Quantification of circulating tumour cells over time in dogs with appendicular osteosarcoma

Enumeration of circulating tumour cells (CTC) has shown promise for prognostication and guidance of therapeutic decisions in human cancers. The objective of this study was to enumerate CTC over time in dogs with naturally occurring osteosarcoma (OSA), and to determine correlation with patient outcome. Twenty-six dogs with OSA and no evidence of metastatic disease at the time of amputation were enrolled. Dogs were assessed for lung metastases and CTC prior to and following amputation, and at each chemotherapy visit. Twenty-one dogs completed the study. Nineteen dogs were euthanized and two were alive and free of metastases. Overall survival time ranged from 88 to 1058 days (median survival time (MST) 374 days). Increased serum alkaline phosphatase activity, advanced age, and higher body weight were significantly associated with lower MST. Dogs with OSA had a mean of 356 (0 to 4443) CTC/10⁶ leukocytes. In 12 of 15 dogs that developed radiographic evidence of metastasis, a pre-metastatic CTC spike was retrospectively detectable on average 36.5 (1–100 days) days prior to metastasis and was associated with significantly shorter MST (301 ± 64 vs. 626 ± 55 days; p = .0107). In a multivariable analysis, dogs with a CTC spike were 10× more likely to die compared with those without. These results suggest that a spike in CTC frequency precedes detection of metastasis in dogs with OSA and is associated with shorter survival. More frequent enumeration of CTC in a larger cohort of dogs with OSA may be warranted.     

Development of an ELISA to detect circulating anti‐asparaginase antibodies in dogs with lymphoid neoplasia treated with Escherichia coli l‐asparaginase

Resistance to Escherichia coli l ‐asparaginase in canine lymphoma occurs frequently with repeated administration, a phenomenon often attributed, without substantiation, to the induction of neutralizing antibodies. To test the hypothesis that treated dogs develop antibodies against the drug, we created an enzyme‐linked immunosorbent assay (ELISA) to measure plasma anti‐asparaginase immunoglobulin G responses. Using samples from dogs that had received multiple doses, specific reactivity against l ‐asparaginase was demonstrated, while naïve patients' samples were negative. The optimized ELISA appeared sensitive, with endpoint titers >1 600 000 in positive control dogs. Intra‐ and inter‐assay coefficients of variation were 3.6 and 14.5%. The assay was supported by the observation that ELISA‐positive plasma could immunoprecipitate asparaginase activity. When clinical patients were evaluated, 3/10 dogs developed titers after a single injection; with repeated administration, 4/7 dogs were positive. l ‐asparaginase antibodies showed reduced binding to the PEGylated drug formulation. The ELISA should prove useful in investigating the potential correlation of antibody responses with resistance.     

Prior joint disease is associated with increased risk of periarticular histiocytic sarcoma in dogs

Periarticular histiocytic sarcoma (PAHS) is the most common synovial tumour in dogs and is characterized by aggressive local disease with a high rate of distant metastasis. Previously, an association between PAHS and prior joint disease has been demonstrated in the Bernese Mountain Dog breed and suggested in the Rottweiler. We hypothesized that this association would be present in other breeds and investigated this via a retrospective, case‐controlled analysis. Cases were dogs diagnosed with PAHS of the stifle or elbow. Controls were age, breed and sex‐matched dogs without a diagnosis of histiocytic sarcoma. Diagnosis of prior joint disease was determined based on review of medical records and direct veterinarian and owner communications. Data were evaluated using logistic regression, 2‐sampled t tests, and chi‐squared analysis. Our study population consisted of 28 cases and 46 controls, including Flat‐Coated, Golden and Labrador Retrievers, Rottweilers, English Bulldogs, Shih Tzus, Australian Shepherds, Staffordshire Terriers and mixed breed dogs. Dogs with PAHS were more likely to have prior joint disease in the tumour‐affected joint compared with the control population (odds ratio [OR] = 13.42, P < .0001, 95% confidence interval [CI] = 4.33‐48.63). A total of 88.2% of dogs with stifle PAHS had prior joint disease in their tumour‐affected joint, most commonly cranial cruciate ligament rupture. This study confirms that the previously noted association between prior joint disease and PAHS in Bernese Mountain Dogs also applies to other breeds. Additional studies are needed to further investigate for a causal relationship.     

