Radioresistance of cancer stem‐like cell derived from canine tumours

Cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye‐based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage‐independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct‐4 and CD133 gene than those of adherent‐cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent‐cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent‐cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.     

Establishment and characterization of a canine soft tissue sarcoma patient‐derived xenograft model

Spontaneously occurring soft tissue sarcoma (STS) is relatively common in canine cancer patients. Because of the similarities to human disease, canine STSs are a valuable and readily available resource for the study of new therapeutics. In this study, a canine patient‐derived xenograft (PDX) model, CDX‐STS2, was established. The CDX‐STS2 model was engrafted and expanded for systemic administration studies with chemotherapeutic agents commonly used to treat STS, including doxorubicin, docetaxel and gemcitabine. Immunohistochemistry for drug‐specific biomarkers and tumour growth measurement revealed tumour sensitivity to doxorubicin and docetaxel, whereas gemcitabine had no effect on tumour growth. Although many human PDX tumour models have been established, relatively few canine PDX models have been reported to date. CDX‐STS2 represents a new STS PDX research model of canine origin that will be useful in bridging preclinical research with clinical studies of STS in pet dogs.     

Effect of hemolysis on canine kaolin‐activated thromboelastography values and ADVIA 2120 platelet activation indices

Background: The impact of hemolysis on thromboelastography (TEG) and platelet activation indices has not been evaluated. Objective: The aim of this study was to investigate the influence of hemolysis induced mechanically (HM) and hemolysis induced by freezing (HF) on TEG, platelet counts (PLT), and platelet activation indicators. Methods: Blood from 17 dogs was divided into the following samples: controls, HM, and HF. HM was induced by 20 repetitions of expulsion of blood through a 23 g needle. Freezing was at −80°C, followed by warming to 37° and dilution with equal parts room temperature blood at 22°C. TEG variables that were examined included reaction time (R), coagulation time (K), angle (α), maximum amplitude (MA), and clot rigidity (G). Platelet indices were measured with the ADVIA 2120 hematology analyzer. Results: Hematocrit (HCT) (mean±SD) for controls, HM, and HF were 0.41±0.02, 0.39±0.03, and 0.25±0.02 L/L, respectively, consistent with decreases in HCT of 4.8% (HM) and 39.0% (HF). HM resulted in decreased R (2.5±0.9 minutes compared with 5.2±1.9 minutes for controls; P<0.001), and HF resulted in increased K (15.2±8.6 minutes compared with 5.3±4.0 minutes in controls; P<0.01) and decreased α (20±11° compared with 46±17° in controls; P<0.001). MA was decreased more in HF samples (26±2 mm) than in HM (38±8 mm) or control samples (49±9 mm; P<0.0001). The same applied to G values. PLT decreased after HM but not after HF. Hemolysis of both types resulted in decreased mean platelet component (MPC) concentration: control, 19.3±2.0, HM 15.5±3.4, and HF 14.3±0.7 g/dL (P<0.0001). Conclusion: In hemolyzed samples decreased MPC and R suggested activated primary and secondary hemostasis, respectively, but decreased MA and G indicated reduced clot firmness, possibly due to hyporeactive platelets. TEG and platelet activation indices should be interpreted cautiously after hemolysis.     

Analysis of radiosensitivity of cancer stem‐like cells derived from canine cancer cell lines

Cancer stem‐like cells (CSCs) are self‐renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere‐forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs‐like properties by expression of CSCs markers (real‐time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53‐binding protein 1) after X‐ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X‐ray irradiation as compared with AC. In addition, the amount of DNA double‐strand breaks after X‐ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs‐like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double‐strand break after X‐ray exposure.     

Analysis of genomic mutation and immunohistochemistry of platelet‐derived growth factor receptors in canine vascular tumours

We examined whether mutation of the platelet‐derived growth factor receptor protein tyrosine kinase (PDGFR)‐α and PDGFR‐β genes contributes to their overexpression in canine vascular tumours. Genomic sequences of trans‐ or juxtamembrane regions of PDGFR‐α and PDGFR‐β were analysed with immunohistochemical staining and polymerase chain reaction‐direct sequencing using DNA from paraffin‐embedded neoplastic tissues of 27 hemangiosarcomas (HSAs) and 20 hemangiomas (HAs). Immunohistochemically, 75% of the HA cases were positive for PDGFR‐α and almost most of the HA cases were negative for PDGFR‐β. Of the HSA cases, 55.6% were negative for PDGFR‐α and 63% were strongly positive for PDGFR‐β. Among the HA cases, 1 missense mutation was detected in PDGFR‐α exon 18 and 1 in PDGFR‐β exon 17. Two HSA cases had missense mutations in exon 14 and 1 in exon 17 of PDGFR‐β. Thus, genomic mutation of trans‐ or juxtamembrane regions of PDGFRs was not the main mechanism driving the activation of receptors in HSA and HA.     

