Kenji Hosoya

Retrospective evaluation of the effectiveness of epsilon aminocaproic acid for the prevention of postamputation bleeding in retired racing Greyhounds with appendicular bone tumors: 46 cases (2003–2008)

OBJECTIVES To determine the frequency of delayed postoperative bleeding in retired racing Greyhounds with appendicular bone tumors undergoing limb amputations. To identify if administration of epsilon-aminocaproic acid (EACA) was effective on the prevention of postoperative bleeding. DESIGN Retrospective study from December 2003 to December 2008. SETTING Veterinary university teaching hospital. ANIMALS Forty-six retired racing Greyhounds (RRGs) diagnosed with primary appendicular bone tumors that underwent limb amputation were included in the study. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Thirteen of 46 RRGs (28%) included in the study had delayed postoperative bleeding starting 48-72 h after surgery. Bleeding episodes included cutaneous, subcutaneous, and external bleeding that extended from the area of the surgical site that became widespread within hours, and that required administration of blood components. A paired t-test suggests that there was a significant decrease in PCV postoperatively for both dogs that bled and dogs that did not bleed (P < 0.0001). Forty of 46 RRGs (86%) received either fresh frozen plasma (FFP) or EACA or both, for the prevention of postoperative bleeding. A logistic regression model determined that dogs that did not receive EACA were 5.7 times more likely to bleed than dogs that did receive EACA, when controlling for whether or not they received FFP (95% CI: 1.02-32.15, P = 0.047). CONCLUSION This retrospective study suggests that preemptive postoperative administration of EACA appears to be efficacious in decreasing the frequency of bleeding in RRGs undergoing limb amputation; however, a prospective study is warranted to corroborate its effectiveness.

Adjuvant CCNU (Lomustine) and Prednisone Chemotherapy for Dogs With Incompletely Excised Grade 2 Mast Cell Tumors

The use of adjuvant 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; lomustine) to treat incompletely excised canine mast cell tumors (MCTs) has not been evaluated. Medical records of 12 dogs with grade 2 MCT treated with incomplete surgical excision and adjuvant CCNU and prednisone chemotherapy were reviewed. Local recurrence rate, metastasis rate, and survival time were evaluated. None of the dogs developed local recurrence or regional/ distant metastases. Two dogs developed fatal liver failure. The 1- and 2-year progression-free rates of surviving dogs were 100% and 77%, respectively. Postoperative adjuvant CCNU appears to be a useful alternative to radiation therapy for incompletely excised canine cutaneous MCTs.

Prevalence of elevated alanine transaminase activity in dogs treated with CCNU (Lomustine).

The purpose of this study was to evaluate prevalence of serum alanine transaminase (ALT) elevation in dogs receiving lomustine (CCNU) and to analyse the pattern of occurrence and potential risk factors. Serum ALT activity in 109 dogs during single-agent CCNU chemotherapy was retrospectively analysed. The median initial dose, dose-intensity and cumulative dose of CCNU were 64 mg m(-2), 21 mg m(-2) week(-1) and 171 mg m(-2), respectively. The overall prevalence of major ALT elevation [> 5-fold upper reference limit (URL)] was 29% (32/109) and developed most commonly after one to three doses of CCNU. These ALT elevations occurred without preceding mild ALT elevation in 53% (17/32) of the cases. Three dogs (2.8%) developed clinical hepatopathy. For severe ALT elevation (>10-fold URL), age < or =5-year-old was associated with higher risk. The findings of this study showed that elevation of ALT is common during CCNU chemotherapy in dogs and severe elevation can develop on a sudden onset.

VAC Protocol for Treatment of Dogs with Stage III Hemangiosarcoma

Hemangiosarcomas (HSAs) are aggressive tumors with a high rate of metastasis. Clinical stage has been considered a negative prognostic factor for survival. The study authors hypothesized that the median survival time (MST) of dogs with metastatic (stage III) HSA treated with a vincristine, doxorubicin, and cyclophosphamide (VAC) chemotherapy protocol would not be different than those with stage I/II HSA. Sixty-seven dogs with HSA in different anatomic locations were evaluated retrospectively. All dogs received the VAC protocol as an adjuvant to surgery (n = 50), neoadjuvant (n = 3), or as the sole treatment modality (n = 14). There was no significant difference (P = 0.97) between the MST of dogs with stage III and stage I/II HSA. For dogs presenting with splenic HSA alone, there was no significant difference between the MST of dogs with stage III and stage I/II disease (P = 0.12). The overall response rate (complete response [CR] and partial response [PR]) was 86%). No unacceptable toxicities were observed. Dogs with stage III HSA treated with the VAC protocol have a similar prognosis to dogs with stage I/II HSA. Dogs with HSA and evidence of metastases at the time of diagnosis should not be denied treatment.

