Effective detection of BRAFV595E mutation in canine urothelial and prostate carcinomas using immunohistochemistry.

Canine urothelial carcinoma (UC) and prostate carcinoma (PC) frequently exhibit the BRAFV595E mutation, akin to the BRAFV600E mutation common in various human cancers. Since the initial discovery of the BRAF mutation in canine cancers in 2015, PCR has been the standard method for its detection in both liquid and tissue biopsies. Considering the similarity between the canine BRAFV595E and human BRAFV600E mutations, we hypothesized that immunohistochemistry (IHC) using a BRAFV600E-specific antibody could effectively identify the canine mutant BRAFV595E protein. We tested 122 canine UC (bladder n = 108, urethra n = 14), 21 PC, and benign tissue using IHC and performed digital droplet PCR (ddPCR) on all 122 UC and on 14 IHC positive PC cases. The results from ddPCR and IHC were concordant in 99% (135/136) of the tumours. Using IHC, BRAFV595E was detected in 72/122 (59%) UC and 14/21 (65%) PC. Staining of all benign bladder and prostate tissues was negative. If present, mutant BRAF staining was homogenous, with rare intratumour heterogeneity in three (4%) cases of UC. Additionally, the BRAFV595E mutation was more prevalent in tumours with urothelial morphology, and less common in glandular PC or UC with divergent differentiation. This study establishes that BRAFV600-specific IHC is a reliable and accurate method for detecting the mutant BRAFV595E protein in canine UC and PC. Moreover, the use of IHC, especially with tissue microarrays, provides a cost-efficient test for large-scale screening of canine cancers for the presence of BRAF mutations. This advancement paves the way for further research to define the prognostic and predictive role of this tumour marker in dogs and use IHC to stratify dogs for the treatment with BRAF inhibitors.

Radiation toxicity grading after chemoradiotherapy of canine urinary tract carcinomas: Comparing VRTOG to VRTOG_v2.0.

Radiation toxicities may be underestimated after treatment of transitional cell carcinoma in dogs' lower urinary tract. Assessing acute and late toxicities and differentiating them from progressive disease (PD) impacts further therapeutic approach. We retrospectively assessed dogs treated with definitive-intent chemoradiotherapy (12 × 3.8 Gy, various first-line chemotherapeutics). Local tumour control, radiation toxicities and survival were evaluated. We classified radiation toxicities according to the previously published radiation toxicity scheme "VRTOG" as well as the updated version, "VRTOG_v2.0". Fourteen dogs with transitional cell carcinoma of bladder ± urethra (n = 8), +prostate (n = 3) or solely urethra (n = 3), were included. Median follow-up was 298 days (range 185-1798 days), median overall survival 305 days (95%CI = 209;402) and 28.6% deaths were tumour-progression-related. Acute radiation toxicity was mild and self-limiting with both classification systems: In VRTOG, 5 dogs showed grade 1, and 1 dog grade 2 toxicity. In VRTOG_v2.0, 2 dogs showed grade 1, 3 dogs grade 2, and 3 dogs grade 3 toxicity. Late toxicity was noted in 14.2% of dogs (2/14) with the VRTOG, both with grade 3 toxicity. With VRTOG_v2.0, a larger proportion of 42.9% of dogs (6/14) showed late toxicities: Four dogs grade 3 (persistent incontinence), 2 dogs grade 5 (urethral obstructions without PD resulting in euthanasia). At time of death, 5 dogs underwent further workup and only 3 were confirmed to have PD. With the updated VRTOG_v2.0 classification system, more dogs with probable late toxicity are registered, but it is ultimately difficult to distinguish these from disease progression as restaging remains to be the most robust determinant.

Reason for euthanasia in dogs with urothelial carcinoma treated with chemotherapy or radiation therapy or both: A retrospective observational study.

