CT features of subcutaneous, intermuscular, and intramuscular mast cell tumors in dogs.

Surgical removal is the treatment of choice for subcutaneous (SC), intermuscular (InterM), and intramuscular (IntraM) mast cell tumors (MCTs). Advanced imaging (CT or MRI) is frequently used for presurgical planning, but InterM and IntraM MCTs can be difficult to identify and delineate on CT. Aims of the current retrospective, diagnostic accuracy, observer agreement study were to describe the imaging features of SC, InterM, and IntraM MCTs on CT and to assess the limitation of CT to identify the full local extent of the MCT. Inclusion criteria for the study were dogs with a cytologically or histologically diagnosed MCTs determined to be SC, InterM, or IntraM MCT based on histology and/or a CT scan performed in the gross disease setting. Two board-certified veterinary radiologists reviewed the CT images and recorded location, contrast enhancement pattern, and delineation between the normal and abnormal tissue. Sensitivity and specificity of CT for determining location (SC/InterM versus IntraM) was 85.71% and 55.56%, respectively, when compared to consensus location based on surgical pathology report/CT/MRI review. There was a low inter-rater agreement for delineation (kappa: 0.150 (-0.070 to 0.370) and measurement had a low/moderate correlation (rho: 0.4667 to 0.5792). Upon review by a surgical oncologist, CT findings were deemed insufficient for curative surgical planning in 13 of 16 due to inadequate definition of tumor depth, compartment boundary (fascial plane) or MCT margins. The use of CT for presurgical planning of SC/InterM/IntraM MCT dogs has limitations, especially when differentiating MCT from the adjacent muscle.

Multiplex genome editing of mammalian cells for producing recombinant heparin.

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.

Carboxypeptidase A3 expression in canine mast cell tumors and tissue-resident mast cells.

Mast cell tumors (MCTs) are one of the most common cutaneous malignancies in dogs. Previous studies have reported expression of mast cell-specific proteases chymase and tryptase in canine cutaneous MCTs and in connective tissue and mucosal mast cells. In humans and rodents, mast cells express an additional specific protease, carboxypeptidase A3 (CPA3). In this article, we describe CPA3 immunoreactivity in connective tissue, visceral, mucosal, and neoplastic mast cells in dogs. Positive immunolabeling for CPA3 was observed in nonneoplastic mast cells in 20/20 formalin-fixed paraffin-embedded normal tissues (skin, liver, spleen, intestine), and in 63/63 MCTs irrespective of their histological grade. CPA3 protein expression was comparable to that of c-kit in both the nonneoplastic and neoplastic mast cells. Three distinct labeling patterns (membranous, diffuse, and focal cytoplasmic) were observed for CPA3 in MCTs. The focal cytoplasmic labeling pattern was associated with high-grade MCTs staged with the Kiupel 2-tier grading criteria. We propose CPA3 as a novel immunohistochemical marker for canine mast cells in health and disease.

[A CHILD CASE OF DIFFUSE CUTANEOUS MASTOCYTOSIS].

Cutaneous mastocytosis (CM) usually appears in childhood and improves substantially before adolescence. The c-KIT mutation of D816V is present in 36% and 20% of patients with childhood-onset CM and diffuse cutaneous mastocytosis (DCM), respectively. In some cases of childhood-onset DCM, the disease can progress to systemic mastocytosis; in others, it resolves spontaneously. Thus, assessing the prognosis is difficult. Herein, we described a case of DCM in an 11-month-old, male patient without a c-KIT mutation. The patient presented with dark brown macules and sporadic erythema topped by bullous lesions. A skin biopsy of the macule on the abdomen revealed accumulation of mast cells which were round to oval-shaped with amphophilic cytoplasm within the upper dermis. The patient had received H1 inhibitor until age 3 years and continued to experience blisters on the trunk. However, no severe symptoms, such as anaphylaxis, occurred. Included in this manuscript is a review of previous reports of childhood-onset DCM in Japan and cases specifically seen at our dermatology clinic.

Case report of a clinically indolent but morphologically high-grade cutaneous mast cell tumor in an adult: Atypical cutaneous mastocytoma or mast cell sarcoma?

