Detection of canine transitional cell carcinoma using a bladder tumor antigen urine dipstick test

Canine transitional cell carcinoma (TCC) carries a poor prognosis in part due to late disease detection. The measurement of specific tumor markers shed in the urine may aid in sensitive, early disease detection and therefore improved prognosis. A 1-year prospective clinical trial was designed to assess the efficacy, sensitivity and specificity of the first generation Bard BTA test to diagnose TCC in dogs. This test is a qualitative, rapid, latex agglutination, dipstick test run on voided urine, which measures a glycoprotein antigen complex associated with bladder cancer in human patients. Sixty-five dogs were entered in the study: 20 TCC confirmed patients, 19 healthy controls and 26 urologic controls with a variety of conditions including urinary tract infection, crystalluria and proteinuria. Overall test sensitivity was 90% and specificity was 78%. False positive test results were noted in the presence of significant glucosuria (4+), proteinuria (4+), and pyuria or hematuria (> 30-40 WBC or RBC per hpf). Urine parameters that had no effect on efficacy included collection method (cystocentesis or free catch), pH, specific gravity, crystalluria, bilirubinuria, bacteriuria and casts. These data indicated that the Bard BTA test was sensitive for the detection of the bladder tumor-associated antigen complex in canine TCC. As evaluated, this test may serve as a useful adjunct to diagnosis, especially when cytology or biopsy is questionable or impractical. Furthermore, because of the high sensitivity of the test, it may be a practical screening test to rule out TCC in geriatric patients or patients with clinical signs related to the lower urinary tract, particularly before pyuria and hematuria develop which may interfere with test results.     

Preclinical evaluation of 5-aminolevulinic acid-based photodynamic therapy for canine transitional cell carcinoma

As a prelude to photodynamic therapy, 5‐aminolevulinic acid (ALA) was given orally to healthy dogs. ALA‐induced protoporphyrin IX (PpIX) fluorescence significantly increased in the mucosa of the urinary bladder in an ALA dose‐dependent fashion. Vomiting occurred after ALA administration in 70% of the dogs but did not affect PpIX fluorescence. ALA‐based photodynamic therapy (PDT) of the urinary bladder in healthy dogs caused only submucosal oedema within the bladder wall. No haematologic or serum biochemistry abnormalities were observed after ALA administration. Microscopic haematuria was observed in all the dogs after PDT but was mild and self limiting. ALA‐based PDT was administered to six dogs with transitional cell carcinoma (TCC) of the lower urinary tract. ALA‐based PDT resulted in tumour progression‐free intervals from 4 to 34 weeks in five dogs; one dog with pre‐existing hydronephrosis died shortly after PDT. Dogs with TCC represent an outbred, spontaneous, tumour model for developing PDT protocols for humans with bladder cancer.     

Laparoscopically implanted tissue expander radiotherapy in canine transitional cell carcinoma

Organ motion and injury to adjacent structures limit curative treatment of intraabdominal tumors with external beam radiotherapy. We evaluated the use of Laparoscopically Implanted Tissue Expander Radiotherapy (LITE-RT) to exclude critical structures during irradiation of the urinary bladder in two dogs with transitional cell carcinoma (TCC) using helical tomotherapy. Dogs had histologically confirmed bladder TCC with no metastasis. A custom-shaped tissue expander was placed between the colon and bladder laparoscopically in one dog and during laparotomy in the other. The prescribed radiation dose was 45 Gy to 98% volume of the bladder in 18 fractions of 2.5 Gy. Tumor response and normal tissue effects were monitored with cystoscopy and colonic biopsies before treatment and 3, 6, and 15 months after treatment. Based on treatment plans from inflated vs. deflated tissue expander CT images, there was a mean dose reduction to the colon of 53% and 31% for the two dogs. Interfractional target repositioning was possible by using volumetric megavoltage computed tomography helical tomotherapy. Both dogs had no clinical signs of chronic colitis but did experience mild cystitis during treatment. Tissue expanders became detached, requiring an additional surgery for reattachment, in both dogs. One dog developed a fibrous adhesion resulting in bladder rupture during inflation, which necessitated early device removal. One dog was euthanized for tumor-associated ureteral obstruction at 8 months while the other is alive at 21 months. We conclude that LITE-RT shows promise in treatment of canine bladder TCC due to lack of acute colitis and enteritis.     

