Mast Cell Tumors and Histiocytomas in Domestic Goats and Diagnostic Utility of CD117/c-Kit and Iba1 Immunohistochemistry.

Cutaneous round cell tumors in goats present a diagnostic challenge. In this article, we provide a description of caprine cutaneous mast cell tumors (MCT) and histiocytomas, and report on the validation of anti-human antibodies to CD117/KIT and Iba1 by immunohistochemistry on a range of caprine tissues. Cells immunolabeled for CD117/KIT included resident mast cells in normal lung and skin, interstitial cells of Cajal (intestine), and neuronal cell bodies (brain). Cells immunolabeled for Iba1 included resident macrophages in many tissues including normal lung, dendritic cells (hemolymphatic tissues), Kupffer cells, and microglia. Of 5 cutaneous MCT, only one had metachromasia of cytoplasmic granules; however, neoplastic cells of all 5 MCT had positive immunolabeling for CD117/KIT. The CD117/KIT immunolabeling pattern was predominately focal paranuclear in 3 cases, and cytoplasmic or membranous in 1 case each. Two histiocytomas were identified and had strong positive immunolabeling for Iba1 but not CD117/KIT. All 7 cutaneous round cell tumors described herein occurred in goats less than 4 years of age; the 2 cutaneous histiocytomas were in goats less than 14 months of age. Neither of the cutaneous histiocytomas recurred within 24 months of surgical removal.

A Role for Angiogenesis in Canine Cutaneous Histiocytoma Regression: Insights into an Old Clinical Enigma.

BACKGROUND/AIM: Canine Cutaneous Histiocytoma (CCH) is a Langerhans' cells benign tumour that undergoes spontaneous regression. The aim of the present study was to investigate the role of angiogenesis, a key step for tumour development, in CCH regression. MATERIALS AND METHODS: 50 CCH samples were classified into 4 histological groups according to a regression scale, and evaluated for expression of vascular endothelial factor-A (VEGF-A) and its receptor VEGFR-2 as well as microvessel density (MVD). RESULTS: Tumours during early stages of the regressive process had a lower MVD compared to later stages, while CCH tumoural cells showed a limited production of VEGF, but higher levels of VEGFR-2. On the contrary, tumours in advanced phases of regression showed a higher number of neovessels, probably associated with the inflammatory state and the healing process. CONCLUSION: Our results suggest that angiogenesis may be compromised at early stages of histiocytoma development and this may be a determinant of regression in this tumour.

FOXP3, CD208, and CD206 Expression in Canine Cutaneous Histiocytoma.

Canine cutaneous histiocytoma (CCH) is a noninfectious tumor that spontaneously regresses. It is suggested that this regression is due to tumor cell maturation, which is responsible for CD8 lymphocyte activation and tumor cell destruction. Nevertheless, the possible role of the immune microenvironment in tumor regression has not been investigated to date. The aim of this study was to investigate the expression of CD208 and FoxP3 as markers of dendritic cells and regulatory T lymphocytes, respectively, and tumor cell expression of CD206 as a marker of Langerhans cell activation, and relate these parameters to the different phases of CCH and to intratumoral T cell infiltration. Formalin-fixed, paraffin-embedded samples from 31 CCH were evaluated. In each case, the mitotic count and regression phase were recorded. Within the tumor, a quantitative evaluation of immunolabeled CD208+ cells, FoxP3+ cells, and CD3+ lymphocytes was performed, as well as the CD206+ tumor cell location. Intratumoral CD208+ cells correlated with CD3+ lymphocytic infiltration. The possible role of dendritic cells in tumor regression was not confirmed since CD208 seemed to be a nonspecific marker for canine dendritic cells. FoxP3+ lymphocyte density was not correlated with any parameter. Neoplastic Langerhans cells presented progressive CD206 expression, from the bottom of the tumor to the epidermis, which correlated with the tumor regression phase and with intratumoral T lymphocyte infiltration. In conclusion, we confirmed a CD206 phenotype change in tumor cells in a spatial group-related pattern, supporting the hypothesis that tumoral Langerhans cells acquire a mature phenotype with tumor regression.

