Spontaneous tumor incidence in rasH2 mice: review of internal data and published literature.

Alternate transgenic mouse models are accepted as replacements for the standard carcinogenicity mouse bioassay by regulatory agencies with a companion 2-year rat bioassay. The slower rate of industry acceptance of these shorter transgenic mouse cancer bioassays has been due to lack of historical data and diagnostic criteria, and the use of nonstandardized terminologies in published data. To address these issues, especially that of generating a large historical database, a retrospective analysis of the spontaneous tumor incidences in rasH2 mice from internally sponsored 6-month carcinogenicity studies was compared to the published literature. Incidences of common spontaneous tumors (incidences > 1%) observed in these studies were lung bronchiolo-alveolar adenomas (mean 3.9-9.9%; range 0-18%), lung bronchiolo-alveolar adenocarcinomas (mean 1.4-2.4%; range 0-5%), splenic hemangiosarcomas (mean 3.0-3.9%; range 0-17%), cutaneous squamous cell papillomas (mean 1.1-1.2%; range 0-4%), Harderian gland adenoma (mean 0.8-1.2%; range 0-4%), and hepatocellular adenomas (mean 1.8%; 0-9% in males only). The remarkable similarity in the tumor incidences in multiple rasH2 studies over a decade and the observed stability of the inserted human gene are important indicators of the minimal drift in this model. Overall, the historical control data for spontaneous neoplasms should assist in the interpretation of future rasH2 mouse studies.

An analysis of pharmaceutical experience with decades of rat carcinogenicity testing: support for a proposal to modify current regulatory guidelines.

Data collected from 182 marketed and nonmarketed pharmaceuticals demonstrate that there is little value gained in conducting a rat two-year carcinogenicity study for compounds that lack: (1) histopathologic risk factors for rat neoplasia in chronic toxicology studies, (2) evidence of hormonal perturbation, and (3) positive genetic toxicology results. Using a single positive result among these three criteria as a test for outcome in the two-year study, fifty-two of sixty-six rat tumorigens were correctly identified, yielding 79% test sensitivity. When all three criteria were negative, sixty-two of seventy-six pharmaceuticals (82%) were correctly predicted to be rat noncarcinogens. The fourteen rat false negatives had two-year study findings of questionable human relevance. Applying these criteria to eighty-six additional chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome. These analyses support a proposal to refine regulatory criteria for conducting a two-year rat study to be based on assessment of histopathologic findings from a rat six-month study, evidence of hormonal perturbation, genetic toxicology results, and the findings of a six-month transgenic mouse carcinogenicity study. This proposed decision paradigm has the potential to eliminate over 40% of rat two-year testing on new pharmaceuticals without compromise to patient safety.

Nonclinical safety evaluation of sunitinib: a potent inhibitor of VEGF, PDGF, KIT, FLT3, and RET receptors.

Sunitinib malate (SUTENT) is a multitargeted receptor tyrosine kinase (RTK) inhibitor that is approved multinationally for the treatment of imatinib-resistant/-intolerant gastrointestinal stromal tumor and advanced renal cell carcinoma. This paper characterizes the organ toxicity of sunitinib in Sprague-Dawley rats and cynomolgus monkeys, and the reversibility of any treatment-induced effects. Rats and monkeys received sunitinib (0-15 and 0-20 mg/kg/day, respectively) orally on a consecutive daily dosing schedule for thirteen weeks or on an intermittent daily dosing schedule for up to nine months. Clinical observations and laboratory parameters were recorded. Necropsy was conducted following treatment/recovery periods, and histologic examinations were performed. In rats, sunitinib was generally tolerated at 0.3 and 1.5 mg/kg/day, and findings were reversible. In monkeys, the level at which there were no observed adverse effects was 1.5 mg/kg/day, and findings were similarly reversible (except for uterine/ovarian weight changes and skin pallor). Data suggest that inhibition of multiple RTK pathways may induce pharmacologic effects on organ systems in nonclinical species. Key pharmacologic effects of sunitinib included reversible inhibition of neovascularization into the epiphyseal growth plate, and impaired corpora lutea formation and uterine development during estrus. Similar observations have been noted with this class of RTK signaling inhibitors and are consistent with pharmacologic perturbations of physiologic/angiogenic processes associated with the intended molecular targets.

