Fixed-dose-rate administration of gemcitabine in cancer-bearing cats: A pilot study.

Gemcitabine is an antimetabolite chemotherapy agent with schedule-dependent metabolism and efficacy. The purpose of this study was to identify the fixed-dose-rate (FDR) of gemcitabine administration in cancer-bearing cats that achieved a target plasma concentration (TPC) of 10 to 20 µM. Fifteen client-owned cats received gemcitabine infusions administered at various FDR for 1 to 6 hours. Plasma gemcitabine and dFdU (2',2'-difluorodeoxyuridine), the major gemcitabine metabolite, were quantitated by high performance liquid chromatography. Cats treated with an FDR less than 2.5 mg/m2 per minute failed to achieve TPC, whereas cats treated with an FDR of 10 mg/m2 per minute quickly exceeded the target range. An FDR of 5 mg/m2 per minute provided the longest duration of exposure without exceeding the upper limit of the TPC. Plasma dFdU concentration mirrored plasma gemcitabine concentrations. These data suggest that in order to maintain TPC of gemcitabine in cats the FDR lies between 2.5 and 5 mg/m2 per minute. A Phase II study to evaluate efficacy and toxicity of this approach is underway.

Administration de gemcitabine à vitesse et à dose fixes chez des chats atteints du cancer : une étude pilote. La gemcitabine est un agent de chimiothérapie antimétabolite ayant un métabolisme et une efficacité qui dépendent du plan thérapeutique. Cette étude visait à identifier la vitesse et la dose fixes (VDF) d'administration de la gemcitabine chez des chats atteints du cancer qui avaient atteints une concentration plasmatique cible (CPC) de 10 à 20 µM. Quinze chats appartenant à des clients ont reçu des infusions de gemcitabine administrées à diverses VDF pendant 1 à 6 heures. La gemcitabine et la dFdU (2',2'-difluorodeoxyuridine) dans le plasma, le métabolite majeur de la gemcitabine, ont été quantifiés par chromatographie liquide à haute performance. Les chats traités à l'aide de VDF de moins de 2,5 mg/m2 par minute n'ont pas réussi à atteindre la CPC, tandis que les chats traités à l'aide de VDF de 10 mg/m2 par minute ont rapidement dépassé la zone cible. Des VDF de 5 mg/m2 par minute ont fourni la durée d'exposition la plus longue sans dépasser la limite supérieure de la CPC. La concentration de dFdU dans le plasma a reflété les concentrations de gemcitabine dans le plasma. Ces données suggèrent qu'fin de maintenir la CPC de la gemcitabine chez les chats, les VDF doivent se situer entre 2,5 et 5 mg/m2 par minute. Une étude de phase II pour évaluer l'efficacité et la toxicité de cette approche est actuellement en cours.(Traduit par Isabelle Vallières).

Using canine osteosarcoma as a model to assess efficacy of novel therapies: can old dogs teach us new tricks?

Since its domestication more than 10,000 years ago, the dog has been the animal that most intimately shares our work and homelife. Interestingly, the dog also shares many of our diseases including cancer such as osteosarcoma. Like the human, osteosarcoma is the most common bone malignancy of the dog and death from pulmonary metastasis is the most common outcome. The incidence of this spontaneous bone neoplasm occurs ten times more frequently that it does so in children with about 8,000-10,000 cases estimated to occur in dogs in the USA. Because there is no "standard of care" in veterinary medicine, the dog can also serve us by being a model for this disease in children. Although the most common therapy for the dog with osteosarcoma is amputation followed by chemotherapy, not all owners choose this route. Consequently, novel therapeutic interventions can be attempted in the dog with or without chemotherapy that could not be done in humans with osteosarcoma due to ethical concerns. This chapter will focus on the novel therapies in the dog that have been reported or are in veterinary clinical trials at the author's institution. It is hoped that collaboration between veterinary oncologists and pediatric oncologists will lead to the development of novel therapies for (micro- or macro-) metastatic osteosarcoma that improve survival and might ultimately lead to a cure in both species.

Targeting canine bladder transitional cell carcinoma with a human bladder cancer-specific ligand.

