title: Studies on equine eosinophilic granuloma and mast cell tumour
Creators: name: Elbahi, AM
Publisher: name: University of Liverpool Repository
Year: 2019

description: Eosinophilic granuloma (EG) and mast cell tumour (MCT) are skin diseases that affect horses as well as other animal species. In equine species they exhibit similar and potentially overlapping morphological features, and it is not clear if they are two different entities or represent a continuum of the same disease. Equine eosinophilic granuloma is the most common inflammatory nodular skin disease in horses and represents a chronic inflammation characterised by intense infiltrates of eosinophils associated with macrophages. The cause of this change is not fully clear. However, different factors are reported to potentially cause it and in turn lead to a hyper sensitivity reactions, dominated by eosinophils. In contrast, equine mast cell tumour is an uncommon neoplastic disease characterised by aggregates of mast cells, admixed with variable numbers of eosinophils and scattered areas presenting with lesions morphologically identical to those observed in equine eosinophilic granuloma. The aim of this study is to investigate the morphological and molecular features of equine eosinophilic granuloma and mast cell tumour in order to better understand their pathogenesis. One hundred and ninety one lesions from 153 horses which had previously been diagnosed with equine eosinophilic granuloma or mast cell tumour were retrieved form the archive of the Department of Veterinary Pathology and Public Health, University of Liverpool. Clinical data were recorded and histomorphological features were investigated. The present study showed that Thoroughbreds with Thoroughbreds cross, and Arabian with Arabian cross are the most affected breeds with EG and MCT. In addition, however, a large number of different breeds were affected which had no sex predilection. With regard to MCT, the Arab breed was significantly predominant and males were overrepresented. The age range was wide, with older horses more affected by MCT than by EG, as expected. The most common location of the lesions was the trunk for eosinophilic granuloma and the head for MCT. The morphological examination for 191 lesions was performed regardless (blind) of initial diagnosis. This analysis revealed variable histological features among cases. Criteria examined were: location of lesions within the skin, lesion size, area and number of eosinophilic granuloma component (EGC) and mast cell aggregates (MCA), presence of individual mast cells infiltrating the lesions, association between EGC and hair follicle structures, presence of embedded collagen in EGC, evidence of mineralisation, evidence of ulceration, density of infiltrating viable eosinophils within the MCA, mitotic activity of the mast cell population, and presence and number of lymphocyte aggregates. Using these criteria, four lesions exhibiting unique morphological features were identified. These cases were characterised by marked eosinophil infiltration and embedded mast cell population with very large pleomorphic mast cells. These four cases were excluded from the study and are described separately in Appendix II section-B. From all 191 examined cases, 95 lesions were selected for further analysis. Five morphological categories were created based on the presence and relationship between the EGC and MCA components as following: category I (EGC only), category II (EGC &gt; MCA), category III (EGC= MCA), category IV (EGC &lt; MCA), and category V (MCA only). Based on these categories, three major groups were created: Group1 (Category I), Group2 (Categories II, III, and IV), and Group3 (Category V). The newly classified eosinophilic granuloma (EG) corresponded to roup 1 (Category I), while mast cell tumour (MCT) was identified through gGroups 2+3 (Categories II, III, IV, and V). EG lesions exhibited more variable histological features among cases within group 1, while MCT lesions were more similar among other groups. There were differences in lesion size and number between the categories with EG being smaller than MCT with size among different categories of MCT being highly variable. Deep dermis was the most common location for MCT, while EG was the lesion found most superficially. However, both EG and MCT stretched to different layers and when a “depth score” was evaluated, there was no difference between categories. Interestingly, a strong association between Category I lesions (EG) and hair follicle structures involvement was identified (52% of cases). Individual mast cells scattered throughout the