BACKGROUND: Clients want to know the ultimate cause of death in their pet after cancer treatment. The cause of euthanasia and investigation of urinary obstruction in treated dogs with urothelial carcinoma (UC) has not been specifically reported in veterinary literature. HYPOTHESIS/OBJECTIVES: Our hypothesis was that the majority of treated dogs with UC are euthanized secondary to primary tumor factors, such as urinary obstruction. ANIMALS: Fifty-nine client-owned dogs diagnosed with UC. METHODS: Retrospective observational study on clinical signs and disease at euthanasia of dogs with UC treated by radiation therapy or chemotherapy or both. RESULTS: The median overall survival time (OST) of all dogs was 339 days (range, 17-1996; 95% confidence interval [CI], 185-392; interquartile range [IQR], 112-505). Of dogs deemed to have been euthanized because of UC (50/59, 85%), the primary cause was considered to be local progression in 31/50 (62%), most often because of perceived complete or partial urinary obstruction (24/31, 77%). No variables were found to be predictive of urinary obstruction. The overall documented metastatic rate was 56%. In dogs euthanized because of UC, metastasis was deemed to be the cause in 19/50 (38%) dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Regardless of the type of treatment, UC in dogs has a poor prognosis and there is a continuing need to improve treatments that focus on local control of the primary tumor, given its high contribution to the decision for euthanasia. Proactive management to avoid the high frequency of urinary obstruction may be worthy of future investigation.

Myeloid derived suppressor cells in peripheral blood can be a prognostic factor in canine transitional cell carcinoma.

Myeloid-derived suppressor cells (MDSCs) are immature cells with immunosuppressive properties found in the tumor microenvironment. MDSCs are divided into two major subsets: polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). Both MDSC subsets contribute to the creation of an immunosuppressive environment for tumor progression. In humans, patients with high levels of MDSCs show worse outcomes for several types of cancers. However, the association between MDSCs and clinical features has rarely been investigated in canine studies. In the present study, we measured the proportion of PMN-MDSCs and M-MDSCs in the peripheral blood and tumor tissue of dogs with hepatocellular carcinoma (HCC), prostate cancer (PC), transitional cell carcinoma (TCC), lymphoma, and pulmonary adenocarcinoma. Additionally, we examined immunosuppressive ability of PMN-MDSCs and M-MDSCs in peripheral blood mononuclear cells of TCC case on CD4+, CD8+ and interferon-γ+ cells and investigated the relationships of MDSCs with clinical features and outcomes. PMN-MDSCs increased in HCC, PC, TCC, and lymphoma. In contrast, M-MDSCs increased in the TCC. Both PMN-MDSCs and M-MDSCs exhibited immunosuppressive effects on CD8+, CD4+ and interferon-γ+ cells. In dogs with TCC, lymph node metastasis was associated with high level of PMN-MDSCs but not with M-MDSCs. High levels of both PMN-MDSCs and M-MDSCs were related to advanced tumor stage. Kaplan-Meier analysis revealed that high levels of both PMN-MDSCs and M-MDSCs were significantly associated with shorter overall survival. In addition, the Cox proportional hazard regression model showed that M-MDSCs and the tumor stage were independent prognostic factors for TCC. These results suggest that PMN-MDSCs and M-MDSCs may be involved in tumor progression and could be prognostic factors and promising therapeutic targets in dogs with TCC.

Urinary and household chemical exposures in pet dogs with urothelial cell carcinoma.

Urothelial cell carcinoma (UCC) has been linked to environmental chemical exposures in people, but these risk factors are not well understood in dogs with UCC. We hypothesised that household chemical exposures contribute to the risk of UCC in pet dogs. This prospective cross-sectional case-control study included 37 dogs with UCC and 37 unaffected breed-, sex-, and age-matched controls. Dog owners completed an environmental questionnaire and household samples were collected and analysed for arsenic (in tap water and room dust) and acrolein (in room air). Urine samples from UCC dogs, control dogs, and consenting owners were analysed for inorganic arsenic species, the acrolein metabolite 3-HPMA, and the phenoxy herbicide 2,4-D. Public data on chlorination byproducts (total trihalomethanes) in municipal drinking water were also compared between case and control households. Dogs with UCC were more likely to swim in a pool (15.2%) compared with control dogs (0%) (OR 1.69, 95% CI = 1.69-∞; p = .02). Dogs with UCC also had more than 4-fold higher reported municipal water concentrations of chlorination byproducts (median 28.0 ppb) compared with controls (median 6.9 ppb; p < .0001). Dust arsenic concentrations were unexpectedly lower in case households (median 0.277 ng/cm2) compared with control households (median 0.401 ng/cm2; p = .0002). Other outcomes were not significantly different between groups. These data suggest that dog owners, especially those of breeds known to be at higher risk for UCC, consider limiting access to swimming pools and installing water filtration units that remove total trihalomethanes.

