Phase I clinical trial of the use of zinc phthalocyanine tetrasulfonate as a photosensitizer for photodynamic therapy in dogs

OBJECTIVE: To determine the threshold for acute toxicosis of parenterally administered zinc phthalocyanine tetrasulfonate (ZnPcS(4)), a candidate second-generation photosensitizer, in mice and evaluate the compound's safety in a phase I clinical trial of ZnPcS(4)-based photodynamic therapy (PDT) in pet dogs with naturally occurring tumors. ANIMALS: Male Swiss-Webster mice and client-owned dogs with naturally occurring neoplasms. PROCEDURES: For the study of acute toxicosis, mice were given graded doses of ZnPcS(4). To determine safety, a rapid-titration phase I clinical trial of ZnPcS(4)-based PDT in tumor-bearing dogs was conducted. RESULTS: In mice, administration of >or= 100 mg of ZnPcS(4)/kg resulted in renal tubular necrosis 24 hours after IP injection. In tumor-bearing dogs, ZnPcS(4) doses 相似文献   

Efficacy and Toxicity of Intracavitary Administration of Pegylated Liposomal Encapsulated Doxorubicin (DOXIL) in Dogs with Hemangiosarcoma

Introduction:  Canine hemangiosarcoma (HSA) is a fatal malignancy and most dogs die within 6–8 months of diagnosis. The spleen is a common primary site, representing 50% of all cases. These dogs typically present with clinical signs due to tumor rupture and intra‐abdominal dissemination; the abdomen is also the main site of disease progression when these patients fail. Direct delivery of chemotherapy into the abdominal cavity may therefore be a rational approach in this malignancy.
Methods:  14 dogs with stage 2 or 3 splenic HSA were recruited. Doxil at a dose of 1 mg/kg was diluted in saline and administered via ultrasound‐guidance into the abdominal cavity. The dogs were scheduled to receive 4 treatments every 3 weeks. Samples of plasma and abdominal fluid were collected for pharmacokinetic analysis. All dogs were monitored for recurrence and complete necropsies were requested at death.
Results:  8 dogs with stage 3 and 6 dogs with stage 2 HSA were enrolled. All 14 dogs have died, 12/14 due to tumor and 2 from other causes. There was no difference in median survival days between stages (stage 2: 244, stage 3: 125, p = .22). All 12 dogs that died due to tumor‐related causes failed with intra‐abdominal recurrence. Necropsies showed that the dogs in this study had relatively fewer extra‐abdominal metastasis compared to dogs treated with systemic chemotherapy. Pk analysis showed detectable plasma doxorubicin 1 and 2 weeks after treatment.
Conclusion:  Direct abdominal administration of Doxil did not prevent intra‐abdominal recurrence; however, it appeared to provide effective systemic coverage.     

Increase in Regulatory T Cells in Dogs with Cancer Undergoing Chemotherapy

Introduction:  Regulatory T cells (Treg) are a naturally occurring population of T cells phenotypically identified by co‐expression of CD4 and the IL‐2 receptor (CD25). Theyplay a critical role in the control of tolerance and autoimmunity and have also been implicated in impairment of anti‐tumor responses. We hypothesized that levels of Treg would be higher in cancer‐bearing dogs than in normal dogs and that they would decrease with chemotherapy.
Methods:  Serial PBMC were isolated from twenty cancer‐bearing dogs receiving either single‐agent doxorubicin or the Madison‐Wisconsin protocol. The following time points were studied: pre‐treatment, day 2, week 1, week 3, 3 months and 6 months after initial treatment. Ten age‐matched, normal dogs were also studied. PBMC were immunostained with directly conjugated antibodies to CD4, CD8, CD44 and IL‐2 receptor and then evaluated by flow cytometry.
Results:  Low numbers of lymphocytes with the CD4+/IL‐2R+ phenotype were detectable in both normal and cancer‐bearing dogs. A statistically significant increase in the percentage of IL2‐R+/CD4+ T cells was observed in the cancer‐bearing dogs beginning two days after chemotherapy and persisting throughout treatment. The percentage of IL‐2R+/CD4+ T cells was also increased in pre‐treated cancer‐bearing dogs compared to control dogs.
Conclusion:  The percentage of IL‐2R+/CD4+ T cells was generally higher in dogs with cancer than in healthy dogs. Unexpectedly, the percentage of IL‐2R+/CD4+ cells increased during chemotherapy which suggests that chemotherapy may exert immunosuppressive effects through a previously undescribed mechanism. The identity of these CD4+/IL‐2R+ T cells as true Treg awaits additional characterization studies.     

