Factors associated with survival in dogs with a histopathological diagnosis of hepatocellular carcinoma: 94 cases (2007-2018).

Background: Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer in dogs. Despite this, relatively few reports of this disease exist pertaining to prognostic factors and outcome. Aim: To evaluate factors associated with survival in dogs with all subtypes of HCC diagnosed on histopathology. Methods: A retrospective single institutional study was carried out on 94 client-owned dogs with a histopathologic diagnosis of HCC between 2007 and 2018 obtained by biopsy (21/94) or attempted definitive resection (73/94). Signalment, preoperative features, surgical findings, and postoperative outcomes were recorded. Associations between survival to discharge data were collected and univariable logistical regression was carried out. Kaplan-Meier survival analysis was carried out to identify negative risk factors for long-term prognosis. Results: The median survival time (MST) for all patients was 707 days (95% CI = 551-842). MST was not significantly different (p > 0.05) between patients who had suspected versus incidentally diagnosed HCC (695 vs. 775 days), between complete versus incomplete surgical margins (668 vs. 834 days), or between patients with massive subtype versus nodular/diffuse subtype (707 vs. 747 days). Logistical regression identified an association with the excision of the right medial lobe and risk of perioperative death (OR = 9.2, CI 1.5-55.9, p = 0.016). An American Society of Anesthesiologists score ≥4, disease present within the quadrate lobe, and elevated blood urea nitrogen, potassium or gamma-glutamyltransferase were identified as negative prognosticators during multivariable Cox regression. Preoperative imaging (ultrasound or CT) agreed with the surgical location in 91% of the cases. Preoperative cytology was consistent with a diagnosis of HCC in 15/32 (46.9%) cases. Conclusion: Type of diagnosis (incidental vs presumed), completeness of excision, and subtype were not associated with MST in this study. Preoperative identification of tumors within the central division may be related to a less favorable outcome. Results of preoperative cytology were not highly sensitive for identifying a malignancy.

Adjuvant Sirolimus Does Not Improve Outcome in Pet Dogs Receiving Standard-of-Care Therapy for Appendicular Osteosarcoma: A Prospective, Randomized Trial of 324 Dogs.

PURPOSE: The mTOR pathway has been identified as a key nutrient signaling hub that participates in metastatic progression of high-grade osteosarcoma. Inhibition of mTOR signaling is biologically achievable with sirolimus, and might slow the outgrowth of distant metastases. In this study, pet dogs with appendicular osteosarcoma were leveraged as high-value biologic models for pediatric osteosarcoma, to assess mTOR inhibition as a therapeutic strategy for attenuating metastatic disease progression. PATIENTS AND METHODS: A total of 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a two-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb, followed by adjuvant carboplatin chemotherapy ± oral sirolimus therapy. The primary outcome measure was disease-free interval (DFI), as assessed by serial physical and radiologic detection of emergent macroscopic metastases; secondary outcomes included overall 1- and 2-year survival rates, and sirolimus pharmacokinetic variables and their correlative relationship to adverse events and clinical outcomes. RESULTS: There was no significant difference in the median DFI or overall survival between the two arms of this trial; the median DFI and survival for standard-of-care (SOC; defined as amputation and carboplatin therapy) dogs was 180 days [95% confidence interval (CI), 144-237] and 282 days (95% CI, 224-383) and for SOC + sirolimus dogs, it was 204 days (95% CI, 157-217) and 280 days (95% CI, 252-332), respectively. CONCLUSIONS: In a population of pet dogs nongenomically segmented for predicted mTOR inhibition response, sequentially administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend DFI or survival in dogs with appendicular osteosarcoma.

A retrospective study of proteinuria in dogs receiving toceranib phosphate.

The incidence of proteinuria in humans receiving tyrosine kinase inhibitors has been well-documented. Reports of proteinuria with this class of drugs are limited in veterinary medicine. This retrospective study describes the incidence, severity, and progression of proteinuria in 55 dogs treated with toceranib phosphate, with or without concurrent glucocorticoid or NSAID (non-steroidal anti-inflammatory drug). Six dogs were proteinuric at baseline. Twelve of the 49 dogs that were not proteinuric at baseline developed proteinuria while receiving toceranib phosphate. Median urine protein:creatinine (UPC) ratio when proteinuria developed was 0.75 (range: 0.6 to 4.9). There was no association with intermittent glucocorticoid or NSAID use and development of proteinuria (P = 0.5 and P = 0.7, respectively). Overall duration of toceranib phosphate treatment ranged from 70 to 802 days in proteinuric dogs and 28 to 1285 days in non-proteinuric dogs. Our results indicate a subset of dogs receiving toceranib phosphate may develop proteinuria; careful monitoring with serial UPCs is recommended.