Sorafenib inhibits tumor cell growth and angiogenesis in canine transitional cell carcinoma

Canine transitional cell carcinoma (cTCC) is the most common naturally occurring bladder cancer and accounts for 1–2% of canine tumors. The prognosis is poor due to the high rate of invasiveness and metastasis at diagnosis. Sorafenib is a multi-kinase inhibitor that targets rapidly accelerated fibrosarcoma (RAF), vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, VEGFR-3, platelet-derived growth factor receptor-β (PDGFR-β), and KIT. In previous studies, a somatic mutation of B-rapidly accelerated fibrosarcoma (BRAF) and expressions of VEGFR-2 and PDGFR-β were observed in over 80% of patients with cTCC. Therefore, in this study, we investigated the anti-tumor effects of sorafenib on cTCC. Five cTCC cell lines were used in the in vitro experiments. All five cTCC cell lines expressed VEGFR-2 and PDGFR-β and sorafenib showed growth inhibitory effect on cTCC cell lines. Cell cycle arrest at the G0/G1 phase and subsequent apoptosis were observed following sorafenib treatment. In the in vivo experiments, cTCC (Sora) cells were subcutaneously injected into nude mice. Mice were orally administered with sorafenib (30 mg/kg daily) for 14 days. Sorafenib inhibited tumor growth compared to vehicle control. The necrotic area in the tumor tissues was increased in the sorafenib-treated group. Sorafenib also inhibited angiogenesis in the tumor microenvironment. Thus, sorafenib may be potential therapeutic agent for cTCC via its direct anti-tumor effect and inhibition of angiogenesis.     

Evaluation of carbon dioxide laser ablation combined with mitoxantrone and piroxicam treatment in dogs with transitional cell carcinoma

CASE DESCRIPTION: 8 dogs that underwent carbon dioxide (CO2) laser ablation of transitional cell carcinoma in the bladder trigone and proximal portion of the urethra and were also treated with mitotranxone and piroxicam. CLINICAL FINDINGS: Transitional cell carcinoma of the bladder frequently involves the trigone and urethra and can be difficult to manage surgically. Dogs underwent laser ablation of the primary tumor and were treated with mitoxantrone at a dosage of 5 mg/m2)every 3 weeks for 4 treatments. Piroxicam was given at a dosage of 0.3 mg/kg (0.14 mg/lb) once daily for the remaining life of the dog. TREATMENT AND OUTCOME: Median and mean disease-free intervals were 200 and 280 days, respectively. Median and mean survival times were 299 and 411 days, respectively. Adverse treatment effects were observed in 2 dogs; signs included mild, self-limiting inappetance and lethargy. The procedure appeared to be well tolerated; all treated dogs had rapid resolution of clinical signs of disease of the lower portion of the urinary tract. CLINICAL RELEVANCE: Although survival times achieved with CO2 laser ablation and treatment with mitoxantrone and piroxicam were similar to survival times associated with chemotherapy alone, resolution of clinical signs was better with the combined treatment.     

Glycoproteomic profiling of serum peptides in canine lymphoma and transitional cell carcinoma

Differential expression of fucosylated glycoproteins has been correlated with malignancy and metastatic potential in various types of neoplasia. Utilizing glycoproteomics techniques, changes in fucosylated serum peptides associated with naturally occurring canine lymphoma and transitional cell carcinoma (TCC) have been evaluated. In both types of neoplasia, the majority of the fucosylated peptides that changed increased with the cancer. In one lymphoma case that was examined over the course of the disease, the same fucosylated peptides that increased during pre-chemotherapy decreased during post-chemotherapy, and then subsequently increased upon recurrence of the lymphoma. When comparing all the fucosylated peptides that increased in both types of cancer, there were only two peptides in common allowing discrimination between lymphoma and TCC based on their peptide profiles. These results emphasize the prospect of glycopeptide profiling in proteomics for use in discovering a panel of non-invasive, diagnostic or prognostic biomarkers of cancer.     