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

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

An hGM‐CSF DNA‐Cationic Lipid Complexed Autologous Tumor Cell Vaccine did not Improve Remission Duration in Dogs with Lymphoma

Introduction:  Lymphoma is one of the most common cancers in dogs and while clinical remission can be induced using chemotherapy, very few dogs are cured. Since cytokine‐adjuvanted autologous whole‐tumor‐cell vaccines (ATCV) can induce potent antitumor immune responses against otherwise non‐immunogenic cancers we initiated a study of such an approach in dogs with lymphoma.
Methods:  Following achievement of a complete remission using a 19‐week CHOP‐based chemotherapy protocol, 51 dogs with B‐cell lymphoma were randomized to receive 8 treatments (4 weekly, then 4 additional at q2wk intervals) of vaccine or lipid‐equivalent placebo. Dogs were followed monthly for assessment of remission duration and survival. Surrogate indices of immune response (delayed‐type hypersensitivity, interferon‐γ quantitative RT‐PCR, lymphocyte proliferation, and flow cytometry for lymphoma‐specific antibodies) were also investigated before and after vaccination.
Results:  No significant difference in median remission duration was observed between dogs receiving vaccine (277 days) or placebo (258 days); the Kaplan‐Meier curves were virtually super‐imposable. No significant differences in surrogate indices of immune response were noted pre‐ and post‐vaccination.
Conclusions:  In this context, an hGM‐CSF DNA‐cationic lipid complexed ATCV vaccine did not enhance remission duration in dogs with lymphoma, likely due to lack of vaccine‐induced tumor‐specific immunity.     

Xenogeneic Human Tyrosinase DNA Vaccination Induces Specific Immune Response in Dogs with Advanced Malignant Melanoma

Introduction:  Xenogeneic melanosomal differentiation antigens, delivered in the form of a plasmid DNA vaccine, can overcome host immune ignorance/tolerance in preclinical animals to melanoma by: 1) generating humoral and cytotoxic T cell responses and 2) inducing protection from tumor challenge. Initial trials of human tyrosinase (huTyr) DNA vaccination of dogs with advanced malignant melanoma (Bergman et al , 2003) demonstrated safety and prolongation of survival with this therapeutic modality. We investigated antigen‐specific immunity in dogs receiving huTyr DNA vaccination.
Methods:  Three cohorts of three dogs each with advanced (WHO stage II‐IV) canine malignant melanoma (CMM) received four biweekly IM injections (dose levels 100, 500, or 1,500 ug, respectively/vaccination) of huTyr plasmid DNA via the Biojector2000 jet delivery device. Sera samples were taken before and after vaccination to detect specific antibody formation to huTyr by Enzyme‐Linked ImmunoSorbent Assays (ELISAs) and flow‐cytometry.
Results:  Three dogs have measurable, 2 to 4 fold huTyr‐specific antibody titers. Preliminary studies by flow‐cytometry have confirmed antibody response to huTyr by positive binding to endogenous human tyrosinase in SK‐Mel188 cells using post‐vaccinate serum.
Conclusions:  Xenogeneic (huTyr) DNA vaccination generates antigen‐specific humoral responses, which may partially explain the previously reported clinical efficacy and anti‐tumor responses. Ongoing studies include: 1) a comprehensive analysis by flow‐cytometry to detect huTyr‐specific antibodies and 2) a quantitative measure of potential cytotoxic T‐cell responses in these dogs by the DNA vaccination.     