lesions were evident in 13 lesions (42%) of Category I (EGC only) while they were present in all lesions of Category II (EG &gt; MCT), and absent in the other categories. The analysis of Von Kossa stained sections indicated that EG exhibited significantly more mineralisation than MCT (P˂0.001). Ulceration was present in both EG and MCT with no difference and was probably a secondary change not linked with the pathogenesis of either lesions. Collagen fibres or their fragments were visualised in almost all EG lesions (93%) within EGC, using the Masson Trichrome Stain. EG showed significantly more fragments of collagen within the EGC cores than MCT. The presence of lymphocytes aggregates was mainly a feature of EG (61% cases) rather than MCT, where only rare lymphocytes aggregates were detected in a few cases. The density of viable eosinophils infiltrating in MCA had an increasing trend among categories. The number of mitotic figures was different among Categories (II to V) with significantly higher numbers in Category V (P˂0.01). Morphological evaluation identified macrophages actively phagocytosing eosinophils; this was only detected in MCT lesions, while no evidence of such phenomenon was noted in EG. Lendrum’s stain, used to identify eosinophils within the sections, successfully highlighted the eosinophils granules, but also demonstrated that eosinophils in EGC cores exhibited different staining colours (purple, intense red, pale red) or were colourless. The presence of this colour pattern demonstrated that the EGC core did not have a regular centrifugal development as expected, but an “irregular onion ring” like arrangement, with different orders of colours change, overlapping each other. Purple Lendrum’s stain overlapped perfectly with von Kossa stained areas suggesting that this chromatic affinity indicates mineralisation. In the present study infiltration of immune cells was investigated using immunohistochemistry. CD3- positive cells, indicative for T lymphocytes were infiltrating in large numbers in EG and MCT, but higher CD3 density was detected in EG lesions compared to MCT. CD79a-postive lymphocytes, indicative for B lymphocytes, which though rare in both MCT and EG lesions were, when present, forming lymphocyte aggregates. These cells were significantly more present in EG lesions compared to MCT (P˂0.05). Histiocytes (Iba1- positive) were more numerous in EG compared to MCT; in both categories they were highly represented. Their observation was associated with EGC but also infiltrating in between mast cells in MCT, possibly representing tumour-associated macrophages (TAMs). Analysis of cytokine expression was performed using in-situ hybridisation on formalin fixed paraffin embedded tissue (Eotaxin, MIP1-alpha, IL-4, IL-5, IL-13, TGF-beta, SCF) or immunohistochemistry (RANTES) in a sub group of lesions (n=10) belonging to EG (n=5) and MCT (n=5). In EG the predominant cytokine expressed by macrophages and lymphocytes including epithelioid macrophages was IL-13, which was significantly more expressed in EG compared to MCT. In MCT the most represented cytokine was IL-5 which was transcribed by mast cells and lymphocytes and was significantly higher in MCT than EG (P˂0.05). Among other cytokines RANTES was expressed in lymphocytes and histiocytes in higher number in EG compared to MCT. Other cytokines investigated such as IL-4, MIP-1a, TGF beta, and SCF showed weaker differences amongst groups, and their role in the two lesions was less clear. Summarising, according to the results of this study: EG were predominantly lesions characterised by EGC with mineralised cores and the presence of collagen fragments. EGC were associated in some cases with lymphocytes aggregates composed of B cells, and often extended to the most superficial layers of the skin with hair follicle involvement. T cells were predominant, and IL-13 transcribed by lymphocytes and macrophages is likely to be predominantly involved in eosinophils recruitment in these lesions. MCT were lesions predominantly located in deep dermis, exhibiting a variable number and variable sizes of EGC or, in some cases, none. MCT exhibited large numbers of infiltrating macrophages consistent with TAMs. Within the MCA, scattered macrophages phagocytosing eosinophils were detected, a unique feature of MCT. EGC associated with MCT were less often mineralised than those of EG, and only very rarely associated with B cells. IL-5 was the most abundant cytokine transcribed in these lesions by mast cells and lymphocytes, and it is believed that it is associated with the recruitment and increase in survival of eosinophils.