OBJECTIVE: To determine if a human bladder cancer-specific peptide named PLZ4 can target canine bladder cancer cells. EXPERIMENTAL DESIGN: The binding of PLZ4 to five established canine invasive transitional cell carcinoma (TCC) cell lines and to normal canine bladder urothelial cells was determined using the whole cell binding assay and an affinitofluorescence assay. The WST-8 assay was performed to determine whether PLZ4 affected cell viability. In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging. RESULTS: PLZ4 exhibited high affinity and specific dose-dependent binding to canine bladder TCC cell lines, but not to normal canine urothelial cells. No significant changes in cell viability or proliferation were observed upon incubation with PLZ4. The in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control. CONCLUSIONS AND CLINICAL RELEVANCE: PLZ4 can specifically bind to canine bladder cancer cells. This suggests that the preclinical studies of PLZ4 as a potential diagnostic and therapeutic agent can be performed in dogs with naturally occurring bladder cancer, and that PLZ4 can possibly be developed in the management of canine bladder cancer.

Evaluation of an extractionless high-performance liquid chromatography-tandem mass spectrometry method for detection and quantitation of rosiglitazone in canine plasma.

OBJECTIVE: To develop a simple extractionless method for detection of rosiglitazone in canine plasma and test the method in a pharmacokinetic study after oral administration of rosiglitazone in dogs. ANIMALS: 3 client-owned dogs with cancer. PROCEDURES: High-performance liquid chromatography-tandem mass spectrometry was performed on canine plasma. The 3 dogs with cancer in the pharmacokinetic study were assessed via physical examination and clinicopathologic evaluation and considered otherwise healthy. Food was withheld for 12 hours, and dogs were administered a single dose (4 mg/m²) of rosiglitazone. Plasma was collected at various times, processed, and analyzed for rosiglitazone. RESULTS: The developed method was robust and detected a minimum of 0.3 ng of rosiglitazone/mL. Mean ± SD maximum plasma concentration was 205.2 ± 79.1 ng/mL, which occurred at 3 ± 1 hours, and mean ± SD elimination half-life was 1.4 ± 0.4 hours. The area under the plasma rosiglitazone concentration-versus-time curve varied widely among the 3 dogs (mean ± SD, 652.2 ± 351.3 ng/h/mL). CONCLUSIONS AND CLINICAL RELEVANCE: A simple extractionless method for detection of rosiglitazone in canine plasma was developed and was validated with excellent sensitivity, accuracy, precision, and recovery. The method enabled unambiguous evaluation and quantitation of rosiglitazone in canine plasma. This method will be useful for pharmacokinetic, bioavailability, or drug-drug interaction studies. Oral rosiglitazone administration was well tolerated in the dogs.

Immunohistochemical and histomorphometric evaluation of vascular distribution in intact canine cranial cruciate ligament.

OBJECTIVES: To (1) describe vascular distribution in the grossly intact canine cranial cruciate ligament (CCL) using immunohistochemical techniques specific to 2 components of blood vessels (factor VIII for endothelial cells, laminin for basement membrane); and (2) compare the vascularity in different areas of interest (craniomedial versus caudolateral bands; core versus epiligamentous regions; and proximal versus middle versus distal portions) in the intact normal canine CCL. STUDY DESIGN: In vitro study. ANIMALS: Large, mature dogs (n=7) of breeds prone to CCL disease that were euthanatized for nonorthopedic conditions. METHODS: Intact CCL were collected from fresh canine cadavers free from stifle pathology. CCL tissue was processed for immunohistochemistry and stained for factor VIII and laminin. Vascular density was determined by histomorphometric analysis. RESULTS: Specific vascular staining was sparsely identified throughout the CCL; however, the proximal portion of the CCL appears to have a greater number of vessels than the middle or distal portion of the ligament. CONCLUSIONS: The CCL is a hypovascular tissue and its vascular distribution is not homogeneous.

Vascular distribution in ruptured canine cranial cruciate ligament.

OBJECTIVE: To (1) determine the microanatomic vascular distribution in ruptured canine cranial cruciate ligaments (CCL) using specific vascular immunohistochemical techniques, and (2) compare vessel density between ruptured and intact canine CCL and between different areas of interest in ruptured CCL using histomorphometric analysis. STUDY DESIGN: In vitro study. ANIMALS: Dogs (n=41) admitted for surgical treatment of ruptured CCL and 19 dogs euthanatized for nonorthopedic conditions. METHODS: Diseased (variable CCL rupture) and intact (normal control) CCL were processed for immunohistochemical staining specific to vessels (factor VIII, laminin). Mean vascular density was assessed and compared in areas of interest (torn end versus remaining core regions of CCL, proximal femoral versus distal tibial core CCL regions). RESULTS: Ruptured CCL was more vascular than intact CCL; however there was no difference in vascular density between the torn end and the remaining core area of the ruptured CCL. Ruptured CCL was vascularized to a greater degree at the proximal portion than the distal portion of the CCL. Partially ruptured CCLs had a higher vessel density than completely ruptured CCLs. CONCLUSIONS: Vascular density is increased in diseased CCL compared with intact CCL. It remains to be determined whether this finding is associated with the cause of CCL rupture or is a result of CCL degeneration and rupture.