Antitumor Activity of Mycobacterial Cell Wall‐DNA Complex (MCC) Against Canine Urinary Bladder Transitional Cell Carcinoma Cells

Introduction:  Mycobacterial cell wall‐DNA complex (MCC) is a bifunctional anticancer agent that induces cancer cell apoptosis and stimulates cytokine synthesis by immune cells. Intravesical MCC is currently being evaluated in humans with high‐grade urinary bladder cancer. Evaluation of MCC in dogs with transitional cell carcinoma (TCC) will allow mechanistic studies in a natural animal model of TCC, and a potentially beneficial therapy for dogs with this cancer. In this study, we have determined the anticancer activity of MCC against canine TCC cells in vitro .
Methods:  Canine TCC cells (K9TCC cell line) were incubated with MCC (0.05–100 μg/ml, 0.5–72 hours). Cellular proliferation was measured by MTT reduction. Cell cycle was analyzed by flow cytometry with propidium iodide. Apoptosis was identified by flow cytometry using anti‐active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies. Apoptosis‐inducing activity of 100 μg/ml MCC in combination with piroxicam (0.1–1.0 uM) was evaluated.
Results:  MCC inhibited K9TCC cell proliferation in a concentration‐dependent manner (maximal activity – 45% at 100 μg/ml MCC) in association with the presence of activated caspase‐3 and cleaved PARP. Inhibition of proliferation and apoptosis‐inducing activities of MCC were independent of cell cycle phase. A thirty‐minute exposure of MCC was sufficient for optimal activity. Piroxicam (0.5 uM) enhanced apoptosis‐inducing activity of MCC.
Conclusions:  MCC induces apoptosis in K9TCC cells. This activity is potentiated by piroxicam. Following positive results in vitro , in vivo studies have been initiated. One dog, treated to date, has had a minor reduction in tumor volume following the first course of treatment with no treatment‐related toxicity.     

Association of p53 Gene Mutation with the Prognosis of Canine Lymphoid Tumors

Introduction:  Many dogs with lymphoid tumors develop resistance to chemotherapy. As a mechanism of drug resistance in canine lymphoma, ATP‐dependent drug efflux by P‐glycoprotein was reported, however, inhibition of apoptosis mediated by P53 inactivation has not been investigated. In this study, we investigated the relationship between p53 gene mutation and clinical drug resistance in canine lymphoid tumors.
Methods:  Tumor specimens were obtained from 44 dogs with lymphoid tumors. Mutations of p53 gene at exon 4–8 of these tumor tissues were examined by PCR‐SSCP (single strand conformational polymorphism) analysis, followed by nucleotide sequencing of the abnormal bands. The cases were treated with UW‐Madison protocol, and its response was evaluated by the tumor size or the number of peripheral leukemic cells.
Results:  Of the 44 dogs, 15 dogs (34%) had p53 mutation, whereas 29 dogs (66%) were devoid of p53 mutation, before or during the chemotherapeutic protocol. Rate of good response (CR and PR) to chemotherapy was significantly lower in the dogs with p53 mutation (20%) than those without p53 mutation (55%)(p = 0.022). Median overall survival duration after examination of p53 mutation was significantly shorter in dogs with p53 mutation (101days) than those without p53 mutation (223days)(p = 0.008).
Conclusions:  Lymphoid tumors with p53 mutations were shown to have worse prognosis than those without p53 mutation.     