Immunohistochemical Labeling of Multiple Myeloma Oncogene 1/Interferon Regulatory Factor 4 (MUM1/IRF-4) in Canine Cutaneous Histiocytoma.

Multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF-4) immunohistochemistry (IHC) is mainly used for diagnostic confirmation of plasma cell tumors (PCTs) in dogs and cats. This article describes MUM1/IRF-4 IHC expression in 20 cases of canine cutaneous histiocytoma (CH) and compares it with 10 cutaneous or mucocutaneous PCTs and 5 cutaneous histiocytic sarcomas (HSs) submitted to the same IHC protocol. All histiocytomas had strong nuclear and variable cytoplasmic immunolabeling for MUM1/IRF-4, whereas all PCTs had strong nuclear and moderate cytoplasmic immunolabeling for MUM1/IRF-4. No MUM1/IRF-4 immunolabeling was detected in the HSs. Although not typically a diagnostic challenge, MUM1/IRF-4 expression may have to be used with caution or in conjunction with additional immunomarkers to differentiate among poorly differentiated round cell tumors, especially when a histiocytic or plasma cell origin is suspected.

Prevalence of Bartonella spp. in Canine Cutaneous Histiocytoma.

Canine cutaneous histiocytoma (CCH) is a common, benign neoplastic proliferation of histiocytes of Langerhans cell origin that often ulcerate, become secondarily infected and regress spontaneously. Bartonella is a fastidious genus of facultative intracellular pathogens that can be transmitted through arthropod bites and epidermal animal scratches and has been identified previously in the cytoplasm of histiocytes within granulomatous lesions and in skin biopsy samples of inflammatory pustules and papules. Based on the established inflammatory and oncogenic properties of Bartonella, we hypothesized that Bartonella spp. DNA could be amplified from CCH more often than from non-lesional skin and bacteria could be localized within skin tumours using indirect immunofluorescence (IIF). Paraffin wax-embedded surgical biopsy samples from dogs with CCH and non-neoplastic skin adjacent to osteosarcomas (control group selected due to wide surgical margins) were retrieved from the archive of the pathology service of North Carolina State University College of Veterinary Medicine. DNA was extracted and regions of the 16S-23S rRNA intergenic transcribed spacer (ITS) region and the pap31 and gltA genes were amplified by polymerase chain reaction (PCR) using Bartonella-specific primers. IIF was performed using a primary Bartonella henselae monoclonal antibody to localize B. henselae in tissues of PCR-positive dogs. Bartonella vinsonii subsp. berkhoffii was amplified from 1/17 (5.8%) control tissues and B. henselae was amplified from 4/29 (13.8%) CCH tissues. The prevalence of B. vinsonii subsp. berkhoffii (P = 0.37) or B. henselae (P = 0.28) did not vary statistically between study groups. B. henselae could be visualized in 2/4 (50.0%) CCH tissues using IIF. Based on this study, Bartonella spp. are unlikely to cause CCH.

Cytomorphometry of canine cutaneous histiocytoma.

A morphometric analysis of tumoral Langerhans cells and activated macrophages was conducted using canine cutaneous tumors (65 cases of canine cutaneous histiocytoma and 7 cases of pyogranuloma). The histiocytic origin of the tumor cells was confirmed using immunohistochemistry. The parameters of the morphometric analysis included cellular and nuclear size and shape and the nuclear:cytoplasmic ratio; the variability of these features was calculated separately for each tumor. The canine cutaneous histiocytoma group was divided into four stages of regression depending on the intensity of the lymphocytic infiltration. Statistical analysis revealed that the anisocytosis, anisokaryosis and cellular pleomorphism of tumoral Langerhans cells increased, while the cellular circularity and nuclear:cytoplasmic ratio decreased with tumor regression. Activated macrophages of the pyogranuloma were significantly larger, and had larger nuclei, than tumoral Langerhans cells. Furthermore, these activated macrophages showed greater anisocytosis and anisokaryosis and a lower nuclear:cytoplasmic ratio than tumoral Langerhans cells in the first stages of tumor regression. These results indicate that tumoral Langerhans cells undergo morphologic changes during the regression of canine cutaneous histiocytoma, reflecting their maturation and differentiation. Morphometry can be a useful method for distinguishing activated macrophages from tumoral Langerhans cells.

