Presumptive malignant transformation of chronic polypoid cystitis into an apical transitional cell carcinoma without BRAF mutation in a young female dog

Abstract A 3‐year‐old spayed female English Springer Spaniel was presented twice 4 months apart for investigation of hematuria and pollakiuria without urinary tract infection. Both ultrasound examinations identified a stable craniodorsal bladder wall thickening. The first cystoscopic biopsy samples indicated lymphoplasmacytic cystitis and the second polypoid cystitis. The dog was represented 8 months later for recurrent clinical signs despite medical management. Although the ultrasound examination showed stable disease, repeat cystoscopic biopsy identified transitional cell carcinoma (TCC), confirmed on tissue removed by partial cystectomy. No BRAF mutation was ever detected in urine or tissue samples. To our knowledge, this case represents the first report of presumptive malignant transformation of polypoid cystitis into an apical TCC in a dog. Dogs with polypoid cystitis should be followed closely and surgical management considered if rapid resolution is not achieved with medical management.


| INTRODUCTION
Polypoid cystitis is a common disease of the urinary bladder in dogs. 1,2 The cause of polyp formation in dogs remains unknown. It may represent an inflammatory and hyperplastic reaction to chronic irritation of the bladder mucosa, 3 and has been most commonly associated with chronic urinary tract infection (UTI) and urolithiasis. [3][4][5][6][7][8][9][10][11] In human medicine, polypoid cystitis is believed to be an inflammatory reaction to injury most commonly caused by longstanding indwelling catheterization. [12][13][14][15] Despite its common occurrence, limited information on polypoid cystitis in dogs has been published. [3][4][5][6][7][8][9][10][11] In 1 case, polypoid cystitis developed after cystoscopic-guided laser ablation of ectopic ureters. 16 Macroscopically, the mucosa is thrown into broad-based folds, often projecting into the bladder lumen, covering a core of proliferating stroma. Occasionally, no macroscopic lesions or a diffuse thickening of the bladder wall is observed rather than a defined mass. 3 Microscopically, epithelial cords project down into the underlying stroma, accompanied by mucosal erosions and ulcerations, stromal edema, inflammation, and hemorrhage. [1][2][3]17 Dysplastic changes of the epithelium, characterized by nuclear atypia and increased mitotic activity, are found in some cases. 2,3 Polypoid cystitis is an important differential diagnosis of bladder cancer both in people and in dogs, and histopathology confirms a final Abbreviations: CIS, carcinoma in situ; hpf, high-power field; RBC, red blood cells; TCC, transitional cell carcinoma; UC, urothelial carcinoma; UTI, urinary tract infection; WBC, white blood cells. diagnosis in most cases. Liquid biopsy using the CADET BRAF test is a new noninvasive diagnostic tool to detect cells and DNA released by transitional cell carcinoma (TCC) in dogs with a sensitivity and specificity of more than 95% and 100%, respectively. [18][19][20][21] In human medicine, polypoid cystitis and other types of hyperplastic cystitis often are accompanied by metaplasia and dysplasia of the epithelium and are considered preneoplastic changes. 17,22,23 . In veterinary medicine, however, the clinical relevance of such changes as preneoplastic is not well established, and a study found such changes could be normal findings in the canine urinary bladder. 24 Despite rare reports of neoplastic transformation of polypoid cystitis, the biologic behavior of polypoid cystitis and its neoplastic potential have not yet been elucidated. 3,5,10 Escherichia coli sensitive to all antibiotics tested was isolated. The dog was prescribed a 6-week course of marbofloxacin (4 mg/kg PO q24h) and meloxicam (0.08 mg/kg PO q24h) with normal urinalysis and negative urine culture findings while on both medications. Treatment completely resolved hematuria and pollakiuria, and only rare episodes of nocturia were reported.

| CASE DESCRIPTION
Ten weeks later, the dog was presented for repeat ultrasound examination. The urinary bladder wall was markedly improved but remained slightly thickened cranially (5.8 mm) with slightly irregular F I G U R E 1 Ultrasonographic examinations before (A) and 10 weeks after (B) initial treatment with a 6-week course of marbofloxacin and meloxicam mucosa ( Figure 1). Urine specific gravity was 1.014 and the reminder of the urinalysis was normal. Urine culture was negative.
One month later, the dog became markedly pollakiuric, and was treated with marbofloxacin (4 mg/kg PO q24h) by the primary veterinarian pending a urine culture, which was negative. A repeat ultrasound examination showed that the urinary bladder wall was still slightly thickened cranially (5.