Étude rétrospective de la protéinurie chez des chiens recevant du phosphate de tocéranibe. L'incidence de protéinurie chez les humains recevant des inhibiteurs de la tyrosine kinase a été bien documentée. Les rapports de protéinurie avec cette classe de médicaments sont limités en médecine vétérinaire. Cette étude rétrospective décrit l'incidence, la gravité et la progression de la protéinurie chez 55 chiens traités à l'aide de phosphate de tocéranibe, avec ou sans des glucocorticoïdes ou des AINS (anti-inflammatoires non stéroïdiens) concomitants. Six chiens étaient protéinuriques au point de référence. Douze des 49 chiens qui n'étaient pas protéinuriques au point de référence ont développé la protéinurie pendant qu'ils recevaient du phosphate de tocéranibe. Le ratio médian de protéine-créatinine au moment du développement de la protéinurie était de 0,75 (fourchette : de 0,6 à 4,9). Il n'y avait aucune association avec l'utilisation intermittente de glucocorticoïdes ou d'AINS et le développement de la protéinurie (P = 0,5 et P = 0,7, respectivement). La durée totale du traitement au phosphate de tocéranibe s'échelonnait de 70 à 802 jours chez les chiens protéinuriques et de 28 à 1285 jours chez les chiens non protéinuriques. Nos résultats indiquent qu'un sous-groupe de chiens recevant du phosphate de tocéranibe peut développer la protéinurie et une surveillance attentive à l'aide d'une série de ratios urinaires protéine-créatinine est recommandée.(Traduit par Isabelle Vallières).

Metabolic Abnormalities Detected in Phase II Evaluation of Doxycycline in Dogs with Multicentric B-Cell Lymphoma.

Doxycycline has antiproliferative effects in human lymphoma cells and in murine xenografts. We hypothesized that doxycycline would decrease canine lymphoma cell viability and prospectively evaluated its clinical tolerability in client-owned dogs with spontaneous, nodal, multicentric, substage a, B-cell lymphoma, not previously treated with chemotherapy. Treatment duration ranged from 1 to 8 weeks (median and mean, 3 weeks). Dogs were treated with either 10 (n = 6) or 7.5 (n = 7) mg/kg by mouth twice daily. One dog had a stable disease for 6 weeks. No complete or partial tumor responses were observed. Five dogs developed grade 3 and/or 4 metabolic abnormalities suggestive of hepatopathy with elevations in bilirubin, ALT, ALP, and/or AST. To evaluate the absorption of oral doxycycline in our study population, serum concentrations in 10 treated dogs were determined using liquid chromatography tandem mass spectrometry. Serum levels were variable and ranged from 3.6 to 16.6 µg/ml (median, 7.6 µg/ml; mean, 8.8 µg/ml). To evaluate the effect of doxycycline on canine lymphoma cell viability in vitro, trypan blue exclusion assay was performed on canine B-cell lymphoma cell lines (17-71 and CLBL) and primary B-cell lymphoma cells from the nodal tissue of four dogs. A doxycycline concentration of 6 µg/ml decreased canine lymphoma cell viability by 80%, compared to matched, untreated, control cells (mixed model analysis, p < 0.0001; Wilcoxon signed rank test, p = 0.0313). Although the short-term administration of oral doxycycline is not associated with the remission of canine lymphoma, combination therapy may be worthwhile if future research determines that doxycycline can alter cell survival pathways in canine lymphoma cells. Due to the potential for metabolic abnormalities, close monitoring is recommended with the use of this drug in tumor-bearing dogs. Additional research is needed to assess the tolerability of chronic doxycycline therapy.

Effects of lycopene on proliferation and death of canine osteosarcoma cells.

OBJECTIVE: To determine the effects of lycopene with and without concurrent chemotherapeutic treatment on growth and apoptosis of canine osteosarcoma cells. SAMPLE POPULATION: Cell cultures of 3 established canine osteosarcoma cell lines (D17, OS 2.4, and HMPOS). PROCEDURES: Growth curve kinetics and cell cytotoxicosis for various treatment combinations were assessed by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Additionally, cell cycle kinetics and colony-forming soft agar assays were performed to determine the influences of lycopene on the cell cycle and anchorage-independent growth. Western immunoblotting of HMPOS cells was performed to examine signaling and apoptotic pathways implicated in lycopene-induced apoptosis. RESULTS: Lycopene alone caused mild to pronounced attenuation of cell proliferation of all 3 cell lines as well as apoptosis in HMPOS cells but did not interfere with cell death in response to doxorubicin. Soft agar anchorage-independent growth assays revealed complete inhibition of cell proliferation in 2 of 3 osteosarcoma cell lines. Further investigation into the apoptotic response revealed activation of mitochondrial-induced apoptosis primarily through expression of truncated Bid and a decrease in protein kinase B (ie, AKT) phosphorylation. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that lycopene may be beneficial during treatment of osteosarcomas. Lycopene did not negatively or positively affect survival of osteosarcoma cells during doxorubicin treatment and independently induced apoptosis in the HMPOS cell line. These findings warrant further in vitro and in vivo studies into the use of this natural compound as an adjuvant antiproliferative, proapoptotic treatment in dogs with osteosarcoma.

