Association of argyrophilic nucleolar organizing regions, Ki-67, and proliferating cell nuclear antigen scores with histologic grade and survival in dogs with soft tissue sarcomas: 60 cases (1996-2002)

OBJECTIVE: To determine whether argyrophilic nucleolar organizing regions (AgNORs), Ki-67, and proliferating cell nuclear antigen (PCNA) scores were associated with histologic grade and survival in dogs with soft tissue sarcomas (STSs). DESIGN: Retrospective study. ANIMALS: 60 dogs with STSs. PROCEDURE: Medical records were examined and histologic specimens were reviewed. Tissue specimens obtained from archival materials were used to prepare sections for histologic staining for AgNOR and immunohistochemical staining for Ki-67 and PCNA labeling. Follow-up monitoring was obtained by reevaluation or telephone conversations with referring veterinarians or owners. RESULTS: 27 (45%) STSs were grade 1, 23 (38%) were grade 2, and 10 (17%) were grade 3. The mean and median AgNOR, Ki-67, and PCNA scores were determined, and significant positive associations among AgNOR and Ki-67 scores with histologic grade and mitotic score were detected. Fifty-four dogs had adequate follow-up examinations and were included in survival analysis and evaluation of prognostic factors. Overall median survival time was > 1,306 days. Twelve of 54 (22%) dogs died of tumor-related causes. Metastatic disease developed in 8 of 54 (15%) dogs. Results of univariate analysis indicated that increased mitotic score, increased AgNOR score, increased Ki-67 score, incomplete surgical margins, noncurative intent surgery, Ki-67 score greater than the median Ki-67 score, and AgNOR score greater than the median AgNOR score were prognostic factors for decreased survival time. Results of multivariate analysis indicated that increased AgNOR score was the only prognostic factor for decreased survival time. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that AgNORs and possibly Ki-67 should be routinely evaluated with histologic grading for STSs in dogs.     

Urine N-telopeptide excretion in dogs with appendicular osteosarcoma

Canine appendicular osteosarcoma (OSA) is a commonly diagnosed cancer that is capable of inducing pathologic bone remodeling. Investigating surrogate indices of bone metabolism may contribute to the diagnostic and therapeutic management of bone malignancies in companion animals. This study evaluated the excretion of N-terminal telopeptide (NTx), a marker of bone resorption that is detected in urine. Sixty-three dogs with appendicular OSA were compared with 29 age-matched healthy dogs. Dogs with appendicular OSA had significantly higher baseline urine NTx excretion than healthy controls (P < .0001). In 17 dogs with OSA treated with either amputation or standardized palliative therapies, significant reductions in urine NTx excretion were observed, suggesting that excessive bone resorption in dogs with OSA may be linked with focal skeletal osteolysis or its consequences. To identify any relationship between indicators of pathologic bone turnover, baseline urine NTx excretion was correlated with serum bone alkaline phosphatase (bALP) or radiographic tumor lengths at diagnosis. No significant correlations were identified between baseline urine NTx excretion and either bALP or tumor length. The findings from this study suggest that high urinary NTx excretion may support the diagnosis of focal skeletal osteolysis in dogs, and reductions in urine NTx excretion after treatment may reflect elimination or minimization of pathologic bone resorption.     

Risk factors for sterile hemorrhagic cystitis in dogs with lymphoma receiving cyclophosphamide with or without concurrent administration of furosemide: 216 cases (1990-1996)

OBJECTIVES: To determine incidence and identify predisposing factors for sterile hemorrhagic cystitis (SHC) in dogs with lymphoma that were treated with cyclophosphamide and to evaluate whether furosemide administered i.v. concurrently with cyclophosphamide decreased the incidence of SHC. DESIGN: Retrospective study. ANIMALS: 216 dogs with lymphoma. PROCEDURE: Medical records of dogs with lymphoma that received cyclophosphamide chemotherapy in accordance with 1 of 2 protocols, with or without concurrent i.v. administration of furosemide, were examined. Data for the 2 groups were analyzed to determine the incidence and predisposing factors (age, breed, sex, weight, previous or preexisting disease, previous or preexisting urinary tract infection, neutropenia, azotemia, dose, and number of cyclophosphamide treatments) for cyclophosphamide-associated SHC. RESULTS: Cyclophosphamide-associated SHC developed in 12 of 133 (9%) dogs that had not received concurrent administration of furosemide and cyclophosphamide treatments; of the 83 dogs that had received furosemide, only 1 (1.2%) developed SHC. Dogs receiving cyclophosphamide and furosemide concurrently were significantly less likely to develop SHC than dogs that did not receive furosemide. Dogs with previous or preexisting immune-mediated disease were significantly more likely to develop cyclophosphamide-associated SHC. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of results suggested an association between i.v. administration of furosemide concurrently with cyclophosphamide and decreased incidence of cyclophosphamide-associated SHC. Incidence of cyclophosphamide-associated SHC was similar in treated dogs that did not receive concurrent furosemide to that observed for other studies in which cyclophosphamide was administered orally. Cyclophosphamide-associated SHC appeared to develop early during the course of chemotherapy when furosemide was not administered concurrently with cyclophosphamide.     

