Mami Murakami

The class A macrophage scavenger receptor CD204 is a useful immunohistochemical marker of canine histiocytic sarcoma.

The immunohistochemical expression of the class A macrophage scavenger receptor CD204, was investigated in 50 canine histiocytic sarcomas (HSs) and compared with that of CD18, CD163, CD11d and class II molecules of the major histocompatibility complex (MHC). Expression of CD204 was also determined in 81 canine round cell tumours and pleomorphic sarcomas including T- and B-cell lymphomas, mast cell tumours, extramedullary plasmacytomas, cutaneous histiocytomas, transmissible venereal tumours, pigmented or amelanotic melanomas, poorly differentiated haemangiosarcomas and rhabdomyosarcomas. All of the 50 HSs expressed CD204, CD18 and MHC class II; 27 were positive for CD163 and seven expressed CD11d. All of the round cell tumours, except for one grade III mast cell tumour, were negative for CD204; however, they showed varying immunoreactivity patterns for CD18 and MHC class II. None of the pleomorphic sarcomas were immunoreactive for CD204. CD204 would appear to be a useful marker for canine HS.

Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas

KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases.

Establishment of canine hemangiosarcoma xenograft models expressing endothelial growth factors, their receptors, and angiogenesis-associated homeobox genes

BackgroundHuman hemangiosarcoma (HSA) tends to have a poor prognosis; its tumorigenesis has not been elucidated, as there is a dearth of HSA clinical specimens and no experimental model for HSA. However, the incidence of spontaneous HSA is relatively high in canines; therefore, canine HSA has been useful in the study of human HSA. Recently, the production of angiogenic growth factors and their receptors in human and canine HSA has been reported. Moreover, the growth-factor environment of HSA is very similar to that of pathophysiological angiogenesis, which some homeobox genes regulate in the transcription of angiogenic molecules. In the present study, we established 6 xenograft canine HSA tumors and detected the expression of growth factors, their receptors, and angiogenic homeobox genes.MethodsSix primary canine HSAs were xenografted to nude mice subcutaneously and serially transplanted. Subsequently, the expressions of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factors (bFGF), flt-1 and flk-1 (receptors of VEGF-A), FGFR-1, and angiogenic homeobox genes HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 were investigated in original and xenograft tumors by histopathology, immunostaining, and reverse transcription polymerase chain reaction (RT-PCR), using canine-specific primer sets.ResultsHistopathologically, xenograft tumors comprised a proliferation of neoplastic cells that were varied in shape, from spindle-shaped and polygonal to ovoid; some vascular-like structures and vascular clefts of channels were observed, similar to those in the original tumors. The expression of endothelial markers (CD31 and vWF) was detected in xenograft tumors by immunohistochemistry and RT-PCR. Moreover, the expression of VEGF-A, bFGF, flt-1, flk-1, FGFR-1, HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 was detected in xenograft tumors. Interestingly, expressions of bFGF tended to be higher in 3 of the xenograft HSA tumors than in the other tumors.ConclusionWe established 6 xenograft canine HSA tumors in nude mice and found that the expressions of angiogenic growth factors and their receptors in xenograft HSAs were similar to those in spontaneous HSA. Furthermore, we detected the expression of angiogenic homeobox genes; therefore, xenograft models may be useful in analyzing malignant growth in HSA.

Expression of the anti-apoptotic factors Bcl-2 and survivin in canine vascular tumours.

To investigate whether anti-apoptotic factors play a role in the malignant growth of canine haemangiosarcomas (HSAs), 83 HSAs and 22 haemangiomas were examined immunohistochemically for bcl-2 and survivin expression. Additionally, bcl-2 and survivin mRNA expression was quantified by semiquantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Immunolabelling for bcl-2 was observed in 50 of the 83 HSA samples (60.2%) but in none of the haemangiomas. The average survivin positive index was 24.7% in the HSAs and 0.6% in the haemangiomas. In contrast to the high average value for survivin mRNA expression, which was approximately six times that for the haemangiomas, no significant difference was observed between HSAs and haemangiomas for the average bcl-2 mRNA expression level. The discrepancy between bcl-2 mRNA and bcl-2 protein expression requires further investigation, but the results suggest that malignant proliferation in canine HSAs is associated with bcl-2 and survivin expression.

MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases

Malignant endothelial proliferative diseases including human angiosarcoma (AS) and canine hemangiosarcoma (HSA) are serious diseases with a grave prognosis. Establishing liquid biopsy-based biomarkers for screening has definite clinical utility; however, plasma miRNAs up- or down-regulated in these sarcomas have been unclear. For identifying possible diagnostic plasma miRNAs for these sarcomas, we investigated whether plasma miR-214 and miR-126, which miRNAs play important roles in angiogenesis and tumorigenesis, were elevated in malignant endothelial proliferative diseases. For this investigation, human angiosarcoma and canine hemangiosarcoma cell lines and clinical plasma samples of canine hemangiosarcoma were examined by performing miRNA qRT-PCR. We report here that human angiosarcoma and canine hemangiosarcoma cell lines over-secreted miR-214 and miR-126 via microvesicles; in addition, their levels in the plasma samples from canines with hemangiosarcoma were increased. Moreover, the surgical resection of primary tumors decreased the levels of plasma miR-214 and miR-126. Our findings suggest that these malignant endothelial proliferative diseases over-secreted miR-214 and miR-126, thus suggesting that these miRNAs have potential as diagnostic biomarkers for malignant endothelial proliferative diseases in canine and possible in human angiosarcoma.

MicroRNA-214 Promotes Apoptosis in Canine Hemangiosarcoma by Targeting the COP1-p53 Axis

MicroRNA-214 regulates both angiogenic function in endothelial cells and apoptosis in various cancers. However, the regulation and function of miR-214 is unclear in canine hemangiosarcoma, which is a spontaneous model of human angiosarcoma. The expression and functional roles of miR-214 in canine hemangiosarcoma were presently explored by performing miRNA TaqMan qRT-PCR and transfecting cells with synthetic microRNA. Here, we report that miR-214 was significantly down-regulated in the cell lines used and in clinical samples of canine hemangiosarcoma. Restoration of miR-214 expression reduced cell growth and induced apoptosis in canine hemangiosarcoma cell lines through transcriptional activation of p53-regulated genes although miR-214 had a slight effect of growth inhibition on normal endothelial cells. We identified COP1, which is a critical negative regulator of p53, as a novel direct target of miR-214. COP1 was overexpressed and the specific COP1 knockdown induced apoptosis through transcriptional activation of p53-regulated genes as well as did miR-214-transfection in HSA cell lines. Furthermore, p53 knockdown abolished the miR-214-COP1-mediated apoptosis; thus, miR-214 and COP1 regulated apoptosis through controlling p53 in HSA. In conclusion, miR-214 functioned as a tumor suppressor in canine hemangiosarcoma by inducing apoptosis through recovering the function of p53. miR-214 down-regulation and COP1 overexpression is likely to contribute to tumorigenesis of HSA. Therefore, targeting miR-214-COP1-p53 axis would possibly be a novel effective strategy for treatment of canine hemangiosarcoma and capable of being applied to the development of novel therapeutics for human angiosarcoma.

Cytologic, histologic, and immunohistochemical features of maxillofacial alveolar rhabdomyosarcoma in a juvenile dog

A 15-month-old castrated male dog with a history of intermittent epistaxis and sneezing was admitted for the examination of a maxillofacial mass. An impression smear of a biopsy sample from the cauliflower-shaped gingival mass contained numerous round cells, 5-25 microm in diameter, which contained a moderate amount of clear to pale blue cytoplasm and resembled lymphoid cells. Mitotic figures were frequently observed. The mass was diagnosed as malignant round cell neoplasia. On histologic examination the tumor was composed of diffusely arranged, small, atypical round cells with a small amount of fibrovascular stroma. Immunohistochemically, the cells were negative for CD3, CD18, CD20, CD79alpha, cytokeratin, melan-A, chromogranin A, alpha-smooth muscle actin, and myoglobin but positive for vimentin and desmin. The cells also had strong positive nuclear staining for myogenin and MyoD1. A diagnosis of solid-pattern alveolar rhabdomyosarcoma was made on the basis of morphologic and immunohistochemical results. Alveolar rhabdomyosarcoma should be considered in the differential diagnosis of tumors in juvenile dogs, especially when cytologic findings reveal round, undifferentiated cells.