Oral bioavailability of etoposide after administration of a single dose to tumor-bearing dogs.

OBJECTIVE: To characterize oral bioavailability and pharmacokinetic disposition of etoposide when the IV formulation was administered orally to dogs. ANIMALS: 8 tumor-bearing dogs. PROCEDURES: An open-label, single-dose, 2-way crossover study was conducted. Dogs were randomly assigned to initially receive a single dose of etoposide (50 mg/m2) IV or PO. A second dose was administered via the alternate route 3 to 7 days later. Medications were administered before IV administration of etoposide to prevent hypersensitivity reactions. Oral administration of etoposide was prepared by reconstituting the parenteral formulation with 0.9% NaCl solution and further diluting the reconstituted mixture 1:1 with a sweetening agent. Plasma samples were obtained after both treatments. Etoposide concentrations were measured with a high-performance liquid chromatography assay, and plasma etoposide concentration-time profiles were analyzed by use of noncompartmental methods. RESULTS: 4 dogs had hypersensitivity reactions during IV administration of etoposide. No adverse effects were detected after oral administration. Plasma etoposide concentrations were undetectable in 2 dogs after oral administration. Oral administration of etoposide resulted in significantly lower values for the maximum plasma concentration and the area under the plasma etoposide concentration-versus-time curve, compared with results for IV administration. Oral bioavailability of etoposide was low (median, 13.4%) and highly variable among dogs (range, 5.7% to 57.3%). CONCLUSIONS AND CLINICAL RELEVANCE-Vehicle-related toxicosis can limit the IV administration of etoposide in dogs. The parenteral formulation of etoposide can be safely administered orally to dogs, but routine use was not supported because of low and variable oral bioavailability in this study.

In vitro and in vivo evaluation of combined calcitriol and cisplatin in dogs with spontaneously occurring tumors.

PURPOSE: Calcitriol potentiates cisplatin-mediated activity in a variety of tumor models. We examine here, the effect of calcitriol and cisplatin pre-clinically and clinically in canine spontaneous tumors through in vitro studies on tumor cells and through a phase I study of calcitriol and cisplatin to identify the maximum-tolerated dosage (MTD) of this combination in dogs with cancer and to characterize the pharmacokinetic disposition of calcitriol in dogs. METHODS: Canine tumor cells were investigated for calcitriol/cisplatin interactions on proliferation using an MTT assay in a median-dose effect analysis; data were used to derive a combination index (CI). Cisplatin was given at a fixed dosage of 60 mg/m2. Calcitriol was given i.v. and the dosage was escalated in cohorts of three dogs until the MTD was defined. Serum calcitriol concentrations were quantified by radioimmunoassay. RESULTS: In vitro, CIs < 1.0 were obtained for all combinations of calcitriol/cisplatin examined. The MTD was 3.75 microg/kg calcitriol in combination with cisplatin, and hypercalcemia was the dose-limiting toxicosis. The relationship between calcitriol dosage and either Cmax or AUC was linear. Calcitriol dosages >1.5 microg/kg achieved Cmax > or = 9.8 ng/mL and dosages >1.0 microg/kg achieved AUC > or = 45 h ng/mL. CONCLUSIONS: Calcitriol and cisplatin have synergistic antiproliferative effects on multiple canine tumor cells and high-dosages of i.v. calcitriol in combination with cisplatin can be safely administered to dogs. Cmax and AUC at the MTD 3.75 microg/kg calcitriol exceed concentrations associated with antitumor activity in a murine model, indicating this combination might have significant clinical utility in dogs.

Comparison of 3 protocols for treatment after induction of remission in dogs with lymphoma.