Pan-tumour analysis of COX-2 expression in dogs.

Cyclooxgenase-2 (COX-2) is associated with inflammatory microenvironment and tumour progression. COX-2 expression was reported in canine tumours, and anti-COX treatment showed therapeutic effects in selected tumour types. Currently, direct comparisons between different tumour types or reports were impossible due to varying evaluation protocols. Additionally, COX-2 expression in relatively uncommon tumours were yet to be evaluated. Here, we analysed COX-2 expression across various tumour types in dogs in a consistent protocol, aiming to revisit accumulated evidence in the field and report novel candidate tumours for anti-COX therapy. COX-2 expression in 32 histological types of tumours, which consisted of 347 samples in total, was investigated using immunohistochemistry followed by the Belshaw's method scoring (range: 0-12). More than the half of the samples expressed COX-2 in mast cell tumours, transitional cell carcinoma in the urinary tract, squamous cell carcinoma, liposarcoma, and melanoma, with COX-2 median scores ranging from 1-8. On the other hand, <20% tissues expressed COX-2 in the half of tumour types investigated. Overall COX-2 positive rate was 27%. In conclusion, the results confirmed COX-2 expression in the well-known COX-2-expresing tumour types and suggested novel candidate tumours for anti-COX-2 therapy. At the same time, overall COX-2 expression was low, and inter- and intra-histology heterogeneity was apparent. This study will provide a foundation reference for future research in canine tumours.

Comparative pharmacokinetics of tyrosine kinase inhibitor, lapatinib, in dogs and cats following single oral administration.

Lapatinib is an orally administered tyrosine kinase inhibitor used to treat human epidermal growth factor receptor 2 (HER2) -overexpressing breast cancers in humans. Recently, the potential of lapatinib treatment against canine urothelial carcinoma or feline mammary tumor was investigated. However, the pharmacokinetic studies of lapatinib in dogs and cats are not well-defined. In the present study, the pharmacokinetic characteristics of lapatinib in both cats and dogs after a single oral administration at a dose of 25 mg/kg were compared with each other. Lapatinib was administered orally to four female laboratory cats and four female beagle dogs. Blood samples were collected over time, and the plasma lapatinib concentrations were analyzed by HPLC. Following a single dose of 25 mg/kg, the averaged maximum plasma concentration (Cmax) of lapatinib in cats was 0.47 µg/mL and achieved at 7.1 hr post-administration, while the Cmax in dogs was 1.63 µg/mL and achieved at 9.5 hr post-administration. The mean elimination half-life was 6.5 hr in cats and 7.8 hr in dogs. The average area under the plasma concentration-time curve of dogs (37.2 hr·µg/mL) was significantly higher than that of cats (7.97 hr·µg/mL). These results exhibited slow absorptions of lapatinib in both animals after oral administration. The Cmax observed in cats was significantly lower and the half-life was shorter than those observed in dogs. Based on these results, a larger dose or shorter dosing intervals might be recommended in cats to achieve similar plasma concentration as dogs.

Canine urothelial carcinoma: a pilot study of microRNA detection in formalin-fixed, paraffin-embedded tissue samples and in normal urine.

We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue (n = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and t-tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.

Association between cigarette smoke exposure and urinary bladder cancer in Scottish terriers in a cohort study.

Canine urothelial carcinoma (UC) initially responds favorably to treatment, but is ultimately lethal in most cases. Research to identify modifiable risk factors to prevent the cancer is essential. The high breed-associated risk for UC, e.g. 20-fold higher in Scottish terriers, can facilitate this research. The objective was to identify environmental and host factors associated with UC in a cohort of Scottish terriers. Information was obtained through dog owner questionnaires for 120 Scottish terriers ≥ 6 years old participating in a bladder cancer screening study, with comparisons made between dogs that did or did not develop UC during the 3 years of screening. Univariable models were constructed, and variables with P < 0.20 were included when building the multivariable model, and then removed using a backward stepwise procedure. P < 0.05 was considered statistically significant. Urine cotinine concentrations were measured by liquid chromatography-mass spectrometry to further investigate potential cigarette smoke exposure. Biopsy-confirmed UC which was found in 32 of 120 dogs, was significantly associated with the dogs living in a household with cigarette smokers (odds ratio [OR], 6.34; 95 % confidence intervals [CI], 1.16-34.69; P = 0.033), living within a mile of a marsh or wetland (OR, 21.23; 95 % CI, 3.64-123.69; P = 0.001), and history of previous bladder infections (OR, 3.87; 95 % CI, 1.0-14.98; P = 0.050). UC was diagnosed in 18 of 51 dogs (35.3 %) with quantifiable cotinine concentrations, and six of 40 dogs (15.0 %) without quantifiable cotinine concentrations in their urine (P = 0.0165). In conclusion, the main modifiable risk factor for UC in this cohort of dogs was exposure to second-hand tobacco smoke.

