Investigating the molecular mechanism of pamidronate‐induced in vitro cytotoxicity in canine osteosarcoma cells

Introduction: Pamidronate is used to treat metastatic bone lesions in human cancer patients. By directly inhibiting the mevalonate pathway, pamidronate disrupts GTPase‐binding protein prenylation, resulting in osteoclast apoptosis. Pamidronate has been demonstrated to exert dose‐ and time‐dependent cytotoxic effects in several canine malignant osteoblastic cell lines. However, the exact cytotoxic mechanism remains speculative. The purpose of this study was to investigate and characterize the molecular mechanism of pamidronate‐induced cytotoxicity in canine malignant osteoblasts. Methods: The involvement of farnesyl or geranylgeranyl pyrophosphate synthase inhibition was evaluated by performing rescue experiments with cells incubated for 48 hours with cytotoxic concentrations of pamidronate (50 and 100 μM), with or without farnesol (FOH) and geranylgeraniol (GGOH). Cell lysates were fluorometrically‐assessed for caspase‐3‐like activity (R&D Systems) following incubation with varying concentrations of pamidronate (control, 50 and 100 μM) for 48 hours. The expression of a prenylated GTPase protein, Rap1 A, was qualitatively evaluated with immunoblotting using a polyclonal goat antibody (Santa Cruz Biotechnology) in cells incubated for 48 hours with varying concentrations of pamidronate (0, 1, 10, 50, and 100 μM). Results: In cells treated with lower cytotoxic concentrations of pamidronate (50 μM) for 48 hours, the addition of GGOH, but not FOH, was able to diminish the degree cytotoxicity, p ≤ 0.05. However, cells incubated with higher cytotoxic concentrations of pamidronate (100 μM), neither GGOH nor FOH were able to reduce the level of pamidronate‐induced cytotoxicity. Caspase‐3‐like activity directly correlated with the degree of pamidronate‐induced cytotoxicity. Cells treated with pamidronate at 50 μM and 100 μM increased caspase‐3‐like activity by 5.3 and 7.1‐fold, respectively over untreated controls. The detection of Rap1A by immunoblotting requires further optimization. Conclusions: Pamidronate‐induced cytotoxicity in canine osteosarcoma cells may share a similar mechanism as has been demonstrated for osteoclasts. Inhibition of the mevalonate pathway appears to contribute to the observed cytotoxicity at lower doses of pamidronate. In addition, caspase‐3‐like activity contributes to the apoptotic process induced by pamidronate in malignant canine osteoblasts.     

Preliminary evaluation of serial (18) FDG-PET/CT to assess response to toceranib phosphate therapy in canine cancer

Palladia(TM) (toceranib phosphate-Pfizer Animal Health) is a novel orally administered receptor tyrosine kinase inhibitor (TKI) approved for treatment of canine mast cell tumors. Receptor tyrosine kinase dysregulation leads to tumor growth, progression, and metastasis. Toceranib's targets include vascular endothelial growth factor receptor (VEGFR-2/Flk-1/KDR), platelet-derived growth factor receptor, and kit. Positron Emission Tomography/Computed Tomography (PET/CT) is used commonly to diagnose, prognosticate, and monitor response to antineoplastic therapy in human patients. In this study, serial PET/CT imaging with (18) F-fluorodeoxyglucose ((18) FDG) was used to assess response to toceranib therapy in dogs with measurable solid malignancies. Six tumor-bearing dogs underwent tumor assessment using both standard RECIST criteria and PET/CT prior to and at a median of 5 weeks postinitiation of toceranib treatment. Toceranib was prescribed initially at a target dose 3.25 mg/kg PO q48 h, with subsequent modifications based on observed toxicity. Treatment was continued in patients achieving stable disease with acceptable drug tolerance. One dog was maintained on drug despite dose modification due to toxicity; measurable clinical and image-based responses were seen after 10 weeks of therapy. All others had stable or progressive disease based on clinical restaging and PET/CT at first recheck. . Due to discordance with anatomic and metabolic imaging, further studies are needed to investigate the role of molecular imaging in assessment of drug response and identify other potential molecular targets of toceranib.     

