Studies on equine eosinophilic granuloma and mast cell tumour

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 > MCA), category III (EGC= MCA), category IV (EGC 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

Travel burden and clinical presentation of retinoblastoma: analysis of 1024 patients from 43 African countries and 518 patients from 40 European countries

BACKGROUND: The travel distance from home to a treatment centre, which may impact the stage at diagnosis, has not been investigated for retinoblastoma, the most common childhood eye cancer. We aimed to investigate the travel burden and its impact on clinical presentation in a large sample of patients with retinoblastoma from Africa and Europe. METHODS: A cross-sectional analysis including 518 treatment-naïve patients with retinoblastoma residing in 40 European countries and 1024 treatment-naïve patients with retinoblastoma residing in 43 African countries. RESULTS: Capture rate was 42.2% of expected patients from Africa and 108.8% from Europe. African patients were older (95% CI -12.4 to -5.4, p<0.001), had fewer cases of familial retinoblastoma (95% CI 2.0 to 5.3, p<0.001) and presented with more advanced disease (95% CI 6.0 to 9.8, p<0.001); 43.4% and 15.4% of Africans had extraocular retinoblastoma and distant metastasis at the time of diagnosis, respectively, compared to 2.9% and 1.0% of the Europeans. To reach a retinoblastoma centre, European patients travelled 421.8 km compared to Africans who travelled 185.7 km (p<0.001). On regression analysis, lower-national income level, African residence and older age (p<0.001), but not travel distance (p=0.19), were risk factors for advanced disease. CONCLUSIONS: Fewer than half the expected number of patients with retinoblastoma presented to African referral centres in 2017, suggesting poor awareness or other barriers to access. Despite the relatively shorter distance travelled by African patients, they presented with later-stage disease. Health education about retinoblastoma is needed for carers and health workers in Africa in order to increase capture rate and promote early referral

Global Retinoblastoma Presentation and Analysis by National Income Level.

Early diagnosis of retinoblastoma, the most common intraocular cancer, can save both a child's life and vision. However, anecdotal evidence suggests that many children across the world are diagnosed late. To our knowledge, the clinical presentation of retinoblastoma has never been assessed on a global scale. To report the retinoblastoma stage at diagnosis in patients across the world during a single year, to investigate associations between clinical variables and national income level, and to investigate risk factors for advanced disease at diagnosis. A total of 278 retinoblastoma treatment centers were recruited from June 2017 through December 2018 to participate in a cross-sectional analysis of treatment-naive patients with retinoblastoma who were diagnosed in 2017. Age at presentation, proportion of familial history of retinoblastoma, and tumor stage and metastasis. The cohort included 4351 new patients from 153 countries; the median age at diagnosis was 30.5 (interquartile range, 18.3-45.9) months, and 1976 patients (45.4%) were female. Most patients (n = 3685 [84.7%]) were from low- and middle-income countries (LMICs). Globally, the most common indication for referral was leukocoria (n = 2638 [62.8%]), followed by strabismus (n = 429 [10.2%]) and proptosis (n = 309 [7.4%]). Patients from high-income countries (HICs) were diagnosed at a median age of 14.1 months, with 656 of 666 (98.5%) patients having intraocular retinoblastoma and 2 (0.3%) having metastasis. Patients from low-income countries were diagnosed at a median age of 30.5 months, with 256 of 521 (49.1%) having extraocular retinoblastoma and 94 of 498 (18.9%) having metastasis. Lower national income level was associated with older presentation age, higher proportion of locally advanced disease and distant metastasis, and smaller proportion of familial history of retinoblastoma. Advanced disease at diagnosis was more common in LMICs even after adjusting for age (odds ratio for low-income countries vs upper-middle-income countries and HICs, 17.92 [95% CI, 12.94-24.80], and for lower-middle-income countries vs upper-middle-income countries and HICs, 5.74 [95% CI, 4.30-7.68]). This study is estimated to have included more than half of all new retinoblastoma cases worldwide in 2017. Children from LMICs, where the main global retinoblastoma burden lies, presented at an older age with more advanced disease and demonstrated a smaller proportion of familial history of retinoblastoma, likely because many do not reach a childbearing age. Given that retinoblastoma is curable, these data are concerning and mandate intervention at national and international levels. Further studies are needed to investigate factors, other than age at presentation, that may be associated with advanced disease in LMICs