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Supplementary Table 3 contains a summary of the outcomes, with statistical comparisons, of the dogs enrolled in the SOC clinical trial arm. This includes the DFI and outcomes of the groups of dogs stratified by tumor location and ALP status.

Supplementary Fig. 1 depicts the chronology of clinical trial visits and associated procedures for dogs enrolled in this clinical trial.

Supplementary Table 2 contains the summary comparison of both the Intent-to-treat and Per-protocol analyses of clinical outcomes for dogs enrolled in Standard of Care (SOC) and Standard of Care + sirolimus

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.

Supplementary Table 4 contains a summary of the outcomes, with statistical comparisons, of the dogs enrolled in the SOC + sirolimus clinical trial arm. This includes the DFI and outcomes of the groups of dogs stratified by tumor location and ALP status.

Supplementary Table 1 contains the inclusion and exclusion criteria for pet dogs considered for enrollment into the SOC and SOC + S clinical trial arms.

Supplementary Fig. 2 shows the walk-in pharmacokinetics, both measured results and simulated values, for dogs treated with oral sirolimus in preparation for the definitive adjuvant trial.

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.

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.

Objective To compare the effect of sentinel lymph node (SLN) histology vs locoregional lymph node (LRLN) fine needle aspiration (FNA) cytology on assigned disease stage and adjunctive treatment recommendations and describe the incidence of anatomic disparity between the LRLN and SLN. Study design A pre-post study refers to a study design type in which subjects are compared pre and post the intervention of interest. Animals Seventeen dogs undergoing primary excision of 20 cutaneous and subcutaneous mast cell tumors (MCT). Methods Client-owned dogs presenting to the Cornell University Hospital for Animals for surgical removal of a cytologically confirmed cutaneous or subcutaneous MCT >1 cm in diameter were enrolled. Cytological examination of FNA from the LRLN was compared with histology of the SLN. The SLN was identified by indirect computed tomographic lymphangiography (ICTL) after peritumoral injection of iopamidol and scanning at 1, 3, 5, 10, and 15 minutes. Histopathologic node score > 1 was considered metastatic. After case review by an oncologist, LRLN FNA cytology was compared with SLN histology for effect on changes in stage assignment and adjunctive treatment recommendations. Results Mast cell tumors were graded as 2 low (n = 11), 2 high (n = 2), and subcutaneous (n = 7). Optimal scan timing was 10 minutes after injection of iopamidol. Sentinel lymph node differed anatomically from LRLN in 5 of 18 scans. Metastases were detected by histology in 9 of 20 SLN compared with in 1 of 20 FNA of LRLN (P = .001), changing stage and adjunctive treatment recommendations 8 of 20 tumors. Only 6 of 19 LRLN FNA samples were diagnostic. Conclusion Sentinel lymph nodes were consistently identified with ICTL and differed from LRLN in one-quarter of tumors. Histopathological examination of SLN altered recommendations in half of the dogs compared with the previous standard of care. Clinical significance Indirect computed tomographic lymphangiography and SLN excision should be considered as a new standard for dogs with MCT.

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.