Procedural description and prospective evaluation of short-term outcome for the use of prostatic artery embolization in dogs with carcinoma of the prostate.

OBJECTIVE: To describe the procedure of prostatic artery embolization (PAE) in dogs with prostatic carcinoma and to evaluate the short-term outcome for treated dogs. ANIMALS: 20 client-owned dogs with prostatic carcinomas between May 2014 and July 2017. PROCEDURES: In this prospective cohort study, dogs with carcinoma of the prostate underwent PAE with fluoroscopic guidance. Before and after PAE, dogs underwent CT and ultrasonographic examinations of the prostate, and each owner completed a questionnaire about the dog's clinical signs. Results for before versus after PAE were compared. RESULTS: Prostatic artery embolization was successfully performed in all 20 dogs. Tenesmus, stranguria, and lethargy were significantly less common 30 days after PAE (n = 2, 1, and 0 dogs, respectively), compared with before PAE (9, 10, and 6 dogs, respectively). Median prostatic volume was significantly less 30 days after PAE (14.8 cm3; range, 0.4 to 48.1 cm3; interquartile [25th to 75th percentile] range, 6.7 to 19.5 cm3), compared with before PAE (21.7 cm3; range, 2.9 to 77.7 cm3; interquartile range, 11.0 to 35.1 cm3). All dogs had a reduction in prostatic volume after PAE, with a median prostatic volume loss of 39.4% (95% CI, 20.3% to 59.3%). CONCLUSIONS AND CLINICAL RELEVANCE: Prostatic artery embolization was associated with decreased prostate volume and improved clinical signs in this cohort. The short-term response to PAE appears promising, and evaluation of the long-term impact on survival time is needed.

Use of percutaneous microwave ablation in the treatment of retroperitoneal neoplasia in three dogs.

CASE DESCRIPTION: 3 dogs with retroperitoneal masses (2 renal and 1 located near the diaphragm) were treated by percutaneous microwave ablation (MWA). CLINICAL FINDINGS: Dogs between 11 and 13 years of age weighing between 13.7 and 43.8 kg had either a renal mass (n = 2) or a mass located in the caudodorsal aspect of the retroperitoneal space near the right side of the diaphragm (1). Cytology revealed that one of the renal masses and the mass located near the diaphragm were malignant neoplasias. Findings on cytologic evaluation of a sample of the other renal mass was nondiagnostic. Maximum mass diameters ranged between 1.4 and 2.5 cm. TREATMENT AND OUTCOME: All dogs were treated by percutaneous MWA. Probes were directed into tumors by use of ultrasound and CT guidance, and microwave energy was applied to each mass. Findings on imaging of each mass following MWA was consistent with successful treatment. No intraprocedural or major postprocedural complications occurred, and all dogs were discharged from the hospital within 3 days of treatment. Two dogs died at 3 and 21 months after MWA with no known local recurrence; 1 dog was still alive 64 months after treatment. CLINICAL RELEVANCE: Although the indications for MWA in the treatment of neoplasia in companion animals are limited, the outcomes of dogs in the present report provided preliminary evidence that percutaneous MWA can be safely used to effectively treat retroperitoneal neoplasia. This procedure was successfully performed with image guidance in all 3 dogs.

Establishment of a dog model for the p53 family pathway and identification of a novel isoform of p21 cyclin-dependent kinase inhibitor.