We present a case of an adult male with a solitary mast cell tumor of the skin with unusual nuclear pleomorphism and mitotic activity. The tumor was excised, recurred within 2 years, was reexcised after 4 years and did not recur >6 years after diagnosis. The tumor showed progressive cytonuclear atypia and a high mitotic and proliferation rate by Ki67-staining from the onset. No KIT mutations were identified in the tumor and bone marrow. Serum tryptase levels and a bone marrow aspirate and trephine biopsy were normal. Although the histomorphology of the skin tumor was consistent with mast cell sarcoma, the clinical behavior without systemic progression argued against this diagnosis. The tumor was finally considered as atypical mastocytoma, borderline to mast cell sarcoma. Currently, the patient is in close follow-up and still in complete remission.

Pleomorphic mastocytoma associated with loss of chromosome 5, PDGFRA, and HRAS mutations: A case of cutaneous mastocytosis with severe atypia and indolent behavior.

A 21-year-old female presented with a 5-year history of an erythematous papule on her right breast. The biopsy showed a dense, dermal nodular infiltrate, extending focally into the subcutaneous tissue. The infiltrate was composed predominantly of pleomorphic cells with bi-lobed, multi-lobed, horseshoe, or ring-shaped nuclei. There was a smaller subset of monomorphous cells characterized by a round, reniform, or elongated single-lobed nucleus. Accompanying cells included few foamy histiocytes, lymphocytes, and numerous scattered eosinophils. No necrosis, vascular invasion, or ulceration was present. The pleomorphic and monomorphic granular cells were positive for Giemsa stain as well as for tryptase, CD117, CD68, CD2, and CD30 immunohistochemistry and negative for S100, CD1a, myeloperoxidase, lysozyme, and CD56. Clinical examination was negative for any additional similar lesions and serum tryptase was within normal limits. The bone marrow was not biopsied. In addition, fluorescent in situ hybridization revealed multiple clones with loss of number 5 chromosome and PDGFRA and HRAS mutations. The lesion did not recur or progress after a 6-year clinical follow-up. To our full knowledge, we report the first case of pleomorphic mastocytoma with loss of chromosome 5 and PDGFRA and HRAS mutations.

Role of Zinc Sulphate in Immune Regulation in Artemisia annua Pollen-challenged P815 Mastocytoma Cells.

Objective This study aimed to investigate the role of zinc sulphate in immune regulation in Artemisia annua pollen-challenged P815 mastocytoma cells. Methods P815 mastocytoma cells were treated with various concentrations of zinc sulphate and Artemisia annua pollen. Cell proliferation was measured using the Cell Counting Kit-8. The amount of ST2 and p38 in the cells were measured using Western blotting. The level of interleukins (IL)-33 in the supernatant was determined using the enzyme-linked immunosorbent assay. The levels of IL-2, IL-4, IL-5, interferon-γ, and tumor necrosis factor were measured using the cytometric bead array. Results Artemisia annua pollen at a concentration >0.001 µg/mL induced allergic response in the P815 mastocytoma cells. Expressions of IL-33, IL-4, ST2, and p38 increased along with higher concentrations of Artemisia annua pollen. Zinc sulphate of 50-200 µmol/L promoted the proliferation of P815 mastocytoma cells. Zinc sulphate attenuated the upregulation of IL-33, IL-4, ST2, and p38 caused by Artemisia annua pollen. Conclusion Zinc sulphate can promote the proliferation of P815 mastocytoma cells. It can also attenuate allergic response in the P815 mastocytoma cells induced by Artemisia annua pollen, which might provide a new treatment method for allergic diseases.

Systemic mastocytosis associated with Hodgkin's lymphoma in a 4-year-old child.

Systemic mastocytosis with associated clonal hematologic non-mast cell lineage disease (SM-AHN) represents a specific subtype of mastocytosis and is extremely rare in children. We describe a 4-year-old child with systemic mastocytosis associated with Hodgkin's lymphoma. The child had cutaneous mastocytosis and lymphadenopathy without other clinical features of SM, which was diagnosed only by bone marrow examination.

Grading Cutaneous Mast Cell Tumors in Cats.