Efficacy of C/B‐OPP rescue for the treatment of relapsed canine lymphoma (1998–2004)

Introduction:  MOPP chemotherapy is useful for relapsed canine lymphoma. This study evaluates the efficacy of this protocol after substitution of CCNU (lomustine) or BiCNU (carmustine) for mechlorethamine (C/B‐OPP).
Methods:  Patient signalment, response to chemotherapy, toxicity and survival data were abstracted from medical records of dogs from receiving C/B‐OPP between 1998 and 2004.
Results:  Fifty‐eight dogs received C/B‐OPP rescue chemotherapy during the study period. The median remission duration after initial chemotherapy, consisting of CHOP‐based therapy in 91% of dogs, was 133 days (range, 10 to 932 days). Thirty‐eight of fifty‐eight dogs (66%) responded to C/B‐OPP rescue after relapse (22 CR, 16 PR), for a median of 48 days (range, 2 to 359 days). Overall, C/B‐OPP extended survival by a median of 90 days (range, 2 to 426 days). Twenty‐four dogs (41%) experienced one or more episodes of Grade II or higher gastrointestinal toxicity. Forty‐one dogs (71%) experienced one or more episodes of Grade II or higher hematologic toxicity. Twelve dogs (20%) developed regenerative anemia with diarrhea consistent with gastrointestinal hemorrhage. Treatment delays due to hematologic toxicity occurred in 37 dogs (63%). There were 16 nonfatal treatment‐related episodes of sepsis requiring hospitalization. 5 dogs died due to sepsis and/or chemotherapy‐related complications.
Conclusions:  C/B‐OPP chemotherapy has activity against relapsed canine lymphoma which is similar to that of traditional MOPP rescue therapy. Moderate to severe hematologic toxicity was observed. Further work is warranted to optimize drug doses and scheduling.     

Serologic response of maned wolves (Chrysocyon brachyurus) to canine and canine parvovirus vaccination distemper virus.

This study evaluated the immune response of 47 (22 males, 25 females) captive maned wolves (Chrysocyon brachyurus) to modified-live canine parvovirus and canine distemper virus (Onderstepoort and Rockborn strains) vaccines. Sera were collected from 33 adults and 14 pups, including five free-ranging pups captured at 1 yr of age or younger. All the adults and four captive-born pups had been vaccinated prior to this first blood collection. Virus neutralization and hemagglutination-inhibition assays were performed for quantitating antibodies against canine distemper and canine parvovirus, respectively. Distemper antibody titers > or = 100 were present in 57% of adults and 14% of pups. All adults and 29% of pups had parvovirus antibody titers > or = 80. After vaccination, 72% of the wolves developed antibody titers > or = 100 against distemper and 98% developed titers > or = 80 against parvovirus. Both vaccines used were safe and immunogenic to juvenile and adult maned wolves, regardless of prior vaccination history.     

Biological behaviour of canine mandibular osteosarcoma. A retrospective study of 50 cases (1999–2007)

The purpose of this retrospective study was to describe the biological behaviour of canine mandibular osteosarcoma (OSA) and to examine factors for their impact on metastasis‐free interval (MFI) and survival time (ST). Records from dogs treated with mandibulectomy for OSA (1999–2007) were reviewed. Archived tumour samples were evaluated for mitotic index (MI) and tumour grade. Fifty dogs were included, 21 received chemotherapy. Twenty‐nine dogs (58%) developed metastatic disease. The median MFI was 627 days, and median ST was 525 days. In univariate analysis MI > 40 was prognostic for decreased MFI and ST. Grade also influenced MFI and ST, with 5/21 (24%) dogs with grade II/III tumours metastasis‐free at one year versus 16/22 (72%) dogs with grade I tumours (P = 0.002); and 5/21 (24%) dogs with grade II/III tumours alive versus 17/22 (77%) dogs with grade I tumours (P = 0.001). In multivariate analysis, histological grade and adjuvant chemotherapy were prognostic for MFI and ST.     