Anti‐Tumor Effect of Adenovirus‐Mediated P53 Gene Therapy on the Growth of Canine Osteosarcoma Transplanted into Nude Mice

Introduction:  It has been reported that 40–50% of canine osteosarcoma cases have p53 mutations. The p53 tumor supressor gene plays a central role in cell cycle regulation and induction of apoptosis. We previously showed that adenoviral vector expressing canine P53 (AxCA‐cp53) inhibited growth of cultured canine osteosarcoma cell lines. Here, we evaluated anti‐tumor effect of adenovirus‐mediated p53 gene therapy on the growth of canine osteosarcomas transplanted into nude mice.
Methods:  Nine nude mice were subcutaneously injected with cells of a canine osteosarcoma cell line (POS) having p53 gene mutation. The transplanted tumors formed into nude mice were injected with AxCA‐cp53, AxCA‐LacZ (adenovirus vector expressing LacZ) or PBS (3 mice each) 7 times during 15 days. Tumor sizes were measured every 3 days for 27 days after injection with the adenovirus vectors. Expression efficiency of the adenovirus‐mediated gene transfer was examined by X‐gal staining and P53 immunostaining. Effects of the P53 expression on cell cycle control were examined by RT‐PCR for expression of p21 gene downstream of P53.
Results:  Significant differences in the tumor size was observed between the transplanted osteosarcoma tissues injected with AxCA‐cp53 and those injected with AxCA‐LacZ or PBS. Expressions of LacZ and P53 were confirmed at the injection sites of the tumors. Moreover, p21 mRNA expression was shown to be induced in the AxCA‐cp53‐injected tumors, indicating the funciton of P53 to induce cell cycle arrest.
Conclusions:  Adenoviral vector expressing canine P53 inhibited the growth of canine ostersarcoma transplanted into nude mice.     

Results of the Combined Treatment with Piroxicam and Carboplatin in Canine Oral Non‐Tonsillar Squamous Cell Carcinoma

Introduction:  Over‐expression of COX‐2 has been observed in several human and animal malignancies and is implicated in carcinogenesis through the conversion of arachidonic acid to PGE‐2. Use of platinum‐containing cytostatic agents and/or (non‐)specific COX‐2 inhibitors, has been reported as a treatment option for canine oral non‐tonsillar squamous cell carcinomas (ONT‐SCC). However, no study describes the effect of a combination of carboplatin and piroxicam on this tumor type.
Methods:  7 dogs with a T3 (WHO‐TNM) ONT‐SCC were treated with piroxicam and carboplatin. Five had bone involvement and no detectable metastasis. Two dogs without bone involvement had metastasis in the regional lymph nodes. Piroxicam was given orally 0.3 mg/kg s.i.d. Each dog was scheduled to receive between 6 and 12 carboplatin infusions (300 mg/m² i.v.) at 3 week intervals. Ondansetron and metoclopramide were used as anti‐emetic agents. The dogs are planned to receive piroxicam on a lifelong basis.
Results:  Complete response (CR) without adjuvant surgery was achieved in 4 of the 7 dogs. Two dogs needed adjuvant surgery to achieve CR. One dog had progressive disease and was euthanised 231 days after start of therapy. All the others were still alive and in CR at date of analysis. Median follow‐up was 335 days (107–689 days).
Conclusions:  Our study suggests that a combination of piroxicam and carboplatin is a useful treatment option for canine ONT‐SCC. All dogs tolerated therapy well and the 57% response rate for reaching a complete and durable remission without adjuvant surgery is promising.     

Phase I & Ib Trials of Murine Tyrosinase ± Human GM‐CSF DNA Vaccination in Dogs with Advanced Malignant Melanoma