CLINICAL APPLICATION OF PATLAK PLOT CT‐GFR IN ANIMALS WITH UPPER URINARY TRACT DISEASE

Glomerular filtration rate (GFR), an important parameter of renal function, is difficult to assess clinically. Serum creatinine and blood urea nitrogen measurements lack sensitivity, whereas radionuclide determination of GFR is not always available and requires postinjection patient isolation. GFR can be determined using computed tomography (CT), most commonly via Patlak plot analysis. Four adult cats, two adult dogs, and a foal underwent abdominal CT under general anesthesia for various diseases of the upper urinary tract. CT‐GFR was measured with a single‐slice dynamic acquisition and Patlak plot analysis. In five animals, the total CT‐GFR appeared to be below normal, corresponding with mild (two animals) and moderate (two animals) increases of serum creatinine in four. In the two animals with normal or increased CT‐GFR, serum creatinine was within the reference values. A significant negative logarithmic relationship was found between CT‐GFR and serum creatinine values (P=0.008; r²=0.75). No complications occurred during or following CT‐GFR. CT examination provided clinically relevant information in 3/5 patients with possible ureteral obstruction and in 3/3 patients with suspected ureteral calculi. Single‐slice dynamic CT‐GFR was practical and provided clinically useful information in this small series of patients undergoing CT of the upper urinary tract. There was a significant relationship between CT‐GFR and serum creatinine values, which supports the clinical potential of CT‐GFR and justifies further investigation of this technique.     

COX‐2 and c‐kit expression in canine gliomas

Gliomas are among the most common primary neural tumours of dogs. Cyclooxygenase‐2 (COX‐2) and c‐kit overexpression are associated with increased aggressiveness of gliomas and decreased survival in human beings. COX‐2 is the inducible form of cyclooxygenase, which catalyzes prostaglandin formation and may increase tumour proliferation and angiogenesis. C‐kit is a tyrosine kinase receptor involved in normal cell physiology; c‐kit is upregulated in some canine tumours. In this retrospective study, 20 canine gliomas were identified: 11 (55%) oligodendrogliomas, including 1 anaplastic variant; 1 (5%) oligoastrocytoma; and 8 (40%) astrocytomas, of which 2 were glioblastoma multiforme. None of the gliomas expressed COX‐2. None of the gliomas were immunoreactive for c‐kit, although all three high‐grade tumours had intramural vascular expression. Consequently, COX‐2 inhibitors would likely be ineffective against canine gliomas. C‐kit inhibitors may have an anti‐angiogenic effect in high‐grade gliomas, but would likely be ineffective in low‐ and medium‐grade tumours.     

Reticulocyte indices in a canine model of nutritional iron deficiency

BACKGROUND: Reticulocyte indices, especially reticulocyte hemoglobin content (CH retic), have shown promise as markers of iron deficiency (ID), but there have been no prospective investigations of reticulocyte indices in experimental models of ID. OBJECTIVES: The objective of this study was to compare reticulocyte indices with conventional hematologic and biochemical indices as markers of ID in dogs. METHODS: Iron deficiency was induced in 7 dogs by feeding an iron-deficient diet, and corrected by restoring dietary iron and by giving iron parenterally. Blood samples were collected at weekly intervals. Results of hematologic and biochemical tests were compared using t-tests and receiver operator characteristic (ROC) curve analysis. RESULTS: Comparing mean values on days 0 and 35, by which time hemoglobin concentration decreased to 90% of baseline in all dogs, % Macro retic, % Hypo retic, % Low CH retic, and % High CH retic differed by greater than 3-fold, whereas no conventional hematologic or biochemical indices differed by as much as 2-fold. Comparing conventional hematologic and reticulocyte indices by ROC curve analysis using 4 different biochemical diagnostic criteria of ID, CH retic, % Hypo retic, % Low CH retic, and % High CH retic had higher areas under the curve (AUC) than either MCV conv or MCHC conv according to all 4 criteria, and % Macro retic and MCV retic had higher AUC values according to 3 of 4 criteria (differences were not always statistically significant). CONCLUSIONS: Results of this study support the value of reticulocyte indices in the diagnosis and monitoring of ID in dogs. Species similarities in the pathophysiology and hematologic manifestations of ID suggest these findings also may be relevant to ID in people.     

Impact of canine atopic dermatitis on the health‐related quality of life of affected dogs and quality of life of their owners

Atopic dermatitis is a chronic or chronically relapsing human and canine skin disease that adversely affects the quality of life of affected individuals. The impact of the disease on affected children and their parents has been extensively studied, but very little is known of its impact on affected animals and their owners. The goal of the present study was to fill this gap. Owners were asked to complete three questionnaires concerning their own quality of life, their perception of the treatment provided and their pets’ quality of life. Clinical features, such as severity index, pruritus score and disease duration, were evaluated in parallel and correlated with the questionnaire answers. The study not only demonstrated that canine atopic dermatitis deeply influenced the way of life of the owners of affected dogs but also in which aspects of life the impact was most severe.     