Cutaneous mast cell tumors (cMCTs) account for approximately 20% of skin neoplasms in cats. As there is no grading system for these tumors, prognosis is difficult to estimate. Although the typical presentation is a benign tumor that can be cured by surgical excision, a small but important proportion of feline cMCTs is biologically aggressive and can spread to local lymph nodes, precede the onset of disseminated cutaneous disease, or be associated with visceral involvement. A number of macroscopic and histologic features were retrospectively evaluated in cases of feline cMCTs treated with surgical excision with or without medical therapy. Cats were divided into 2 groups based on the clinical outcome. Group 1 included cats alive with no mast cell tumor-related disease at 1000 days from surgery; group 2 included cats developing histologically confirmed metastatic or cutaneous disseminated disease. The criteria allowing the best differentiation between the groups were used to develop a grading scheme. Groups 1 and 2 were composed by 48 (76%) and 15 (24%) cases, respectively. Tumors were classified as high grade if there were >5 mitotic figures in 10 fields (400×) and at least 2 of the following criteria: tumor diameter >1.5 cm, irregular nuclear shape, and nucleolar prominence/chromatin clusters. According to this scheme, the 15 (24%) high-grade cMCTs had significantly reduced survival time (median, 349 days; 95% CI, 0-739 days) as compared with the 48 low-grade tumors (median not reached; P < .001). Further studies are warranted to validate this grading system and test reproducibility on a larger case series.

Perioperative anaphylaxis in a patient with a solitary mastocytoma.

Children with more extensive cutaneous mastocytosis have a higher risk for symptoms secondary to release of mast cell mediators. However, the remote possibility of anaphylaxis in patients with a solitary lesion suggests the need for cautious use of general anesthesia in these children. We describe an unusual case of a patient with a solitary mastocytoma who experienced an anaphylactic reaction during a surgical procedure and make recommendations to reduce the risk of intraoperative anaphylaxis in mast cell disease.

Pleomorphic mastocytoma in an adult.

Cutaneous mastocytoma (CM) is a localized variant of mastocytosis, characterized by an over-accumulation of mast cells in the skin, without extra-cutaneous organ involvement. It is defined as the presence of up to 3 isolated mast-cell skin lesions and commonly develops in newborns and children. We report the case of a 35-year-old healthy Caucasian woman presenting with a 4-year history of a pruritic brown plaque on her left breast. Hematoxylin-eosin staining showed a dense dermal infiltrate of atypical mast cells extending to the subcutis. The cells presented a marked nuclear pleomorphism with bilobed and multilobed nuclei. Immunohistochemical studies revealed strongly expressed KIT (CD117) and CD25 proteins. Serum tryptase levels and bone marrow biopsy were normal. The diagnosis was a solitary cutaneous pleomorphic mastocytoma. This case can be added to 17 other cases of adult mastocytoma documented in the literature, although, unlike other reported cases, and as far as we are aware, this is the first case of pleomorphic mastocytoma in an adult.

Nanog Expression and Proliferation Indices in Canine Cutaneous Mast Cell Tumors.

Mast cell tumors are one of the most frequent skin tumors in dogs. Treatment decisions often depend on a wide range of clinical information and the main criteria for prognostic formulation are histological grade, mitotic count, Ki67 index, and KIT immunostaining pattern. NANOG is a pluripotency factor expressed by normal and cancer stem cells, which is a prognostic marker and a potential therapeutic target for several human tumors. In the present study, mast cell tumor samples from 41 dogs were evaluated for NANOG and Ki67 by immunohistochemistry. All samples were positive for NANOG but its expression was not correlated with Ki67 index and no significant differences were found with respect to histopathological grades, disease-related mortality, or survival. Our results suggest that, although related to pluripotency, NANOG expression does not correlate with proliferative activity, and is not a reliable prognostic factor for canine cutaneous mast cell tumors.

Assessment of Canine Mast Cell Tumor Mortality Risk Based on Clinical, Histologic, Immunohistochemical, and Molecular Features.