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

Background Thyroid dysfunction is associated with the use of tyrosine kinase inhibitors (TKI) in people. Hypothesis/Objectives To determine whether dysfunction in the hypothalamic‐pituitary‐thyroid axis occurs in dogs receiving the TKI, toceranib phosphate. Animals Forty‐three client‐owned dogs with cancer. Methods Prospective, observational study. Concentrations of total thyroxine (TT4), free thyroxine (FT4), total triiodothyronine (TT3), and thyroid‐stimulating hormone (TSH) were evaluated on day 0, 30, and 90. Dogs also were evaluated for the presence of thyroglobulin autoantibodies. Results The proportion of dogs with low TT4, low FT4, low TT3, high TSH, or primary hypothyroidism (increased TSH and decreased TT4, FT4 or both) did not change over 90 days. Hormone concentrations remained within laboratory reference intervals, but FT4 (P = 0.0032) and TSH (P < 0.0001) changed over time. Mean FT4 was 1.22 ng/dL (95% confidence interval [CI], 1.10–1.34) on day 0 and 1.00 ng/dL (95% CI, 0.86–1.16) on day 90. Mean TSH was 0.17 ng/mL (95% CI, 0.13–0.23) on day 0 and 0.34 ng/mL (95% CI, 0.24–0.48) on day 90. Furthermore, TT4/TT3 ratio also changed over time (P = 0.0086). Mean TT4/TT3 ratio was 2.57 (95% CI, 2.26–2.88) on day 0 and 2.02 on day 90 (95% CI, 1.61–2.44). Thyroglobulin autoantibodies were not detected in any dog. Conclusions and Clinical Importance Toceranib phosphate can disrupt the hypothalamic‐pituitary‐thyroid axis in dogs. Periodic evaluation of TT4, FT4, TT3, and TSH should be carried out in dogs receiving long‐term treatment with this medication.

The goal of the current study was to determine the efficacy of oral docetaxel in combination with cyclosporine in the treatment of canine epithelial cancer. Requirements for eligibility were histological confirmation of epithelial neoplasia, measurable disease, no chemotherapy treatment within 2 weeks, and a life expectancy of ≥ 3 months. Fifty-one dogs were enrolled. All dogs received 1.625 mg kg(-1) of docetaxel with 5 mg kg(-1) of cyclosporine (DT/CSA) by gavage. Ten dogs had progressive disease at 2 weeks, one dog died, and one dog was withdrawn from the study. Thirty-nine dogs were given a second dose of DT/CSA, three each receiving a third or fourth dose. Eight dogs had a dose reduction (1.5 mg kg(-1)) and six dogs had treatment delays primarily for gastrointestinal toxicity. The overall response rate was 16.7% (8/48 had a partial response there were no complete responses). The highest response rate was seen in dogs with oral squamous cell carcinoma (50%; 6/12).

An In Vitro study was conducted to investigate docetaxel as a radiation sensitizer in four canine (mammary carcinoma—CMT12 and CMT25, osteosarcoma—OS2.4, and transitional cell carcinoma—PTCC), and one feline cancer cell line (oral squamous cell carcinoma—SCCF1) to provide a basis for combination therapy in clinical patients. Cells were exposed to docetaxel followed by a single dose of radiation. The percent surviving fraction was determined by MTT assay. The combination index (CI) method determined synergistic cytotoxicity for the CMT12, CMT25 and OS2.4 cell lines with median CI values of 0.35, 0.47, 0.63 respectively. The SCCF1 cell line had moderate synergistic cytotoxicity with a median CI of 0.76, while the PTCC cell line resulted in antagonistic cytoxicity with a median CI of 2.75. The results indicated that docetaxel was a radiation sensitizer in 4 out of the 5 cancer cell lines tested.

An L-CHOP protocol with interposed treatments of CCNU and MOPP (L-CHOP-CCNU-MOPP) was evaluated in 66 dogs with stages III-V lymphoma. Results were compared with a historical group of 71 dogs treated with an L-CHOP protocol. Complete remission (CR) rates (85 and 80%, respectively) did not differ significantly between protocols (P = 0.48). First CR duration for dogs treated with L-CHOP-CCNU-MOPP was significantly longer: median, 317 days; 2-year CR rate, 35% versus median, 298 days; 2-year CR rate, 13%, P = 0.05). For the L-CHOP-CCNU-MOPP protocol, dogs in substage-b had a 4.3 times greater hazard of having a relapse than dogs in substage-a (P = 0.002). Frequency of adverse chemotherapy-associated gastrointestinal effects did not differ between protocols (P = 0.77). Neutropenia (primarily after CCNU) occurred more frequently in dogs treated with L-CHOP-CCNU-MOPP (P < 0.001). In summary, the L-CHOP-CCNU-MOPP protocol showed an improved duration of first CR as compared with an L-CHOP protocol, but the relevance of this finding might be subject to clinical judgement.