Proteomics of canine lymphoma identifies potential cancer-specific protein markers.

PURPOSE: Early diagnosis of cancer is crucial for the success of treatment of the disease, and there is a need for markers whose differential expression between disease and normal tissue could be used as a diagnostic tool. Spontaneously occurring malignancies in pets provide a logical tool for translational research for human oncology. Lymphoma, one of the most common neoplasms in dogs, is similar to human non-Hodgkin's lymphoma and could serve as an experimental model system. EXPERIMENTAL DESIGN: Thirteen lymph nodes from normal dogs and 11 lymph nodes from dogs with B-cell lymphoma were subjected to proteomic analysis using two-dimensional PAGE separation and matrix-assisted laser desorption/ionization time-of-flight analysis. RESULTS: A total of 93 differentially expressed spots was subjected to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis, and several proteins that showed differential expression were identified. Of these, prolidase (proline dipeptidase), triosephosphate isomerase, and glutathione S-transferase were down-regulated in lymphoma samples, whereas macrophage capping protein was up-regulated in the lymphoma samples. CONCLUSIONS: These proteins represent potential markers for the diagnosis of lymphoma and should be further investigated in human samples for validation of their utility as diagnostic markers.

Comparison of distributions of survivin among tissues from urinary bladders of dogs with cystitis, transitional cell carcinoma, or histologically normal urinary bladders.

OBJECTIVE: To compare distributions of survivin among tissues from urinary bladders of dogs with cystitis, transitional cell carcinoma (TCC), or histologically normal urinary bladders. SAMPLE POPULATION: 24 archived and 7 fresh-frozen specimens of urinary bladders from dogs with cystitis. PROCEDURES: Immunohistochemical analysis of archived tissue specimens was performed to identify survivin protein in the nucleus and cytoplasm of cells by use of polyclonal rabbit anti-survivin antibody. Tissues that contained > or = 5% immunoreactive cells were considered positive for survivin protein. Reverse-transcription PCR analysis was performed on fresh-frozen tissues to identify survivin mRNA. Data on tissues from dogs with TCC or histologically normal urinary bladders that were obtained during another study were used for statistical comparisons. RESULTS: Twelve of 24 (50%) cystitic tissues were positive for nuclear survivin, compared with 28 of 41 (68%) TCC tissues and 0 of 46 (0%) normal tissues. Two of 24 (8%) cystitic tissues were positive for cytoplasmic survivin, compared with 7 of 41 (17%) TCC tissues and 17 of 46 (37%) normal tissues. Proportions of specimens that contained nuclear or cytoplasmic survivin were significantly different between cystitic and normal tissues but not between cystitic and TCC tissues. Four of 7 cystitic tissues were positive for survivin mRNA, which was comparable with results for TCC and normal tissues. CONCLUSIONS AND CLINICAL RELEVANCE: Nuclear survivin was detected in TCC and cystitic tissues but not in normal urinary bladder tissues. Additional studies are needed to determine whether nuclear survivin contributes to the development or progression of TCC.

Hemangiosarcoma in cats: 53 cases (1992-2002).