Inhibition of Survivin Influences the Biological Activities of Canine Histiocytic Sarcoma Cell Lines

Canine histiocytic sarcoma (CHS) is an aggressive malignant neoplasm that originates from histiocytic lineage cells, including dendritic cells and macrophages, and is characterized by progressive local infiltration and a very high metastatic potential. Survivin is as an apoptotic inhibitory factor that has major functions in cell proliferation, including inhibition of apoptosis and regulation of cell division, and is expressed in most types of human and canine malignant neoplasms, including melanoma and osteosarcoma. To investigate whether survivin was expressed at high levels in CHS and whether its expression was correlated with the aggressive biological behavior of CHS, we assessed relation between survivin expression and CHS progression, as well as the effects of survivin inhibition on the biological activities of CHS cells. We comparatively analyzed the expression of 6 selected anti-apoptotic genes, including survivin, in specimens from 30 dogs with histiocytic sarcoma and performed annexin V staining to evaluate apoptosis, methylthiazole tetrazolium assays to assess cell viability and chemosensitivity, and latex bead assays to measure changes in phagocytic activities in 4 CHS cell lines and normal canine fibroblasts transfected with survivin siRNA. Survivin gene expression levels in 30 specimens were significantly higher than those of the other 6 genes. After transfection with survivin siRNA, apoptosis, cell growth inhibition, enhanced chemosensitivity, and weakened phagocytic activities were observed in all CHS cell lines. In contrast, normal canine fibroblasts were not significantly affected by survivin knockdown. These results suggested that survivin expression may mediate the aggressive biological activities of CHS and that survivin may be an effective therapeutic target for the treatment of CHS.

Comparative analysis of mRNA expression of surface antigens between histiocytic and nonhistiocytic sarcoma in dogs.

Background Definitive diagnosis of histiocytic sarcoma (HS) in dogs is relatively difficult by conventional histopathological examination because objective features of HS are not well defined. Hypothesis Quantitative analysis of mRNA expression of selected cellular surface antigens (SAs) specific to HS in dogs can facilitate objective and rapid diagnosis. Animals Dogs with HS (n = 30) and dogs without HS (n = 36), including those with other forms of lymphoma (n = 4), inflammatory diseases (n = 6), and other malignant neoplasias (n = 26). Methods Retrospective clinical observational study. Specimens were collected by excisional biopsy, needle core biopsy, or fine needle aspiration. To determine HS detection efficacy, mRNA expression levels of selected SAs specific to HS in dogs, including MHC class IIα, CD11b, CD11c, and CD86, were quantitatively analyzed using real‐time quantitative polymerase chain reaction. Results Each SA mRNA expression level was significantly higher in HS dogs than in non‐HS dogs (P = .0082). Cutoff values for discriminating between HS and non‐HS dogs based on these expression levels were calculated on the basis of receiver‐operating characteristic analysis. Accuracy of the cutoff values, including MHC class IIα, CD11b, CD11c, and CD86, was 87.9, 86.4, 86.4, and 84.8%, respectively. Conclusions and Clinical Importance Our results suggest that quantitative analysis of mRNA expression of the selected SAs could be an adjunctive diagnostic technique with high diagnostic accuracy for HS in dogs. Substantial investigation is required for exclusion of diseases with similar cell types of origin to lymphoma.

Postoperative adjuvant combination therapy with doxorubicin and noncytotoxic suramin in dogs with appendicular osteosarcoma.

Although conventional treatment of dogs with osteosarcoma (OSA) by amputation and chemotherapy results in reported survival times (STs) of 262-413 days, no major improvements in STs have occurred in the past 2 decades. Suramin is a polysulfonated napthylurea, which at noncytotoxic concentrations in vitro, increases tumor sensitivity to chemotherapy, including doxorubicin. The study authors evaluated the combination of noncytotoxic suramin and doxorubicin after amputation in dogs with OSA. The hypothesis was that treatment of dogs with appendicular OSA with amputation, adjuvant doxorubicin, and noncytotoxic suramin would be well tolerated and result in STs at least comparable to those of doxorubicin alone. Forty-seven dogs received 6.75 mg/kg of suramin IV followed by 30 mg/m(2) of doxorubicin IV 4 hr later. Treatment was repeated q 2 wk for five doses. The median disease free time (DFI) was 203 days (range, 42-1,580+ days) and the median ST for all dogs was 369 days (range, 92-1,616+ days). There was no statistical difference in ST and DFI between greyhounds and nonngreyhounds. Adjuvant doxorubicin and noncytotoxic suramin was well tolerated in dogs with OSA following amputation. Additional studies are needed to determine if this combination treatment protocol provides additional clinical benefit compared with doxorubicin alone.