In vitro evaluation of metacarpophalangeal joint loading during simulated walk

Reasons for performing study: Insight into the loading pattern of the articular cartilage surface during the complete stride is important as biomechanical factors play a pivotal role in the pathogenesis of joint trauma and osteoarthritis (OA). Objectives: To determine the loading pattern in the equine MCP articulation in vitro during simulated walk. Methods: Eight cadaveric limbs from mature Dutch Warmblood horses were loaded in a pneumatic loading device in 6 different positions (A1‐A6). The pressure distribution on the articular surface of the proximal phalanx (P1) was measured at 7 sites (S1‐7) using intra‐articularly placed pressure sensitive films, which were analysed by scanning and densitometry. Results: Pressures recorded after mid‐stance (A4, 5, 6) were significantly (P<0.05) higher than those before (A1, 2, 3) and showed the biphasic loading pattern of the walk at all sites, except for the site halfway along the sagittal groove (S7). At S7, there was a linear increase in pressure during the progress of the stance phase of the stride in most horses. Medially (S4, 5, 6) the pressure was significantly higher than laterally (S1, 2, 3) (P<0.05). Conclusions and potential relevance: The heavier medial loading coincides with the location where articular cartilage degeneration in the process of OA in the equine MCP joint is known to start. The discrepancy between the loading of the central groove and the other parts of the joint may result in large stress differences at the end of the stance phase, which might be related to the pathogenesis of stress fractures in the first phalanx and distal third metacarpal bone.     

In vitro mechanical evaluation of torsional loading in simulated canine tibiae for a novel hourglass-shaped interlocking nail with a self-tapping tapered locking design

OBJECTIVE: To describe a novel interlocking nail (ILN) and locking system and compare the torsional properties of constructs implanted with the novel ILN or a standard 8-mm ILN (ILN8) by use of a gap-fracture model. SAMPLE POPULATION: 8 synthetic specimens modeled from canine tibiae. PROCEDURES: An hourglass-shaped ILN featuring a tapered locking mechanism was designed. A synthetic bone model was custom-made to represent canine tibiae with a 50-mm comminuted diaphyseal fracture. Specimens were repaired by use of a novel ILN or an ILN8 with screws. Specimens were loaded for torsional measurements. Construct compliance and angular deformation were compared. RESULTS: Compliance of the ILN8 was significantly smaller than that of the novel ILN. Mean +/- SD maximum angular deformation of the ILN8 construct (23.12 +/- 0.65 degrees) was significantly greater, compared with that of the novel ILN construct (9.45 +/- 0.22 degrees). Mean construct slack for the ILN8 group was 15.15 +/- 0.63 degrees, whereas no slack was detected for the novel ILN construct. Mean angular deformation for the ILN8 construct once slack was overcome was significantly less, compared with that of the novel ILN construct. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of results of this study suggests that engineering of the locking mechanism enabled the novel hourglass-shaped ILN system to eliminate torsional instability associated with the use of current ILNs. Considering the potential deleterious effect of torsional deformation on bone healing, the novel ILN may represent a biomechanically more effective fixation method, compared with current ILNs, for the treatment of comminuted diaphyseal fractures.     

Clinical outcome of partial cystectomy for transitional cell carcinoma of the canine bladder

Canine transitional cell carcinoma (TCC) of the bladder has historically been treated with a combination of chemotherapy, cyclooxygenase inhibitors and radiation therapy. While surgery has been used to treat TCC of the bladder, its efficacy has yet to be established. Thirty‐seven client owned dogs that underwent partial cystectomy +/? various nonsurgical treatments for TCC were retrospectively evaluated. The overall median progression‐free interval (PFI) was 235 days and the median survival time (ST) was 348 days. Prognostic factors identified on univariate analysis significant for ST were age, tumor location, full thickness excision and frequency of piroxicam administration. Prognostic factors significant for PFI were full thickness excision and frequency of piroxicam administration. The median ST with partial cystectomy and daily piroxicam therapy, with or without chemotherapy, was 772 days. Dogs with non‐trigonal bladder TCC treated with full thickness partial cystectomy and daily piroxicam (+/? chemotherapy) may have improved outcome compared to dogs treated with medical therapy.     