Introduction:  Canine malignant melanoma (CMM) is an aggressive neoplasm treated with surgery and/or fractionated RT; however, metastatic disease is common and chemoresistant. Preclinical and clinical studies by our laboratory and others have shown that xenogeneic DNA vaccination with tyrosinase family members can produce immune responses resulting in tumor rejection or protection and prolongation of survival. The potency of DNA vaccines can be further enhanced by adding DNA encoding cytokine genes. We have shown in preclinical mouse models that GM‐CSF DNA enhances immune responses and tumor protection. These studies provided the impetus for murine tyrosinase (muTyr) ± human GM‐CSF (huGM‐CSF) DNA vaccination in CMM.
Materials & Methods:  Two groups of five dogs each with advanced (WHO stage II‐IV) CMM received four biweekly IM injections (100 ug or 500 ug, respectively/vaccination) of plasmid DNA encoding muTyr via the Biojector2000 jet delivery device. Subsequently, three groups of nine dogs each with advanced CMM received four biweekly IM injections of plasmid DNA encoding muTyr (50 ug), huGM‐CSF (3 dogs each at 100/400/800 ug) or both.
Results:  Minimal to mild pain was noted on vaccination and no toxicity or induction of autoimmunity was seen. The KM median survival time was 224 days (100/500 ug muTyr), 278 days (50 ug muTyr), 140 days (huGM‐CSF) and >265 days (muTyr & huGM‐CSF; 6 dogs still alive).
Conclusions:  The results of these trials demonstrate that xenogeneic DNA vaccination continues to be a safe and potentially therapeutic modality for CMM. These results also warrant further evaluation of this novel therapeutic in a Phase II setting.     

The Biologic Behavior of Mast Cell Tumors in Pug Dogs

Introduction:  The purpose of this study was to quantitate the risk and to describe the behavior of mast cell tumors (MCT) in Pugs.
Methods and Materials:  Using the Veterinary Medicine Database, the frequency of MCT in Pugs was compared to the frequency in other dogs using a Chi‐square test. To describe the biologic behavior of MCT in pugs, cases with histologically confirmed diagnosis were identified through the University of Minnesota (UMN) Diagnostic Laboratory and Veterinary Medical Center. Histology was reviewed by a single pathologist. Survival analysis was performed to determine the impact of clinical and histologic data on survival.
Results:  The frequency of MCT diagnosis in Pugs was significantly increased compared to other dogs (OR = 2.28, 95% CI = 1.81–2.86). Twenty‐five purebred Pugs with a diagnosis of MCT were identified through UMN. Multiple cutaneous tumors were documented in 14 (56%) of the dogs. Most tumors were low to intermediate grade. Only three dogs have died of their disease. Sixteen are still living (median follow‐up = 660 days). The only factors predicting survival were grade, mitotic index and tumor size.
Discussion:  Our data confirms MCT predisposition in Pugs and suggests that mast cell tumors in Pugs are relatively benign, despite the presence of multiple cutaneous tumors in most cases. Multiple tumors in breeds with predisposition to MCT may indicate separate primaries rather than advanced stage disease.
This work was supported in part by NCI grants R03‐CA101030 and K08‐CA89530.     

131‐I therapy for Advanced, Unresectable Thyroid Tumors in Dogs

Introduction:  Greater than 50% of dogs with thyroid tumors present with surgically unresectable disease for which external beam radiotherapy has been reported to prolong survival. The success of ¹³¹I for control of thyroid tumors in cats and in humans suggests such therapy may also play a role in the management of canine thyroid cancer.
Methods:  Thirty‐nine dogs with WHO stage II/III (invasive or ectopic; n = 32) or IV (metastatic; n = 7) thyroid tumors were treated with ¹³¹I alone. Changes in thyroid function, ^99MTc‐pertechnetate (^99MTc) scintigraphic changes, and tumor response were recorded. Dogs with ventral cervical tumors were evaluated for feasibility of surgical resection following ¹³¹I.
Results:  Median overall survival was 839 days and 366 days for dogs with stage II/III and stage IV tumors, respectively. Thyroid hormone status, site and surgical resection were not associated with outcome in dogs with stage II/III tumors. Three dogs developed severe bone marrow suppression.
Conclusions:  These findings suggest ¹³¹I should be investigated more thoroughly in dogs with thyroid tumors not considered surgical candidates to more clearly characterize the indications for therapy and followup recommendations. ¹³¹I dosimetry in dogs with thyroid tumors remains problematic. Administration of ¹³¹I is currently based on empiric recommendations and, in general, the treatment is well tolerated although additional studies are indicated to optimize response and minimize toxicity.     