Changes in Urinary Exosomal Nuclear Matrix Protein-22 in Dogs With Urothelial Carcinoma: A Pilot Study.

BACKGROUND/AIM: Nuclear matrix protein-22 (NMP-22) is widely used in human medicine as a prognostic and diagnostic tool for urothelial carcinoma (UC). In addition, the use of urinary exosomes as a liquid biopsy tool is emerging for the diagnosis of certain types of cancer in human medicine. This study aimed to investigate the change in urinary exosomal NMP-22 for the diagnosis of UC in dogs. PATIENTS AND METHODS: Among canine patients who visited the veterinary hospital, urine was collected from those whose owners provided consent. A total of 23 dogs (UC group, n=6; control group, n=17) were included in the analysis. After exosomes were isolated from the urine, NMP-22 was measured using enzyme-linked immunosorbent assay. RESULTS: In the UC group, the expression of NMP-22 in urinary exosomes was significantly higher than that in non-UC groups (p<0.0001). CONCLUSION: NMP-22 is significantly increased in exosomes in the urine of dogs diagnosed with UC, suggesting that urinary exosome NMP-22 can be considered as one of the liquid biopsy tools for diagnosing UC in dogs.

CD204⁺ tumor-associated macrophages are associated with clinical outcome in canine pulmonary adenocarcinoma and transitional cell carcinoma.

Tumor-associated macrophages are abundant infiltrating cells in the tumor microenvironment (TME). Macrophages can be classified into several types of subsets based on their immune responses. Among those subsets, M2 macrophages contribute to anti-inflammatory responses and create an immunosuppressive environment that promotes tumor cell proliferation. In a previous study, human cancer patients with high M2 macrophages showed a worse prognosis for many types of tumors. However, studies examining the relationship between M2 macrophages and clinical outcomes in canine tumors are limited. In the previous human and canine studies, CD204 has been used as the marker for detecting M2 macrophages. Then we evaluated CD204+ and total macrophages infiltration and its association with clinical outcomes in canine solid tumors. In this study, we examined dogs with oral malignant melanoma (OMM), pulmonary adenocarcinoma (PA), hepatocellular carcinoma (HCC), and transitional cell carcinoma (TCC). Compared to healthy tissues, CD204+ and total macrophages were increased in OMM, PA, and TCC, but not in HCC. High CD204+ macrophage levels were significantly associated with lung metastasis in TCC (P = 0.030). Kaplan-Meier analysis revealed that high CD204+ macrophage levels were associated with shorter overall survival (OS) in canine patients with PA (P = 0.012) and TCC (P = 0.0053). These results suggest that CD204+ macrophages contribute to tumor progression and could be a prognostic factor in dogs with PA and TCC.

Whole exome sequencing analysis of canine urothelial carcinomas without BRAF V595E mutation: Short in-frame deletions in BRAF and MAP2K1 suggest alternative mechanisms for MAPK pathway disruption.