Molecular diagnosis of feline sporotrichosis

SPOROTRICHOSIS is a chronic infection caused by Sporothrix schenckii, and usually presents as a fixed cutaneous or lymphocutaneous infection in animals (Kwon-Chung and Benett 1992, Rosser and Dunstan 1998). Since a number of diseases can resemble sporotrichosis quite closely (KwonChung and Bennett 1992), it should be diagnosed definitively on the basis of histopathological and mycological findings. Direct microscopic examination is rapid, but can be quite insensitive and is reliant on the operator’s skills. Kano and others (2003) developed a PCR method to detect S schenckii directly in biopsy samples from human patients with sporotrichosis using specific oligonucleotide primers based on the chitin synthase 1 (CHS1) gene of S schenckii (Kano and others 2001). The primer pair (S2 and R2) did not amplify DNAs from other pathogenic fungi (Aspergillus fumigatus, Blastomyces dermatitidis, Candida species, Cryptococcus neoformans, dermatophytes, Malassezia species and Histoplasma capsulatum), bacteria (Staphylococcus aureus), normal human skin cells or normal feline skin cells (Kano and others 2001). The sensitivity of the PCR was equivalent to the detection of 10 pg of S schenckii genomic DNA (Kano and others 2001). This short communication describes a study to identify S schenckii DNA directly from biopsy specimens obtained from a case of feline sporotrichosis. A 23-month-old male crossbred cat, weighing 5·4 kg, was referred to the Gifu University Animal Medical Center with abscesses and a draining puncture wound on the left foreleg and right hindleg which had been present for seven months. The lesions had been drained surgically and treated with chitin and chitosan. A subcutaneous nodule, 18 x 6 x 15 mm in size, was found on the right hindleg. Histopathological analysis of a biopsy of the nodule revealed granulomatous inflammation and many globular and ovoid budding yeast cells (Fig 1), with no asteroid bodies. Therefore, molecular diagnosis of sporotrichosis was performed. DNA was extracted from the biopsy. The tissue sample (approximately 100 mg) was digested at 37°C for 16 hours with 100 μg of proteinase K in 400 μl of a lysis buffer containing 0·1mM Tris-HCl (pH 8·0) and 0·3 per cent 2-mercaptoethanol. The DNA was obtained by phenol and chloroform extraction and then dissolved in Tris-EDTA buffer (10mM Tris-HCl [pH 8·0] and 1mM EDTA) and used directly for PCR amplification. A sample (200 ng) of DNA was amplified by PCR in a 30 μl reaction mixture containing 10mM Tris-HCl (pH 8·3), 50mM potassium chloride, 1·5mM magnesium chloride, 0·001 per cent gelatin, 200μM of each deoxynucleoside triphosphate, 1·0 U Taq polymerase (Takara) and 0·5 μg of the primer pair. To detect S schenckii rapidly, species-specific primers for PCR based on its CHS1 gene sequence were used (Kano and others 2001, 2003). The sequence of the forward primer (S2) was 5′-TGGGCGTCTACCAAGAGGGTATTGC-3′ (nucleotides 173 to 197 of the S schenckii CHS1 gene) (GenBank accession number L24908) and the sequence of the reverse primer (R2) was 5′-GCACATGGGCTCAAGATCAAAGGCC-3′ (nucleotides 468 to 492 of the S schenckii CHS1 gene). With these primers, a 318 base pair (bp) fragment of the CHS1 gene was expected to be amplified. The PCR amplification was carried out for 40 cycles consisting of template denaturation (one minute at 94°C), primer annealing (two minutes at 68°C) and polymerisation (two minutes at 72°C). Then, 10 μl of PCR products were separated in a 2 per cent (w/v) agarose gel, stained with ethidium bromide and visualised with ultraviolet light. The S2-R2 primer pair amplified approximately 320 bp fragments of the DNA from the clinical specimen and a control sample of S schenckii DNA (Fig 2). The primer pair did not amplify 320 bp fragments from normal feline skin DNA (Fig 2). Therefore, the case was diagnosed molecularly as sporotrichosis. The biopsy material from the nodule on the right hindleg was cultured on sunflower seed agar at 27°C for one week and then examined morphologically. The clinical isolate developed a black colony with a grey aerial surface after incubation for two weeks on sunflower seed agar. Microscopic examination of the isolate revealed one-cell, dark brown, thick-walled, subglobular, elliptical to cylindrical, smooth and hyaline conidia, which were 2 to 3 x 3 to 6 μm in size, produced on hyphae and in a bouquet at the tip of conidiophores. The conidiophores were slender and tapered from 1 to 2 μm diameter at the base to 0·5 to 1 μm at the tip. Since these findings were consistent with the description of S schenckii by Kwon-Chung and Bennett (1992), the isolate was identified as S schenckii. The cat was treated with itraconazole (Itrizole; Janssen Pharmaceutical) administered orally at a dose of 15 mg/kg once a day. After two months of treatment, the nodule had healed and the itraconazole was discontinued. The cat was in Veterinary Record (2005) 156, 484-485