Spontaneous tumors in the dog offer a unique opportunity as models to study human cancer etiology and therapy. p53, the most commonly mutated gene in human cancers, is found to be altered in dog cancers. However, little is known about the role of p53 in dog tumorigenesis. Here, we found that on exposure to DNA damage agents or MDM2 inhibitor nutlin-3, canine p53 is accumulated and capable of inducing its target genes, MDM2 and p21. We also found that on DNA damage, canine p53 is accumulated in the nucleus, followed by MDM2 nuclear translocation and increased 53BP1 foci formation. In addition, we found that canine p63 and p73 are up-regulated by DNA damage agents. Furthermore, colony formation assay showed that canine tumor cells are sensitive to DNA damage agents and nutlin-3 in a p53-dependent manner. Surprisingly, canine p21 is expressed as two isoforms. Thus, we generated multiple canine p21 mutants and found that amino acids 129 to 142 are required, whereas amino acid 139 is one of the key determinants, for the expression of two p21 isoforms. Finally, we showed that although the full-length human p21 cDNA expresses one polypeptide, amino acid 139 seems to play a similar role as that in canine p21 for various migration patterns. Taken together, our results indicate that canine p53 family proteins have biological activities similar to human counterparts. These similarities make the dog an excellent outbred spontaneous tumor model, and the dog can serve as a translation model from benchtop to cage side and then to bedside.

Biological behaviour and ezrin expression in canine rhabdomyosarcomas: 25 cases (1990-2012).

There are few published reports of canine rhabdomyosarcomas. In human paediatrics, rhabdomyosarcomas account for 5%-10% of all tumours and >50% of soft tissue sarcomas. They have an aggressive biologic behaviour; most patients develop diffuse metastatic disease. Ezrin, a cytoskeleton linker protein, has been correlated with metastasis in a number of tumours, including rhabdomyosarcomas. The goal of this study was to describe dogs with non-urinary rhabdomyosarcomas including clinical findings, ezrin expression and outcome. Twenty-five dogs with rhabdomyosarcomas were identified from two institutions' databases. Signalment, primary tumour location, cytologic and histologic findings, metastatic sites, treatments, survival time and necropsy results were recorded. Immunohistochemical staining for ezrin expression was performed on archived samples; cellular localization of ezrin was characterized. The mean and median age of all patients was 4.3 and 2 years, respectively. Subcutaneous and retrobulbar/orbital were the most common primary tumour locations. Sixteen dogs had metastatic disease at diagnosis. Three dogs presented with diffuse disease where a primary tumour could not be identified. A round cell tumour was the initial diagnosis in 32% of cases, and 76% of cases required immunohistochemistry to establish the diagnosis. The median survival was 10 days. Twenty-one cases had archived samples available for ezrin staining; all but one was positive and exhibited both membranous and cytoplasmic localization. Rhabdomyosarcomas occur in young dogs, may have a round cell appearance, and exhibit aggressive biologic behaviour. Given ezrin's defined role in metastasis, its observed expression in the tumours in this study suggest its possible role in canine rhabdomyosarcoma's aggressive biologic behaviour.

Substitution of mitoxantrone for doxorubicin in a multidrug chemotherapeutic protocol for first-line treatment of dogs with multicentric intermediate- to large-cell lymphoma.

OBJECTIVE To evaluate effects of substituting mitoxantrone for doxorubicin in a cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapeutic protocol for first-line treatment of dogs with multicentric intermediate- to large-cell lymphoma. DESIGN Retrospective cohort study. ANIMALS 44 dogs treated with cyclophosphamide, mitoxantrone, vincristine, and prednisone (CMOP) and 51 dogs treated with CHOP at 12 referral institutions. PROCEDURES Medical records were reviewed to determine response to treatment, progression-free survival time, and overall survival time. For dogs treated with CMOP, adverse events were also recorded. RESULTS All 44 (100%) dogs treated with CMOP and 37 of 38 (97.4%) dogs treated with CHOP had a complete or partial response. Median progression-free survival time for dogs treated with CMOP was 165 days (95% confidence interval [CI], 143 to 187 days), and median overall survival time was 234 days (95% CI, 165 to 303 days). For dogs treated with CHOP, median progression-free survival time was 208 days (95% CI, 122 to 294 days), and median overall survival time was 348 days (95% CI, 287 to 409 days). Progression-free and overall survival times were not significantly different between groups. Overall, 9 of the 44 (20%) dogs treated with CMOP had adverse events likely or probably related to mitoxantrone, but all of these adverse events were mild. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that mitoxantrone may be a reasonable substitution in a CHOP protocol for treatment of dogs with multicentric intermediate- to large-cell lymphoma when doxorubicin is contraindicated.

Association of macrophage and lymphocyte infiltration with outcome in canine osteosarcoma.