Molecular profiling studies have shown that 85% of canine urothelial carcinomas (UC) harbor an activating BRAF V595E mutation, which is orthologous to the V600E variant found in several human cancer subtypes. In dogs, this mutation provides both a powerful diagnostic marker and a potential therapeutic target; however, due to their relative infrequency, the remaining 15% of cases remain understudied at the molecular level. We performed whole exome sequencing analysis of 28 canine urine sediments exhibiting the characteristic DNA copy number signatures of canine UC, in which the BRAF V595E mutation was undetected (UDV595E specimens). Among these we identified 13 specimens (46%) harboring short in-frame deletions within either BRAF exon 12 (7/28 cases) or MAP2K1 exons 2 or 3 (6/28 cases). Orthologous variants occur in several human cancer subtypes and confer structural changes to the protein product that are predictive of response to different classes of small molecule MAPK pathway inhibitors. DNA damage response and repair genes, and chromatin modifiers were also recurrently mutated in UDV595E specimens, as were genes that are positive predictors of immunotherapy response in human cancers. Our findings suggest that short in-frame deletions within BRAF exon 12 and MAP2K1 exons 2 and 3 in UDV595E cases are alternative MAPK-pathway activating events that may have significant therapeutic implications for selecting first-line treatment for canine UC. We developed a simple, cost-effective capillary electrophoresis genotyping assay for detection of these deletions in parallel with the BRAF V595E mutation. The identification of these deletion events in dogs offers a compelling cross-species platform in which to study the relationship between somatic alteration, protein conformation, and therapeutic sensitivity.

Immunohistochemical analysis of expression of VEGFR2, KIT, PDGFR-ß, and CDK4 in canine urothelial carcinoma.

Urothelial carcinomas (UCs), also known as transitional cell carcinomas, are the most common canine urinary tract neoplasms. Tyrosine kinases (TKs) are enzymes that tightly regulate cell growth and differentiation through phosphorylation. Receptor TK (RTK) inhibitors are currently used to treat UCs. Toceranib phosphate (Palladia; Pfizer) is an RTK inhibitor that blocks the activity of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor-alpha and -beta (PDGFR-α, -ß), FMS-like tyrosine kinase 3, stem cell factor receptor (KIT, kinase inhibitor targeting), and colony stimulating factor receptor. To better understand UCs and validate treatment targets, we performed immunohistochemical staining for RTKs, as well as a novel target, cyclin-dependent kinase 4 (CDK4, a central regulator of the mammalian cell cycle), on formalin-fixed, paraffin-embedded tissues from bladder biopsies from 17 dogs with UCs, 17 dogs with cystitis (diseased controls), and 8 normal dogs (negative controls). Although immunohistochemical scores could not be extrapolated to prognostic value, response to treatment, and outcome of patients with UC, we demonstrated expression of PDGFR-ß and VEGFR2 in UCs; all UC samples staining positively for VEGFR2. Minimal positive staining for KIT was noted in the tumor samples. CDK4 staining intensity was significantly weaker in UCs compared with normal and cystitis bladder samples. The intense staining of VEGFR2 in UC cells suggested that VEGFR2 may be of prognostic and/or therapeutic value in dogs with UC. Overexpression of VEGFR2 in UC cells validates this receptor as a treatment target in UC.

Urothelial carcinoma in a Russian tortoise (Testudohorsfieldii).

An adult female Russian tortoise (Testudo horsfieldii) was admitted to a veterinary clinic with a 1-week history of anorexia and lethargy. Physical examination revealed significant dehydration and generalized dysecdysis. Blood parameters were all within species-reported limits. Radiography revealed an increased amount of soft tissue opacity within the coelomic cavity and a distinct loss of serosal detail. A large cavitated mass was detected ultrasonographically in the right coelom and a tentative diagnosis of kidney pathology was made. Using computed tomography and magnetic resonance imaging, a partly mineralized heterogeneous mass was seen in the right coelom. After stabilization, a transplastron coeliotomy was performed. A 7 × 5 cm mass, including associated cysts, was removed. The tortoise died 8 h postoperatively and post-mortem examination revealed a urothelial carcinoma arising from the renal pelvis. To the authors' knowledge, this is the first report in a tortoise of urothelial carcinoma, which has rarely been reported in reptiles.

Establishment and characterization of urothelial carcinoma cell lines with and without BRAF mutation (V595E) in dogs.