Evaluation of a point-of-care hematology analyzer for use in dogs and cats receiving chemotherapeutic treatment

OBJECTIVE To compare WBC, neutrophil, and platelet counts and Hct values obtained with a point-of-care hematology analyzer with values obtained by a reference method for dogs and cats receiving chemotherapy. DESIGN Cross-sectional study. ANIMALS 105 dogs and 25 cats undergoing chemotherapy. PROCEDURES Blood samples were analyzed with a point-of-care hematology analyzer and with an impedance- and laser-based analyzer with manual differential WBC counts. Results for WBC, neutrophil, and platelet counts and Hct were compared. Sensitivity and specificity of the point-of-care analyzer to detect leukopenia, neutropenia, and anemia were calculated. RESULTS 554 canine and 96 feline blood samples were evaluated. Correlation coefficients for dogs and cats, respectively, were 0.92 and 0.95 for total WBC count, 0.91 and 0.88 for neutrophil count, 0.95 and 0.92 for Hct, and 0.93 and 0.71 for platelet count. Sensitivity and specificity, respectively, of the point-of-care analyzer to detect leukopenia were 100% and 75% for dogs and 100% and 68% for cats; to detect neutropenia were 80% and 97% for dogs and 100% and 80% for cats; to detect anemia were 100% and 80% for dogs and 100% and 66% for cats; and to detect thrombocytopenia were 86% and 95% for dogs and 50% and 87% for cats. CONCLUSIONS AND CLINICAL RELEVANCE The point-of-care analyzer was reliable for monitoring CBCs of dogs and cats receiving chemotherapy. It had good to excellent correlation for WBC and neutrophil counts and Hct and accurately detected leukopenia, neutropenia, and anemia. Sensitivity of the analyzer for detecting thrombocytopenia was lower but acceptable.

Survivin suppressor (YM155) enhances chemotherapeutic efficacy against canine histiocytic sarcoma in murine transplantation models

Histiocytic sarcoma (HS) in dogs exhibits aggressive clinical and biological behavior. Currently, no effective treatments are available for dogs with HS. Survivin, a member of a family of apoptosis protein inhibitors, could serve as a potential therapeutic target in several canine cancers. Sepantronium bromide (YM155) has recently been established as a novel survivin-targeting agent. The aim of this study was to use YM155 as a tool for evaluating survivin-targeted therapies against dogs with HS, and to investigate how YM155 treatment affects antitumor and chemotherapeutic efficacies in murine xenograft models using canine HS cells. The results showed that in HS cells with lomustine (CCNU) resistance, YM155 treatment suppressed both the cell-growth potential and cell resistance to CCNU, which essentially increases the chemotherapy efficacy in the murine models. The evidence presented here supports the favorable preclinical evaluation that survivin-targeted therapies might be effective against HS in dogs.

Compensatory Cellular Reactions to Nonsteroidal Anti-Inflammatory Drugs on Osteogenic Differentiation in Canine Bone Marrow-Derived Mesenchymal Stem Cells

ABSTRACT The suppressive effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the bone healing process have remained controversial, since no clinical data have clearly shown the relationship between NSAIDs and bone healing. The aim of this study was to assess the compensatory response of canine bone marrow-derived mesenchymal stem cells (BMSCs) to several classes of NSAIDs, including carprofen, meloxicam, indomethacin and robenacoxib, on osteogenic differentiation. Each of the NSAIDs (10 µM) was administered during 20 days of the osteogenic process with human recombinant IL-1β (1 ng/ml) as an inflammatory stimulator. Gene expression of osteoblast differentiation markers (alkaline phosphatase and osteocalcin), receptors of PGE2 (EP2 and EP4) and enzymes for prostaglandin (PG) E2 synthesis (COX-1, COX-2, cPGES and mPGES-1) was measured by using quantitative reverse transcription-polymerase chain reaction. Protein production levels of alkaline phosphatase, osteocalcin and PGE2 were quantified using an alkaline phosphatase activity assay, osteocalcin immunoassay and PGE2 immunoassay, respectively. Histologic analysis was performed using alkaline phosphatase staining, von Kossa staining and alizarin red staining. Alkaline phosphatase and calcium deposition were suppressed by all NSAIDs. However, osteocalcin production showed no significant suppression by NSAIDs. Gene expression levels of PGE2-related receptors and enzymes were upregulated during continuous treatment with NSAIDs, while certain channels for PGE2 synthesis were utilized differently depending on the kind of NSAIDs. These data suggest that canine BMSCs have a compensatory mechanism to restore PGE2 synthesis, which would be an intrinsic regulator to maintain differentiation of osteoblasts under NSAID treatment.