Multiple cutaneous histiocytomas treated with lomustine in a dog.

BACKGROUND: Histiocytoma is a common benign neoplasm of young dogs. Multiple histiocytomas are rare. Surgical or medical treatment of solitary tumours is not required in the majority of cases because the tumour usually undergoes spontaneous regression. Therapy is required when lesions are persistent, recurrent, ulcerated or in uncomfortable locations. HYPOTHESIS/OBJECTIVES: To describe a case of canine multiple cutaneous histiocytomas treated with lomustine. ANIMAL: A 5-year-old miniature Pinscher dog was presented with multiple, disseminated, alopecic cutaneous nodules, with no associated systemic signs on initial presentation. METHODS: Histopathological examination of skin biopsies and immunocytochemistry of biopsy imprints were performed. Inguinal lymph node, liver, spleen and bone marrow cytological examination and abdominal ultrasound examination were also performed. RESULTS: The clinical, histopathological and immunocytochemical findings supported a diagnosis of canine multiple cutaneous histiocytomas. Owing to the increasing number and size of the nodules, medical treatment was initiated. Prednisone and ciclosporin resulted in worsening of lesions. Lomustine orally once monthly led to complete resolution followed by relapse. Metabolic disorders such as increased serum alanine transaminase and alkaline phosphatase activities were recorded, and therapy was stopped. Increase in size of the tumours, severe dullness and anorexia led the owner to elect euthanasia. CONCLUSIONS AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first case report of canine multiple cutaneous histiocytomas treated with lomustine. Lomustine is effective in histiocytic diseases, but adverse effects must be considered because they can be severe and life threatening.

Canine cutaneous histiocytomas are clonal lesions as defined by X-linked clonality testing.

Canine cutaneous histiocytoma (CCH) is the most common skin tumour of young dogs. CCH is assumed to be a tumour, but as these lesions often undergo complete regression, they have also been proposed to represent hamartomas or unregulated hyperplasia of polyclonal histiocytic cells. To demonstrate a monoclonal origin of CCH, and thereby a probable neoplastic nature, a clonality assay was established based on the heterozygosity of a polymorphic microsatellite within the canine androgen receptor located on the X chromosome. Analysis of 11 histiocytomas identified a non-random X chromosome inactivation of one androgen receptor allele. CCH may, therefore, be a clonal lesion and of neoplastic nature.

Regression of canine cutaneous histiocytoma: reduced proliferation or increased apoptosis?

BACKGROUND/AIM: Canine cutaneous histiocytoma (CCH) is a tumour that undergoes spontaneous regression. The aim of this study was to establish a possible relationship between regression of CCH and tumoural cell proliferation and apoptosis. MATERIALS AND METHODS: Immunostaining with Ki-67 antigen and the terminal deoxytransferase (TdT) deoxyuridine-5'-triphosphated (dUTP) nick-end labelling (TUNEL) method were performed on 93 specimens of CCH, grouped into four histological groups. RESULTS: The proliferative index evaluated with Ki-67 antigen expression was on average 23.56 ± 7.91%. The apoptotic index determined by the TUNEL method was on average 39.37 ± 5.87%. Neither the proliferative nor apoptotic index differed between histological groups. Moreover, the proliferative and apoptotic indices did not correlate significantly. However, apoptotic activity was higher than proliferative activity in almost all tumours. CONCLUSION: A reduction of proliferation or an increase of apoptosis does not appear to justify regression of CCH. However, our results suggest that an imbalance between cell proliferation and apoptotic cell death plays a significant role in spontaneous regression of CCH.