Each 5 urothelial carcinoma (UC) cell lines with and without the v-Raf murine sarcoma virus oncogene homolog B (BRAF) gene mutation (V595E) were established and examined V595E-related tumorigenic characteristics in dogs. No typical morphological features were observed in cloned cells with and without V595E. The cell proliferation of both cloned cells showed logarithmic growth curve and those doubling time were 24.9 ± 4.1 h in V595E ( +) and 29.3 ± 11.3 h in V595E ( -). On the growth curve of xenotransplanted tumor in severe combined immunodeficiency mice, 3 out of 5 V595E ( +) and 2 out of 5 V595E ( -) cloned cells revealed gradually and remarkably increasing curve, indicating clearly tumorigenicity. The xenotransplanted tumors with V595E ( +) showed typical features of UC, such as solid proliferation of pleomorphic tumor cells, formation of papillary structure, and glandular structure. Additionally, various vascular formation was observed, probably indicating an advanced growth phase of UC. In mitogen-activated protein kinase (MAPK) signaling pathway, cytoplasmic phosphorylated-BRAF (pBRAF) and cytoplasmic and nuclear phosphorylated-ERK1/2 (pERK1/2) were detected in all 4 tumors with V595E ( +), whereas only cytoplasmic and nuclear pERK1/2 was detected in tumors with V595E ( -). Since V595E can directly activate MAPK signaling pathway, coincidence of V595E with pBRAF (phosphor Thr598/Ser601) indicates acquired resistance to BRAF inhibitors. These established UC cell lines, especially V595E ( +) cell lines, are useful tool for understanding pathophysiological states and controlling therapeutic manners of UC in dogs.

Vaginal swab cytology as a diagnostic tool for neoplasia of the lower urinary tract in 5 dogs.

Urothelial carcinoma (UC), or transitional cell carcinoma, is the most common canine urothelial malignancy in dogs. Females are predisposed and provide a challenge for diagnostic tumor sampling. The objectives of this study were to investigate the use and tolerability of vaginal swab cytology for UC diagnosis. Five dogs were identified with non-diagnostic urine sediment cytology and UC diagnosed on vaginal cytology with confirmation by another means. All patients tolerated the vaginal swab with minimal restraint. This study confirms the potential of vaginal swab cytology as a simple, inexpensive, and well-tolerated means for lower urinary tract UC diagnosis in female dogs. Key clinical message: Vaginal swab cytology is a non-invasive, low-cost method of obtaining a sample for cytological assessment for UC.

La cytologie vaginal par écouvillonnage comme outil de diagnostic de la néoplasie des voies urinaires inférieures chez cinq chiennes. Le carcinome urothélial (CU), ou carcinome à cellules transitionnelles, est la tumeur maligne urothéliale canine la plus courante. Les femelles sont prédisposées et constituent un défi pour l'échantillonnage diagnostique des tumeurs. Les objectifs de cette étude étaient d'étudier l'utilisation et la tolérabilité de la cytologie vaginale par écouvillonnage pour le diagnostic du CU. Cinq chiennes ont été identifiées avec une cytologie des sédiments urinaires non diagnostique et un CU diagnostiqué sur la cytologie vaginale avec confirmation par un autre moyen. Toutes les patientes ont toléré le prélèvement vaginal avec un minimum de contention. Cette étude confirme le potentiel de la cytologie vaginale par écouvillonnage en tant que moyen simple, peu coûteux et bien toléré pour le diagnostic du CU des voies urinaires inférieures chez les chiennes.Message clinique clé :La cytologie vaginale par écouvillonnage est une méthode non-invasive et peu coûteuse pour obtenir un échantillon pour évaluation cytologique du CU.(Traduit par les auteurs).

Investigating PSMA differential expression in canine uroepithelial carcinomas to aid disease-based stratification and guide therapeutic selection.

BACKGROUND: In male dogs, uroepithelial cancers include invasive urothelial carcinoma (iUC) and prostate carcinoma (PCA). The inability to distinguish iUC involving the prostate from PCA results in indiscriminate clinical management strategies that could be suboptimal as first-line chemotherapy for iUC (cisplatin) and PCA (docetaxel) differ in people. Prostate specific membrane antigen (PSMA) is a transmembrane protein, and its overexpression has been identified in human prostate carcinoma and neovasculature associated with solid tumor growth. This study investigates whether differential PSMA expression exists between presumptive canine iUC and PCA among cell lines and archived patient samples, which might allow for improved accuracy in disease-based stratification and optimal chemotherapy selection. Additionally, in vitro sensitivities of reported canine iUC and PCA cell lines to uroepithelial directed chemotherapeutic agents were characterized. RESULTS: Normalized PSMA gene and protein expressions were not significantly different between 5 iUC and 4 PCA cell lines. PSMA protein expression was uniformly observed in uroepithelial cancers regardless of anatomic origin from archived patient samples, further confirming that PSMA cannot differentiate iUC from PCA. In vitro sensitivity of cell lines to uroepithelial directed chemotherapeutics revealed that vinblastine exerted the broadest cytotoxic activity. CONCLUSIONS: Differential expression of PSMA was not identified between canine iUC and PCA cell lines or archived patient samples, and PSMA alone cannot be used for disease stratification. Nonetheless given its conserved overexpression, PSMA may be a targetable surface marker for both canine iUC and PCA. Lastly, in uroepithelial carcinomas, vinblastine might exert the broadest anticancer activity regardless of cellular origin.

