mRNA sequencing analysis and growth inhibitory effects of palbociclib on cell lines from canine histiocytic proliferative disorders.

Canine histiocytic proliferative disorders include aggressive and fatal diseases, such as histiocytic sarcoma (HS) and histiocytosis (SyH). The molecular mechanisms underlying cell proliferation need to be elucidated for the development of effective treatments. In the present study, mRNA expression levels were comprehensively analysed in cell lines derived from localized HS, disseminated HS, SyH and Langerhans cell histiocytosis (LCH) in dogs. Based on the results obtained, the growth inhibitory effects of palbociclib, a CDK4/6 inhibitor, were verified with the cell lines in vitro and in xenograft mouse model. Hierarchical clustering and principal component analysis plots of mRNA expression profiles divided the cell lines into three groups: a localized HS group, disseminated HS/SyH group, and LCH. The results of an ingenuity pathway analysis suggested that the MAPK signalling pathway was activated in the localized HS and LCH cell lines, and the PI3K signalling pathway in the disseminated and localized HS cell lines. In all cell lines, the expression of the tumour suppressor genes TP53, CDKN2A and CDKN1A was down-regulated, whereas that of Rb was preserved. In vitro assessments revealed the growth inhibitory effects of palbociclib in all cell lines examined. In a xenograft mouse model using a cell line from disseminated HS, palbociclib exerted significant growth inhibitory effects. These results suggest the potential of palbociclib as a therapeutic drug candidate for the treatment of malignant histiocytic proliferative disorders of the dog.

Giant Cell Sarcomas in Domestic Rabbits (Oryctolagus cuniculus).

Multinucleated giant cells (MGCs) are a prominent histological feature of various mesenchymal neoplasms and are often considered a criterion of malignancy. Mesenchymal neoplasms with MGCs for which the cell lineage is unclear generally are referred to as giant cell sarcomas. Here we characterize the gross, histologic, and immunohistochemical features of 90 giant cell sarcomas in domestic pet rabbits. Based on the anatomic location and histologic and immunohistochemical findings, 18 cases were classified as histiocytic sarcomas (HS) and 72 cases as anaplastic sarcomas (AS). At postmortem examination, HS was either localized HS (n = 7) always affecting the lungs, or disseminated HS (n = 10) that affected the lungs (n = 10), liver (n = 6), kidneys (n = 4), pleura (n = 2), mediastinum (n = 2), heart (n = 4), skeletal muscle (n = 1), adipose tissue (n = 1), and lymph node (n = 1). Additionally, one cecal biopsy was consistent with HS. Microscopically, HS were characterized by sheets of neoplastic polygonal to round cells that contained single to several, often greatly enlarged nuclei as well as abundant cytoplasm. HS were always positive for CD204 and always negative for SMA and desmin. In contrast, AS arose most commonly from the skin or subcutis (n = 62) and rarely the skeletal muscle (n = 8) or abdominal organs (n = 2). In 29% of extra-abdominal AS, the tumor deeply invaded into surrounding connective tissue, skeletal muscle, tendons, and bone causing pathological fractures. Five of 9 postmortem cases metastasized to various organs often including the lungs. Microscopically, AS were characterized by sheets of spindle or pleomorphic cells admixed with variable numbers of MGCs. Immunohistochemically, AS were always negative for CD204 and often (71%) positive for SMA and/or desmin.

Genome-wide DNA copy number analysis and targeted transcriptional analysis of canine histiocytic malignancies identifies diagnostic signatures and highlights disruption of spindle assembly complex.

Canine histiocytic malignancies (HM) are rare across the general dog population, but overrepresented in certain breeds, such as Bernese mountain dog and flat-coated retriever. Accurate diagnosis relies on immunohistochemical staining to rule out histologically similar cancers with different prognoses and treatment strategies (e.g., lymphoma and hemangiosarcoma). HM are generally treatment refractory with overall survival of less than 6 months. A lack of understanding regarding the mechanisms of disease development and progression hinders development of novel therapeutics. While the study of human tumors can benefit veterinary medicine, the rarity of the suggested orthologous disease (dendritic cell sarcoma) precludes this. This study aims to improve the understanding of underlying disease mechanisms using genome-wide DNA copy number and gene expression analysis of spontaneous HM across several dog breeds. Extensive DNA copy number disruption was evident, with losses of segments of chromosomes 16 and 31 detected in 93% and 72% of tumors, respectively. Droplet digital PCR (ddPCR) evaluation of these regions in numerous cancer specimens effectively discriminated HM from other common round cell tumors, including lymphoma and hemangiosarcoma, resulting in a novel, rapid diagnostic aid for veterinary medicine. Transcriptional analysis demonstrated disruption of the spindle assembly complex, which is linked to genomic instability and reduced therapeutic impact in humans. A key signature detected was up-regulation of Matrix Metalloproteinase 9 (MMP9), supported by an immunohistochemistry-based assessment of MMP9 protein levels. Since MMP9 has been linked with rapid metastasis and tumor aggression in humans, the data in this study offer a possible mechanism of aggression in HM.

