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Ugent, faculteit diergeneeskunde, departement morfologie

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Abstract Bachelor Project FBT 2020-2021: Immunohistochemical detection of various antibodies as a prognostic marker in canine mast cell tumours

The neoplastic proliferation of mast cells leads to the formation of a mast cell tumour (MCT), which is the most common cutaneous tumour in dogs. After surgery, these tumours are usually graded according to the Patnaik and Kiupel grading system, followed by a conclusion of the degree of malignancy. This determination is important because it affects the therapy and prognosis of the dog. To get a better view on the morphology and to confirm the degree in addition to the histopathological research, multiple prognostic biomarkers can be used. In this study, an immunohistochemical (IHC) technique is used as a method to visualize tumours and determine their malignancy. Therefore, multiple MCT samples of different dogs are collected on which the markers were tested. The purpose of this study is to link the positivity of the biomarkers with the tumour grade and prognosis. First, CD-30 was optimized as a marker for canine neoplastic mast cells. After that, a marker which plays a role in cellular damage repair was tested and a comparison was made between the positivity of the staining and proliferation and other differentiation markers such as Ki67 and the KIT staining pattern. In addition, the average number of positive cells per high-power field (HPF) were investigated. The same protocol was used for both markers consisting of immunohistochemical staining of formalin-fixed paraffin-embedded (FFPE) samples. After optimalisation, CD-30 has an optimal dilution of 1/100 for 16 hours on 4 degrees. Grade I had a greater number of positive cells than a high grade tumour. The higher the Ki-67, the stronger the expression of the cellular marker. A granular, cytoplasmic stain occurs in a greater number of cells compared to the other KIT patterns. The higher the degree, the greater the mean value of the mitotic count. When there is a metastasis, there are clearly fewer positive cells for the cellular marker. To conclude, no correlation has yet been found between the intensity of the CD-30 stain and the grade of tumours. More research must be done with multiple MCT of different degree. No significant correlation between the expression of the cellular marker and the tumour grade, Ki-67, KIT staining pattern, mitotic count and metastasis was found.

Abstract Bachelor Project FBT 2019-2020: Immunohistochemical visualization of blood vessels in canine mast cell tumours

Mast cell tumours (MCT) are the most malignant skin tumours in dogs. They can be divided into three grades following the Patnaik grading system and two grades following the Kiupel grading system where high-grade MCT (or grade III) have a worse prognosis. Due to the malignancy of these tumours, it is essential to know the tumour grade. Up to now, this is only possible after tumour excision and histopathological examination. To gain more insight in the malignancy of these tumours, several markers, such as von Willebrand factor (vWF) for the staining of tumour vascularization and macrophage marker for tissue macrophage staining, are examined by using immunohistochemistry (IHC). In addition, patients that were treated for MCT are followed-up during a couple of months. The purpose of the study is to test the presence of these markers in different grades of cutaneous MCT and to investigate if they are linked with survival time (ST). Prior to IHC, all tumours are fixed in 4 % buffered formalin for 24 hours, placed in 70 % alcohol and embedded in paraffin. Small sections are cut with a microtome. Von Willebrand factor was tested on 66 tumours and MAC-387 was tested on 27 tumours. After the immunohistochemical staining of the vWF, the vessels were counted in five ad random regions in each tumour section. For the calculation of ST, the owners of 25 patients were contacted and asked if their dog was still alive at the time of the study. The results of this study do not match with the literature that suggested that there is a positive correlation between number of tumoral vessels and the grade. In this study there is no significant correlation between tumour grade and the tumour vascularization. After the staining with MAC-387, the tumour sections were screened. There were sections with no staining, little staining, diffuse staining or hotspots. The results of the macrophage marker match with those in the literature that suggest that there is no correlation between the tissue macrophages and the tumour grade. The results of the follow-up show that ST decreases with an increasing tumour grade. This correlates with the literature. It can be concluded that there is no link between the tumour grade and vascularization. Furthermore, there is no link between the macrophage marker and the tumour grade. The survival time decreases with an increasing tumour grade. Out of this, there can be concluded there is no link between the tested markers and the ST.



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


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