Meta-analysis of normal canine echocardiographic dimensional data using ratio indices

ObjectivesTo investigate the dependence of echocardiographic ratio indices (ERIs) on age, body weight (BW) and breed/study group using individually contributed and published summarized data in dogs.BackgroundERIs allow for narrow prediction intervals of M-mode echocardiographic measurements in generic adult dogs. Breed and age-specific differences have not been examined systematically using ERI methods.Animals, materials and methodsIndividual M-mode measurements were contributed by 15 published investigators from 661 dogs, allowing direct calculation of ERIs and summary statistics for each of these breed/study groups. M-mode ERI summary statistics were estimated from published summaries of 22 additional groups that included 527 adult and 36 growing dogs. Individual two-dimensional (2DE) left atrial (LA) and aortic root (Ao) measurements were contributed from 36 dogs. ERIs were analyzed for dependence on BW, breed/study group and age.ResultsThe majority of variation among ERIs was due to differences in the breed or study technique with comparatively little dependence on BW. Age dependence of ERIs was seen in the early growth phases of young dogs, but expected values for each ERI became static long before maturity, roughly at 10–12 weeks of age. ERIs derived from individual 2DE LA and Ao measurements showed no significant dependence on BW.ConclusionsERIs are well normalized for body size and may be useful for clinical evaluation of individuals, prediction of expected M-mode and 2DE cardiac dimensions, and investigation of age or breed-specific cardiac shape changes.     

The animal‐dependent risk factors in canine T‐cell lymphomas

Malignant lymphomas are one of the most common malignancies occurring in dogs; among them T‐cell tumours are less commonly recognized. Recently, many authors have recommended cytology as a sufficient diagnostic method for canine lymphomas, especially if supported by immunocytochemistry or flow cytometry. The aim of the study was to characterize animal‐dependent risk factors in canine T‐cell lymphomas (TCLs) in Poland, including specific cytological subtypes. Determination of the type and subtype of the tumour was made based on the updated Kiel cytological classification adopted for dogs as previously described. Two breeds turned out predisposed to TCL (dog de Bordeaux and Boxer) while no predisposition to B‐cell lymphoma could be evidenced. Dogs with low‐grade lymphoma were significantly older than those with high‐grade lymphoma.     

Efficacy of doxorubicin‐based chemotherapy for non‐resectable canine subcutaneous haemangiosarcoma

Eighteen dogs with measurable subcutaneous haemangiosarcoma (SQHSA) were treated with doxorubicin‐based chemotherapy. Response assessment was evaluated and compared using World Health Organization (WHO), Response Evaluation Criteria in Solid Tumours (RECIST) and tumour volume criteria. The overall response rate for all dogs was 38.8% using WHO criteria, 38.8% using RECIST criteria and 44% using tumour volume criteria. One dog had a complete response. The median response duration for all dogs was 53 days (range 13–190 days). Four dogs had complete surgical excision after neoadjuvant chemotherapy. The median progression‐free interval for dogs with complete surgical excision after neoadjuvant chemotherapy was significantly longer than those not having surgical excision (207 days versus 83 days, respectively) (P = 0.003). No significant difference in metastasis‐free interval or survival time was found between the groups. Doxorubicin‐based chemotherapy appears to be effective for non‐resectable canine SQHSA, although the response duration is relatively short.     

Clinicopathologic features of lingual canine T‐zone lymphoma

Canine T‐zone lymphoma (TZL) is a subtype of T‐cell lymphoma characterized by unique histologic pattern and cytomorphology, immunophenotypic loss of CD45 expression, and an indolent clinical behaviour. Dogs with TZL typically present with 1 or more enlarged lymph nodes and/or lymphocytosis. We describe a novel extranodal presentation of TZL involving the tongue. Twelve dogs with tongue masses were diagnosed with lingual TZL based on a variable combination of immunophenotyping via flow cytometry, cytology, histopathology, immunohistochemistry and/or PCR for antigen receptor rearrangement (PARR) assay. Eleven dogs exhibited concurrent lymphocytosis and/or lymph node enlargement. Three cases were initially diagnosed as plasma cell tumours based on histology alone, thereby revealing a potential diagnostic challenge. Seven dogs achieved clinical remission and 4 achieved stable disease following variable treatment, consistent with the indolent nature of typical TZL involving the lymph nodes and peripheral blood. In 1 case the TZL resulted in progressive disease and failure to respond to treatment. In this case, the TZL exhibited histologic features of a higher grade neoplasm. This case series highlights a unique presentation of TZL and identifies a new differential diagnosis for lingual neoplasia. In this study, we characterize the clinical presentation, diagnostic features and patient outcomes of 12 dogs with lingual TZL.