Tissue S100/calgranulin expression and blood neutrophil-to-lymphocyte ratio (NLR) in dogs with lower urinary tract urothelial carcinoma.

BACKGROUND: Urothelial carcinoma (UC) is the most common neoplasm of the canine lower urinary tract, affecting approximately 2% of dogs. Elderly female patients of certain breeds are predisposed, and clinical signs of UC can easily be confused with urinary tract infection or urolithiasis. Diagnosis and treatment are challenging given the lack of disease-specific markers and treatments. The S100A8/A9 complex and S100A12 protein are Ca2+-binding proteins expressed by cells of the innate immune system and have shown promise as urinary screening markers for UC. The neutrophil-to-lymphocyte ratio (NLR) can also aid in distinguishing certain neoplastic from inflammatory conditions. Our study aimed to evaluate the tissue expression of S100/calgranulins and the blood NLR in dogs with UC. Urinary bladder and/or urethral tissue samples from dogs with UC (n = 10), non-neoplastic inflammatory lesions (NNUTD; n = 6), and no histologic changes (n = 11) were evaluated using immunohistochemistry. Blood NLRs were analyzed in dogs with UC (n = 22) or NNUTD (n = 26). RESULTS: Tissue S100A12-positive cell counts were significantly higher in dogs with lower urinary tract disease than healthy controls (P = 0.0267 for UC, P = 0.0049 for NNUTD), with no significant difference between UC and NNUTD patients. Tissue S100A8/A9-positivity appeared to be higher with NNUTD than UC, but this difference did not reach statistical significance. The S100A8/A9+-to-S100A12+ ratio was significantly decreased in neoplastic and inflamed lower urinary tract tissue compared to histologically normal specimens (P = 0.0062 for UC, P = 0.0030 for NNUTD). NLRs were significantly higher in dogs with UC than in dogs with NNUTD, and a cut-off NLR of ≤ 2.83 distinguished UC from NNUTD with 41% sensitivity and 100% specificity. Higher NLRs were also associated with a poor overall survival time (P = 0.0417). CONCLUSIONS: These results confirm that the S100/calgranulins play a role in the immune response to inflammatory and neoplastic lower urinary tract diseases in dogs, but the tissue expression of these proteins appears to differ from their concentrations reported in urine samples. Further investigations of the S100/calgranulin pathways in UC and their potential as diagnostic or prognostic tools and potential therapeutic targets are warranted. The NLR as a routinely available marker might be a useful surrogate to distinguish UC from inflammatory conditions.

Palliative radiation therapy as a treatment for feline urinary bladder masses in four cats.

CASE SERIES SUMMARY: Urinary bladder masses in four cats were treated with palliative radiation therapy (RT). Three cats were previously diagnosed with chronic kidney disease (CKD): International Renal Interest Society (IRIS) stage 2 in two cats and IRIS stage 3 in one cat. One cat had a diagnosis of hyperthyroidism and inflammatory bowel disease. Three cats had urinary tract infections diagnosed by urine culture and susceptibility testing prior to or during treatment. All patients had urine cytospin cytology performed; one case showed suspect urothelial carcinoma and three had no cytological evidence of neoplasia. All clients declined further sampling from the bladder masses. Therefore, cytologic/histologic diagnosis in all cases was not available. An abdominal ultrasound was performed in all cats, which revealed urinary bladder mass(es) prior to referral for RT. Three cats had pretreatment thoracic radiographs, which revealed no evidence of pulmonary metastasis. An abdominal CT was performed in all cases and one case had thoracic CT performed for staging. The thoracic CT showed a focal lesion of unknown etiology in the right caudal lung lobe. Palliative RT was performed with four weekly 6 Gy fractions (24 Gy in total). The urinary signs in all cats resolved over the course of RT: after the first RT treatment in two cats and after the second RT treatment in two cats. There were two Veterinary Radiation Therapy Oncology Group grade 1 gastrointestinal and one grade 2 genitourinary acute RT side effects. RELEVANCE AND NOVEL INFORMATION: This is the first report in the literature of a standardized RT protocol as a treatment option for feline urinary bladder masses.