Effects of gemcitabine and gemcitabine in combination with carboplatin on five canine transitional cell carcinoma cell lines

Objective-To evaluate in vitro effects of gemcitabine alone and in combination with carboplatin on canine transitional cell carcinoma (TCC) cell lines. Sample-In vitro cultures of 5 canine TCC cell lines. Procedures-Cells were treated with gemcitabine, carboplatin, or a combination of both at various concentrations. Cell proliferation was assessed via a fluorescence-based microplate cell proliferation assay. Cell cycle was evaluated via propidium iodide staining, and apoptosis was assessed by measurement of caspase 3 and 7 enzymatic activity. Synergy between gemcitabine and carboplatin was quantified via combination index analyses. Results-Treatment of 5 canine TCC cell lines with gemcitabine or carboplatin decreased cell proliferation, increased apoptosis, and induced cell cycle arrest. Cell cycle arrest and apoptosis were markedly increased when cell lines were treated with both gemcitabine and carboplatin simultaneously or sequentially. Order of administration during sequential treatment did not consistently affect cell proliferation results in TCC cell lines. When TCC cell lines were treated with gemcitabine and carboplatin in combination at therapeutically relevant concentrations (gemcitabine concentration, < 10μM; carboplatin concentration, < 250μM), a significant decrease in cell proliferation was observed, compared with cell proliferation following treatment with gemcitabine or carboplatin alone. In combination, the effects of gemcitabine and carboplatin were synergistic in 3 of 5 cell lines and additive in the other 2. Conclusions and Clinical Relevance-Gemcitabine had antitumor effects on canine TCC cells in vitro, and the combination of gemcitabine and carboplatin had synergistic activity at biologically achievable concentrations.     

In vitro effects of Yunnan Baiyao on canine hemangiosarcoma cell lines

Yunnan Baiyao is a Chinese herbal medicine that has been utilized for its anti‐inflammatory, haemostatic, wound healing and pain relieving properties in people. It has been utilized in the veterinary profession to control bleeding in dogs with hemangiosarcoma (HSA) and has been anecdotally reported to prolong survival times in dogs with this neoplasm. This study evaluated the in vitro activity of Yunnan Baiyao against three canine HSA cell lines after treatment with increasing concentrations of Yunnan Baiyao (50, 100, 200, 400, 600 and 800 µg mL^?1) at 24, 48 and 72 h. Mean half maximum inhibitory concentration (IC₅₀) at 72 h for DEN, Fitz, SB was 369.9, 275.9 and 325.3 µg mL^?1, respectively. Caspase‐3/7 activity increased in correlation with the IC₅₀ in each cell line which was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, APO‐BRDU Kit; BD Biosciences, San Jose, CA, USA) assay. VEGF in cell supernatant was also quantified. Overall, the study found that Yunnan Baiyao causes dose and time dependent HSA cell death through initiation of caspase‐mediated apoptosis, which supports future studies involving Yunnan Baiyao.     

In vitro evaluation of five canine tibial plateau leveling methods

OBJECTIVE: To compare application time, accuracy of tibial plateau slope (TPS) correction, presence and magnitude of rotational and angular deformities, and mechanical properties of 5 canine tibial plateau leveling methods. SAMPLE POPULATION: 27 canine tibial replicas created by rapid prototyping methods. PROCEDURE: The application time, accuracy of TPS correction, presence and magnitude of rotational and angular deformation, and construct axial stiffness of 3 internal fixation methods (tibial plateau leveling osteotomy, tibial wedge osteotomy, and chevron wedge osteotomy [CWO]) and 2 external skeletal fixation (ESF) methods (hinged hybrid circular external fixation and wedge osteotomy linear fixation [WOLF]) were assessed. RESULTS: Mean bone model axial stiffness did not differ among methods. Mean application time was more rapid for WOLF than for other methods. Mean TPSs did not differ from our 5 degrees target and were lower for ESF methods, compared with internal fixation methods. Mean postoperative rotational malalignment did not differ from our target or among groups. Mean postoperative medio-lateral angulation did not differ from our target, except for CWO. Internal fixation methods lead to axially stiffer constructs than ESF methods. Reuse of ESF frames did not lead to a decrease in axial stiffness. CONCLUSIONS AND CLINICAL RELEVANCE: The 5 tibial plateau leveling methods had acceptable geometric and mechanical properties. External skeletal fixation methods were more accurate as a result of precise data available for determining the exact magnitude of correction required to achieve a 5 degrees TPS.     