OBJECTIVE: To characterize the biological behavior and prognostic factors associated with hemangiosarcoma in cats. DESIGN: Retrospective case series. ANIMALS: 53 cats with hemangiosarcoma. PROCEDURES: Data were retrieved from a state veterinary diagnostic laboratory, 3 veterinary colleges, and a private practice. RESULTS: Cutaneous and subcutaneous tumor locations were more common than visceral (abdominal and thoracic) and oral locations. Surgical excision was the primary treatment in 47 cats. Tumor-free surgical margins were more likely in cutaneous than subcutaneous lesions and were associated with longer survival times. Local recurrence was observed in 6 of 12 cats with subcutaneous lesions for which follow-up was available. Metastatic disease was detected in 5 of 13 cats with adequate staging at initial diagnosis. A sixth cat had pulmonary metastases at the time of euthanasia. In 4 of 10 cats with visceral hemangiosarcoma, the diagnosis was made at necropsy or they were euthanized at the time of diagnosis. Adjuvant therapy was uncommonly used. Eighteen of the 21 known deaths or euthanasias were tumor-related. Higher mitotic counts (> 3 in 10 hpfs) were associated with shorter survival times. CONCLUSIONS AND CLINICAL RELEVANCE: Subcutaneous hemangiosarcoma was more biologically aggressive than the cutaneous form and was more likely to recur locally and result in euthanasia or death of the cat. Metastatic potential of the cutaneous and subcutaneous forms may be greater than previously reported. Visceral hemangiosarcoma is associated with a grave prognosis.

Primary renal neoplasia of dogs.

BACKGROUND: Primary renal tumors are diagnosed uncommonly in dogs. HYPOTHESIS: Signs and survival will differ among different categories of primary renal tumors. ANIMALS: Data were collected from the medical records of 82 dogs with primary renal tumors diagnosed by examination of tissue obtained by ultrasound-guided biopsy, needle aspiration, surgery, or at postmortem examination. METHODS: This was a multi-institutional, retrospective study. RESULTS: Forty-nine dogs had carcinomas, 28 had sarcomas, and 5 had nephroblastomas. The dogs were geriatric (mean 8.1 years; range: 1-17) with a weight of 24.9 kg (range: 4.5-80). Tumors occurred with equal frequency in each kidney with 4% occurring bilaterally. Initial signs included one or more of hematuria, inappetance, lethargy. weight loss, or a palpable abdominal mass. Pain was reported more frequently in dogs with sarcomas (5/28). The most common hematologic abnormalities were neutrophilia (22/63), anemia (21/64), and thrombocytopenia (6/68). Polycythemia was present in 3 dogs and resolved with treatment. Hematuria (28/49), pyuria (26/49), proteinuria (24/50), and isosthenuria (20/56) were the most frequently observed abnormalities on urinalysis. Pulmonary metastases were noted on thoracic radiographs in 16% of dogs at diagnosis. Seventy-seven percent of dogs had metastatic disease at the time of death. Median survival for dogs with carcinomas was 16 months (range 0-59 months), for dogs with sarcomas 9 months (range 0-70 months), and for dogs with nephroblastomas 6 months (range 0-6 months). CONCLUSIONS AND CLINICAL IMPORTANCE: Primary renal tumors in dogs are generally highly malignant with surgery being the only treatment that improves survival.

Clinical evaluation of mitoxantrone and piroxicam in a canine model of human invasive urinary bladder carcinoma.

PURPOSE: Cyclooxygenase inhibitors show promise in chemoprevention and therapy of certain carcinomas, an effect that may be additive to that of standard chemotherapy. The purpose of this study was to evaluate the efficacy of combined therapy using the cyclooxygenase inhibitor, piroxicam, and mitoxantrone against a relevant canine model of human invasive bladder cancer. EXPERIMENTAL DESIGN: Fifty-five dogs with transitional cell carcinoma of the urinary bladder were enrolled in this nonrandomized one-armed prospective multi-institutional clinical trial. Mitoxantrone was administered i.v. (5 mg/m(2)) every 21 days for four treatments, and piroxicam was administered p.o. (0.3 mg/kg/day) for the study duration. Tumor staging was performed at baseline, day 42 and every 3 months after protocol completion. Endpoints included time-to-treatment failure and survival time (ST). RESULTS: Response data were available for 48 dogs and included one complete response, 16 partial responses, 22 with disease stabilization, and 9 with progressive disease for an overall 35.4% measurable response rate. Subjective improvement occurred in 75% of treated dogs. Median time-to-treatment failure and ST were 194 and 350 days, respectively. Using censoring and end point definitions similar to those of previous reports of dogs treated with piroxicam alone, the median ST in this study was 291 days, compared with 181 days with piroxicam alone. Diarrhea and azotemia were the most common treatment complications. CONCLUSIONS: Mitoxantrone/piroxicam induced remission more frequently than previously reported for either drug as a single agent in this canine model of invasive human transitional cell carcinoma. Additional evaluation of these drugs in combination protocols should be explored.

Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses.

In previous studies, novel putative viral pathogens designated that asinine herpesvirus 4 (AsHV4) and asinine herpesvirus 5 (AsHV5) were associated with fatal interstitial pneumonia in donkeys (Equus asinus). Nucleotide sequence analysis of a portion of the DNA polymerase gene identified these putative pathogens as herpesviruses and possibly as members of the Gammaherpesvirinae subfamily. Although similar to equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5), sequence diversity was observed among the detected viruses. In this study, novel sequence is reported for a DNA-packaging protein gene of EHV5 plus AsHV4, AsHV5, and a newly described putative pathogen herein designated asinine herpesvirus 6 (AsHV6). Phylogenetic analysis of these sequences suggested that the equine gammaherpesviruses may form a separate clade within the Gammaherpesvirinae subfamily. Based on the sequence of EHV2 and the novel sequences reported in this study, a PCR assay was developed to detect equine gammaherpesviruses. Products of the predicted size were produced after amplification of DNA from EHV2, EHV5, AsHV4, AsHV5, and AsHV6. This nonnested assay was shown to consistently amplify approximately 10 genomic copies of EHV2. Amplification products were not produced from DNA template of other alpha- and gammaherpesviruses. Because the role of gammaherpesviruses has not been well defined in equine disease, it is envisioned that a single, sensitive PCR assay to detect these potential pathogens will facilitate further assessment of their role in disease.

Exophthalmia associated with paranasal sinus osteoma in a Quarterhorse mare.

An 11-year-old Quarterhorse mare developed a paranasal sinus osteoma that extended into the right orbit and led to ipsilateral exophthalmia. Although the tumor was radiographically evident in the paranasal sinuses, ultrasonography was used to demonstrate extension of the tumor into the retrobulbar space, and endoscopy was used to identify its extension into the nasopharynx. Biopsies were obtained using both fine-needle aspiration and paranasal sinus trephination. Despite numerous antemortem diagnostic tests, only postmortem histologic analysis of the mass afforded the diagnosis of osteoma.

Association of two newly recognized herpesviruses with interstitial pneumonia in donkeys (Equus asinus).

Over a period of 6 years, antemortem and postmortem examinations were performed on a number of donkeys suffering from respiratory disease. For many cases, initial diagnostic efforts failed to identify an etiology consistent with the pathologic findings. However, retrospective examination of these cases using consensus primer polymerase chain reaction, designed to recognize herpesviruses from all 3 subfamilies of the Herpesviridae, amplified a fragment of the highly conserved herpesvirus DNA polymerase gene from a number of these animals. Two novel herpesviruses, herein designated asinine herpesvirus 4 (AHV4) and asinine herpesvirus 5 (AHV5), were consistently detected in lung tissue from donkeys in which the histopathology was characterized by interstitial pneumonia and marked syncytial cell formation but not in lung tissue from donkeys with evidence of bacterial or verminous pneumonia. Nucleotide sequence and phylogenetic analysis places these new viruses within the Gammaherpesvirinae subfamily and indicates that they are most closely related to the recently identified zebra herpesvirus and wildass herpesvirus as well as equine herpesviruses 2 and 5.

Uterine neoplasia in 13 cats.

