Cancer of the urinary bladder is a neoplasm with considerable importance in veterinary medicine, given its high incidence in several domestic animal species and its life-threatening character. Bladder cancer in companion animals shows a complex and still poorly understood biopathology, and this lack of knowledge has limited therapeutic progress over the years. The development and validation of Transitional cell carcinoma (TCC) molecular markers is of great importance for scientists and clinicians alike. Somatic and hereditary BRAF mutations received much attention and can now be detected via multiple types of tests, sometimes in useful combinations with CNA tests. Urine-based tests for detecting BRAF may allow the early detection of post-treatment relapse.
|Primary Canine Urinary Bladder Tumours|
|Transitional cell carcinoma||75–90||Papilloma||2|
|Squamous cell carcinoma||3|
|Canine Transitional Cell Carcinoma Classification|
|Often multiple and may cover large regions of the mucosa. Form papillary or exophytic growths that project into the lumen of the bladder. Invade the stalk and wall of the bladder, lamina propria, and muscle layers and may be transmural. Mild to marked cellular atypia. Likely to metastasise.|
|Do not invade the stroma of their own stalk, do not go beyond the lamina propria, so unlikely to metastasise. Differentiation from papilloma is subjective and based on criteria such as overall size, cellular atypia, small branches off the main lesion, among others. Non-invasive tumours may be adjacent to invasive TCC, and additional sections should be searched for invasion.|
|Form plaques and flat nodules, which can cover large regions of the mucosa. Surfaces are often ulcerated, tumour infiltrates into muscle layers, so high tendency to metastasise. Marked histological and cytological variability.|
|Rare. Additionally, defined as carcinoma in situ; confined to the epithelium and do not form papillae. Neoplastic epithelium more intensely eosinophilic than non-neoplastic cells; cells may be dysplastic to mildly anaplastic. Loss of intercellular cohesion. Usually located adjacent to invasive carcinoma; if seen, additional section analysis recommended to look for invasion.|
|Biomarker||Sample||Method||Diagnostic Utility, Commercial Availability||Utility as a Prognostic and/or Therapeutic Target||Power of the Test|
|BRAF mutation||Tissue, urine, blood
|Determination of cBRAFV595E mutation status in DNA retrieved from cells, using ddPCR analysis or other molecular methods.||Highly sensitive test for detecting TCC cells bearing the BRAF mutation.
Could be used as a first, non-invasive screening test.
Commercially available for dogs, for use in free-catch urine samples—CADET® BRAF mutation detection assay.
Provides qualitative results (positive vs. negative for V595E) and quantitative data of tumour-derived mutation load in urine DNA.
Reported to detect TCC in free-catch urine samples up to several months before development of clinical signs.
The test is not affected by the presence of blood or bacteria in the urine.
~20% of tumours of canine TCC and PC patients do not possess the mutation, which limits the sensitivity of the ddPCR assay to ~80%.
A more recent test that detects chromosomal copy number variation can be added in BRAF mutation-negative patients, increasing combined sensitivity to ~95% (CADET® BRAF-PLUS).
|BRAF mutation was not a predictor for histological grade, nor for survival.
Measuring levels of BRAF mutation in urine or blood samples may be useful for monitoring treatment response and relapse.
Potential target for treatment.
(TCC, tissue and urine)
|Rapid latex agglutination dipstick colorimetric test for qualitative detection of tumour analytes in urine. The test uses antibodies to detect a urinary bladder tumour-associated glycoprotein complex.||Useful as a screening test to rule out TCC, especially in dogs at high risk of developing TCC.
False positive test results reported in dogs with non-neoplastic urinary tract disease, e.g., in the presence of significant glycosuria, proteinuria, and pyuria or haematuria. Presence of lower urinary tract malignant tumours other than TCC may yield positive results. Discrepancies with results may be observed over time, while reading the test.
Commercially available—V-BTA Test. Results are either positive or negative.
Not recommended as a confirmatory/definitive diagnostic test for urinary tract TCC in dogs, and should not be indiscriminately used in every patient presenting clinical signs of urinary tract disease.
|N.A.||88–90%||35–41% in dogs with non-malignant urinary tract disease; 84–94% in healthy dogs or unhealthy dogs due to non-urinary tract diseases|
|ELISA urine test for human and canine bFGF. A quantitative sandwich enzyme immunoassay technique has also been developed using an antibody for canine bFGF.||Urine bFGF could be useful as a diagnostic tumour marker, helping to distinguish dogs with UTI from those with TCC.