Total Skin Electron Beam Irradiation for Generalized Cutaneous Lymphoma

Introduction:  Generalized cutaneous T‐cell lymphoma (CTCL) in canines has many similarities to CTCL in humans. In humans, early stage mycosis fungoides (MF) patients that do not have lymph node or any systemic involvement can do very well with total skin electron beam irradiation (TSE). In fact, if these early stage patients were treated with 30–35 Gy, the median overall survival was not reached at 15 years. Only one dog with CTCL has been treated with total skin irradiation using orthovoltage irradiation. The patient died the night of the 2nd fraction of RT. Now that more veterinary RT facilities have linear accelerators with electron beam capabilities, it is possible canines with CTCL can be treated with TSE. This paper describes an adaptation of the Modern TSE technique used in human MF patients and its application in canine patients with MF/CTCL.
Methods:  The modified Modern TSE technique and its application for canine MF/CTCL patients involves 20 treatments given M‐F over 4 weeks. The entire skin surface is treated from multiple directions.
Results:  Four canine patients with CTCL have been treated with this TSE technique. All patients responded to the treatment initially with the first patient having tumor control for >20 months. One patient has failed the therapy at this date.
Conclusions:  The TSE technique adapted for use in canines with CTCL was well tolerated with no significant toxicity noted. Development of a collaborative study with other veterinary RT centers having electron beam capabilities is being evaluated through VRTOG.     

Immunohistochemical Differentiation and Clinicopathological Features of Canine Gastrointestinal Stromal Tumors and Leiomyosarcomas

Introduction:  Gastrointestinal stromal tumors (GIST) and leiomyosarcomas (LMS) have recently been differentiated by immunohistochemical staining techniques and have been shown to have different biological behaviors in humans. Expression of the c‐kit protein, a transmembrane tyrosine kinase receptor, occurs in nearly all GISTs. The aim of this study was to differentiate canine GIST from LMS, and to compare their clinicopathological features.
Methods:  Archived blocks of previously diagnosed LMS were analyzed. Immunohistochemical staining using antibodies against c‐kit, smooth muscle actin (SMA), desmin, vimentin and S100 was performed. GISTs were diagnosed based on positive c‐kit staining. LMS were diagnosed based on absence of c‐kit staining and positive SMA staining. Follow‐up information was obtained from medical records and telephone interviews with owners.
Results:  Forty‐two dogs were included in the study. Mean age was 10.9 yrs (range 5–15 yrs). There were 18 females and 24 males. Twenty‐eight tumors were GISTs, 10 were LMS and 4 stained negatively for c‐kit, SMA and S100 (sarcomas). GISTs were more likely to occur in the large intestine and LMS were more common in the small intestine (p = 0.01). All were surgically excised and only two were treated with adjunctive chemotherapy. Only two GISTs and one sarcoma had metastasized at the time of surgery. Survival time in dogs discharged after surgery for GIST, LMS and sarcomas was 1123, 233 and 88 days respectively (p = 0.08).
Conclusions:  Many previously diagnosed LMS should be reclassified as GIST based on the results of immunohistochemical staining. The biological behavior of these tumors appears to be different.     