Immunohistochemical and immunoelectron study of major histocompatibility complex class-II antigen in canine cutaneous histiocytoma: its relation to tumor regression.

In order to investigate the immune mechanisms involved in regression of canine cutaneous histicytoma (CCH), major histocompatibility complex (MHC) class-II immuno-expression and the number of T- and B-lymphocytes and macrophages were analyzed in 93 cases of CCH. MHC class-II was also studied in 16 cases of CCH by immunoelectron microscopy. All tumors expressed MHC class-II, and two major staining patterns were identified: focal juxtanuclear cytoplasmic staining and rim-like staining along the cell periphery. The MHC class-II labelling pattern and T- and B-lymphocyte infiltrates were associated with tumor regression. In regressing lesions, MHC class-II molecules shift to the cell surface and an increase of both T- and B-lymphocytes were noted. The increasing expression of MHC class-II molecules on the cell surface could be a significant factor for the onset and progression of tumour regression.

Histomorphological and immunohistochemical characterization of 172 cutaneous round cell tumours in dogs / Caracterização histomorfológica e imuno-histoquímica de 172 neoplasias cutâneas de células redondas em cães

This paper describes the use of a panel of antibodies (CD117, CD3, CD79a, CD45, cytokeratin, vimentin and E-cadherin) on formalin-fixed, paraffin-embedded sections of canine cutaneous round cell tumours. Neoplastic tumours were diagnosed by histology and histochemical stains and included 107 mast cell tumours, 31 cutaneous histiocytomas, two localized histiocytic sarcomas, 21 cutaneous lymphomas, three plasma cell tumours, one transmissible venereal tumour and seven unclassified round cell tumours. The histologic diagnosis was modified in 39.5% of the total 172 neoplasms. The staining for CD45 and Ecadherin were variable, and therefore, the final diagnoses of cutaneous histiocytoma and localized histiocytic sarcoma were made based on histology in association with negative results for CD3, CD79a, CD117 and cytokeratin. The cellular origin of unclassified round cell tumours was defined in all cases. Cutaneous B-cell lymphoma and plasma cell tumours were CD79a-positive and could be distinguished from each other by the morphological characteristics. Mast cell tumours and T cell lymphoma were CD117 and CD3 positive, respectively. The positive staining for vimentin and the negative staining for CD3, CD79a, CD117 and cytokeratin favoured the diagnosis of transmissible venereal tumours. Thus, the final diagnosis of cutaneous round cell tumours should be based on the interpretation of immunohistochemical results together with the cellular morphology observed by histology. Therefore, more studies to optimize the specific markers in formalin-fixed, paraffinembedded tissues (especially for histiocytes) are required for definitive diagnosis of round cell tumours in dogs.

Este trabalho descreve o uso de um painel de anticorpos (CD117, CD3, CD79a, CD45, citoqueratina, vimentina e e-caderina em tecidos formalizados e parafinizados para o diagnóstico de neoplasias de células redondas em cães. Os tumores foram diagnosticados usando-se a histopatologia e a marcação imuno-histoquímica. Foram incluídos 107 mastocitomas, 31 histiocitomas cutâneos, 2 sarcomas histiocíticos localizados, 21 linfomas cutâneos, 3 plasmocitomas, 1 tumor venéreo transmissível e 7 tumores de células redondas não classificados. O diagnóstico histológico foi modificado em 39,5% do total de 172 neoplasias. A marcação do anticorpo CD45 e E-caderina foi variável e, nesse sentido, o diagnóstico final de histiocitoma cutâneo e sarcoma histiocítico localizado foi baseado na histologia em associação com os resultados negativos para CD3, CD79a, CD117 e citoqueratina. A origem celular dos tumores de células redondas não classificados foi definida em todos os casos. Linfoma cutâneo de célula B e plasmocitoma foram positivos para CD79a e foram distinguidos entre si pelas características morfológicas. Marcação positiva para vimentina e negativa para CD3, CD79a, CD117 e citoqueratina favoreceram o diagnóstico dos tumores venereos transmissíveis. Assim, o diagnóstico final dos tumores de células redondas foram baseados na interpretação dos resultados da imuno-histoquímica em conjunto com a avaliação das características morfológicas observadas na histologia. Finalmente, mais estudos em relação à padronização de marcadores específicos para tecidos parafinizados (especialmente para histiócitos) são necessários para o diagnóstico definitivo das neoplasias de células redondas em cães.

