This study aims to evaluate the safety and feasibility of electrochemotherapy (ECT) with intravenous bleomycin (BLM) as a local therapy for bladder UC. This prospective study included 21 dogs with spontaneous bladder UC. Regional/distant metastases and neoplastic infiltration of the serosa was considered the main exclusion criteria. We had no deaths during ECT or in the immediate postoperative period, and no 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 cases (13/21), while partial response (PR) was achieved in 24% (5/21). The median survival and disease-free survival times were 284 and 270 days, respectively. Overall survival was significantly better in the dogs who achieved a CR. In conclusion, 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.


Intraoperative histopathological evaluation
Intraoperative histopathological analysis was performed for the 26 dogs initially enrolled (Fig. 1A).Neoplastic infiltration of the serosa was not identified in 21 cases, which received ECT treatment immediately after evaluation (Fig. 1B and 2; Supplementary Video 1).Conversely, five dogs presented serosal involvement, which were excluded from the study and redirected to other therapies.Information about other histopathological features are described in Table 3. High grade papillary UC represented 76% of the cases (16/21) and the majority of them were classified as grade 3 (13/16).Three cases had non-papillary tumors and all of them were high grade.Only one case was classified as UC with divergent differentiation (microcystic).Inflammation was present in almost all cases (95%; 20/21) and lymphatic invasion was identified in four cases (19%) (Fig. 1C).

Safety evaluation
None of the dogs died during the surgical procedure, ECT treatment, or in the immediate postoperative period.Furthermore, none of the dogs had suture dehiscence in the bladder or uroperitoneum.All the dogs were evaluated for adverse events (Table 4).Nineteen dogs (90%) presented with mild adverse effects during the postoperative period, including pollakiuria, stranguria, dysuria, and transitory urinary incontinence.The degree of toxicity for these dogs was classified as grade 1 according to the "Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events" (VCOG-CTCAE v2) 49 .Two dogs had more serious complications (VCOG = 3), developing ureteral stenosis 56 and 69 days after the procedure.These dogs presented ultrasonographic alterations consistent with ureter dilation, i.e. megaureter, pyelectasis, and/or hydronephrosis (Fig. 3).Thereafter, they underwent a surgical procedure for the placement of a temporary "double-J" ureteral stent which was removed after recovery of ureteral patency.
Life-threatening abnormalities on blood tests were detected during follow-up in four dogs, who died within 34 days (cases #3, #12, #13, #21) due to causes unrelated to the ECT.These alterations included anemia, thrombocytopenia, leukocytosis, and elevated levels of serum creatinine and BUN, and were associated with their respective causes of death (Table 2).www.nature.com/scientificreports/

Quality of life
According to the quality of life questionnaire filled by the owners and the oncologist's clinical evaluations, all 19 dogs who developed mild adverse events had no relevant negative impact on their quality of life.
As expected, the two dogs that developed ureteral stenosis manifested a more severe alteration of their life quality, including a higher level of pain and changes in behavior.However, this negative impact on the quality of life was transitory, lasting until surgical resolution of the complication.The procedures were well tolerated by the dogs, who completely recovered their baseline quality of life score according to subsequent assessments.

Cause of death
Three dogs died of disease progression.Of these, two dogs developed local progression of the tumor inside the bladder (case #1) and to the prostate (case #18), and the other dog developed distant metastatic spread to the lungs (case #8).
Sixteen dogs died of causes unrelated to the disease or the ECT, which included mainly other types of neoplasia (4/16; 25%), and chronic kidney disease (3/16; 18.7%).The remaining causes of death included ehrlichiosis, sepsis, pulmonary thromboembolism, peliosis hepatis, perforated gastric ulcer, acute kidney injury and cardiomyopathy (Table 2).It was not possible to determine the cause of death in one dog.
Necropsy was performed only in two dogs (case #3 and #4).In the remaining cases it was not performed due to owners' refusal, and the cause of death was presumed according to the clinical condition and blood and imaging tests.

Local response
Sixty-two percent of the dogs (13/21) achieved a complete response (CR) (Fig. 4), and 24% (5/21) had partial response (PR) (Fig. 5).An objective response rate (ORR) of 86% was achieved.The remaining three dogs 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).www.nature.com/scientificreports/Among the dogs who achieved a CR, most of them achieved it in one session (77%).Among the dogs who required two sessions (15%), a median of 56 days (range 22-71) passed between the two sessions.For the dogs 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 dogs who had a CR, which underwent a new ECT session.However, the owners of the remaining eight dogs with CR refused any additional surgery.
Of the five dogs who achieved PR, no additional ECT sessions were performed because of the owner's refusal.Two dogs showed local disease progression.
The median duration of response (DOR) was 270 days (average 371, range 30-981 days).Tumor recurrence occurred in five dogs (38%) in which CR was achieved, and the median time to its development was 257 days (average 354, range 170-790 days) (Table 2).
At the end of the study, two dogs remained alive, one with CR and the other with tumor recurrence.Among the 19 dogs who died, six (31.5%) died without tumor evidence (CR), 10 (52.6%) died with macroscopic or microscopic tumor evidence, and three (15.7%)died before the period of response evaluation (Table 2).