Monitoring of canine parvovirus (CPV) strains detected in vaccinated puppies in Brazil

The objective of this study was to investigate, by partial sequencing of VP2 protein, the variability of CPV detected in 37 fecal samples collected from vaccinated puppies with enteritis. Laboratorial diagnosis of CPV was confirmed by HA/HI and PCR and, for sequencing analyses, two different regions of the VP2 gene were amplified by PCR. From 1995 to 2004, all strains were characterized as CPV-2a. After that, both CPV-2a and CPV-2b were detected. All CPV-2a showed a non-synonymous mutation in the residue 297 (Ser → Ala). A synonymous substitution at the AA 574 was also observed in 15/37 samples. Our findings indicate that the cases of vaccine failure are most likely not associated to the mutations detected in the sequenced regions. However, the monitoring of genotyping mutations that led to new CPV strains is essential to determinate if current vaccines will keep providing protection against all new future variants.     

Radiotherapy for canine chronic superficial keratitis using soft X-rays (15 kV)

Objective  To evaluate the effect of soft X-ray therapy in the treatment of refractory chronic superficial keratitis (CSK).
Animals studied  Thirteen dogs with severe CSK, that had been refractory to prior medical and/or surgical therapy were treated with soft X-ray therapy.
Procedures  Both corneas of each dog were irradiated with soft X-rays (15 kV), to a total dose of 30 Gy, administered as two fractions over 48–96 h. Treatment was carried out under deep sedation in all dogs. Three dogs were treated by superficial lamellar keratectomy 48 h prior to radiotherapy. Changes in the extent of corneal pigmentation, pigment density and corneal vascularization were documented using a semi-quantitative grading scheme, schematic drawings and clinical photographs.
Results  Only minor, transient adverse effects of treatment, such as photophobia, epiphora and blepharitis were noted. Overall the effect of soft X-rays on the course of the keratitis was superior when compared to the effect of Sr-90 irradiation that had been determined in a previous study.
Conclusion  Soft X-ray irradiation combined with keratectomy is a safe and effective new treatment option for severe and advanced CSK with significant visual impairment due to corneal pathology.     

Low concentrations of cerebrospinal fluid GABA correlate to a reduced response to phenobarbital therapy in primary canine epilepsy

In this study, we investigated whether pretreatment cerebrospinal fluid (CSF) neurotransmitter concentrations of gamma-aminobutyric acid (GABA) and glutamate (GLU) were correlated with response to phenobarbital treatment in dogs with primary epilepsy. Eleven untreated dogs, 6 males and 5 females, with a median age of onset of seizures of 3 years (range: 0.5-5 years) were selected for therapy based on progressive or serious seizure patterns. The median interval between the first observed seizure and start of phenobarbital therapy was 485 days (range: 101-1,765 days). All dogs were purebred, with the exception of I male dog. Oral phenobarbital was started at 2.5 mg/kg every 12 hours. Trough serum phenobarbital concentrations were measured at 15, 45, 90, 180, 360, 540, and 720 days after the start of treatment. There was no difference in the mean trough serum concentration or in the mean number of seizures recorded between each time period of phenobarbital measurement over the 2-year evaluation. No correlation was found between CSF GLU, GABA, or GLU: GABA ratio and the total number of seizures recorded before or after initiation of phenobarbital therapy. Lower CSF GABA concentration, however, was correlated with a lower seizure frequency difference (the total number of seizures before phenobarbital therapy minus the total number of seizures after phenobarbital therapy for an identical time period of evaluation) and lower percentage reduction in seizures: ([total number of seizures before phenobarbital therapy minus the total number of seizures after phenobarbital therapy] divided by the total number of seizures before phenobarbital therapy) x 100. There was no correlation between CSF GLU and the seizure frequency difference and percentage reduction in seizures. A negative correlation between the CSF GLU:GABA ratio and seizure frequency difference was found. Thus, dogs with an initial lower CSF GABA concentration before phenobarbital therapy did not respond as well as did dogs with a higher CSF GABA concentration.     

