Angiogenesis in Transitional cell carcinoma of the bladder in dogs

In questo studio è stato valutato il potenziale angiogenico espresso dal carcinoma a cellule transizionali della vescica del cane misurando alcuni marcatori nelle urine di soggetti affetti da tale neoplasia. In particolare è stata misurata l'attività delle Gelatinasi MMP2 e MMP9 ed è stata misurata la concentrazione di VEGF urinario. E' stata inoltre stabilita la presenza di mutazione somatica cBRAFV595E. I risultati evidenziano un aumento significativo di molecule angiogeniche nei soggetti affetti da carcinoma a cellule transizionali della vescica rispetto ad un gruppo di controllo. Inoltre, nel 90% dei casi esaminati è presente mutazione cBRAFV600E. I risultati ottenuti aprono prospettive nel campo terapeutico per il trattamento di questa neoplasia del cane.The aims of this study was to establish the angiogenic potential of the transitional cell carcinoma (TCC) of the bladder in dogs by assessing some urinary markers known to reflects the angiogenesis mechanisms. In particular, MMP2 and MMP9 activities were measured by gelatin zimography as well as the VEGF urinary concentration was mesasured using a canine specific ELISA assay. Furthermore, the cBRAFV595E somatic mutation was evaluated. Our findings show that angiogenesis markers are significantly increased in TCC when compared to healthy control dogs. Finally, the cBRAFV595E mutation was highly prevalent and present in the 90% of dogs. Our results support the rationale for a change in the paradigm of the TCC treatment in dogs

Validation of a Liquid Biopsy Protocol for Canine BRAFV595E Variant Detection in Dog Urine and Its Evaluation as a Diagnostic Test Complementary to Cytology

A significant proportion of canine urothelial carcinomas carry the driver valine to glutamic acid variation (V595E) in BRAF kinase. The detection of V595E may prove suitable to guide molecularly targeted therapies and support non-invasive diagnosis of the urogenital system by means of a liquid biopsy approach using urine. Three cohorts and a control group were included in this multi-step validation study which included setting up a digital PCR assay. This was followed by investigation of preanalytical factors and two alternative PCR techniques on a liquid biopsy protocol. Finally, a blind study using urine as diagnostic sample has been carried out to verify its suitability as diagnostic test to complement cytology. The digital PCR (dPCR) assay proved consistently specific, sensitive, and linear. Using the dPCR assay, the prevalence of V595E in 22 urothelial carcinomas was 90.9%. When compared with histopathology as gold standard in the blind-label cases, the diagnostic accuracy of using the canine BRAF (cBRAF) variation as a surrogate assay against the histologic diagnosis was 85.7% with 92.3% positive predictive value and 80.0% negative predictive value. In all the cases, in which both biopsy tissue and the associated urine were assayed, the findings matched completely. Finally, when combined with urine sediment cytology examination in blind-label cases with clinical suspicion of malignancy, the dPCR assay significantly improved the overall diagnostic accuracy. A liquid biopsy approach on urine using the digital PCR may be a valuable breakthrough in the diagnostic of urothelial carcinomas in dogs

Neoangiogenesis markers in canine urothelial carcinomas: A cross‐sectional study

Background: In humans, there is a growing body of evidence that neoangiogenesis is crucial for tumour growth and progression in urothelial carcinomas (UC) which also typically exhibit overactivation of the RAS-MAPK pathway. In canine UC (cUC), the same pathway has been aberrantly activated due to V595E BRAF variant and BRAF inhibitors has been evaluated as more effective treatment. However, BRAF inhibition is hampered in humans by rapidly occurring of chemoresistance. Targeting angiogenesis has been speculated to increase the effectiveness of BRAF inhibitors and to delay the development of chemoresistance. Objectives: This study aimed to investigate the level of angiogenic markers in urine samples of UC affected dogs (n = 15) in comparison to an unmatched control group (n = 16) along with the clinical, morphological and molecular features. Methods: In urine, both vascular endothelial growth factor (VEGF) concentration, using an ELISA assay, and MMP2 and 9 activities, using the zymographic assay, were measured. BRAF analysis was carried out using a digital PCR method. Results: Urinary VEGF concentration (mean pg/g_uCrea 6.9 +/- 27.7 vs. 1074 +/- 1797, p < 0.01) and MMP activity (mean 6.8 x 10(6) +/- 9.2 x 10(6) vs. 2.5 x 10(7) +/- 2.3 x 10(7), p < 0.05) were higher in affected dogs than in healthy controls. Urinary active MMP9 was significantly correlated with T3 stage, it was absent in dogs with undetectable VEGF and it correlated well with urinary VEGF concentration. In this cohort, 10/10 UC affected dogs exhibited the V595E BRAF variation. Conclusion: The findings are consistent with the presence of overactive neoangiogenesis in cUC. Urinary active MMP9 may be suitable for use as tumour progression biomarker. The addition of angiogenesis targeting may be rationale for novel therapeutic strategies