Safety and efficacy of a protocol of alternating 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; 70 mg m(-2)) and vinblastine (3.5 mg m(-2)), and prednisone (1-2 mg kg(-1); CVP) in dogs with mast cell tumours (MCT) were evaluated. A total of 17 dogs had nonresectable MCTs and 35 received CVP as adjunctive treatment to locoregional control of metastatic MCTs or grade III MCTs. Neutropenia with fever occurred in 8% of dogs after treatment with vinblastine and in 2% after treatment with CCNU. Persistent elevation of serum alanine transaminase, suggestive of hepatotoxicity, occurred in 9% of the dogs. Response rate in dogs with nonresectable MCTs was 65%; five achieved a complete response (median, 141 days) and six achieved a partial response (median, 66 days). Overall median progression-free survival (PFS) time in dogs treated in the adjuvant setting was 489 days. Dogs with grade III MCTs had shorter PFS compared with dogs with metastatic grade II MCTs (190 days versus 954 days; P < 0.001). Phase III studies are needed to provide reliable information about the comparative efficacy of this protocol.

In mice and people, administering corticosteroids before chemotherapy can reduce the severity of myelosuppression without reducing antitumour effects. This study investigated whether pretreatment with dexamethasone would reduce the incidence of grade 4 neutropenia in dogs receiving CCNU. Twenty-five dogs received dexamethasone [0.1 mg kg(-1) per os (PO) every 12 h] for 5 days and on the sixth day received CCNU (90 mg m(-2) PO). Historical dogs (n = 67) received CCNU alone (90 mg m(-2) PO). Forty-five percent of historical dogs had grade 4 neutropenia, while 64% of dogs pretreated with dexamethasone had grade 4 neutropenia (P = 0.16). Dexamethasone plasma levels were quantified by enzyme-linked immunosorbent assay in three healthy dogs. Peak plasma concentrations after a single oral 0.1-mg kg(-1) dose were

Background: The optimal dosage and clinical efficacy of vinblastine (VBL) for treatment of mast cell tumors (MCTs) in dogs has not been established. Hypothesis: Single-agent VBL has antitumor activity against MCTs in dogs. Animals: Fifty-one dogs with nonresectable grade II or III cutaneous MCTs. Methods: Prospective, open clinical trial. Dogs were systematically allocated (by hospital record number) to receive IV treatment with VBL at a dosage of 2.0 mg/m2 (weekly for 4 treatments then biweekly for 4 treatments; VBL 2.0) or treatment with VBL at a dosage of 3.5 mg/m2 (biweekly for 5 treatments; VBL 3.5). The primary outcome measure was reduction in tumor size. Results: Twenty-five dogs were allocated to the VBL 2.0 group and 26 were allocated to the VBL 3.5 group. In the VBL 2.0 group, 3 (12%) had a partial response (PR) for a median of 77 days (range, 48–229 days). Overall response rate in the VBL 3.5 group was 27%. One dog (4%) had a complete response for 63 days and 6 dogs (23%) had a PR for a median of 28 days (range, 28–78 days). Toxicoses were uncommon in the VBL 2.0 group. Twelve (46%) dogs in the VBL 3.5 group had