Commercially available (for research use, only): Quantikine® HS ELISA, Human FGF basic Immunoassay, Canine BFGF ELISA Kit, Nori® Canine FGF Basic ELISA Kit.
|Quantification of urine bFGF could be useful as a non-invasive indicator of treatment response.||N.S.||N.S.|
|Chromosomal CNAs||Tissue, urine
|Assessment of urothelial cell ploidy/DNA copy number status in biopsy sections and in urine sediment by FISH.||Non-invasive method for canine TCC diagnosis.
Potentially high-sensitivity and high-specificity FISH-based method/assay for the detection of canine TCC diagnosis utilising low-volume, free-catch urine specimens. Expensive and high effort method/labour intensive, expertise, time-consuming, increased cost, which may limit its application as routine diagnostics in a clinical environment.
Not commercially available for canine TCC. Available for in vitro diagnostic use in human samples. A multicolour FISH-based assay for detection of aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus through FISH in urine specimens—UroVysion Bladder Cancer Kit.
|Multiplexed ddPCR assay for the detection and quantification of DNA copy number imbalances/changes characteristic to canine TCC.||Accurate, high-throughput method for evaluation of copy number changes in dogs with TCC. In this study, changes in copy number were not detected in 33% of urine DNA samples from dogs with TCC, which was probably due to the presence of inflammatory cells. Thus, additional techniques to improve sensitivity in those samples may be required. In such cases, FISH will still provide a more accurate evaluation.
Commercially available for dogs, for use in free-catch urine samples: CADET® BRAF-PLUS. Can be used in BRAF mutation-negative patients. Could be added to CADET® BRAF, increasing combined sensitivity to ~95%.
|PCR study of a panel of 22 microsatellite DNA sequences from exfoliated urothelial cells and blood cells; comparison of microsatellites genotypes.||The technique added little value as a diagnostic test for TCC in dogs.
High rate of false positives (32%, 12 of 38).
|N.A.||55% (48% *)||68% (76%, vs. V-BTA)|
|* When compared with results of V-BTA from the same study.|
|MicroRNAs||Tissue, cell lines
|QPCR of specific miRNAs involved in the pathophysiology of TCC in humans.||MiR-34a, miR-16, miR-103b and miR-106b could be useful diagnostic biomarkers for the identification of dogs with TCC.
More studies are required, with a larger sample.
|MiR-103b and miR-16 are potential non-invasive diagnostic biomarkers for TCC; particularly for distinguishing LUTD and TCC in canine urine samples.
Urine tests seem to be superior in distinguishing TCC from LUTD.
|Telomerase||Canine TCC cell line, urine
|PCR-based telomeric repeat amplification protocol for detection/measurement of telomerase activity.||Telomerase activity may be useful in diagnosing canine TCC in urine samples in a clinical context. Results of the assay are either telomerase-positive or telomerase-negative. Urine samples containing other telomerase-positive cells may yield false-positive results (e.g., presence of activated lymphocytes in dogs with bacterial cystitis). False-negative results may occur with unappropriated urine samples storage.||N.A.||91%||89%|
|Diagnostic sensitivity/specificity of the TRAP assay applied to clinical canine urine samples.|
|Species-specific radioimmunoassays to measure urine concentrations of canine calgranulins S100A8/A9 and S100A12.||Results presented as normalised to urine specific gravity levels (S100A8/A9USG) and as S100A8/A9-to-S100A12 ratio (UcalR). Provides quantitative results.
S100A8/A9USG could be a good a screening test for TCC/PC in dogs, especially in those where a UTI has been ruled out as a cause of clinical signs of lower urinary tract disease (due to a moderate rate of false positives observed for dogs ≥6 years of age with UTI).
UcalR can help differentiate patients with a UTI from those with TCC/PC, even though a moderate false negative rate was seen in dogs ≥ 6 y.o. with a UTI.
A combination of S100A8/A9USG and uCalR improved diagnostic accuracy for the detection of canine TCC/PC.