Clinical and serological response of wild dogs (Lycaon pictus) to vaccination against canine distemper,canine parvovirus infection and rabies

Wild dogs Lycaon pictuis (n = 8) were vaccinated 4 times against canine distemper (n = 8) (initially with inactivated and subsequently with live attenuated strains of canine distemper) and canine parvovirus infection (n = 8) over a period of 360 days. Four of the wild dogs were also vaccinated 3 times against rabies using a live oral vaccine and 4 with an inactivated parenteral vaccine. Commercially-available canine distemper, canine parvovirus and parenteral rabies vaccines, intended for use in domestic dogs, were used. None of the vaccinated dogs showed any untoward clinical signs. The inactivated canine distemper vaccine did not result in seroconversion whereas the attenuated live vaccine resulted in seroconversion in all wild dogs. Presumably protective concentrations of antibodies to canine distemper virus were present in all wild dogs for at least 451 days. Canine parvovirus haemagglutination inhibition titres were present in all wild dogs prior to the administration of vaccine and protective concentrations persisted for at least 451 days. Vaccination against parvovirus infection resulted in a temporary increase in canine parvovirus haemagglutination inhibition titres in most dogs. Administration of both inactivated parenteral and live oral rabies vaccine initially resulted in seroconversion in 7 of 8 dogs. These titres, however, dropped to very low concentrations within 100 days. Booster administrations resulted in increased antibody concentrations in all dogs. It was concluded that the vaccines were safe to use in healthy subadult wild dogs and that a vaccination protocol in free-ranging wild dogs should at least incorporate booster vaccinations against rabies 3-6 months after the first inoculation.     

Serologic response of domestic ferrets (Mustela putorius furo) to canine distemper and rabies virus vaccines

Nine unrelated 12-week-old naive domestic ferrets (Mustela putorius furo) were used to evaluate the serologic responses to commercial canine distemper virus (CDV) and rabies virus (RV) vaccines. Five of the ferrets (group 1) were inoculated 3 times at 2-week intervals with a multivalent modified-live virus vaccine of canine cell-line origin, containing CDV and an inactivated RV vaccine. Four of the ferrets (group 2) were inoculated once with the multivalent modified-live virus vaccine containing CDV and were not inoculated with the RV vaccine. Both group-1 and group-2 ferrets seroconverted to the CDV component of the vaccine. Group-1 ferrets also seroconverted after RV vaccination and maintained serum antibody titers to both CDV and RV for at least 7 months. Domestic ferret sera were found to have IgG epitopes similar to sera of domestic dogs and cats. Domestic ferret sera did not contain antibodies to feline coronavirus or FeLV antigens.     

Correlation of histologic grading of canine mast cell tumors with Ki67/PCNA/AgNOR/c‐Kit scores: 38 cases (2002–2003)

Introduction: The Patnaik grading system for canine cutaneous MCT is currently one of the best determinants of prognosis; however, clinical outcome does not always correlate with histologic grade. The development of molecular markers offers a potential advantage and may complement subjective grading. The primary purpose of this study was to correlate histologic grading to Ki67/PCNA/AgNOR/c‐Kit scores. Methods: Thirty‐eight dogs with cutaneous MCT underwent surgical resection. Tumors were graded, with expansion of grade II MCT to low, medium (or II only) and high. For statistical purposes, MCT grade I, II (low, medium, high) and III were assigned a score of 1, 2, 3, 4, or 5, respectively. Sections were processed for AgNOR staining and expression of PCNA, Ki67 and c‐Kit as previously described (modified biotin‐strepavidin with DAB substrate). Paraffin‐embedded canine tissue arrays were used as positive and negative controls (primary antibody replaced with pre‐immune sera). Parametric statistical testing was performed using Statview statistical software with P ≤ .05 as significant. Results: There were 12, 20, 5 and 1 grade II low, grade II medium, grade II high and grade III MCT, respectively. The mean Ki67 score was 9.114 (median 8.0, range 1–28), mean PCNA score was 26.25 (median 24.0, range 5–65), mean AgNOR score was 1.499 (median 1.35, range 1.02–2.76) and c‐Kit scores were +1 (9/37), +2 (19/37) and +3 (9/37). With parametric statistical testing, significantly positive correlations were found for Ki67/Grade, PCNA/Grade, AgNOR/Grade, Ki67/PCNA, Ki67/AgNOR and PCNA/AgNOR (all P < .0001). No significant correlation was found for c‐Kit and grade; however, +3 c‐Kit scores had statistically significantly higher grades than +2 c‐Kit scores (P = .0458). Conclusions: Ki67/PCNA/AgNOR scores are positively correlated to grade in dogs with MCT. Further studies to correlate Ki67/PCNA/AgNOR/c‐Kit scores with clinical variables are ongoing.     