Retropective Evaluation of Survival Time of Dogs with Stage III Osteosarcoma that Undergo Treatment

Introduction:  Stage III osteosarcoma generally carries a grave prognosis. Despite this, some owners elect to treat using palliative or curative intent protocols. The purpose of this study was to determine the survival times for dogs with stage III osteosarcoma that undergo treatment and to evaluate the variables that affect survival time.
Methods:  Retrospective study using the CSU Animal Cancer Center osteosarcoma data base. Search criteria included dogs diagnosed with osteosarcoma 1985–2004 with metastasis at the time of presentation. Dogs were excluded if they were euthanized at the time of diagnosis (13 dogs) or lost to follow‐up (10 dogs). There were 90 cases for analysis. The survival times were compared based on the primary tumor site, site of metastasis, treatment given, age, sex and breed.
Results:  Survival times in days ranged from 0 to 1583 days. The overall median survival time was 76 days. The one‐year, two‐year and three‐year percent survival were 7%, 4.7% and 3.5%, respectively. Treatment included various combinations of chemotherapy, surgery, radiation therapy, bisphosphonates and NSAIDs. Findings included an increased survival time with surgery and adjuvant therapy compared with surgery alone and a decreased survival time with metastasis to the lung or lymph node.
Conclusions:  Treatment of dogs with stage III osteosarcoma can result in variable survival times. Multimodality therapy appears to result in longer survival times. Metastasis to the lung or lymph node correlated with a decreased survival time.     

Induction of Chemoresistance by Transfection of the Full‐Length Canine mdr1 Gene in Canine Cell Lines

Introduction:  In the chemotherapy for treatment of lymphoid tumors in dogs, myelosuppression is a frequently encountered dose‐limiting factor. One possible approach to overcome myelosuppression is induction of chemoresistance in hematopoietic stem cells through expression of the mdr1 gene. A full‐length canine mdr1 cDNA clone was isolated in our laboratory. The present study was carried out to assess whether it confers multidrug resistance in canine cell lines.
Materials and methods:  The full‐length canine mdr1 cDNA was inserted into an expression plasmid vector. A canine mammary tumor cell line (CIPP) and osteosarcoma cell line (OOS) were transfected with the canine mdr1 expression vector. Expression of P‐gp was examined by immunoblotting. Function of ATP‐dependent drug efflux was measured by flow cytometric analysis using Rhodamine 123. Sensitivity to chemotherapeutic drugs was shown by estimation of 50% inhibitory concentrations (IC₅₀) of vincristine or doxorubicin.
Results:  Immunoblotting of the transfected cells revealed a strong band of P‐gp detected by a monoclonal antibody directed to P‐gp. Rhodamine 123 efflux test showed an apparent drug efflux activity in the transfected cells. From the IC₅₀ of the chemotherapeutic agents, the transfected cells showed a remarkable increase (20 to 60‐fold) in the resistance to vincristine or doxorubicin.
Conclusion:  Transfection of canine mdr1 gene induced P‐gp expression and strong drug resistance in canine cell lines.     

Evaluation of Intracavitary Chemotherapy Delivery for the Treatment of Mammary Carcinoma

Introduction:  The purpose of this study was to evaluate tolerability and efficacy of local (intracavitary) delivery of paclitaxel from a gel polymer (poloxamer 407) following marginal (histologically incomplete) resection of mammary carcinoma in a mouse model
Methods:  In vitro sensitivity to paclitaxel as well as poloxamer‐taxol mixture (polotax) was determined for 4 human breast tumor cell lines using the MTS‐assay (Promega). Nude mice were then injected with MCF‐7/ADR (Adriamycin^R resistant) cells. Tumor growth was monitored by imaging of luciferase activity with a CCD camera (IVIS system, Xenogen). Primary tumors were allowed to grow to 3 different size ranges. At the time of primary tumor resection, animals were randomly assigned to treatment groups comparing intracavitary (in the wound) polotax to intravenous paclitaxel. Mice were imaged weekly to evaluate tumor regrowth and metastasis.
Results:  All cells lines demonstrated sensitivity to paclitaxel and three of the four cell lines demonstrated improved cytotoxicity with polotax compared to drug delivered alone. One of 9 mice treated with polotax had local regrowth (by 60 days) compared to 6 of 9 mice treated with intravenous paclitaxel. One of 9 mice treated with polotax had distant metastasis at 60 days compared to 5 of 8 mice treated with intravenous paclitaxel. These effects were seen with tumors of all sizes.
Discussion/Conclusions:  Poloxamer delivery of paclitaxel appears to result in improved efficacy compared to paclitaxel alone. Improved local and systemic control of mammary carcinoma is seen following intracavitary poloxamer delivery of paclitaxel compared to paclitaxel alone in this mouse model.     