Discussion
To date, two preliminary studies have reported the application of ECT in canine bladder UC; however, both studies used a small series of cases 48,50 .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.
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 700fold 32,33,[51][52][53] .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 by mitotic cell death process.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 32,52 .
Thus, the process of cell death 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, 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.
In addition to the evaluation of tumor invasion, other relevant histopathological information was obtained from each case.Papillary infiltrative UC was considered the most prevalent histological subtype in this study, agreeing with the results of Govoni et al. 54 .Regarding tumor differentiation, 20 cases (95%) in this study were classified as conventional UC, while only one case was classified as divergent UC.Similarly, in the study of De Brot et al. 1 , 90% of the dogs had conventional UC, and 6% divergent UC.
Recently, lymphatic invasion has been proposed as a relevant negative prognostic factor for canine UC 54 .In the present study, only four cases showed lymphatic invasion and due to this small sample size, it is difficult to determine whether this feature affected the OS of the dogs.
None of the cases 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. 48erformed 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 dogs developed any of these complications.
The vast majority of dogs 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. 48also reported transient urinary incontinence in dogs after the treatment, lasting 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 dogs (9.5%) developed ureteral stenosis at 56 and 69 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 front of this, we believe that the tumor proximity to the ureter could be a risk factor for the development of ureteral stenosis.Additional care should be taken in those patients as well as more frequent clinical and imaging reassessments.
One of these dogs (case #17) achieved a PR and developed ureteral stenosis after 56 days from the second session of ECT.At the time of the stent placement, the tumor was not macroscopically identifiable, however, there was a thick area of fibrosis in the bladder wall, close to the ureteral ostium, leading to its stenosis.Intraoperative pathology evaluation of this area revealed the presence of superficial microscopic UC confined to the mucosa associated with inflammation.However, we did not perform a third session of ECT because of the concurrent scarring process in the same area, minimizing the amount of inflammatory tissue (Supplementary Fig. 1).
Studies evaluating partial or total cystectomy in dogs with bladder UC have found more significant complication rates than we did (range 43-70%), including suture dehiscence, ureteral obstruction, pyelonephritis, and uroperitoneum 6,11,14 .
In particular, 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 three dogs (14%) died of UC, one due to distant metastasis and the other two due to local tumor progression.The dog who died due to tumor progression inside the bladder (case #1) achieved a PR and died 517 days after the only ECT session performed.No additional sessions or other treatments were performed due to owner refusal.However, the survival time was above the average for dogs treated with conventional therapies (surgery and/or chemotherapy) 6,8,55,56 .
The other case who died due to local progression (case #18) was an intact dog who presented prostate enlargement with mineralization foci, non responsive to castration.Prostate cytological examination was inconclusive and the owner declined to perform prostate biopsy and necropsy after death.Abdominal ultrasound did not reveal urethral abnormalities.Although it is difficult to determine if it was a primary prostate tumor or a local spread of the bladder tumor to the prostate, we considered it as a local progression to the prostate, as the primary tumor was a grade 4, infiltrative UC with lymphatic invasion.In this study only one dog (case #8) developed distant metastasis to the lungs, identified by chest x-rays, meaning a metastasis rate of 4.7% In contrast, according to a study of Knapp et al. 5 , the rate of distant metastasis at the time of death was 49% among 80 dogs with www.nature.com/scientificreports/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, decreasing the probability of metastasis.However, future studies including a control group could confirm this.The remaining 16 cases died from causes unrelated to UC.The dogs included in this study were old with multiple comorbidities.The treatment of bladder cancer provided very good results and prevented the death of the animals from the disease.However, other concomitant pathologies, i.e. cardiac alterations, gastric ulcers, other tumors, among others, continued progressing and ended up provoking their death.
The case with undetermined cause of death (case #13) had a history of hyperadrenocorticism and diabetes, controlled with trilostane and insulin applications at home.No complications occurred during the ECT procedure and during the first 24 h of hospitalization.However, the dog was withdrew from the clinic by the owner's decision before the completion of the minimum 48 h stay.After a few days the dog presented signs of dyspnea leading to a sudden death.The owners declined necropsy, so it is impossible to determine accurately the cause of death.We consider that the death is not related to the ECT procedure because abdominal ultrasound was unremarkable for urinary tract abnormalities and the dog was urinating normally before death.The cause could be attributed to pulmonary thromboembolism, as a complication of hyperadrenocorticism, considering the sudden clinical signs.Dogs with hyperadrenocorticism have an increased risk of developing blood hypercoagulability due to the increased procoagulant activity and/or decreased fibrinolysis, which can culminate in pulmonary thromboembolism 57,58 .
The case which died due to acute kidney injury (AKI; case #12) had a previous history of chronic kidney disease, however, the renal panel (BUN and creatinine) and urinalysis, performed prior to ECT, were unremarkable, as well as the dog was in good general condition.We believe that this dog may have developed an acute injury to the kidneys due to an aggravation of the previous CKD related to the use of NSAIDs during the postoperative period.Although AKI is not reported as a complication of ECT, caution should be taken with elderly dogs with some grade of kidney disease.