Antibody response to modified-live canine adenovirus vaccine in African hunting dogs (Lycaon pictus).

Nine hunting dogs were bled pre and post vaccination with modified-live virus vaccine. Their antibody responses to canine adenovirus type 1 was measured by an indirect immunofluorescent antibody test. The vaccine proved to be both effective and safe.     

Immunization of pups with maternally derived antibodies to canine parvovirus (CPV) using a modified-live variant (CPV-2b)

The results of vaccination trials carried out on pups with maternally derived antibodies (MDA) to canine parvovirus (CPV), using a modified-live CPV-2b variant vaccine (29-97/40 strain), are reported. The vaccine was able to overcome the obstacle of MDA, and to elicit protective immunity in 100% of the pups whose antibody titres were 1:10-1:40, 83% of the pups with titres of 1:80, 57% of the pups with titres of 1:160, and even in 60% of the pups with antibody titres of 1:320.     

Prevalence of positive antibody test results for canine parvovirus (CPV) and canine distemper virus (CDV) and response to modified live vaccination against CPV and CDV in dogs entering animal shelters

Canine parvovirus (CPV) and canine distemper virus (CDV) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. This study aimed to determine the prevalence of positive antibody test results for CPV and CDV in incoming dogs aged ≥ 4 months and to measure antibody response over 2 weeks following vaccination with a modified live vaccine (MLV). Dogs aged 4-24 months entering an adoption-guarantee shelter (Shelter 1, n=51) and aged ≥ 4 months entering a limited admission shelter (Shelter 2; n=51) were enrolled. Dogs from Shelter 1 had been vaccinated with MLV at a municipal shelter 5 days before enrollment, whereas dogs from Shelter 2 had no known history of vaccination at enrollment. Sera were obtained on day 1, immediately prior to CPV/CDV MLV, and tested using an in-clinic ELISA kit to detect CPV/CDV antibodies. Dogs negative for CPV and/or CDV were retested at day 6-8 and those dogs still negative at day 6-8 were retested at day 13-15. Prior to CPV/CDV MLV on day 1, more dogs tested positive for CPV (Shelter 1 - 68.6%; Shelter 2 - 84.3%) than for CDV (Shelter 1 - 37.3%; Shelter 2 - 41.2%). On day 1, prior to MLV, all spayed/neutered animals tested CPV antibody-positive (n=17/102) and CPV antibody-positive dogs were older than serologically negative dogs (Shelter 1, P=0.0029; Shelter 2, P=0.0042). By day 13-15, almost all dogs were CPV antibody-positive (Shelter 1 - 97.9%; Shelter 2 - 100.0%) and CDV antibody-positive (Shelter 1 - 93.8%; Shelter 2 - 97.8%). MLV induces protective antibody titers against CPV/CDV in almost all dogs after 13-15 days.     

Linac‐based stereotactic radiation therapy for canine non‐lymphomatous nasal tumours: 29 cases (2013‐2016)

Twenty‐nine dogs were treated with linac‐based stereotactic radiation therapy (SRT) for non‐lymphomatous nasal tumours. Only dogs with a follow‐up time >365 days were included in this retrospective analysis. No dogs had evidence of distant metastasis at diagnosis. Treatment was planned and a total of 30 Gy in 3 daily 10 Gy fractions was delivered using intensity‐modulation, cone‐beam CT‐based image guidance and a robotic treatment couch. Clinical signs improved in all cases. Nineteen dogs had CT scans 3‐4 months post‐SRT and all had partial or complete tumour response. Minimal acute toxicities were detected. Clinically significant late toxicities included oronasal or nasocutaneous fistulas (N = 3) and biopsy‐confirmed fungal rhinitis with no evidence of tumour progression (N = 2). The median progression‐free survival (PFS) was 354 days, with 49% and 39% progression‐free at 1 and 2 years post‐SRT, respectively. The median survival time (ST) was 586 days, with 69% and 22% alive 1 and 2 years post‐SRT, respectively. Neither the clinical parameters evaluated (modified Adams’ stage, histopathology, presence of intracranial extension of the tumour) nor dosimetric data were predictive for PFS or ST. This SRT protocol appears to be well tolerated, and PFI and ST are comparable or superior to those reported in other definitive‐intent radiotherapy protocols.