Ugent, faculteit diergeneeskunde, departement morfologie
Abstract Bachelor Project FBT 2018-2019: PERFORMING VARIOUS IMMUNOHISTOCHEMICAL TECHNIQUES ON CANINE MAST CELL TUMOURS
Mast cell tumours are the most common malignant skin tumours in dogs. These tumours are dependent on angiogenesis to quarantee the supply of oxygen and nutrients. To investigate the morphology and malignancy of these tumours, various factors can be investigated such as angiopoietin 1 (ANGPT1) for angiogenesis, KI67 for cell proliferation and von Willebrand factor (vWF) and cluster of differentiation 31 (CD31) to monitor vascularization. The purpose of this study is to test these factors on multiple mast cell tumours of different grades. The goal is to investigate whether these factors have a link with the tumoral grade. First, ANGPT1 (Cloud- Clone Corp USA) was optimized by using immunohistochemistry and then used on different tumours. The optimization was done on a cutaneous mast cell tumour embedded in paraffin.
Different protocols were applied, using different dilutions of this antibody. The conclusion of this optimization was that ANGPT1 does not bind specifically enough. As a result, this antibody could not be used for further research. Because ANGPT1 did not work, this study uses antibodies that were already optimized and that work effectively, namely KI67 clone MIB-1 (Dako® Denmark) and vWF (Dako® Denmark). These factors identify the proliferation and vascularization in tumoral tissue. Seven tumours were fixed in 3.5 % buffered formalin for 24 hours and subsequently placed in a 70 % alcohol solution before being paraffin embedded.
Histopathological grade was determined following the Patnaik and Kiupel grading system. One tumour was diagnosed as a grade I, four as low-grade II, one as high-grade II and one as grade III. Sections are cut and stained with immunohistochemistry. Of the positive results, five at random regions are counted for proliferation and vascularization. The findings of KI67 are in line with current literature. However, these preliminary data do not support the findings of a previous study suggesting a positive correlation between blood vessel density and grade of tumour malignancy. It can be hypothesized that the low number of vessels in combination with a high KI67 expression can be explained by a hypoxic microenvironment that triggers cellular proliferation. More canine mast cell tumour need to be investigated to confirm these preliminary data. In addition, CD31 (Biorbyt UK) can also be used to visualize vascularization. Before CD31 can be used effectively for this study, it is first compared with vWF. For this, a highly vascularized tumour is fixed in buffered formalin 3.5 % and embedded in paraffin. The immunohistochemical staining of CD31 is non-specific and shows a lot of background signal. Because vWF has a good coloration, CD31 is not used for this research.
Key words: dog, mast cell tumour, immunohistochemistry, angiopoietin 1, KI67, von Willebrandfactor, CD31
Ward De Spiegelaere
Shana De Vos