Preliminary investigation of blood concentrations of insulin‐like growth factor,insulin, lactate and β‐hydroxybutyrate in dogs with lymphoma as compared with matched controls

It is well established that tumour cells have metabolic differences when compared with normal cells. This is particularly true for energy metabolism in which dogs with cancer have been reported to have higher blood insulin and lactate concentrations than control dogs. Moreover, some human and animal studies suggest that the insulin‐like growth factor 1 (IGF‐1) signalling pathway may play a role in tumorigenesis and tumour progression. At present, IGF‐1 has not been evaluated in dogs with multicentric lymphoma. In this prospective, cross‐sectional study, blood levels of IGF‐1, as well as other markers of energy metabolism—insulin, glucose, lactate, and β‐hydroxybutyrate—were measured in 16 dogs with histologically or cytologically confirmed treatment‐naïve lymphoma. These results were compared with 16 age‐, sex‐ and weight‐matched healthy controls. Dietary histories were collected, and protein, fat and carbohydrate intake were compared between groups. Results demonstrated that IGF‐1, insulin, glucose and insulin:glucose ratio were not different between groups. However, lactate and β‐hydroxybutyrate were higher in the dogs with lymphoma than that in the control dogs (1.74 ± 0.83 mmoL/L vs 1.08 ± 0.27 and 2.59 ± 0.59 mmol/L vs 0.77 ± 0.38 mmol/L, respectively). Median dietary protein, fat and carbohydrates did not differ between the groups. This preliminary study suggests that higher insulin and IGF‐1 levels relative to controls may not be a consistent finding in dogs with lymphoma. The significance of increased β‐hydroxybutyrate in dogs with lymphoma warrants further investigation in a larger prospective study.     

The growth of the canine glioblastoma cell line D‐GBM and the canine histiocytic sarcoma cell line DH82 is inhibited by the resveratrol oligomers hopeaphenol and r2‐viniferin

Vineatrol^®30 is a grapevine‐shoot extract, which contains resveratrol as well as considerable amounts of so‐called resveratrol oligomers such as hopeaphenol and r2‐viniferin. In this study, we analysed whether the two above‐mentioned resveratrol oligomers were able to inhibit the growth of the canine glioblastoma cell line D‐GBM and the canine histiocytic sarcoma cell line DH82, compared their potency to inhibit tumour cell growth with that of resveratrol and determined whether the induction of apoptosis via caspase 9 and 3/7 activation underlies the tumour cell growth‐inhibiting effect of hopeaphenol and r2‐viniferin. Vineatrol^®30, resveratrol, hopeaphenol and r2‐viniferin inhibited the growth of D‐GBM and DH82 cells in a concentration‐dependent manner, whereby hopeaphenol and r2‐viniferin were more potent than resveratrol itself in inhibiting the growth of the canine tumour cell lines. Moreover, the anti‐proliferative effect of both resveratrol oligomers in D‐GBM cells is based on their capacity to induce caspase 9 and 3/7 activation.     