Immunohistochemical expression of E-cadherin does not distinguish canine cutaneous histiocytoma from other canine round cell tumors.

Immunohistochemistry for E-cadherin (ECAD) has been used to distinguish canine cutaneous histiocytoma from other leukocytic neoplasms ("round cell tumors"). To determine the specificity of this test, 5 types of canine cutaneous round cell tumors were evaluated for immunohistochemical expression of ECAD. Tumors of all 5 types had variable cytoplasmic, plasma membrane, and/or paranuclear ECAD expression: All 13 cutaneous histiocytomas were ECAD+; all but 1 of 14 mast cell tumors expressed ECAD; 10 of 12 epitheliotropic lymphomas reacted with E-cadherin antibody; of 72 plasmacytomas, 54 were ECAD+; and 5 of 5 histiocytic sarcomas were positive. Conclusions based on these results include the following: First, immunoreactivity for ECAD is not limited to leukocytes of cutaneous histiocytoma; second, antibody to ECAD also labels neoplastic cells in most mast cell tumors, plasmacytomas, cutaneous histiocytic sarcomas, and epitheliotropic lymphomas; third, although most histiocytomas have membranous ECAD expression, the immunoreactivity varies among round cell tumors and is frequently concurrent in different cellular compartments; fourth, the distinctively paranuclear ECAD expression pattern in epitheliotropic lymphomas might distinguish them from other round cell tumors; and, fifth, ECAD should be used with other markers (eg, MUM1 for plasmacytomas, KIT for mast cell tumors, CD3 and CD79a for lymphomas) to distinguish among canine round cell tumors.

Decrease of E-cadherin expression in canine cutaneous histiocytoma appears to be related to its spontaneous regression.

BACKGROUND: Canine cutaneous histiocytoma (CCH) is an epidermotropic tumour of Langerhans cells, most frequent in young dogs, which undergoes spontaneous regression. MATERIALS AND METHODS: E-cadherin immunoexpression was analysed in ninety-three CCH, categorized according to Cockerell and Slauson (1976) criteria into four histological groups, representing different stages of tumour regression. RESULTS: All tumours expressed membranous E-cadherin both in keratinocytes, and in tumoural cells and no differences were noted among the histological groups analysed. However, the intensity of the E-cadherin immunolabeling decreased in the presence of lymphoid infiltration and varied significantly among the groups considered (p<0.0001). CONCLUSION: This study strongly suggests a down-regulation of E-cadherin expression in CCH pathogenesis and progression. The loss of E-cadherin expression might represent an activation/ maturation process of the tumoural cells constituting a switch for CCH regression.

E-cadherin expression in canine cutaneous histiocytomas.

Canine cutaneous histiocytoma is a common skin tumour of Langerhans cell origin. Langerhans cells are members of the dendritic cell family of antigen-presenting cells and are located in the epidermis. They are unique among the dendritic cell lineage in that they express high levels of the adhesion molecule E-cadherin. The expression of E-cadherin by the neoplastic Langerhans cells in 37 dogs with cutaneous histiocytoma was studied by flow cytometry and immunohistochemistry. In all the cases, these cells expressed E-cadherin, whereas the infiltrating lymphocytes did not.

CCH cells are potent stimulators in the allogeneic mixed leucocyte reaction.