Background: Vinblastine (VBL) is commonly used in dogs at a dosage of 2.0 mg/m2. The minimal toxicity observed at this dosage indicates that higher dosages might be well tolerated. Hypothesis: The maximum tolerated dosage (MTD) for a single VBL treatment is higher than the previously published dosage of 2.0 mg/m2. Animals: Twenty-three dogs with lymphoma or cutaneous mast cell tumors. Methods: Dogs received 1 single-agent VBL treatment IV. The starting dosage was 3.0 mg/m2, and dosages were increased in increments of 0.5 mg/m2 in cohorts of 3 dogs. Hematologic toxicity was assessed with weekly CBCs. Gastrointestinal toxicity was assessed from medical histories from owners. Once the MTD was determined, additional dogs were treated with VBL at that dosage. Dogs whose cancers responded to VBL continued to receive treatments q2–3 weeks. Results: VBL dosages ranged from 3.0 to 4.0 mg/m2. Neutropenia was the dose-limiting toxicity, with the nadir identified 7 days after treatment and resolving by 14 days after treatment. The MTD was 3.5 mg/m2. Sixteen dogs were treated at this dosage, and 3 experienced severe toxicity characterized by asymptomatic grade 4 neutropenia, febrile grade 4 neutropenia, and death. Gastrointestinal toxicity was mild and self-limiting. Preliminary evidence of antitumor activity was identified in 2 of 12 dogs with lymphoma treated at the MTD. Conclusions and Clinical Importance: In dogs, single-agent VBL is well tolerated at a dosage of 3.5 mg/m2 IV. At this dosage, the minimum safe treatment interval is q2 weeks, and adjunct treatment with prophylactic antibiotics should be considered.

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.

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 > .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.

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.

This study evaluated the constitutive insulin-like growth factor-I (IGF-I) gene expression pattern in spontaneously healing cartilage defects over the course of 16 weeks, and correlated the tissue morphology and matrix gene expression with IGF-I mRNA levels. Full-thickness 15 mm cartilage defects were debrided in the femoral trochlea of both femoropatellar joints of 8 horses and the healing defects examined 2, 4, 8, or 16 weeks after surgery. Samples were harvested for histologic assessment of tissue healing using H&E staining, toluidine blue histochemical reaction for proteoglycan deposition, and in situ hybridization and immunohistochemistry procedures to demonstrate collagen type II mRNA and protein expression. Total RNA was isolated for Northern analysis to measure cartilage matrix molecule expression, and for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) to determine IGF-I gene expression patterns in healing cartilage defects. Full-thickness cartilage defects in horses were slow to heal compared to smaller lesions in similar locations in other animals. However, a progressive decline in tissue cellularity and vascularity, and increased tissue organization were observed on H&E stained specimens over the 16-week experiment. Evidence of early chondrogenic repair was detected through collagen type II in situ hybridization and immunohistochemistry. However, levels of collagen type II and aggrecan mRNA in lesions were not abundant on Northern analysis indicating incomplete chondrogenesis. IGF-I message expression followed a cyclic pattern with low levels at 2 weeks, followed by an increase at 4 and 8 weeks, and a subsequent decline at 16 weeks. There was no direct correlation between the stage of healing and cartilage matrix message expression, and the abundance of IGF-I mRNA in the healing lesions. In conclusion, this study demonstrated that the spontaneous healing of articular defects was accompanied by a temporal fluctuation in IGF-I gene expression which was discoordinate to the steady rise in expression of cartilage matrix molecules such as procollagen type II.

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 anchorageindependent 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.

Second-opinion histopathology is intended to detect clinically significant discrepancies that have a direct impact on patient care. We sought to determine if this practice at our institution affected patient management and prognosis. First- and second-opinion histopathology reports from cases were retrospectively reviewed. Reports were considered to be in diagnostic agreement, partial diagnostic disagreement or complete diagnostic disagreement. Four hundred and thirty cases were studied. In 70% of cases there was a diagnostic agreement. In 20% of cases, there was partial diagnostic disagreement, where diagnoses were the same but information such as grade or lymphatic and/or vascular invasion was changed. In 10% of cases, complete diagnostic disagreement resulted from a change in degree of malignancy (malignant to benign, or converse; 7%) or a change in cell type (3%). In 17% of the cases evaluated, the histopathology review prompted a change in treatment or prognosis. These findings support the use of second-opinion histopathology as an important part of patient care.