Three-dimensional printing of orbital and peri-orbital masses in three dogs and its potential applications in veterinary ophthalmology.

OBJECTIVE: To describe the technique and utility of three-dimensional (3D) printing for orbital and peri-orbital masses and discuss other potential applications for 3D printing. ANIMALS STUDIED: Three dogs with a chronic history of nonpainful exophthalmos. PROCEDURES: Computed tomography (CT) and subsequent 3D printing of the head was performed on each case. CT confirmed a confined mass, and an ultrasound-guided biopsy was obtained in each circumstance. An orbitotomy was tentatively planned for each case, and a 3D print of each head with the associated globe and mass was created to assist in surgical planning. RESULTS: In case 1, the mass was located in the cranioventral aspect of the right orbit, and the histopathologic diagnosis was adenoma. In case 2, the mass was located within the lateral masseter muscle, ventral to the right orbit between the zygomatic arch and the ramus of the mandible. The histopathologic diagnosis in case 2 was consistent with a lipoma. In case 3, the mass was located in the ventral orbit, and the histopathologic diagnosis was histiocytic cellular infiltrate. CONCLUSIONS: Three-dimensional printing in cases with orbital and peri-orbital masses has exceptional potential for improved surgical planning and provides another modality for visualization to help veterinarians, students, and owners understand distribution of disease. Additionally, as the techniques of 3D printing continue to evolve, the potential exists to revolutionize ocular surgery and drug delivery.

Clinical outcome, PDGFRß and KIT expression in feline histiocytic disorders: a multicentre study.

Information about histiocytic disease in cats is limited. The aim of this study was to document clinical findings and outcome in feline histiocytic disorders, and characterize the expression of PDGFRß and KIT in order to identify potential treatment targets. Morphologically diagnosed feline histiocytic tumours were reviewed and characterized by immunohistochemistry (IHC). Five cases of feline progressive histiocytosis (FPH), eight histiocytic sarcomas (HS) and two haemophagocytic histiocytic sarcomas (HaeHS) were confirmed. PDGFRß was variably positive in most histiocytic cases, while KIT was negative in all. Clinical presentation, treatment and outcome were also evaluated. Partial responses were recorded in measurable disease with tyrosine kinase inhibitors and lomustine, and radiotherapy achieved long-term control in some cases. Survival times were shortest in HaeHS and disseminated disease. PDGFRß, but not KIT, may represent a therapeutic target in feline histiocytic disorders but more studies are needed to investigate other potential treatment targets.

Cytologic and immunocytochemical characterization of feline progressive histiocytosis.

BACKGROUND: Feline Progressive Histiocytosis (FPH) is a cutaneous dendritic cell neoplasm characterized by slow progression and spread to internal organs in the terminal stage. FPH is often misdiagnosed as an inflammatory reaction and has not been fully characterized from a cytologic diagnostic perspective. OBJECTIVES: The purpose of the study was to characterize the cytologic and immunocytochemical aspects useful for FPH diagnosis. METHODS: Fine-needle aspiration cytologic samples of 5 cases of FPH confirmed by skin biopsy and necropsy were evaluated. Immunocytochemistry with antibodies recognizing CD1a, CD1c, CD3, CD11b, CD18, CD21, and MHCII was performed on air-dried, acetone-fixed smears. E-cadherin expression was assessed on paraffin-embedded skin biopsies. Transmission electron microscopy (TEM) was performed in one case. RESULTS: Main cytologic findings on variably cellular samples were characterized by single to cohesive large, round to polygonal cells with intermediate to low N/C ratio, abundant clear homogeneous cytoplasm, and round to oval nuclei with rare bi- to multinucleated atypical cells, associated with low numbers of small lymphocytes and/or neutrophils. Neoplastic cells expressed CD1a, CD1c, CD11b, CD18, and MHCII. Anti-CD3 antibodies identified reactive T cells admixed with the neoplastic cells. E-cadherin expression was observed in all but one case. TEM failed to identify Birbeck granules in one case. CONCLUSIONS: FPH is a distinctive neoplastic lesion composed of nonphagocytizing histiocytes variably admixed with neutrophils and small mature lymphocytes. Immunocytochemical analysis with CD1 is mandatory to confirm a dendritic cell origin. Immunocytochemistry and cytomorphology allowed the specific and rapid diagnosis of FPH on cytologic samples.