Mast cell tumor (MCT) is a frequent cutaneous neoplasm in dogs that is heterogeneous in clinical presentation and biological behavior, with a variable potential for recurrence and metastasis. Accurate prediction of clinical outcomes has been challenging. The study objective was to develop a system for classification of canine MCT according to the mortality risk based on individual assessment of clinical, histologic, immunohistochemical, and molecular features. The study included 149 dogs with a histologic diagnosis of cutaneous or subcutaneous MCT. By univariate analysis, MCT metastasis and related death was significantly associated with clinical stage ( P < .0001, rP = -0.610), history of tumor recurrence ( P < .0001, rP = -0.550), Patnaik ( P < .0001, rP = -0.380) and Kiupel grades ( P < .0001, rP = -0.500), predominant organization of neoplastic cells ( P < .0001, rP = -0.452), mitotic count ( P < .0001, rP = -0.325), Ki-67 labeling index ( P < .0001, rP = -0.414), KITr pattern ( P = .02, rP = 0.207), and c-KIT mutational status ( P < .0001, rP = -0.356). By multivariate analysis with Cox proportional hazard model, only 2 features were independent predictors of overall survival: an amendment of the World Health Organization clinical staging system (hazard ratio [95% CI]: 1.824 [1.210-4.481]; P = .01) and a history of tumor recurrence (hazard ratio [95% CI]: 9.250 [2.158-23.268]; P < .001]. From these results, we propose an amendment of the WHO staging system, a method of risk analysis, and a suggested approach to clinical and laboratory evaluation of dogs with cutaneous MCT.

Cellular effects of a turmeric root and rosemary leaf extract on canine neoplastic cell lines.

BACKGROUND: The use of nutraceuticals is gaining in popularity in human and canine oncology with a relatively limited understanding of the effects in the vastly different tumor types seen in canine oncology. We have previously shown that turmeric root (TE) and rosemary leaf (RE) extracts can work synergistically to reduce neoplastic cell growth, but the mechanisms are poorly understood and require further elucidation. RESULTS: Three different canine cell lines (C2 mastocytoma, and CMT-12 mammary carcinoma, D17 osteosarcoma) were treated with 6.3 µg mL-1 extract individually, or 3.1 µg mL-1 of each extract in combination based on studies showing synergy of these two extracts. Apoptosis, antioxidant effects, cellular accumulation of curcumin, and perturbation of signaling pathways were assessed. The TE + RE combination treatment resulted in Caspase 3/7 activation and apoptosis in all cell lines, beyond the effects of TE alone with the CMT-12 cell line being most susceptible. Both extracts had antioxidant effects with RE reducing reactive oxygen species (ROS) by 40-50% and TE reducing ROS by 80-90%. In addition RE treatment enhanced the c-jun N-terminal kinase (JNK) activity in the C2 cell line and TE + RE exposure increased activated JNK by 4-5 times in the CMT-12 cell line. Upon further examination, it was found that RE treatment caused a significant increase in the cellular accumulation of curcumin by approximately 30% in the C2 and D17 cell lines, and by 4.8-fold in the CMT-12 cell line. This increase in intracellular curcumin levels may play a role in the synergy exhibited when using TE and RE in combination. CONCLUSIONS: The use of RE in combination with TE induces a synergistic response to induce apoptosis which is better than either extract alone. This appears to be related to a variable increased TE uptake in cells and activation of pathways involved in the apoptotic response.

Effects and synergy of feed ingredients on canine neoplastic cell proliferation.

BACKGROUND: Adjunctive use of nutraceuticals in human cancer has shown promise, but little work has been done in canine neoplasia. Previous human research has shown that polyphenols and carotenoids can target multiple pathways in vitro and in vivo. These compounds could synergize or antagonize with currently used chemotherapies, either increasing or decreasing the effectiveness of these drugs. Considering the routine and well controlled feeding practices of most dogs, the use of nutraceuticals incorporated into pet food is attractive, pending proof that the extracts are able to improve remission rates. The aim of this study was to examine five feed ingredients for antiproliferative effects, as well as the interaction with toceranib phosphate and doxorubicin hydrochloride, when treating canine neoplastic cell lines in vitro. RESULTS: Screening using MTT proliferation assays showed that green tea, turmeric, and rosemary extracts were the most effective. Turmeric extract (TE) was the most potent and exhibited synergy with a rosemary extract (RE) at concentrations from 1 to 25 µg mL(-1). This combination had an additive or synergistic effect with chemotherapeutic agents at selected concentrations within each cell line. No significant effects on cell viability were observed when the combination therapy was used with normal primary cells. CONCLUSIONS: The use of turmeric and rosemary extracts in combination may be worthwhile to investigate in the pre-clinical and clinical neoplastic considering there are no negative effects on traditional chemotherapy treatment. Further studies into the pharmacokinetics and mechanisms of action of these extracts should be investigated.