Patient characteristics,prognostic factors and outcome of dogs with high‐grade primary mediastinal lymphoma

The goals of this retrospective study were to determine the patient characteristics of dogs with high‐grade primary mediastinal lymphoma and to determine outcome and associated prognostic factors. A total of 42 dogs were identified, in which 36 received treatment and had follow‐up information available. The most common clinical signs included lethargy, anorexia and polyuria/polydipsia. Hypercalcemia and pleural effusion were common findings at diagnosis. The phenotype was almost exclusively T‐cell, most often in association with lymphoblastic cytomorphology as defined by the World Health Organization (WHO) lymphoma classification scheme. The overall progression‐free survival (PFS) and overall survival (OS) were 133 and 183 days, respectively. Treatment with a CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) protocol was associated with an improved PFS (144 days) and OS (194 days) when compared with dogs that received other medical therapies (P = .005 and P = .002, respectively); the absence of pleural effusion at diagnosis was associated with an increased OS but not PFS. These results suggest that while the prognosis for dogs with mediastinal lymphoma is poor, survival may be improved with treatment using a CHOP‐based protocol.     

Anti‐tumour activity of oncolytic reovirus against canine histiocytic sarcoma cells

Canine histiocytic sarcoma is an aggressive, fatal neoplastic disease with a poor prognosis. Lomustine is generally accepted as the first‐line systemic therapy, although this compound does not provide complete regression. Therefore, research into a novel approach against canine histiocytic sarcoma is needed. However, anti‐tumour effects of oncolytic therapy using reovirus against histiocytic sarcoma are unknown. Here, we showed that reovirus has oncolytic activity in canine histiocytic sarcoma cell lines in vitro and in vivo. We found that reovirus can replicate and induce caspase‐dependent apoptosis in canine histiocytic sarcoma cell lines. A single intra‐tumoural injection of reovirus completely suppressed the growth of subcutaneously grafted tumours in NOD/SCID mice. Additionally, we demonstrated that susceptibility to reovirus‐induced cell death was attributable to the extent of expression of type I interferons induced by reovirus infection in vitro. In conclusion, oncolytic reovirus appears to be an effective treatment option for histiocytic sarcoma, and therefore warrants further investigation in early clinical trials.     

Cytochemical staining characteristics of lymph nodes from normal and lymphoma-affected dogs

Frozen sections and imprint smears were used to evaluate the presence and pattern of cytochemical staining reactions in the B- and T-cell regions of lymph nodes from normal dogs and dogs with lymphoma. Staining procedures evaluated included peroxidase (PER), Sudan black B (SBB), naphthol AS-D chloroacetate esterase (CAE), alpha-naphthyl butyrate esterase (NBE), acid phosphatase (ACP), and leukocyte alkaline phosphatase (LAP). In normal lymph nodes, macrophages and some lymphocytes within the interfollicular (T-cell) region and medulla stained positive with ACP and NBE. Smaller numbers of macrophages also occurred sporadically within the germinal follicles. Cells positive for PER, SBB, and CAE were scattered infrequently throughout all regions of the normal lymph node, consistent with granulocytes and mast cells. The LAP stained cells were predominantly and prominently located within the mantle zone of secondary follicles and to a much lesser extent within the germinal centers, compatible with B-cell lymphocytes derived from follicular center cells. Of the 12 dogs with lymphoma, 7 cases (4 immunoblastic, 2 large noncleaved, 1 small noncleaved) stained diffusely positive with LAP, 4 cases (all lymphoblastic) had numerous focally positive lymphocytes using ACP and NBE, and 1 case (immunoblastic) did not stain positive with any of the cytochemical reactions. Cytochemical staining of canine lymph nodes with NBE, ACP, and LAP proved useful in distinguishing between B- or T-cell regions and detecting different cell types of canine lymphoma.     

Serum concentrations of monocyte chemoattractant protein‐1 in healthy and critically ill dogs