[Reactive and neoplastic histiocytic diseases in the dog]. / Reaktive und neoplastische histiozytäre erkrankungen beim hund.

There are different histiocytic diseases in dogs that are characterized by the proliferation of histiocytic cells (macrophages and myeloid dendritic cells). Histiocytic diseases can be devided into neoplastic (cutaneous histiocytoma complex, histiocytic sarcoma, dendritic cell leukaemia) and reactive forms (reactive histiocytosis, haemophagocytic syndrome). All subtypes of the cutaneous histiocytoma complex (cutaneous histiocytoma, metastatic histiocytoma and Langerhans' cell histiocytosis) are of Langerhans' cell origin. Histiocytoma, which is a solitary tumour of the skin in young dogs, shows spontaneous regression in most cases. Occasionally, metastasis to lymph nodes can be seen (metastatic histiocytoma). Only one dog with Langerhans' cell histiocytosis has been described and was euthanized. Histiocytic sarcoma, which arises from myeloid dendritic cells, can be classified as localised histiocytic sarcoma or disseminated histiocytic sarcoma. Another form of histiocytic sarcoma - haemophagocytic histiocytic sarcoma - is derived from macrophages. Histiocytic sarcoma displays a very aggressive clinical course and has a poor prognosis. Breed predispositions have been reported for the disseminated and haemophagocytic form of histiocytic sarcoma in Bernese mountain dogs, Rottweilers and varoiusretrievers. In contrast, reactive histiocytosis (cutaneous and systemic forms) develops by reactive proliferation of interstitial dendritic cells. In systemic histiocytosis, breed predilections are similar to histiocytic sarcoma. Haemophagocytic syndrome develops as a consequence of proliferation of activated macrophages in different tissues. Prognosis in general is moderate to poor and depends on the origin of the underlying disease process.

Mortality in a cohort of flat-coated retrievers in the UK.

A cohort study of 174 flat-coated retrievers was undertaken to establish the importance of cancer in flat coat mortality in terms of the prevalence of neoplasia in the breed and also the relative effect of cancer on lifespan in relation to other forms of mortality. Dogs aged 2-7 years were recruited in 1996 and followed until 2007. An annual health census was used to collect the data. Two dogs were lost to follow-up and 72 dogs (42%) died from confirmed neoplasia. Twenty dogs (11.6%) died of unconfirmed tumours and 61 (35%) died from non-neoplastic conditions. The cause of death was unknown for 19 dogs. Soft tissue sarcoma (especially histiocytic sarcoma) was the predominant cancer type, affecting 32 dogs (44% of neoplasms). Six dogs died with malignant melanoma and three with lymphoma. Median age at death was 9 years for dogs with tumours (eight for sarcoma patients) and 12 years for non-neoplastic fatalities. The results confirm that soft tissue sarcoma, particularly histiocytic sarcoma, is a major cause of mortality in this breed.

Canine ocular histiocytic sarcoma.

OBJECTIVE: To describe and characterize histiocytic sarcoma (HS) first detected in the eyes of dogs using the large database at the comparative ocular pathology laboratory of Wisconsin (COPLOW). METHODS: Cases diagnosed as HS were selected from the COPLOW database. Slides were reviewed to describe the cellular morphology, localize the tumor within the globe, record the tumor distribution and measure the size of the tumor. Further sections were taken to perform immunohistochemistry for Melan-A, CD18 and S-100, and for ferric iron staining. The following clinical information was recorded: breed, age, gender, laterality, clinical signs upon presentation and follow-up information obtained by response to a mailed survey and phone contact. RESULTS: Twenty-six cases were confirmed as being HS according to the immunohistochemical results (CD18 positive and Melan-A negative). The most prevalent breed was Rottweiler (eight cases), followed by Retriever breeds (seven Golden Retrievers and five Labrador Retrievers). The mean age was 8.61 +/- 2.43 years. There were three intact male, eight castrated male, one intact female and 14 spayed female dogs. In 15 dogs there were no concurrent systemic clinical signs at the time of diagnosis. Sixteen of 19 dogs with follow-up information available died as a result of causes related to the tumor, although only three of them received a necropsy. Survival time varied between 5 days and 6 months after enucleation. Three of the dogs were alive at the time the information was gathered. Mean tumor surface was 0.613 +/- 0.38 cm(2). S-100 was diffusely positive in 10 cases, isolated positive cells were found in 11 cases and five cases were completely negative. Seven of the cases were positive for ferric iron. CONCLUSIONS: Histiocytic sarcoma must be considered in the differential diagnosis of dogs with intraocular masses, especially in Rottweilers and Retriever breeds. Because it carries poor prognosis, it must be distinguished from melanoma. A good discriminator for this purpose in paraffin-embedded tissues is finding CD18-positive cells and no reactivity against Melan-A. S-100 and ferric iron staining does not seem to be useful. Ocular HS is considered to be a manifestation of a systemic disease even when the disease is first recognized in the eye.

