Expression of vascular endothelial growth factor receptor-2, epidermal growth factor receptor, cyclooxygenase-2, survivin, E-cadherin and Ki-67 in canine nasal carcinomas and sarcomas - a pilot study

Abstract

Background: Malignant (intra-) nasal tumors (NTs) are the most common cause of chronic nasal discharge in dogs. Besides radiation therapy, palliative therapy is necessary in some dogs. Therefore, studies on receptor expression have supported the utility of tyrosine kinase inhibitors (TKI) in dogs with nasal carcinomas. However, studies on receptor expression in nasal sarcomas are lacking.

Materials and methods: This study evaluated the expression of vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), cyclooxigenase-2 (COX-2), Ki-67, survivin and E-cadherin in nasal carcinomas and sarcomas and compared it with tumor (T) categories based on computed tomography (CT).

Results: In 26 dogs with NTs, cross sectional imaging and upper airway endoscopy with guided biopsy collection were performed, followed by histopathological examination of NTs, revealing 19 epithelial and 7 mesenchymal tumors. While EGFR and E-cadherin were only expressed by carcinomas, the following markers were expressed by both carcinomas and sarcomas without significant differences between tumor types and T-categories: VEGFR-2 (carcinomas and sarcomas 100%), COX-2 (carcinomas 63%, sarcomas 57%), survivin (carcinomas 100%, sarcomas 86%) and Ki-67 (median expression of 28.5% in carcinomas and 17.3% in sarcomas).

Conclusion: Based on similarities in marker expression between canine carcinomas and sarcomas, clinical studies should further elucidate the use of TKI or COX-2 inhibitors as additional therapy in dogs with nasal sarcomas.

Keywords: T-categories; chronic nasal discharge; computed tomography; cyclooxygenase inhibitors; dogs; immunohistochemistry; nasal tumors; tyrosine kinase inhibitors.

Figure 1

Illustration of the evaluation of immunohistochemical staining with the different antibodies based on published analysis methods. EGFR, COX-2, VEGFR-2, survivin and E-cadherin were evaluated semiquantitatively, in contrast to Ki-67, where the positive tumor cells were counted using image analysis software (Fiji Image J, Fiji contributors) on digital images taken by a photomicroscope. For EGFR, COX-2, VEGFR-2 and survivin, a total score (intensity score multiplied by the individual percentage score) was used. For E-cadherin, the percentage score was evaluated only. For Ki-67 the proliferation index was evaluated.

Figure 2

Expression of immunohistochemical markers in canine nasal carcinomas (n = 19) and sarcomas (n = 7). Carcinomas: 100% were positive for VEGFR-2, 63.2% for COX-2, 100% for survivin, 68.4% for EGFR and 100% for E-cadherin. Sarcomas: 100% were positive for VEGFR-2, 57.1% for COX-2, 85.7% for survivin and 0% for EGFR and E-Cadherin. The proportion of positive tumor cell nuclei for Ki-67 of the total number of counted tumor cell nuclei (proliferation index, PI) was 28.5% of nasal carcinomas and 17.3% of sarcomas. Because of the different meaning of [%] in this context-Ki-67 was separated in the bar diagram by a dashed line.

Figure 3

Comparison of the expression of immunohistochemical markers between canine nasal tumors of different T-categories (mean ± SD; order: T1, T2, T3, and T4 - with light gray bars representing carcinomas (C) and dark gray bars representing sarcomas (S)). (A) EGFR, survivin and COX-2. (B) E-Cadherin and Ki-67. Sarcomas are considered in (A) and (B) separately (dark gray bars with orange dots). There is no significant difference in tumor expression according to tumor size or tumor entity in the same T-category. (B) Because of the different meaning of [%] as PI in the context of Ki-67 - it was separated in the bar diagram by a dashed line.

Figure 4

Duration of clinical signs in 25/26 dogs with nasal tumors in months in correlation to tumor size in cross sectional imaging (T = T-category; CT in 24/25 dogs, MRI in 1/25 dogs). Duration of clinical signs of the dogs is presented in bar diagrams with mean ± SD. Time was calculated by the owners from onset of clinical signs until the timepoint when diagnostics in anesthesia were performed at the ENT-Unit of the Small Animal Department of the Leipzig University due to nasal discharge. There was no significant difference of different T-categories in duration of clinical signs (Ordinary one-way ANOVA, p = 0.4350) or between carcinomas and sarcomas (Two tailed Mann–Whitney test, p = 0.0526). Therefore, tumor size at presentation was supposed not only to be correlated with duration of disease, but also with different speed of growths.

Figure 5

Examples of immunohistochemical stainings for the detection of EGFR (A,B), VEGFR-2 (C,D) and COX-2 (E,F) in malignant canine nasal tumors. Representative pictures are displayed for low target protein expression (A,C,E) and high target protein expression (B,D,F). Different histopathological tumor types are shown at a magnification of 200x. (A) Carcinoma with membranous-cytoplasmic staining pattern, total score 6. (B) Squamous cell carcinoma with membranous staining pattern, total score 12. (C) Transitional cell carcinoma with cytoplasmic staining pattern, total score 3. (D) Carcinoma with cytoplasmic staining pattern, total score 6. (E) Carcinoma with cytoplasmic perinuclear staining pattern, total score 4. (F) Carcinoma with cytoplasmic perinuclear staining pattern, total score 9.