Hepatobiliary tumors are uncommon in dogs and cats. They generally occur in older animals with nonspecific clinical signs, usually relating to the gastrointestinal tract. Liver enzyme concentrations are commonly elevated. Early detection for massive-type lesions may allow for surgical resection and prolonged survival especially for hepatocellular carcinomas. Chemotherapy, in general, is not effective for primary liver tumors.

Background: Pleotropic-glycoprotein (P-gp)–mediated resistance is the usual cause of relapse in dogs with lymphoma. 1-(2-chloroethyl)3-cyclohexyl-1-nitrosurea (CCNU) and 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) are alkylating agents that are not affected by P-gp and lack cross-resistance to each other. A combination protocol offers the advantage of improved summation dose and synergistic activity. Hypothesis: A combination of CCNU and DTIC that is well tolerated can be used to treat dogs with lymphoma that developed resistance or failed to respond to previously administered chemotherapy. Animals: Fifty-seven dogs with lymphoma that were resistant to treatment with standard chemotherapy (l-CHOP; l-asparaginase, cyclophosphamide, doxorubicin, vincristine, prednisone). Methods: Prospective phase I and II trials were performed. CCNU was given PO immediately before a 5-h IV infusion of DTIC. Concurrent antiemetics and prophylactic antibiotics were used. Treatments were administered every 4 weeks. Results: Based on the results of 8 dogs in the phase I study, CCNU at 40 mg/m2 PO combined with DTIC at 600 mg/m2 IV was used to treat 57 dogs with resistant lymphoma. Thirteen (23%) dogs had a complete response (CR) for a median of 83 days and 7 (12%) had a partial response for a median of 25 days. The median l-CHOP CR duration of the dogs that did not respond to CCNU-DTIC was significantly longer than that of the dogs that did achieve remission with CCNU-DTIC (225 days versus 92 days, P= .02). The principal toxic event was neutropenia; the median neutrophil count 7 days after treatment was 1,275 cells/μL. Increases in alanine transaminase activity, possibly associated with hepatotoxicity, were detected in 7 dogs. Conclusions and Clinical Importance: A combination of CCNU and DTIC can be an effective option to rescue dogs with resistant lymphoma.

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.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.In vitro, CIs1.0 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 dosages1.5 microg/kg achieved Cmaxor = 9.8 ng/mL and dosages1.0 microg/kg achieved AUCor = 45 h ng/mL.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.

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, singledose, 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 docetaxelCSA 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.

Tissue transglutaminase II (TGase II) is a dual function protein with both transamidating and guanidine triphosphate (GTP)-binding capabilities. Previous studies have implicated TGase as a pro-apoptotic molecule; however, our recent findings indicate that TGase II may act as a survival factor in various cell types. The purpose of this study was to survey TGase II expression in normal tissue and spontaneous tumours of dogs and cats, by Western blotting and immunohistochemistry. Bladder, liver and adrenal gland exhibited prominent expression of TGase II while other tissues, including mammary gland, displayed limited expression and activity. TGase II GTP-binding in normal tissues was proportional to the level of expression in all tissues examined. Normal mammary tissue and that showing benign hyperplasia did not express TGase II. However, 11/25 (44%) of canine mammary carcinomas and 10/12 (83%) of feline mammary carcinomas strongly expressed TGase II in either a stromal, cellular or combined pattern. The pattern of expression was not related to the classification of mammary carcinoma (solid, tubulopapillary, complex or anaplastic), except that two anaplastic canine mammary carcinomas showed prominent TGase II expression. Two canine mammary carcinoma cell lines showed prominent TGase expression, and when the TGase activity was inhibited, the cells became more sensitive to doxorubicin-induced cell death. Thus, TGase II was significantly expressed in mammary cancers from dogs and cats and immunoreactivity of TGase II was similar to that reported in humans beings. The pro-survival effect of TGase II in canine mammary carcinoma cell lines was similar to that previously reported in humans patients.

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. (J Am Vet Med Assoc 2003;222: 1368–1375)