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. 2019 Mar 19;6(1):31.
doi: 10.3390/vetsci6010031.

Correlation of BRAF Variant V595E, Breed, Histological Grade and Cyclooxygenase-2 Expression in Canine Transitional Cell Carcinomas

Julia M Grassinger  1 , Sophie Merz  2 , Heike Aupperle-Lellbach  3 , Hanna Erhard  4 , Robert Klopfleisch  5
Affiliations

Correlation of BRAF Variant V595E, Breed, Histological Grade and Cyclooxygenase-2 Expression in Canine Transitional Cell Carcinomas

Julia M Grassinger et al. Vet Sci. .

Abstract

The presence of BRAF variant V595E, as well as an increased cyclooxygenase-2 (COX-2) expression in canine transitional cell carcinoma (TCC) are well-described in the literature. The aim of the present study was to investigate the correlation between breed (terrier versus non-terrier dogs), histological grade, COX-2 expression, and BRAF mutation in canine TCC. Therefore, transmural TCC biopsies from 65 dogs (15 terriers, 50 non-terriers) were graded histologically into low- and high-grade. Immunohistochemical evaluation of the intensity of COX-2 expression was performed using an immunoreactive score (IRS). Exon 15 of chromosome 16 was examined for the BRAF variant c.1799T>A by TaqMan® SNP assay. TCC was low-grade in 20 cases (one terrier, 19 non-terriers) and high-grade in 45 cases (14 terriers, 31 non-terriers). Contrary to humans, histological grade was not significantly correlated to the intensity of COX-2 expression. BRAF mutation was detected in 11/15 (73%) TCC of terriers and in 18/50 (36%) TCC of non-terriers. Histological grade and BRAF mutation were not correlated significantly (p = 0.2912). Terriers had a considerably higher prevalence of high-grade tumors (p < 0.0001), as well as of BRAF mutation (p ≤ 0.05) compared to non-terriers. In non-terriers, neoplasms with BRAF mutation showed a significantly higher intensity of COX-2 expression than those without BRAF mutation (p ≤ 0.05). In conclusion, in contrast to humans, testing for BRAF mutation in canine TCC is a sensitive diagnostic method especially in terriers (73%) and may be recommended as a screening test. However, evidence of BRAF mutation in canine TCC is not a predictor for the histological grade. Moreover, a positive correlation between histological grade and the intensity of COX-2 expression was not found. Further studies are necessary to clarify the clinical and prognostic relevance of the elevated intensity of COX-2 expression of TCC with BRAF mutation detected in non-terriers.

Keywords: BRAF mutation; COX-2; dog; histological grading; terrier; urothelial carcinoma.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Histology of canine transitional cell carcinoma specimens. (a) Low-grade transitional cell carcinoma of an 11-year-old male mongrel. The epithelium (E) is markedly thickened and multi-layered, and the irregularly-arranged cells show mild anisocytosis and anisokaryosis. The basement membrane is intact (HE, bar = 250 μm). (b) High-grade transitional cell carcinoma of an eight-year-old castrated male mongrel with marked anisocytosis and anisokaryosis. The tumor intensively invades the submucosa, and additionally, an intravascular tumor cell embolus (arrow) is detectable (HE, bar = 100 μm).
Figure 2
Figure 2
Correlation between histological grade and breed: A significantly higher percentage of high-grade transitional cell carcinoma (TCC) compared to low-grade TCC was observed in terriers (**** p < 0.0001) and in non-terriers (* p ≤ 0.05). Furthermore, a considerably higher percentage of high-grade transitional cell carcinoma was observed in terriers compared to non-terriers (* p ≤ 0.05) (Fisher’s exact test).
Figure 3
Figure 3
Cyclooxygenase-2 immunohistochemistry. (a) Normal bladder epithelium (E) of a 10-year-old female mongrel. There is no apparent cyclooxygenase-2 expression (COX-2, bar = 25 μm). (b) High-grade transitional cell carcinoma from an 11-year-old female Scottish terrier with an IRS of 7.6. Intensity of cyclooxygenase-2 expression varied from mild (*) to marked (#) within the specimen (COX-2, bar = 100 μm). (c) Area with minimal intensity of cyclooxygenase-2 expression in high-grade transitional cell carcinoma from an 11-year-old castrated female bracke (COX-2, bar = 25 μm). (d) Area with mild intensity of cyclooxygenase-2 expression in low-grade transitional cell carcinoma from a 12-year-old castrated female beagle (COX-2, bar = 25 μm). (e) Area with moderate intensity of cyclooxygenase-2 expression in high-grade transitional cell carcinoma from a 13-year-old castrated male mongrel (COX-2, bar = 25 μm). (f) Area with marked intensity of cyclooxygenase-2 expression in low-grade transitional cell carcinoma from an 11-year-old castrated male podenco (COX-2, bar = 25 μm).
Figure 3
Figure 3
Cyclooxygenase-2 immunohistochemistry. (a) Normal bladder epithelium (E) of a 10-year-old female mongrel. There is no apparent cyclooxygenase-2 expression (COX-2, bar = 25 μm). (b) High-grade transitional cell carcinoma from an 11-year-old female Scottish terrier with an IRS of 7.6. Intensity of cyclooxygenase-2 expression varied from mild (*) to marked (#) within the specimen (COX-2, bar = 100 μm). (c) Area with minimal intensity of cyclooxygenase-2 expression in high-grade transitional cell carcinoma from an 11-year-old castrated female bracke (COX-2, bar = 25 μm). (d) Area with mild intensity of cyclooxygenase-2 expression in low-grade transitional cell carcinoma from a 12-year-old castrated female beagle (COX-2, bar = 25 μm). (e) Area with moderate intensity of cyclooxygenase-2 expression in high-grade transitional cell carcinoma from a 13-year-old castrated male mongrel (COX-2, bar = 25 μm). (f) Area with marked intensity of cyclooxygenase-2 expression in low-grade transitional cell carcinoma from an 11-year-old castrated male podenco (COX-2, bar = 25 μm).
Figure 4
Figure 4
Fluorescent signals detected by the TaqMan® SNP assay of a canine transitional cell carcinoma. (a) In transitional cell carcinoma without BRAF mutation, there is only a signal from the wild-type probe (homozygous thymine, dotted curve) and no signal from the variant probe (curve without dots). (b) In transitional cell carcinoma with BRAF mutation (heterozygous thymine and adenine), signal curves of both the wild-type probe (thymine, dotted curve) and the variant (adenine, curve without dots) can be seen.
Figure 5
Figure 5
Correlation between breed and the presence or absence of BRAF mutation: BRAF mutation occurs significantly more often in transitional cell carcinoma in terriers than in transitional cell carcinoma in non-terriers (Fisher’s exact test, p ≤ 0.05).
Figure 6
Figure 6
Correlation between the intensity of cyclooxygenase-2 expression and the presence or absence of BRAF mutation: a significant difference in the cyclooxygenase-2 expression was only detected in non-terrier breeds comparing BRAF mutation-positive and -negative transitional cell carcinoma (Mann–Whitney U-test, p ≤ 0.05).
Figure 7
Figure 7
Interpretation algorithm for BRAF mutation in terriers and non-terrier breeds in correlation with the histological grade and cyclooxygenase-2 expression.
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