Orbital Embryonal Rhabdomyosarcoma with Metastasis in a Young Dog

A 2-year-old male Welsh corgi dog was brought to an animal hospital because of left upper eyelid enlargement with lachrymal gland protrusion. The lachrymal and orbital cavity mass was removed surgically. Microscopically, the orbital mass consisted of a mixture of large rhabdomyoblastic and small round tumour cells. Immunohistochemically, the rhabdomyoblastic cells expressed desmin and myoglobin and the small round cells expressed desmin, myogenin and MyoD1. A diagnosis of embryonal rhabdomyosarcoma (ERS) was made. One month later, multiple masses throughout the body were identified, in particular around the cervical region. One of these lesions was sampled and diagnosed as metastatic ERS. The dog died 84 days after the time of first admission.

Outcomes of dogs undergoing radiotherapy for treatment of oral malignant melanoma: 111 cases (2006–2012)

OBJECTIVE To evaluate the characteristics and outcomes of dogs with stage I, II, III, or IV oral malignant melanoma treated by various types of radiotherapy. DESIGN Retrospective case series. ANIMALS 111 dogs. PROCEDURES Medical records of dogs with oral malignant melanoma treated by radiotherapy (with or without adjunctive treatments) at a veterinary medical center between July 2006 and December 2012 were reviewed. Information regarding signalment, tumor location, disease stage, treatment protocols, adverse effects, and survival time were obtained from medical records and by telephone follow-up. Associations between variables of interest and outcome were analyzed. RESULTS Dogs received orthovoltage x-ray (n = 68), megavoltage x-ray (39), or electron beam (4) radiotherapy. Adjunctive treatments included debulking surgery (n = 18), chemotherapy (39), or both (27). Median survival times for dogs with stage I, II, III, and IV melanoma were 758 days (n = 19), 278 days (24), 163 days (37), and 80 days (31), respectively, and differed significantly between dogs with stage I disease and those with all other disease stages. Among dogs with stage III melanoma, risk of death was significantly higher in those that received orthovoltage x-ray treatment than in those that received megavoltage x-ray treatment. Severe (primary or secondary) adverse effects were identified in 9 dogs. CONCLUSIONS AND CLINICAL RELEVANCE Median survival time was significantly longer for dogs with stage I oral malignant melanoma than for dogs with more advanced disease at the time of staging. The staging system used may be a useful tool for prognosis prediction in dogs undergoing similar treatment protocols for oral malignant melanomas.