Thirteen uterine tumors were diagnosed in 13 cats and accounted for 0.29% of all feline neoplasms received during a 9.6-year period. Age at diagnosis ranged from 3 to 16 years; median 9 years. Six were Domestic Shorthair cats, and 7 were purebred cats of 5 different breeds. Eight adenocarcinomas and 1 mixed Müllerian tumor (adenosarcoma) comprised the endometrial tumors. Myometrial tumors included 3 leiomyomas and 1 leiomyosarcoma. One of the adenocarcinomas developed in the uterine stump of an ovariohysterectomized cat; the other cats were sexually intact. Concurrent mammary adenocarcinoma was diagnosed in 1 cat with uterine adenocarcinoma and in another with uterine leiomyoma. Tumors were discovered during elective ovariohysterectomy in 2 cats, but at least 3 others had experienced reproductive problems (infertility or pyometra). Five cats presented for abdominal or pelvic masses. Endometrial adenocarcinomas were positive immunohistochemically for cytokeratins and negative for smooth muscle actin (SMA): 1 of 6 cats was positive for vimentin and 4 of 8 were positive for estrogen receptor-alpha (ER alpha). Adenosarcoma stromal cells were positive for vimentin and ER alpha but negative for cytokeratins and SMA. Smooth muscle tumors were positive for vimentin and SMA and negative for cytokeratins. Leiomyomas, but not the leiomyosarcomas, were positive for ER alpha. Adenocarcinomas in 4 cats had metastasized by the time of ovariohysterectomy. Two other cats were euthanized 5 months after ovariohysterectomy; at least one of these cats had developed an abdominal mass that was not examined histologically. Only 2 cats with endometrial adenocarcinoma had disease-free intervals longer than 5 months after surgery. Metastasis was not detected in any mesenchymal tumor; however, these cats were either euthanized on discovery of the tumor or the tumor was first detected at necropsy.

Immunohistochemical analysis of matrix metalloproteinase-1, -3, and -13 in naturally occurring cartilaginous tumors of dogs.

OBJECTIVE: To determine immunoreactivity of matrix metalloproteinase (MMP)-1, -3, and -13 in cartilaginous tumors of dogs, correlate expression of MMP with histologic grade of tumors and clinical outcome of dogs, and compare MMP immunoreactivity between chondrosarcomas and chondromas. SAMPLE POPULATION: Formalin-fixed, paraffin-embedded tissues obtained from samples of naturally occurring chondrosarcomas (n = 31) and chondromas (8) of dogs that were submitted to our veterinary medical diagnostic laboratory. PROCEDURE: Histologic sections from each sample were stained with H&E and monoclonal antibody to MMP-1, -3, and -13 by use of an avidin-peroxidase immunohistochemical technique. For each section, histologic grade (I, II, or III) and immunohistochemical expression (0, 1, 2, or 3) were evaluated. Clinical outcome was obtained from medical records or interviews with referring veterinarians and scored as a good outcome, moderate outcome, or poor outcome. Correlations among variables and differences between chondrosarcomas and chondromas were analyzed. RESULTS: Samples from chondrosarcomas had significantly higher immunoreactivity of MMP-1 and -13, compared with immunoreactivity in samples from chondromas. In chondrosarcomas, a significant positive correlation (r, 0.386) was found between MMP-1 and -13 immunoreactivities, and a significant negative correlation (r, -0.390) was detected between MMP-3 and -13 immunoreactivities. CONCLUSIONS AND CLINICAL RELEVANCE: A significant increase in expression of collagenases (MMP-1 and -13) in chondrosarcomas, compared with expression in chondromas, suggests that collagenases may play an important role in tumor progression, and possibly metastasis, in chondrosarcomas of dogs.

Surgical treatment of noncommunicating duplication of the colon in a dog.

A 4-month-old sexually intact male Jack Russell Terrier was evaluated because of stranguria and tenesmus. A tubular abdominal mass was palpable abdominally and rectally. Radiographic examination of the abdomen revealed a soft tissue mass located laterally and to the left of the descending colon, which was associated with extraluminal colonic obstruction and urethral compression. During abdominal exploratory surgery, a large cystic mass that was adhered to the antimesenteric border of the descending colon was removed. Porcine small intestinal submucosa was used to reinforce repair of the excision site. Histologic examination of samples of excised tissue identified normal colonic epithelium supported by submucosa and muscular tunics, which was consistent with duplication of the colon. The embryologic etiology of alimentary duplication is poorly understood, and colonic duplication is an extremely rare congenital anomaly.