Evaluation of Ki-67 expression in feline non-ocular melanocytic tumours

Fifty tumours located in skin (n = 33) and mucosae (n = 17) were included. Forty-eight percent and 95% of amelanotic tumours (n = 21) stained positive for Melan A and S100, respectively. Most achromic tumours were mucosal (P  5, Ki-67 > 20% and lack of treatment administration. On multivariable analysis, only tumour histotype and treatment retained prognostic significance. Conclusions Although the majority of feline NOMs behave aggressively, Ki-67 index, together with other parameters, may contribute to prognostic assessment. Prospective studies on homogeneous populations are warranted to identify reliable threshold values for this marker

TREATMENT OF CUTANEOUS SQUAMOUS CELL CARCINOMA ON THE HEAD REGION WITH ELECTROCHEMOTERAPY IN A GROUP OF 19 CATS.

Cutaneous squamous cell carcinoma (SCC) accounts for 10% of all feline cutaneous tumours, and it is the third most common malignancy in cats. [1] SCC is locally invasive with rare metastasis to the regional lymph nodes. [2] Standard treatment approaches include surgery and radiation therapy. [1,2] Electrochemotherapy (ECT) uses the application of electric pulses combined with chemotherapeutic drugs (bleomycin) causing cytotoxic effects on the treated area. ECT also influences the immune system and tumour blood flow. [2,3] The aim was to evaluate the feasibility and efficacy of ECT in the treatment of SCC on the head. Nineteen cats were retrospectively enrolled (December 2004-March 2019). SCCs were diagnosed with cytology and/or histology. ECT was combined with IV bleomycin (15000 UI/m2) alone in 16/19 cases, post-surgery in 2/19, and before surgery in one case. Parameters considered were tumour site and size, electroporation parameters, response rate (response complete [CR] or partial [PR], stable disease [SD]), local recurrence rate (RR), disease-free interval (DFI), survival time, median survival time (MST), treatment outcome and local treatment toxicity (6-point scale). [4] Tumours were mostly located on the nasal planum (14/19). Median tumour size was 0.7 cm. Three different electroporators were used: Cytopulse Oncovet (12/19), Leroy Biotech Electrovet S13 (6/19) and Cliniporator, IGEA (1/19). Electroporation frequencies were 1 Hz or 5kHz and pulse amplitude to electric distance ratio was ranging 1000, 1200 or 1300 V/cm. Response rate was 94.7% (18/19; 13 CR and 5 PR). One cat had SD. Additional ECT was performed for 7 cats; 4 had a 2nd ECT, one a 3rd ECT and two a 4th ECT. For two cats with PR, RR was 10.5%, DFI was 44 and 694 days and survival time was 184 and 751 days respectively. MST for cats with recurrence was 467 days. At the end of the observation period 14 cats died and 5 were still alive. MST for cats dead without tumour (n=8) was 520 days and for cats dead with tumour (n=5) was 228 days. One cat with SD died with tumour 9 days after ECT. Treatment toxicity was ≤2 in 15/19 cases, two cats experienced toxicity score 3 and one each toxicity scores 4 and 5. Two cats were FIV+ and required multiple ECTs (PR), they had recurrence and no further response to treatment after the 4th ECT. All cats with tumours <1 cm achieved CR. A possible reduction of the treatment response in FIV + cats was noticed. However, ECT seems to be a good alternative to excisional surgery, especially in smaller tumours. Treatment toxicity was low and survival time considerably long. [1] Hauck ML. Tumors of the Skin and Subcutaneous Tissues. In: Withrow & MacEwen’s Small Animal Clinical Oncology. 5th ed. Elsevier saunders. [2] Tozon et al. Electrochemotherapy with intravenous bleomycin injection: An observational study in superficial squamous cell carcinoma in cats, J Feline Med Surg, 16(4):291-299, 2014. [3] Čemažar et al. Electrochemotherapy in veterinary oncology, J Vet Intern Med, 22(4):826-831, 2008. [4] Lowe et al. The treatment of canine mast cell tumours with electrochemotherapy with or without surgical excision, Vet Comp Oncol, 15(3):775-784, 2017