Immunotherapeutic strategies have shown promise for the treatment of canine osteosarcoma (cOSA). Very little is known about the immune microenvironment within cOSA, however, limiting our ability to identify potential immune targets and biomarkers of therapeutic response. We therefore prospectively assessed the disease-free interval (DFI) and overall survival time (ST) of 30 dogs with cOSA treated with amputation and six doses of adjuvant carboplatin. We then quantified lymphocytic (CD3+, FOXP3+) and macrophage (CD204+) infiltrates within the primary tumours of this cohort using immunohistochemistry, and evaluated their association with outcome. Overall, the median DFI and ST were 392 and 455 days, respectively. The median number of CD3+ and FOXP3+ infiltrates were 45.8 cells/mm2 (4.6-607.6 cells/mm2 ) and 8.5 mm2 (0-163.1 cells/mm2 ), respectively. The median area of CD204+ macrophages was 4.7% (1.3%-23.3%), and dogs with tumours containing greater than 4.7% CD204+ macrophages experienced a significantly longer DFI (P = 0.016). Interestingly, a significantly lower percentage of CD204+ macrophages was detected in cOSA arising from the proximal humerus compared to other appendicular bone locations (P = 0.016). Lymphocytic infiltrates did not appear to correlate with outcome in cOSA. Overall, our findings suggest that macrophages may play a role in inhibiting cOSA progression, as has been suggested in human osteosarcoma.

In vitro effects of doxorubicin and tetrathiomolybdate on canine hemangiosarcoma cells.

OBJECTIVE To assess the in vitro effects of doxorubicin and tetrathiomolybdate (TM) on cells from a canine hemangiosarcoma cell line. SAMPLE Cultured cells from the canine hemangiosarcoma-derived cell line DEN-HSA. PROCEDURES Cells were treated with TM (0 to 1.5µM), doxorubicin (0 to 5µM), or both with or without 24 hours of pretreatment with ascorbic acid (750µM). Degree of cellular cytotoxicity was measured with a colorimetric assay. Long-term growth inhibition was assessed with a 10-day colony-formation assay. Induction of apoptosis was quantitated by fluorometric assessment of caspase-3 and -7 activation. Formation of reactive oxygen species (ROS) was also detected fluorometrically. RESULTS Exposure of cells to the combination of TM and doxorubicin resulted in a greater decrease in proliferation and clonogenic survival rates than exposure to each drug alone. This treatment combination increased ROS formation and apoptosis to a greater extent than did doxorubicin or TM alone. Ascorbic acid inhibited both TM-induced ROS formation and apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the enhancement in cytotoxic effects observed with DEN-HSA cell exposure to the combination of doxorubicin and TM was achieved through an increase in ROS production. These findings provide a rationale for a clinical trial of this treatment combination in dogs with hemangiosarcoma.

In vitro and in vivo activity of liposome-encapsulated curcumin for naturally occurring canine cancers.

Curcumin has well-established anti-cancer properties in vitro, however, its therapeutic potential has been hindered by its poor bioavailability. Lipocurc is a proprietary liposome-encapsulated curcumin formulation that enables intravenous delivery and has been shown to reach its highest concentration within lung tissue. The goal of this study was to characterize the anti-cancer and anti-angiogenic activity of Lipocurc in vitro, in addition to evaluating Lipocurc infusions in dogs with naturally occurring cancer. We therefore evaluated the effect of Lipocurc, relative to free curcumin, on the viability of canine osteosarcoma, melanoma and mammary carcinoma cell lines, as well as the ability of Lipocurc to inhibit endothelial cell viability, migration and tube formation. We also undertook a pilot clinical trial consisting of four weekly 8-hour Lipocurc infusions in 10 cancer-bearing dogs. Tumour cell proliferation was inhibited by curcumin at concentrations exceeding those achievable in the lung tissue of dogs. Similarly, equivalent high concentrations of Lipocurc and curcumin also inhibited endothelial cell viability, migration and tube formation. Four out of six dogs completing planned infusions of Lipocurc experienced stable disease; however, no radiographic responses were detected.

Outcome and clinical, pathological, and immunohistochemical factors associated with prognosis for dogs with early-stage anal sac adenocarcinoma treated with surgery alone: 34 cases (2002-2013).