Lymph node involvement was assessed through fine-needle aspiration and intraoperative cytology.Even apparently normal nodes were evaluated.Although histopathology is the gold standard for definitive diagnosis of lymph node metastasis, we elected cytological evaluation because it is faster, either for sample collection or intraoperative evaluation, and it is associated with lower intraoperative complication rates.It is important to note that all dogs 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 dogs with bladder UC who undergo cystotomy.Higuchi et al. 59 and Marvel et al. 6 , reported the occurrence of abdominal wall metastasis during a follow-up at 121 and 230 days respectively.We had longer follow-up times of around 1000 days, and in contrast, none of the dogs in the present study developed this complication after cystotomy and ECT.We attribute this to the careful manipulation of the bladder and to the fact that the whole bladder was treated with ECT.
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 of unrelated causes before the minimum period for response.This overall response rate (86%) was greater than those reported in studies evaluating different chemotherapy protocols for dogs with bladder UC, ranging from 3 to 71%, in which partial responses were predominant 7 .However, to confirm this, comparative studies are needed.
For the assessment of local response, ultrasound images sections obtained from each examination should be standardized, for ideal comparative purposes.However, as a limitation of the study, we could not standardize the cross-sections of the images depicted in the ultrasound report for some cases, as we can see in Fig. 4A (transverse section) and 4B (sagittal section).
The median DOR obtained was 270 days (average 371, range 30-981 days), which was higher than the 235 and 85 days obtained by Marvel et al. and Bradburry et al. 6,8 , respectively.The median DOR of this study was also higher than that reported in several studies evaluating medical therapy, which ranged from 96 to 199 days [60][61][62] .
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 the urine, making it more susceptible to the development of multiple microscopic foci of malignant transformation, ultimately leading to macroscopic UC 6,8,63 .
The recurrence rate of this study is 38%, which is considerably inferior when compared to the aforementioned studies.The median time to recurrence in these dogs was 257 (average 354, range 130-790 days) which is longer than the one reported by Marvel et al. and Bradburry et al. 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.In the study of Bradburry et al. 8 , dogs predominantly in stage T2 were included as were in this study.In the study of Marvel et al. the TNM stage of the dogs was not reported.
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 microscopic neoplastic foci along the urothelium, minimizing the development of tumor recurrence and the risk of tumor implantation in other regions of the bladder.This is possible thanks to the selective mechanism of action of BLM, which leads to the preservation of the healthy urothelium 32,33,52 .
Regarding survival data, studies evaluating partial cystectomy in bladder tumors away from the trigone have reported a median OS varying from 348 to 498 days 8,13,14 .For most dogs 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 OS.In contrast, the median OS achieved by the dogs in this study was 284 days (average 420, range 7-1021 days), which is lower to those obtained in the aforementioned studies 8,13,14 .However, it is important www.nature.com/scientificreports/ to consider two main points in the present study.First, ECT was not combined with any other adjuvant therapy.
Second, the majority of cases (76%) had tumors located in the bladder trigone.Tumors in this location naturally have a worse prognosis and shorter OS because of the complex surgical approach.Furthermore, tumor location was not correlated with DFS and survival time in the present study.The median OS achieved was similar or superior to that obtained in studies that evaluated the use of chemotherapy (and/or NSAIDs) as a single treatment, with median OS ranging from 108 to 338 days 55,56,60,62,64,65 .In regard to targeted therapies, clinical trials with single-agent vemurafenib (BRAF inhibitor), mogamulizumab (CCR4 inhibitor) and lapatinib (HER-2 and EGFR inhibitor), alone or in combination with piroxicam, resulted in median OS of 354, 474 and 435 days, respectively [22][23][24] .These survival outcomes were very promising, encouraging further research to compare the effectiveness of targeted therapies with ECT, alone or in combination.In addition, there is an evidence of higher PDGFR-ß expression in bladder UC samples comparing with normal bladder and cystitis samples 66 , suggesting a potential clinical use of toceranib phosphate (VEGFR-2 and PDGFR-ß inhibitor).In a retrospective study 67 , the use of single-agent toceranib phosphate in dogs with UC resulted in a clinical benefit of 86.7%, however, the median OS was 149 days.
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 dogs 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 replication rate of the tumor cells.This varies among tumors taking a longer time to achieve a response in tumors that replicate slower [51][52][53] .The current recommendation suggests that new ECT sessions should be performed when macroscopic evidence of the tumor (or relapse) is seen 33 .In this study, however, as we had intraoperative histopathological analysis, we indicated additional procedures even when macroscopic CR is achieved.In summary, according to our experience and considering the time to tumor response as an individual and variable factor, it is possible to determine the preferable interval between ECT sessions to range from 45 to 60 days.
In conclusion, ECT is a safe and feasible therapy for dogs with T1 and T2 bladder UC without serosal, urethral, or ureteral involvement or metastasis.The therapy was well tolerated by the dogs, 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 comparing ECT with conventional treatments for 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.Another limitation of this study is the small number of dogs requiring two, and three sessions of ECT.For this reason, the comparison of the survival times among these groups is difficult.A case series with more dogs is needed to address this aspect.
In addition, we have that most of the cases were in stage II, it is an early stage and surgery can provide good results when performed correctly.Also, we have to consider that dogs with serosal, urethral, or ureteral invasion were excluded.This can be considered a selection bias, however, due to our knowledge and experience with ECT, we anticipated serious complications if we proceeded with the treatment of those cases.ECT mainly eliminates tumoral cells, as they are replicating faster.If the whole thickness of the bladder wall is replaced with tumoral tissue, then no normal tissue is present in that precise point.When the response is achieved, and the tumoral cells are eliminated, a perforation in the bladder wall can occur, producing the leakage of urine to the peritoneum.For those reasons, we consider it is mandatory to refrain from treating with ECT cases where serosal invasion is present.Future studies are needed to contrast the results of ECT with other local therapies.