Canine cutaneous histiocytoma (CCH) has been identified as a tumour of epidermal Langerhans cells (LCs) on the basis of immunophenotypic studies. Neoplastic Langerhans cells (CCH-LCs) were isolated from lesions of canine cutaneous histiocytoma. The CCH-LC cells expressed CD1b, CD11/18, CD45, MHC-I, and MHC-II. The CCH-LC cells were potent stimulators of the mixed leucocyte reaction (MLR) in vitro when compared to PBMCs from the tumour-bearing animals. This provides evidence that the neoplastic cells in CCH have functional as well as immunophenotypic characteristics of Langerhans cells.

The regression of a canine Langerhans cell tumour is associated with increased expression of IL-2, TNF-alpha, IFN-gamma and iNOS mRNA.

Canine cutaneous histiocytoma is a benign epidermal neoplasm of Langerhans cell origin, which usually displays spontaneous regression. Based on the degree of lymphocytic infiltration, 30 histiocytomas were classified into four groups representing different stages of tumour regression. To elucidate further the mechanisms of the antitumour immune response CD3+, CD21+, CD4+, CD8+ and myeloid/histiocyte antigen+ inflammatory cells were differentiated by immunohistochemistry and quantified. In addition, the number of apoptotic cells was detected using the TdT-mediated biotin-dUTP nick-end labelling (TUNEL) method. Furthermore, the expression of interleukin- (IL-2), IL-12(p40), tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-10 and transforming growth factor-beta (TGF-beta) as well as inducible nitric oxid synthase (iNOS) mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR). Phenotyping of inflammatory cells revealed a significantly increased infiltration of all lymphocyte subsets and myeloid/histiocytic cells with the onset of tumour regression. The latter was significantly correlated to up-regulation of IL-2, TNF-alpha, IFN-gamma and iNOS mRNA expression. Expression of remaining cytokines and percentage of apoptotic cells showed no group-specific changes. The results indicate an initial infiltration of CD4+ T cells followed by increased expression of Th1 cytokines and recruitment of antitumour effector cells as the principal mechanism for tumour regression. Canine cutaneous histiocytoma is a unique example for an effective immune response in a naturally occurring neoplasm derived from epidermal Langerhans cells and might represent a valuable animal model to investigate tumour immunity.

Immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors.

Immunohistochemical and histochemical stains are useful adjunct techniques in the diagnosis of canine cutaneous round cell tumors, which can appear histologically similar. We applied a panel of monoclonal antibodies (recognizing tryptase, chymase, serotonin for mast cells; CD1a, CD18, MHC class II for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells) and one histochemical stain (naphthol AS-D chloroacetate for chymase activity) to formalin-fixed, paraffin-embedded sections of canine cutaneous mast cell tumors, histiocytomas, lymphosarcomas, plasmacytomas, and unidentified round cell tumors. Of 21 tumors with a histologic diagnosis of mast cell tumor, 7/7 (100%) grade I, 6/7 (85.7%) grade II, and 3/7 (42.9%) grade III tumors were diagnosed as mast cell tumors based on positive staining for tryptase antigen and chymase activity. Mast cells were positive for both tryptase antigen and chymase activity, indicating equal efficacy of tryptase immunohistochemistry and chymase histochemistry. Chymase was detected immunohistochemically in both tumor and nontumor cells, while serotonin was not detected in most mast cell tumors, and thus, neither was useful in the diagnosis of mast cell tumors. Immunohistochemistry to detect CD18 and MHC class II was equally effective in staining histiocytomas, although lymphosarcoma must be ruled out through the use of CD3 and CD79a immunohistochemistry. Immunohistochemistry using three different monoclonal antibodies to human CD1a showed no cross-reactivity in canine histiocytomas and was not useful. A final diagnosis was obtained for 4/5 (80%) of the unidentified tumors, indicating the usefulness of multiple stains in poorly differentiated round cell tumors.