OBJECTIVE To determine survival time and metastatic rate for dogs with early-stage anal sac adenocarcinoma (ASACA) treated with surgery alone and assess whether specific clinical, pathological, or immunohistochemical factors were predictive of outcome for those dogs. DESIGN Retrospective case series. ANIMALS 34 dogs with early-stage, nonmetastatic ASACA that were treated with surgery only. PROCEDURES Medical record databases of 2 referral hospitals were searched to identify dogs examined between 2002 and 2013 that had a diagnosis of nonmetastatic ASACA that was < 3.2 cm at its largest diameter. Only dogs that received surgical treatment alone were included in the study. For each dog, information extracted from the medical record included signalment, clinical and diagnostic test findings, tumor characteristics, and outcome. When available, archived tumor specimens were histologically reviewed and tumor characteristics were described; Ki-67 and E-cadherin expressions were evaluated by use of immunohistochemical methods. Clinical, pathological, and immunohistochemical factors were assessed for associations with survival time and tumor recurrence and metastasis rates. RESULTS Median survival time was 1,237 days. Seven dogs had tumor recurrence and 9 dogs developed metastatic disease at a median of 354 and 589 days, respectively, after primary tumor removal. Cellular pleomorphism was positively associated with development of metastatic disease. No other factors evaluated were associated with outcome. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated dogs with early-stage nonmetastatic ASACA generally had a favorable outcome following surgical removal of the primary tumor alone. Routine rectal examination may be a simple and useful method for detection of dogs with early-stage ASACA.

Adrenalectomy and caval thrombectomy in a cat with primary hyperaldosteronism.

A 13-year-old, castrated male, domestic longhaired cat was diagnosed with primary hyperaldosteronism from an adrenal gland tumor and a thrombus in the caudal vena cava. Clinical signs included cervical ventriflexion, lethargy, weakness, inappetence, and diarrhea. Laboratory tests revealed hypokalemia, normonatremia, hyperglycemia, hypophosphatemia, and elevated creatine kinase activity. Hypokalemia worsened despite oral potassium supplementation. An adrenalectomy and caval thrombectomy were successfully performed utilizing deliberate hypothermia followed by progressive rewarming.

Results of a phase II clinical trial on the use of ifosfamide for treatment of cats with vaccine-associated sarcomas.

OBJECTIVE: To determine clinical activity and toxic effects of ifosfamide when used to treat cats with vaccine-associated sarcoma (VAS). ANIMALS: 27 cats with a nonresectable, recurrent, or metastatic VAS. PROCEDURE: Each cat received ifosfamide (900 mg/m(2) of body surface area) as an IV infusion during a 30-minute period. Diuresis by infusion of saline (0.9% NaCl) solution and administration of mesna were used to prevent urothelial toxicosis. Treatments were administered every 3 weeks, and tumor response was assessed after the second treatment. All ifosfamide-associated toxic effects were graded in accordance with predetermined criteria. RESULTS: 61 treatments were administered to 27 cats (median, 2 treatments/cat; range, 1 to 4 treatments/cat). After ifosfamide treatment, 1 cat had a complete response and 10 had partial responses for an overall response rate of 11 of 27 (41%; 95% confidence interval [CI], 25% to 59%). Responses lasted from 21 to 133 days (median, 70 days; 95% CI, 60 to 113 days). The acute dose-limiting toxicosis was neutropenia, which was detected 5 to 28 days (median, 7 days) after treatment. Median nadir neutrophil count was 1,600 cells/muL (range, 200 to 5,382 cells/microL). Nine (33%) cats had adverse gastrointestinal effects (primarily salivation during the ifosfamide infusion and inappetence after treatment). Two cats were euthanatized because of severe nephrotoxicosis, and 1 cat developed pulmonary edema during diuresis. CONCLUSIONS AND CLINICAL RELEVANCE: Ifosfamide has antitumor activity against VAS in cats and is tolerated well by most cats. Ifosfamide should be evaluated as an adjuvant treatment for cats with VAS.

RNA-directed actions of 8-chloro-adenosine in multiple myeloma cells.