Update on Mastocytosis (Part 1): Pathophysiology, Clinical Features, and Diagnosis.

Mastocytosis is a term used to describe a heterogeneous group of disorders characterized by clonal proliferation of mast cells in various organs. The organ most often affected is the skin. Mastocytosis is a relatively rare disorder that affects both sexes equally. It can occur at any age, although it tends to appear in the first decade of life, or later, between the second and fifth decades. Our understanding of the pathophysiology of mastocytosis has improved greatly in recent years, with the discovery that somatic c-kit mutations and aberrant immunophenotypic features have an important role. The clinical manifestations of mastocytosis are diverse, and skin lesions are the key to diagnosis in most patients.

Update on Mastocytosis (Part 2): Categories, Prognosis, and Treatment.

Mastocytosis is a term used to describe a heterogeneous group of disorders characterized by clonal proliferation of mast cells in different organs. The organ most often affected is the skin. The World Health Organization classifies cutaneous mastocytosis into mastocytoma, maculopapular cutaneous mastocytosis, and diffuse mastocytosis. The systemic variants in this classification are as follows: indolent systemic mastocytosis (SM), aggressive SM, SM with an associated clonal hematological non-mast cell lineage disease, mast cell leukemia, mast cell sarcoma, and extracutaneous mastocytoma. The two latest systemic variants are rare. Although the course of disease is unpredictable in children, lesions generally resolve by early adulthood. In adults, however, the disease tends to persist. The goal of treatment should be to control clinical manifestations caused by the release of mast cell mediators and, in more aggressive forms of the disease, to reduce mast cell burden.

[Cutaneous mastocytosis: A case report]. / Mastocitosis cutánea: reporte de un caso.

INTRODUCTION: Mastocytosis represents a group of diseases characterised by an excesive accumulation of mastocytes in one or multiple tissues. It can affect only the skin, or have a systemic involvement. It has a low prevalence, and the prognosis is benign in children. OBJECTIVE: To report a case of urticaria pigmentosa as a subtype of cutaneous mastocytosis, and present a literature review focused on clinical findings, diagnosis and initial basic management. CLINICAL CASE: A child of six months of age presenting with multiple blemishes and light brown papules located on the trunk, arms and legs. The symptoms were compatible with urticaria pigmentosa, and was confirmed by biopsy. Tests to rule out systemic involvement were requested. The patient was treated with general measures, education, and antihistamines, with favourable results. CONCLUSIONS: Cutaneous mastocytosis is a rare disease with a good prognosis. In childhood general measures and education are usually enough to obtain favourable results. Histamine H1 antagonists are the first line drug treatment.

Development of a photoreactive probe-based system for detecting heparin.

We previously identified a peptide heparin-associated peptide Y (HappY) that binds specifically to heparin. In this article, we report a novel heparin detection system using chemically modified HappY as a probe. The photoreactive HappY probe was serially diluted and dispensed into a 96-well plate coated with biotinylated heparin. After ultraviolet irradiation, the HappY probe crosslinked to the heparin on the plate was detected with fluorescein isothiocyanate-conjugated streptavidin. Furthermore, the photoreactive HappY probe was used to stain cutaneous tissue sections obtained from dermatitis-affected or mastocytoma-affected cats and dogs. The photoreactive HappY probe stained limited resident mast cells in the connective tissue of skin compared with the anti-heparan sulfate monoclonal antibody 10E4, suggesting that the probe can be used to distinguish the structure of heparin in tissues. The interactions between glycosaminoglycans and proteins in vivo tend to be weak. Therefore, our method for enhancing such weak interactions may be a promising tool for intermolecular interaction studies in glycobiology research.