Background: The chemokine monocyte chemoattractant protein‐1 (MCP‐1) is a primary regulator of monocyte mobilization from bone marrow, and increased concentrations of MCP‐1 have been associated with sepsis and other inflammatory disorders in critically ill people. The relationship between MCP‐1 and disease in dogs has not been evaluated previously. Objective: The purpose of this study was to assess serum concentrations of MCP‐1 in healthy dogs, dogs in the postoperative period, and critically ill dogs. We hypothesized that MCP‐1 concentrations would be significantly increased in critically ill dogs compared with postoperative or healthy dogs. Methods: Serum concentrations of MCP‐1 were measured in 26 healthy control dogs, 35 postoperative dogs, and 26 critically ill dogs. Critically ill dogs were further subgrouped into dogs with sepsis, parvovirus gastroenteritis, immune‐mediated hemolytic anemia, and severe trauma (n=26). MCP‐1 concentrations were determined using a commercial canine MCP‐1 ELISA. Associations between MCP‐1 concentrations and disease status were evaluated statistically. Results: MCP‐1 concentration was significantly higher in critically ill dogs (median 578 pg/mL, range 144.7–1723 pg/mL) compared with healthy dogs (median 144 pg/mL, range 4.2–266.8 pg/mL) and postoperative dogs (median 160 pg/mL, range 12.6–560.4 pg/mL) (P<.001). All subgroups of critically ill dogs had increased MCP‐1 concentrations with the highest concentrations occurring in dogs with sepsis. However, differences among the 4 subgroups were not statistically significant. Conclusion: Critically ill dogs had markedly increased serum concentrations of MCP‐1 compared with postoperative and healthy dogs. These results indicate that surgery alone is not sufficient to increase MCP‐1 concentrations; thus, measurement of MCP‐1 may be useful in assessing disease severity in critically ill dogs.     

Successful use of prednisolone and radiation therapy in a dog with intracranial histiocytic sarcoma

The ideal treatment for intracranial histiocytic sarcoma (HS) remains unclear. Herein, we report a case of intracranial HS that was successfully treated using prednisolone and radiation therapy. The patient was a 9-year-old spayed female Pembroke Welsh Corgi that presented with epileptic seizures. Magnetic resonance imaging revealed a contrast-enhancing mass adjacent to the right piriform lobe. Prednisolone administration (1 mg/kg/day) decreased the lesion size. Additional palliative radiation therapy (total dose, 37 Gy) resulted in complete disappearance of the lesion. However, on day 164, the dog’s neurological signs deteriorated, and she was euthanized. Necropsy revealed an intracranial metastasis of HS via the cerebrospinal fluid without any extracranial metastasis. Nonetheless, combined prednisolone and radiation therapy might be effective in treating intracranial HS.     

B‐cell lymphoma with Mott cell differentiation in two young adult dogs

Abstract: Two young adult dogs with gastrointestinal signs were each found to have an intra‐abdominal mass based on physical examination and diagnostic imaging. On exploratory laparotomy, small intestinal masses and mesenteric lymphadenopathy were found in both dogs; a liver mass was also found in dog 1. Cytologic and histologic examination of intestinal and liver masses and mesenteric lymph nodes revealed 2 distinct lymphoid cell populations: lymphoblasts and atypical Mott cells. With Romanowsky stains, the atypical Mott cells contained many discrete, clear to pale blue cytoplasmic inclusions consistent with Russell bodies that were positive by immunohistochemistry for IgM and CD79a in both dogs and for IgG in dog 2. The Mott cells and occasional lymphoblasts stained strongly positive with periodic acid‐Schiff. Using flow cytometric immunophenotyping in dog 1, 60% of peripheral blood mononuclear cells and 85% of cells in an affected lymph node were positive for CD21, CD79a, IgM, and MCH II, indicative of B‐cells. With electron microscopy, disorganized and dilated endoplasmic reticulum was seen in Mott cells in tumors from both dogs. Antigen receptor gene rearrangement analysis of lymph node and intestinal masses indicated a clonal B‐cell population. Based on cell morphology, tissue involvement, and evidence for clonal B‐cell proliferation, we diagnosed neoplasms involving Mott cells. To the authors' knowledge, this is the second report of Mott cell tumors or, more appropriately, B‐cell lymphoma with Mott cell differentiation, in dogs. More complete characterization of this neoplasm requires further investigation of additional cases. This lymphoproliferative disease should be considered as a differential diagnosis for canine gastrointestinal tumors.     