Bone marrow hypoplasia associated with fenbendazole administration in a dog.

A 1.5-year-old Doberman pinscher was presented with sudden-onset of fever and malaise. Twelve days prior to presentation, fenbendazole therapy was initiated for a suspected lungworm infection. Results of a complete blood count on presentation showed pancytopenia, while histopathological evaluation of a bone marrow core sample revealed bone marrow hypoplasia of undetermined etiology. Bactericidal antibiotics and fluid therapy, as well as discontinuation of fenbendazole administration, led to a complete resolution of clinical and hematological abnormalities within 15 days. An idiosyncratic reaction to fenbendazole was suspected based on the absence of infectious, neoplastic, autoimmune, and toxic etiologies, as well as resolution of clinical signs and pancytopenia upon drug withdrawal.

Inhibition of intimal hyperplasia by direct thrombin inhibitors in an animal vein bypass model.

Many functions of the coagulation system have nonthrombotic effects. The indirect thrombin inhibitor heparin has been previously shown to be effective in limiting intimal hyperplasia (IH). We sought to study the effect of thrombin on IH by using two direct thrombin inhibitors (DTIs), argatroban and lepirudin. Sprague-Dawley rats underwent interposition vein grafting to the carotid artery. Vein grafts were treated with either saline (n = 6) or one of the two DTIs (n = 6 for both). At 30 days, the rats were sacrificed and vessels were perfusion fixed. Sections of the proximal carotid artery, graft, and both anastomoses were stained with both hematoxlyin/eosin and von Gieson's elastin stain. Sections were examined and compared for luminal area and intima-to-media (IM) ratio. The vessels treated with DTIs had less (p < 0.05) IH (IM ratio for proximal anastomosis: control 1.036 +/- 0.857, lepirudin 0.373 +/- 0.21, argatroban 0.182 +/- 0.118) and better lumen preservation than the control vessels (lumen area of proximal anastomosis: control 1.69 +/- 0.9, lepirudin 2.45 +/- 0.74, argatroban 2.81 +/- 0.78). There were no thromboses in the DTI-treated vessels. Dilatation of the graft segment was noted in the argatroban group. Thus, DTIs are effective at reducing IH in a small-animal model, suggesting that inhibition of thrombin has a protective role in IH. In addition, a difference of action between DTIs is suggested by the dilatation seen only in the argatroban-treated graft sections.

Detection and multigenic characterization of a herpesvirus associated with malignant catarrhal fever in white-tailed deer (Odocoileus virginianus) from Missouri.

Between 1998 and 2001, tissues from four captive white-tailed deer were observed to have histologic lesions of systemic lymphocytic vasculitis. These lesions suggested malignant catarrhal fever, although epizootic hemorrhagic disease and bluetongue were included in the differential diagnosis. Initial diagnostic efforts, including virus isolation and reverse transcription-PCR for epizootic hemorrhagic disease virus and bluetongue virus, failed to identify an etiologic agent. However, consensus primer PCR targeted to the herpesvirus DNA polymerase gene detected viral genomic DNA in each of these four cases. Nucleotide sequence analysis of the amplified product demonstrated that the detected virus was identical over the compared region to the recently described malignant catarrhal fever virus of white-tailed deer (H. Li, N. Dyer, J. Keller, and T. B. Crawford, J. Clin. Microbiol. 38:1313-1318, 2000). Additional nucleotide sequencing of both the DNA polymerase gene and DNA packaging gene followed by phylogenetic analysis solidified this newly recognized herpesvirus as a member of the Gammaherpesvirinae and suggests that this virus, along with ovine herpesvirus 2, alcelaphine herpesvirus 1, alcelaphine herpesvirus 2 and caprine herpesvirus 2, may be part of a separate clade within this subfamily.