The purine analogue, 8-chloro-adenosine (8-Cl-Ado), induces apoptosis in a number of multiple myeloma (MM) cell lines. This ribonucleoside analogue accumulates as a triphosphate and selectively inhibits RNA synthesis without perturbing DNA synthesis. Cellular RNA is synthesized by one of three polymerases (Pol I, II, or III); thus, the inhibition of one or more RNA polymerases may be mediating 8-Cl-Ado cytotoxicity. Here, we have addressed this question by dissecting the RNA-directed actions of 8-Cl-Ado in MM cells. Differential alterations in [(3)H]uridine incorporation were found in the three major classes of RNA after a 20-h exposure with 10 microM 8-Cl-Ado. The synthesis rate of Pol III transcripts, 5 S and tRNA, remained unchanged, whereas Pol I-mediated rRNA synthesis decreased by approximately 20%. In contrast, mRNA synthesis, which is transcribed by Pol II, rapidly declined within 4 h and reached a 50% decrease, which was maintained for 20 h. Parallel to RNA synthesis inhibition, 8-Cl-Ado was maximally incorporated in the mRNA (>13 nmol/mg RNA), which was 5-fold higher than the tRNA and rRNA incorporation. Electrophoretic and radiographic analysis of newly synthesized and processed [(14)C]uridine-labeled transcripts indicated that the analogue blocks transcription elongation. Consistent with that result, high-performance liquid chromatography analysis of micrococcal nuclease and spleen phosphodiesterase-digested RNA demonstrated that the analogue incorporation is at the 3' terminus. In conclusion, our data demonstrate that in MM cells, 8-Cl-Ado is preferentially incorporated into mRNA, suggesting a propensity toward Pol II, and inhibits RNA synthesis by premature transcriptional chain termination.

Arabinosylguanine is phosphorylated by both cytoplasmic deoxycytidine kinase and mitochondrial deoxyguanosine kinase.

The prodrug of 9-beta-D-arabinosylguanine (ara-G), nelarabine, demonstrated efficacy against T-cell acute lymphoblastic leukemia, and its effectiveness correlated with the accumulation of the triphosphate form (ara-GTP). Although in vitro investigations using purified deoxycytidine kinase (dCK) or deoxyguanosine kinase (dGK) suggested that ara-G is a substrate for both enzymes, controversy exists in regard to the role of these enzymes in whole cells. In this work, we used a CEM mutant cell line containing low endogenous levels of dGK and deficient in dCK (dCK-) to assess the role of these kinases in ara-G phosphorylation. Using a retroviral vector system, we infected the dCK- mutant cell line to obtain cell lines with overexpression of dCK (dCK+) or dGK (dGK+). Only the dCK+ cell line phosphorylated 1-beta-D-arabinofuranosylcytosine (used as a substrate for dCK) in a cell-free system; phosphorylation of this compound by dGK+ was below the limit of detection. Again in in vitro assays, the dCK-and dCK+ cell lines phosphorylated dGuo to similar levels (0.91 +/- 0.15 and 0.93 +/- 0.19 pmol/mg/min, respectively), whereas dGK+ phosphorylated dGuo more efficiently (150 pmol at 60 min). When ara-G was used as a substrate in a cell-free system, the maximum accumulation of phosphorylated product was observed in dGK+ extracts at low ara-G levels (10 microM) and in dCK+ extracts at high concentrations of ara-G (100 microM). Thus, both dCK and dGK can phosphorylate ara-G, but at low ara-G concentrations, dGK seems to predominate, whereas at higher ara-G concentrations, dCK seems to be the preferred enzyme. In whole-cell systems after a 3-h incubation with 10 microM ara-G, both dCK+ and dGK+ cells accumulated ara-GTP; however, the levels were significantly (P = 0.0008) higher in dGK+ cells. In contrast, at 100 microM ara-G, intracellular ara-GTP accumulated to similar levels (P = 0.5529) in these cell types; 25 +/- 3.7 microM in dCK+, and 27.8 +/- 2.7 microM in the dGK+ cells. These results from whole-cell experiments are consistent with those from the cell-free system and strongly suggest that ara-G is phosphorylated by both kinases, and at low substrate concentrations, dGK is preferred enzyme. Evaluation of the expression of each of these kinases in primary leukemia cells may reveal a biochemical basis for the pharmacological differences in the accumulation of ara-GTP.

Evaluation of cardiovascular risks for non cardiac surgery.

Physicians are frequently concerned with the management of the surgical risk in patients with heart disease requiring non cardiac surgery. A preoperative evaluation helps to assess the cardiac risk for the planned surgery and helps to take measures to reduce that risk. We summarize the essentials in evaluating patients with coronary artery disease, valvular heart disease, arterial hypertension, arrhythmias, permanent pacemaker bearers, and those with congestive heart failure in order to prevent cardiac complications during the required surgery. Special attention has been given to the functional capacity, cardiac risk present, presence or absence of left ventricular dysfunction and the institution of protective measures. The usefulness of the ACC AHA guidelines has been summarized.