Canine hemophagocytic histiocytic sarcoma: a proliferative disorder of CD11d+ macrophages.

Histiocytic disorders of dogs include histiocytoma, localized histiocytic sarcoma (HS), disseminated HS (malignant histocytosis), and the reactive histiocytoses: cutaneous and systemic. A common element to these diseases is proliferation of dendritic cells (DC) of either Langerhans cell (epithelial DC) or interstitial DC lineage. In this report, 17 dogs with hemophagocytic HS are described. Breeds affected included Bernese Mountain Dog (6), Golden Retriever (4), Rottweiler (3), Labrador Retriever (2), a mixed-breed dog, and a Schnauzer, which were from 2.5 to 13 years old. The dogs presented with Coombs negative responsive anemia in 16/17 dogs (94%), thrombocytopenia in 15/17 dogs (88%), hypoalbuminemia in 16/17 dogs (94%), and hypocholesterolemia in 11/16 dogs (69%). All dogs died or were euthanized. The clinical course ranged from 2 to 32 weeks (mean 7.1 weeks). Diffuse splenomegaly with ill-defined masses was consistently present. Microscopic lesions were prevalent in spleen, liver, lung, and bone marrow. Metastasis occurred by insidious intravascular invasion with minimal mass formation. Histiocytes were markedly erythrophagocytic and accompanied by foci of extramedullary hemopoiesis. Cytologically, the histiocytes varied from well differentiated to atypical, with atypia more prevalent in spleen than bone marrow. These tumors arose from splenic red pulp and bone marrow macrophages, which expressed major histocompatibility complex class II and the beta2 integrin, CD11d. They had low and/or inconsistent expression of CD1 and CD11c, which are dominantly expressed by canine nonhemophagocytic HS of DC origin. Canine histiocytic proliferative diseases now encompass proliferation of all members of the myeloid histiocytic lineage: Langerhans cells, interstitial DC, and macrophages.

Feline progressive histiocytosis.

Histiocytic proliferative diseases include reactive and neoplastic proliferations of dendritic cells (DC) or macrophages. Various forms of DC proliferations have been documented in humans and dogs; their etiology is largely unknown. With the exception of a few case reports, histiocytic proliferations have not been characterized in cats. This study summarizes clinical, morphologic, and immunophenotypic features of a feline progressive histiocytosis (FPH) in 30 cats. There was no breed or age predilection. Females were more often affected than males. Solitary or multiple nonpruritic firm papules, nodules, and plaques had a predilection for feet, legs, and face. Lesions consisted of poorly circumscribed epitheliotropic (13/30) and nonepitheliotropic (17/30) histiocytic infiltrates of the superficial and deep dermis, with variable extension into the subcutis. The histiocytic population was relatively monomorphous early in the clinical course. With disease progression, cellular pleomorphism was more frequently encountered. Histiocytes expressed CD1a, CD1c, CD18, and major histocompatibility complex class II molecules. This immunophenotype suggests a DC origin of these lesions. Coexpression of E-cadherin, a feature of cutaneous Langerhans cells, was only observed in 3 cats. FPH followed a progressive clinical course; the lesions, however, were limited to the skin for an extended period of time. Terminal involvement of internal organs was documented in 7 cases. Treatment with chemotherapeutics or immunosuppressive and immunomodulatory drugs was not successful. The etiology of FPH remains unknown. FPH is best considered an initially indolent cutaneous neoplasm, which is mostly slowly progressive and may spread beyond the skin in the terminal stage.