Adverse Effects of Concurrent Carboplatin Chemotherapy and Radiation Therapy in Dogs

Background: Concurrent chemo- and radiotherapy improves outcome of certain human neoplasms but with increased signs of toxicity. Reports on adverse effects of concurrent chemo- and radiotherapy in the veterinary literature are scant.
Objective: To report adverse hematologic and gastrointestinal effects of combined carboplatin and radiation therapy in dogs.
Animals: Client-owned dogs with spontaneously occurring neoplasia.
Methods: Retrospective case study. Medical records of 65 dogs were reviewed. Criteria for inclusion were administration of radiation according to 1 of 3 fractionation schemes (19 × 3, 16 × 3, or 12 × 4 Gy) and administration of at least 1 concurrent carboplatin treatment at a dosage of 200–300 mg/m². Dog and treatment-related variables were analyzed for association with signs of intoxication.
Results: Median carboplatin dosage was 200 mg/m² (range, 200–250 mg/m²). Twelve of 58 dogs (21%) developed grade 3 or 4 neutropenia. Eleven of 56 dogs (20%) developed grade 3 or 4 thrombocytopenia. Six of 62 dogs (10%) developed grade 3, 4, or 5 gastrointestinal toxicosis. Analysis of association of dog and treatment-related variables with signs of intoxication was hampered by the small numbers of dogs in individual groups, and no statistically significant associations were found.
Conclusions and Clinical Importance: Combined modality therapy resulted in myelosuppression and gastrointestinal toxicosis. Future studies are needed to determine whether the potential benefit of combined modality therapy outweighs the risk of decreasing chemotherapy and radiation treatment intensity.     

The effect of deracoxib and piroxicam on viability of canine osteosarcoma cells in vitro

Introduction:  Cyclooxygenase‐2 (COX‐2) inhibitors are being used increasingly in cancer therapy. Although the effects of COX‐2 inhibitors have been evaluated extensively in carcinomas, less is known about their effects in sarcomas. Since the majority of dogs with appendicular osteosarcoma (OSA) are treated for pain with a non‐steroidal anti‐inflammatory drug (some COX‐2 selective) prior to definitive treatment, it is important to determine the effects that commonly used NSAIDS have on tumor cell growth.
Methods:  Established canine osteosarcoma (POS, HMPOS and COS31) and canine fibroblast cell lines were maintained in culture under standard conditions. Cells were incubated with either deracoxib (1 uM to 500 uM) or piroxicam (1 uM to 1000 uM). Cell viability was assessed at 72 hours by cell counts and the MTT assay. The DNA fragmentation analysis was utilized to assess for apoptosis induction.
Results:  Deracoxib concentrations ≥100 uM and piroxicam concentrations ≥500 uM significantly reduced mean cell viability of all three OSA cell lines (lowest cell viability percentages 20% and 32%, respectively). Deracoxib concentrations ≥250 uM and piroxicam concentrations ≥500 uM also reduced viability of fibroblasts; however, the cell viability percent was reduced to only 54% and 68%, respectively, of the control value. Exposure of OSA cells to cytotoxic concentrations of deracoxib and piroxicam did not result in DNA fragmentation.
Conclusions:  Deracoxib and piroxicam demonstrated a cytotoxic effect on canine osteosarcoma cells. There was no evidence of apoptosis induction at the concentrations evaluated. Further investigation will need to be performed to determine whether either drug exhibits anti‐tumor effects in vivo .