Hyperferritinemia is associated with short survival time in dogs with multicentric lymphoma

In the present study, we examined the relationship between serum ferritin concentration before treatment and survival time in dogs with multicentric lymphoma. Eighteen dogs with multicentric lymphoma were enrolled in the study. When the dogs were classified into high and low ferritin groups on the basis of their serum ferritin concentration (3,000 ng/ml cut-off value), the median survival time of dogs with high concentrations (≥3,000 ng/ml, n=7) was 40 days, whereas it was 360 days among dogs with low concentrations (<3,000 ng/ml, n=11). This difference was statistically significant (P=0.001). This finding suggests that the initial high level of serum ferritin indicates short survival time in dogs with multicentric lymphoma. Large-scale research is necessary to confirm this finding.     

Clinical characteristics and outcome in dogs with small cell T‐cell intestinal lymphoma

Small cell intestinal lymphoma has not been well characterized in dogs. The objective of this study was to describe clinical characteristics and outcome in dogs with small cell intestinal lymphoma. We hypothesized that affected dogs would have prolonged survival compared with high‐grade gastrointestinal (GI) lymphoma. Pathology records were searched for dogs with histologically confirmed small cell GI lymphoma. Seventeen dogs with confirmed small cell intestinal lymphoma were identified, and clinical and outcome data were retrospectively collected. Histopathology was reviewed by a board‐certified pathologist, and tissue sections were subjected to immunophenotyping and molecular clonality assessment. All dogs had small cell, T‐cell, lymphoma confirmed within various regions of small intestine, with 1 dog also having disease in abdominal lymph nodes. All dogs had clinical signs attributable to GI disease; diarrhoea (n = 13) was most common. Ultrasonographic abnormalities were present in 8 of 13 dogs with abnormal wall layering (n = 7) and hyperechoic mucosal striations (n = 7) representing the most common findings. In total, 14 dogs received some form of treatment. The median survival time (MST) for all dogs was 279 days and the MST for the 14 dogs that received any treatment was 628 days. Dogs with anaemia and weight loss at presentation had significantly shorter survival times and dogs that received a combination of steroids and an alkylating agent had significantly longer survival times. Small cell, T‐cell, intestinal lymphoma is a distinct disease process in dogs, and those undergoing treatment may experience prolonged survival.     

Prospective evaluation of flow cytometric characteristics,histopathologic diagnosis and clinical outcome in dogs with naïve B‐cell lymphoma treated with a 19‐week CHOP protocol

Canine B‐cell lymphoma is a clinically heterogenous disease; however, it is generally treated as a single disease entity. The purpose of this clinical trial was to prospectively evaluate naïve canine B‐cell lymphoma patients using histopathology, flow cytometry (FC) and a standardized chemotherapy protocol to better define subsets of this disease that may respond differently to treatment. Sixty‐four dogs with naïve multicentric B‐cell lymphoma were treated with a standardized 19‐week CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy protocol. Most of the dogs (84.3%) were diagnosed with diffuse large B‐cell lymphoma (DLBCL), followed by nodal marginal zone (7.8%), small B‐cell (4.7%), Burkitt‐like (1.6%) and follicular lymphoma (1.6%). FC confirmed the diagnosis of B‐cell lymphoma in all cases. There were no clear phenotyping differences between the subtypes of B‐cell lymphoma detectable by our FC panel. The histologic subtypes in this study exhibited a range of forward scatter values on flow cytometry, but all of the DLBCL cases were higher than a value of 469, while the only cases with a lower forward scatter value were follicular lymphoma and diffuse small B‐cell lymphoma. Dogs with DLBCL had a significantly better objective response rate to the CHOP protocol (96.3%) than the non‐DLBCL subtypes (70%, P = .024). The median progression‐free survival time for patients with DLBCL (233 days) was significantly longer than that of all other histopathologic subgroups combined (163 days, P = .0005).