Evaluation of dysregulation of the receptor tyrosine kinases Kit, Flt3, and Met in histiocytic sarcomas of dogs.

OBJECTIVE: To evaluate canine histiocytic sarcoma cell lines and tumor samples for dysregulation of the Kit/stem-cell factor (SCF), Flt3/Flt3 ligand (Flt3L), and Met/hepatocyte growth factor (HGF) receptor tyrosine kinase signaling pathways, as these are known to contribute to the differentiation and survival of normal dendritic cells as well as malignant transformation of dendritic cells in mouse models. SAMPLE POPULATION: 4 histiocytic sarcoma tumor cell lines and 35 formalin-fixed histiocytic sarcoma specimens obtained from dogs. PROCEDURE: Histiocytic sarcoma cell lines were evaluated for expression of Kit/SCF, Flt3/Flt3L, and Met/HGF by use of reverse transcriptase-PCR procedures. Histiocytic sarcoma cell lines and tumor samples were evaluated for mutations in Kit, Flt3, and Met by use of PCR analysis of genomic DNA, followed by both sequencing and fluorescent PAGE for deletions or internal tandem duplications. The ability of the multi-targeted split-kinase inhibitor SU11654 to block proliferation and induce apoptosis of histiocytic sarcoma cell lines was also evaluated. RESULTS: No mutations in Kit, Flt3, and Met were identified in any of the cell lines or tumor samples evaluated. Furthermore, SU11654 did not induce cell-cycle arrest or apoptosis of histiocytic sarcoma lines, even at supratherapeutic doses. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that dysregulation of Kit/SCF, Flt3/Flt3L, and Met/HGF signaling pathways is unlikely to occur in histiocytic sarcomas of dogs and that inhibitors of the Kit, Flt3, and Met pathways are unlikely to provide clinical benefit to dogs with histiocytic sarcomas.

Establishment of a new canine cell line (CCT) originated from a cutaneous malignant histiocytosis.

A new canine cell line, named CCT, was established from the cutaneous malignant histiocytosis in a 4-year-old male Borzoi. CCT proliferated with loose adherence and doubling time was approximately 30 hr. When co-cultured with latex beads, CCT phagocytized beads vigorously. Lysozyme and vimentin were positive by immunostaining, and non-specific esterase and acid phosphatase were positive by cytochemical staining. These features indicated the cells had a histiocytic nature. Furthermore, by subcutaneous injection to nude mice CCT could successfully form tumors with the morphological and immunohistochemical features similar to the original tumor.

Flow cytometric evaluation of hemophagocytic disorders in canine.

BACKGROUND: Hemophagocytic macrophages in canine bone marrow are observed in malignant histiocytosis as well as benign hemophagocytic histiocytosis. Cytomorphologic evaluation alone may be inadequate to consistently differentiate between benign and malignant forms of hemophagocytic disorders. OBJECTIVE: The purpose of this study was to evaluate the ability of flow cytometry and immunophenotyping to differentiate between benign and malignant types of hemophagocytic disorders in dogs. METHODS: Blood smears and bone marrow differential cell counts were evaluated for 10 dogs with hemophagocytic disorders. Bone marrow samples were labeled with monoclonal antibodies to CD18, MCH class-II, Thy-1, CD14, CD3, and CD21. Using flow cytometry, forward-angle versus side-angle light scatter plots were analyzed and immunophenotypes were determined. RESULTS: Scatter plots from 3 dogs with a necropsy diagnosis of malignant histiocytosis revealed 2 atypical cell clusters. One cluster contained cells of similar size or larger than immature myeloid cells and metamyelocytes. Cells in the other cluster were highly granular, with granularity similar to or greater than that of metamyelocytes. In bone marrow from dogs with malignant histiocytosis that was labeled with anti-CD14 antibody, macrophages represented 29-48% of nucleated cells. Seven dogs had a clinical or histopathologic diagnosis of benign hemophagocytic syndrome. Three of the dogs had normal cell distribution in scatter plots. Two dogs had 2 abnormal cell clusters: 1 within the immature myeloid and metamyelocyte gates and the other with granularity similar to or greater than that of metamyelocytes. The remaining 2 dogs had an atypical cell population, mostly within the immature myeloid gate. For dogs with benign hemophagocytic syndromes, 6-17% of cells in the bone marrow were CD14 positive. CONCLUSIONS: The cellular distribution in scatter plots and the total number of macrophages in bone marrow may be useful in differentiating malignant histiocytosis from benign hemophagocytic syndromes in dogs.