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 were performed in accordance with relevant guidelines and regulations, complying with the ARRIVE guidelines.

Study design and case 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. 68 for reporting clinical studies of ECT were followed.
Before selection, the dogs 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 screening of metastasis and for evaluating the bladder www.nature.com/scientificreports/

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. 71 .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 72 , the local response was assessed 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 rejected them.
The local response and the need for additional ECT sessions were determined by ultrasound assessment performed at each visit by the same radiologist (Figs. 4 and 5).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 73 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 indicated.
Survival time was defined as the time between the first ECT and death, or the end of the study period.DFS was calculated only for cases 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.PFS was calculated only for dogs who achieved PR or stable disease, and was defined as the period from PR achievement (or from the first ECT, for stable disease cases) until progression or death.Finally, the DOR was calculated for all dogs 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.Cases who obtained a PD after the ECT, were reported and redirected to other therapy.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.

Figure 1 .
Figure 1.Intraoperative histological photomicrograph by frozen section of bladder UC (case #15) (A) Central region of a papillary UC (toluidine blue).(B) Muscular (white arrow) and serosal (black arrow) layers of the bladder free of neoplastic infiltration (toluidine blue).(C) Lymphatic infiltration of tumoral cells (black arrow) (toluidine blue).

Figure 3 .
Figure 3. Ultrasonographic evaluation of the urinary tract of case #17.(A) Moderate to severe pyelectasis of right kidney; renal pelvis measuring 2 cm.(B) Ureter dilation (i.e.megaureter) due to stenosis of the ureteral ostium, after 56 days from the second ECT session.

Figure 4 .Figure 5 .
Figure 4. Local response assessment by ultrasonography of case #15.(A) Bladder tumor before the first ECT session.(B) Complete tumor remission after one ECT session.

Table 1 .
Demographic characteristics of the dogs treated.

Table 2 .
General case 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, UC urothelial carcinoma, CKD chronic kidney disease, AKI acute kidney injury.*Days, **Inside the bladder.

Table 3 .
70stopathological features of all the cases evaluated in this study.*AccordingtoChengetal.69and Meuten70.

Table 4 .
Adverse events and treatment related toxicity (veterinary comparative oncology group criteria) in dogs in the clinical trial.