A comparison of two pamidronate dosing protocols on bone turnover in normal dogs and dogs with osteosarcoma

Introduction: Dogs with appendicular osteosarcoma (OSA) excrete higher concentrations of urine cross‐linked N‐telopeptide of type I collagen (NTx) than normal dogs. NTx is a specific biochemical marker of osteoclastic activity. Pamidronate is a bone‐modulating agent that exerts potent inhibitory effects on osteoclasts. The use of pamidronate is currently being evaluated for the management of osteolytic bone pain in dogs with appendicular osteosarcoma. Despite pamidronate's increasing usage in veterinary oncology, optimal dosing has yet to be determined. Commonly utilized dosages range from 1–2 mg/kg, given intravenously (IV) as a 2‐hour constant rate infusion every 28 days. The purpose of this prospective study was to compare the biological activity of two pamidronate doses (1 mg/kg vs. 2 mg/kg) in the suppression of urine NTx excretion in normal dogs and dogs with appendicular osteosarcoma. Methods: Seventeen OSA dogs receiving single‐agent pamidronate as palliative therapy were evaluated. Group 1A (n = 10) received a dose of 1 mg/kg and group 2A (n = 7) received a dose of 2 mg/kg IV. Urine NTx level were measured at day 0 and 28 using a commercial ELISA (Ostex International). Urine NTx level were also measured in 6 normal dogs: Group 1B (n = 3) received a dose of 1 mg/kg and group 2B (n = 3) received a dose of 2 mg/kg. In normal dogs, urine NTx levels were recorded weekly for six consecutive weeks. Results: In dogs with osteosarcoma, greater reductions in urine NTx excretion from baseline values were demonstrated at 2 mg/kg versus 1 mg/kg (57% and 23%, respectively). Likewise, in normal dogs, urine NTx excretion was suppressed to a greater extent with a dosage of 2 mg/kg versus 1 mg/kg (69% and 23%, respectively). Conclusion: Pamidronate possesses biologic activity in both normal dogs and in dogs with osteosarcoma, as assessed by reductions in urine NTx excretion. Based upon reductions in urine NTx excretion, a dosage of 2 mg/kg appears more effective than 1 mg/kg.     

Differentiating effect of pamidronate on canine osteo‐sarcoma cell lines in vitro

Introduction: Pamidronate has been traditionally used to manage pathologic osteoclastic disorders. In addition to its effects on osteoclasts, pamidronate has also been demonstrated to promote phenotypic maturation and inhibition of proliferation in osteoblasts. Canine osteosarcoma (OSA) consists of malignant, undifferentiated osteoblasts. The objective of this study was to determine if micromolar concentrations of pamidronate could induce malignant osteoblastic differentiation as evaluated by an increase in alkaline phosphatase (ALP) activity and/or osteocalcin (OC) production, two specific markers of normal osteoblastic activity. Methods: Two canine OSA cell lines (HMPOS and COS 31) were used for all experiments. Cells were incubated for 48 or 72 hours with various pamidronate concentrations (0, 0.1, 1, 5, 10, and 20 μM). After incubation, the supernatants were sampled and the relative amounts of viable cells were determined with a cell proliferation assay (Cell Titer 96^® AQ_uous, Promega). An ALP detection kit (Starbright^®, Sigma^®) was used to measure the ALP activity and an ELISA (Osteocalcin EIA kit, Biomedical Technologies) was used to determine the concentration of osteocalcin in the supernatants. The ALP and osteocalcin values were corrected for the amount of viable cells. Results: Pamidronate induced a dose‐dependent reduction in the number of viable COS 31 and HMPOS cells at both 48 and 72 hours. A dose‐dependent elevation in ALP activity from baseline was observed. At 20 μM, a 2.3‐fold increase was observed for HMPOS at 72 hours, while a 1.43‐fold increase was observed for COS 31 at 72 hours. Very low level (less than 2 ng/ml) of osteocalcin pre‐ and post‐pamidronate treatment was detected for both COS 31 and HMPOS. Conclusion: The data suggests that pamidronate increases alkaline phosphatase activity in canine OSA cells in a dose‐dependent manner. However, cytotoxic assays are needed in order to accurately characterize any concurrent decrease in the number of viable cells. The potential differentiating effect of pamidronate on malignant osteoblasts provides an additional argument for its use in the palliative treatment of OSA.