BACKGROUND: The optimal treatment after inducing complete remission (CR) in dogs with lymphoma has not been established. HYPOTHESIS: After inducing CR with L-asparaginase, vincristine, cyclophosphamide, doxorubicin, prednisone (L-CHOP); consolidation with either half-body radiation therapy (HBRT); or lomustine (CCNU) and mechlorethamine, vincristine, procarbazine, prednisone (MOPP) would improve first remission duration compared with continuing a CHOP-based protocol for an additional 4 months. ANIMALS: Dogs with stage III-V lymphoma. METHODS: Prospective clinical trial in which dogs initially were treated with an 8-week induction protocol that consisted of L-CHOP. Dogs in CR after induction were then allocated to 1 of 2 consolidation arms. A chemotherapy consolidation arm consisted of 2 treatments with CCNU and 1 cycle of MOPP. A HBRT arm consisted of 2 sequential 8.0-Gy fractions to the cranial and caudal half-body separated by 30 days. Vincristine was given between fractions. Results of the consolidation arms also were compared with a historical group treated with the same 8-week induction protocol followed by CHOP therapy until week 24. RESULTS: Overall, 67% of the dogs were in CR after 8 weeks of induction chemotherapy and were compared. Fifty-two dogs were in the historical arm, 23 in the CCNU/MOPP arm, and 27 in the HBRT arm. No difference in first remission duration was found among groups. Median first remission duration for the historical, CCNU/MOPP, and HBRT arms were 307, 274, and 209 days, respectively (P = .28). Overall second CR rate was 82% and was not different among groups (all P > or = .58). Overall remission duration (P = .28) and survival time (P = .48) were not different among groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Consolidation with either CCNU/MOPP or HBRT showed no advantage over a standard CHOP-based protocol.

Phase I and pharmacokinetic evaluation of the combination of orally administered docetaxel and cyclosporin A in tumor-bearing dogs.

OBJECTIVE: To determine the maximum tolerated dose and characterize the pharmacokinetic disposition of an orally administered combination of docetaxel and cyclosporin A (CSA) in dogs with tumors. ANIMALS: 16 client-owned dogs with metastatic or advanced-stage refractory tumors. PROCEDURES: An open-label, dose-escalation, single-dose, phase I study of docetaxel administered in combination with a fixed dose of CSA was conducted. Docetaxel (at doses of 1.5, 1.625, or 1.75 mg/kg) and CSA (5 mg/kg) were administered concurrently via gavage twice during a 3-week period. Plasma docetaxel concentrations were quantified by use of high-performance liquid chromatography, and pharmacokinetic disposition was characterized by use of noncompartmental analysis. Dogs' clinical signs and results of hematologic and biochemical analyses were monitored for evidence of toxicosis. RESULTS: No acute hypersensitivity reactions were observed after oral administration of docetaxel. Disposition of docetaxel was dose independent over the range evaluated, and pharmacokinetic variables were similar to those reported in previous studies involving healthy dogs, with the exception that values for clearance were significantly higher in the dogs reported here. The maximum tolerated dose of docetaxel was 1.625 mg/kg. Gastrointestinal signs of toxicosis were dose limiting. CONCLUSIONS AND CLINICAL RELEVANCE: The absence of myelosuppression suggested that the docetaxel-CSA combination may be administered more frequently than the schedule used. Further studies are warranted to evaluate combination treatment administered on a biweekly schedule in dogs with epithelial tumors.

Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells.

Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.

Evaluation of urine sulfated and nonsulfated bile acids as a diagnostic test for liver disease in dogs.

OBJECTIVE: To evaluate 3 methods for measuring urine bile acids (UBA) and compare their diagnostic performance with that of the serum bile acids (SBA) test and other routine screening tests in dogs with hepatic disorders. DESIGN: Prospective study. ANIMALS: 15 healthy dogs, 102 dogs with hepatic disorders, and 9 dogs with clinical signs of hepatic disorders that were found to have nonhepatic disorders. PROCEDURES: Blood and urine samples were collected from sick dogs and healthy dogs for serum biochemical analyses, and determination of concentrations of SBA and UBA. Urine samples were obtained from 15 healthy dogs to establish an upper cutoff value for UBA concentrations. The UBA were measured by use of a quantitative-linked enzymatic colorimetric method. Three analytical modifications were evaluated; 1 quantified only urine sulfated bile acids (USBA), 1 only urine nonsulfated bile acids (UNSBA), and 1 quantified both (USBA plus UNSBA). The UBA values were standardized with the urine creatinine concentration. RESULTS: The UNSBA-to-creatinine ratio and USBA plus UNSBA-to-creatinine ratio tests had the best diagnostic performance of the UBA tests; each had a substantially higher specificity, slightly higher positive predictive value, slightly lower negative predictive value, and lower sensitivity than the SBA test. These UBA-to-creatinine values were positively correlated with SBA values. The USBA-to-creatinine ratio had poor sensitivity, indicating a low rate of bile acid sulfation in dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The UBA can be measured in dogs with sufficient repeatability and accuracy for clinical application. The UNSBA-to-creatinine ratio and USBA plus UNSBA-to-creatinine ratio identified dogs with hepatic disorders nearly as well as the SBA test.