Coincidence of v-raf murine sarcoma viral oncogene homolog B mutation (V595E) with phosphorylated v-raf murine sarcoma viral oncogene homolog B in urothelial carcinoma in dogs.

Expression of phosphorylated v-raf murine sarcoma viral oncogene homolog B (pBRAF) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) were investigated in urothelial carcinoma (UC) in dogs with or without the BRAF gene mutation (V595E). Among the 10 cases of UC with V595E (-), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was reported in 8, with 7 displaying moderate reactivity and 1 displaying intense reactivity. Nuclear immunoreactivity against pBRAF was detected in 5 cases; however, these reactivities were non-specific, due to pBRAF being limited in the cytoplasm. In addition, positive cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in 7 cases and nuclear immunoreactivity against ERK1/2 was detected in 6 cases. Among the 13 cases of UC with V595E (+), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was detected in all 13 cases and nuclear immunoreactivity against pBRAF was detected in 10 cases; however, the nuclear immunoreactivity was non-specific. Cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in all 13 cases and nuclear immunoreactivity against pERK1/2 was also detected in all cases. As nuclear pERK1/2 indicates a progressive signaling process in the mitogen-activated protein kinase pathway, V595E (+) UC might be in its growing stage. Probable phosphorylated sites of pBRAF at Thr598/Ser601, detected in this study, are major and essential sites of the upstream rat sarcoma viral oncogene homolog (RAS) signaling pathway. In human cancers, the BRAF mutation never coincides with oncogenic RAS. To our knowledge, this is the first report on the simultaneous occurrence of the BRAF mutation (V595E) and pBRAF expression (at Thr598/Ser601) in dogs with UC with V595E (+).

L'expression de l'homologue B de l'oncogène viral du sarcome murin phosphorylé v raf (pBRAF) et de la kinase1/2 régulée par le signal extracellulaire phosphorylé (pERK1/2) ont été étudiées dans le carcinome urothélial (CU) chez des chiens avec ou sans la mutation du gène BRAF (V595E). Parmi les 10 cas de CU avec V595E (−), une immunoréactivité cytoplasmique contre pBRAF de cellules néoplasiques a été rapportée chez huit, sept présentant une réactivité modérée et un présentant une réactivité intense. L'immunoréactivité nucléaire contre pBRAF a été détectée dans cinq cas; cependant, ces réactivités n'étaient pas spécifiques, car pBRAF était limité dans le cytoplasme. De plus, une immunoréactivité cytoplasmique positive contre pERK1/2 des cellules néoplasiques a été détectée dans sept cas et une immunoréactivité nucléaire contre ERK1/2 a été détectée dans six cas. Parmi les 13 cas de CU avec V595E (+), une immunoréactivité cytoplasmique contre pBRAF de cellules néoplasiques a été détectée dans les 13 cas et une immunoréactivité nucléaire contre pBRAF a été détectée dans 10 cas; cependant, l'immunoréactivité nucléaire était non spécifique. L'immunoréactivité cytoplasmique contre pERK1/2 des cellules néoplasiques a été détectée dans les 13 cas et l'immunoréactivité nucléaire contre pERK1/2 a également été détectée dans tous les cas. Comme le pERK1/2 nucléaire indique un processus de signalisation progressif dans la voie de la protéine kinase activée par les mitogènes, V595E (+) UC pourrait être dans sa phase de croissance. Les sites phosphorylés probables de pBRAF à Thr598/Ser601, détectés dans cette étude, sont des sites majeurs et essentiels de la voie de signalisation de l'oncogène viral (RAS) du sarcome de rat en amont. Dans les cancers humains, la mutation BRAF ne coïncide jamais avec le RAS oncogène. À notre connaissance, il s'agit du premier rapport sur la survenue simultanée de la mutation BRAF (V595E) et de l'expression de pBRAF (à Thr598/Ser601) chez des chiens atteints de CU avec V595E (+).(Traduit par Docteur Serge Messier).