| DISCUSSION
To the best of our knowledge, this represents the first case report of a young dog with presumptive malignant transformation of polypoid cystitis into TCC without BRAF mutation.
In human medicine, some polypoid lesions eventually undergo neoplastic transformation, but the causative factors are not clear. 25 Inactivation of tumor suppression genes p53 and fragile histidine triad (FHIT) occurs in the majority of bladder TCCs in humans, 2,25 and some studies have shown that p53 is also suppressed in some types of cystitis. 25 However, information on genetic mutations in bladder lesions of dogs is lacking. The malignant potential of polypoid cystitis and bladder polyps in veterinary medicine previously has been suspected but never confirmed, and whether mutations in benign polypoid lesions can lead to neoplasia is unknown. 3,5,10 There are rare reports in the veterinary literature of polypoid cystitis or bladder polyps that have presumably progressed to urothelial carcinoma (UC), 3,5 but none has done so over the course of 1 year. Additional studies are needed to determine the relationship between chronic cystitis and development of TCC. 3 Presently, we assume that UC in dogs progresses through stages of carcinoma in situ (CIS), low grade to high grade, and that we only recognize the tumor when it is advanced. 2 In our case, the young age of the dog and location of the TCC are unusual. The most common location of UC in dogs is the trigone area of the urinary bladder. 2 Urothelial carcinoma is a neoplasm of older dogs (average age, 9-11 years) 2,26,27 and is most common in females. 2,26 Polypoid cystitis has been reported to arise from the cranioventral bladder mucosa in most cases. 3 Younger male dogs are reported to be the predominant patient group in some studies, 2 whereas in the only case series (17 dogs), a strong female predisposition was found, and ages ranged from 2 to 13 years. 3 On ultrasound examination, at cystoscopy, or on gross examination, polypoid cystitis may be mistaken for a urothelial neoplasm. 2,3,17 The mucosal surface of the bladder is elevated by a single polyp or multiple nodular to polypoid lesions protruding into the lumen. On cut surface, the proliferations do not extend below the mucosa. 2 Microscopically, the fronds of polypoid cystitis typically are much broader than those of a papillary carcinoma 17 and the urothelium is hyperplastic, but usually not stratified as in carcinomas. 17 In addition, the fibrovascular cores of the papillae in TCC typically lack the prominent inflammation that characterizes both papillary and polypoid cystitis, and the edema seen in the latter, at least in human patients. 17 Lymphoid aggregates may be prominent in both polypoid lesions and UC. 1,2,17,26,27 Crucial to the diagnosis of TCC in dogs are location in the urinary bladder, large epithelial cells with multiple cellular and nuclear features of atypia, Melamed-Wolinska bodies, and invasion to some extent. 2 In our case, the clinical history of UTI, undetected BRAF mutation, location of the lesions, patient age and gross and histological appearance of the lesions all were compatible with polypoid cystitis. However, we cannot rule out that CIS was already beginning to develop at the time when the first biopsy sample was taken. The apparent initial ultrasonographic improvement of the bladder after medical management in part may have been affected by operator variability and bladder volume. [28][29][30] All types of UC consist of neoplastic transitional cell epithelium that is in various stages of differentiation. 2 Carcinoma in situ is the most well-differentiated form, and it is accepted by some pathologists that if CIS or low-grade UC are the only lesions present in biopsy specimens, a more invasive neoplasm likely is present in different F I G U R E 6 Partial cystectomy histopathology. A, Polypoid cystitis is still visible in this section, accompanied by typical changes: hemorrhage, proprial edema, and inflammation. B, Area of transition from the same biopsy, showing development of TCC (on the left) adjacent to polypoid proliferation of the mucosa (on the right). Note invasion into the lamina propria of neoplastic epithelium. A lymphoid follicle is observed as well, a common finding in polypoid cystitis (arrow) regions that may not have been included in the biopsy. 2 Carcinoma in situ is a recognized precursor to invasive UC in humans, and therefore a similar progression may occur in animals. 2 In our patient, the mutation in exon 15 of the canine BRAF gene present in >85% of TCC cases in dogs 20 was not present. This characteristic complicated the final diagnosis for a substantial period of time and prevented confirmation of the time at which a malignant transformation occurred. This case emphasizes that, despite the high sensitivity of the CADET BRAF 19 and CADET BRAF Plus, 20  After the confirmed diagnosis of TCC on cystoscopic biopsy, partial cystectomy was performed. Analysis of the full thickness bladder wall allowed us to confirm the diagnosis of TCC despite the previously conflicting results of cystoscopic biopsies and undetected BRAF mutation. Surgery also presumably alleviated the discomfort related to the chronically inflamed and neoplastic tissue. A recent study in dogs with bladder TCC found the best outcomes (median survival time, 772 days) for dogs with nontrigonal bladder TCC treated by full thickness partial cystectomy and daily piroxicam treatment, with or without chemotherapy. 31 At the time of writing, our patient is still alive and has a normal appearing bladder and urethra on ultrasound examination, 431 and 180 days after first diagnosis of TCC and partial cystectomy, respectively. This report should increase the awareness of potential rapid malignant transformation from polypoid cystitis into TCC. Histopathology is the gold standard for final diagnosis when the BRAF mutation is not detected, and TCC remains a differential diagnosis. Surgical management should be considered if rapid resolution is not achieved with medical management.

ACKNOWLEDGMENT
No funding was received for this study.

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Authors declare no IACUC or other approval was needed because this was retrospective.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.