Urethral transitional cell carcinoma in a cat

A 15-year-old, male neutered cat was referred for investigation of dysuria. A retrograde urethrography was performed which showed two space-occupying masses within the lumen of the mid-to-proximal urethra. Exploratory coeliotomy revealed two urethral masses. Segmental urethrectomy was performed to resect the mass, and the lower urinary tract was reconstructed by vesico-urethral anastomosis. Histopathology showed the mass to be a transitional cell carcinoma with incomplete surgical margins. Tumour regrowth was suspected when dysuria was found approximately 318 days after surgery. Clinical signs were palliated by radiation using weekly fractions of 6 Gy for three weeks. The cat died of unknown causes 386 days postoperatively.     

Hedgehog signaling is activated in canine transitional cell carcinoma and contributes to cell proliferation and survival

Transitional cell carcinoma (TCC) is the most commonly diagnosed tumor of the canine urinary system. Hedgehog (HH) signaling represents one possible novel therapeutic target, based on its recently identified central role in human urothelial carcinoma. The purpose of this study was to determine if HH mediators are expressed in canine TCC and the effect of inhibition of this pathway on cell growth and survival. HH pathway mediators were found to be expressed in five canine TCC cell lines. Indian HH was expressed in tumor cells in five canine bladder tumor tissues, but not in normal canine bladder tissue. Inhibition of HH signaling with cyclopamine and GANT61 led to significantly decreased cell proliferation but had a smaller effect on apoptosis. These results support future investigation of inhibitors of HH signaling in the treatment of canine TCC.     

Expression of cyclooxygenase-2, P-glycoprotein and multi-drug resistance-associated protein in canine transitional cell carcinoma

Cyclooxygenase-2 (COX-2), P-glycoprotein (P-gp) and multi-drug resistance-associated protein (MRP) are considered important tumor-associated proteins in humans and dogs. In the present study, we immunohistochemically evaluated the expression of these proteins in canine patients with transitional cell carcinoma (TCC). Of 52 cases, 30 (57.7%) were positive for COX-2, 40 (76.9%) for P-gp, and only 10 (19.2%) for MRP. In addition, 27 samples (27/52, 51.9%) were positive for two markers, while 3 (5.7%) and 5 (9.6%) cases were positive and negative, respectively, for all three markers. No significant correlations were seen for COX-2 and P-gp on Fisher's exact test and Mann-Whitney's test, but a significance was seen on Spearman's rank correlation analysis using the IHC scoring system (P=0.043). These results suggest that P-gp expression is induced by overexpression of COX-2 in canine patients with TCC.     

Phototoxic effects of 635-nm light on canine transitional cell carcinoma cells incubated with 5-aminolevulinic acid

OBJECTIVE: To determine whether transitional cell carcinoma (TCC) cells incubated in media containing 5-aminolevulinic acid (ALA) would produce sufficient protoporphyrin IX (PpIX) to cause lethal phototoxic effects when exposed to 635-nm light. SAMPLE POPULATION: Canine TCC cells (K9TCC). PROCEDURE: Cultured K9TCC cells were exposed to graded doses of ALA, and PpIX concentrations were determined. Cells then were exposed to various doses of 635-nm light from a diode laser, and cell viability was assayed. RESULTS: Production of PpIX was dependent on time and dose of ALA. The K9TCC cells incubated with ALA produced sufficient PpIX to cause lethal phototoxic effects when exposed to 635-nm light. Phototoxic effects were dependent on time and dose of ALA. Increasing laser power density and energy density decreased cell survival. CONCLUSIONS AND CLINICAL RELEVANCE: ALA is an effective photosensitizer for in vitro photodynamic treatment of K9TCC cells. Further studies are warranted to assess the safety and efficacy of ALA as a photosensitizer for use in treating dogs with TCC. Impact for Human Medicine-On the basis of this study, dogs with TCC may be useful in the development of protocols for ALA-based photodynamic therapy of humans affected with muscle-invasive bladder cancer.