Test levels are not affected by haematuria.
|N.A.||96% S100A8/A9USG *
|66% S100A8/A9USG *
|* For detection of TCC/PC in dogs ≥ 6 y.o.; ** to distinguish dogs with TCC/PC from dogs with UTI in dogs ≥ 6 y.o.|
|Characterisation of the canine urinary proteome by using liquid chromatography tandem mass spectrometry and immunoblot.||A protein signature was identified, that could distinguish between healthy patients and those with TCC or UTIs. A statistical model using a biomarker multiplex for categorising samples as TCC or non-TCC was developed, predicting the presence of disease with 90% confidence. Potential relevance of the identified proteins as biomarkers for the diagnosis of TCC in dogs. Preliminary study, high-throughput technique. A more direct assay will be useful for clinical diagnosis.||N.A.||N.S.||N.S.|
|Nuclear magnetic resonance spectroscopy-based metabolite profiling analysis.||Six metabolites showed significantly higher levels in dogs with TCC compared to controls: urea, choline, methylguanidine, citrate, acetone and β-hydroxybutyrate. Good sensitivity to predict the healthy control and disease samples. Potential for early detection of bladder cancer. Preliminary study, high-throughput technique.||N.A.||86%||78%|
|Imaging analysis to examine lipidome/lipid profiles, using desorption electrospray ionisation mass spectrometry.||Differentiation of canine cancerous bladder tissue and cutaneous metastasis from noncancerous canine bladder tissue samples. Different lipid distributions between healthy and diseased tissues.
DESI-MS imaging could be useful in diagnosing TCC by using a multimarker approach based on the lipid profiles and intensities of tissue samples.
Further studies are required with larger populations and additional control groups, i.e., with other lower urinary diseases.
Still requires invasive techniques for tissue collection.
|Analysis of lipid profiles using liquid chromatography-mass spectrometry.||Unique lipid profiles were found among dogs with TCC, dogs with UTI, and healthy dogs. Specific statistical analyses allowed their differentiation. Concentrations of the specific lipids could not be determined, and thus the study did not conclude which lipid families were up or downregulated. Foundation for further research on urinary lipids as potential biomarkers for TCC. Non-invasive method.||N.A.||N.S.||N.S.|
|Immunohistochemistry for detection of survivin, an apoptosis-inhibiting protein; RT-PCR analysis for the survivin gene.||Initial phases of investigational development with limited samples. Additional research needed to investigate potential role of nuclear survivin as an early marker for bladder tumours, as well as in the development, progression and as a therapeutic target.||N.A.||N.S.||N.S.|
|IHC and qPCR analysis for EGFR.||EGFR expression could potentially be used as a marker to aid canine TCC diagnosis. It may improve the sensitivity of urine cytological diagnosis when provisional diagnosis is needed.||Not useful for predicting prognosis of TCC.||72%||100%|
|IHC for HER-2.||N.A.||Potential maker of malignancy and therapeutic target in canine TCC.||N.S.||N.S.|
|Tissue, cell lines
|IHC for expression of VEGFR2, PDGFR-β, c-KIT.||PDGFR-β could play a role in canine TCC tumourigenesis.||PDGFR-β and VEGFR2 might be involved in mediating clinical response of TCC to toceranib.||N.S.||N.S.|
|Granzyme B, CD3||Tissue
|IHC and PCR assay for CD3 and granzyme B.||N.A.||Granzyme B+ tumour-infiltrating cells could be involved in inhibition of tumour progression, and a favourable prognosis.
Presence of granzyme B+ tumour-infiltrating cells might be an independent prognostic factor.
|P63, Ki67, β-catenin||Tissue
|IHC for p63.||P63 could potentially be used as a clinical marker for diagnosing canine TCC.||P63 could potentially be used as a clinical marker for predicting prognosis in canine TCC.||N.S.||N.S.|
|IHC for UP III, CK 7 and CK20.||UP III is the most common marker of urothelial differentiation used in dogs. It was considered the marker of choice in canine urothelial neoplasms. Although UP III is not a specific marker for TCC itself (it does not differentiate neoplastic from non-neoplastic lesions), it can be useful e.g., to rule in TCC in a biopsy from a tumour of unknown origin and to identify metastatic carcinomas in the skin.
CK 7 was more sensitive than UP III for canine TCC, but CK 7 is expressed in several non-urothelial tumours and also in normal tissues, as is CK 20. CK 7 should be used for tumours negative for UP III but suspected of being TCC.
CK 20 alone did not prove to be useful for diagnosis of urothelial tumours.
Some urothelial carcinomas might not be positively labelled when using UP III and CK 7 as diagnostic markers.
COX-2 has been found to be expressed in canine TCCs but not by normal urothelium of the urinary bladder.
|UP III, CK 7, COX-2:
Significant associations between specific patterns of expression and tumour classification, depth of neoplastic cell infiltration.
Intensity of COX-2 expression did not correlate with grading.
Nonselective COX and COX-2 specific inhibitors have been used for treating TCC.
Still unclear whether it could be useful as a predictive factor for treatment response.