E. J. Ehrhart

Incidence of acute myeloid leukemia and hepatocellular carcinoma in mice irradiated with 1 GeV/nucleon 56Fe Ions

Abstract Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the leukemogenic efficacy of one such HZE species, 1 GeV 56Fe ions, a component of space radiation, in a mouse model for radiation-induced acute myeloid leukemia. CBA/CaJ mice were irradiated with 1 GeV/nucleon 56Fe ions or 137Cs γ rays and followed until they were moribund or to 800 days of age. We found that 1 GeV/nucleon 56Fe ions do not appear to be substantially more effective than γ rays for the induction of acute myeloid leukemia (AML). However, 56Fe-ion-irradiated mice had a much higher incidence of hepatocellular carcinoma (HCC) than γ-irradiated mice, with an estimated RBE of approximately 50. These data suggest a difference in the effects of HZE iron ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis.

Immunohistochemistry of Atrial and Brain Natriuretic Peptides in Control Cats and Cats with Hypertrophic Cardiomyopathy

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones involved in electrolyte and fluid homeostasis. Our laboratory has investigated the use of ANP and BNP as diagnostic markers of cardiac disease in cats. We hypothesize that the cardiac distribution of ANP and BNP increases in cats with hypertrophic cardiomyopathy (HCM). Accordingly, we evaluated the immunohistochemical distribution of ANP and BNP in hearts of four cats with naturally occurring HCM relative to five healthy controls. Indirect immunoperoxidase was performed with polyclonal immunoglobulin G against feline ANP (1–28) and proBNP (43–56). In control cats, ANP and BNP immunoreactivity was restricted to the atria. Staining for both peptides was most intense adjacent to the endocardial surface. Auricles stained more diffusely than atria for both peptides. The interstitial capillaries and nerve fibers within the heart were positive only for BNP. Atrial immunoreactivity for ANP and BNP was more diffuse and had a less distinctly layered pattern in HCM than in control cats. Ventricular cardiomyocytes of HCM cats were negative for ANP but stained lightly and diffusely for BNP. The capillaries and nerve fibers remained positive for BNP. We conclude that in cats with HCM, the cardiac distribution of ANP and BNP is more diffuse in the atria and that novel expression of BNP in the ventricular cardiomyocytes occurs.

Recommended Guidelines for Submission, Trimming, Margin Evaluation, and Reporting of Tumor Biopsy Specimens in Veterinary Surgical Pathology

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

Expression and Function of Survivin in Canine Osteosarcoma

Osteosarcoma has a high mortality rate and remains in need of more effective therapeutic approaches. Survivin is an inhibitor of apoptosis family member protein that blocks apoptosis and drives proliferation in human cancer cells where it is commonly elevated. In this study, we illustrate the superiority of a canine osteosarcoma model as a translational tool for evaluating survivin-directed therapies, owing to the striking similarities in gross and microscopic appearance, biologic behavior, gene expression, and signaling pathway alterations. Elevated survivin expression in primary canine osteosarcoma tissue correlated with increased histologic grade and mitotic index and a decreased disease-free interval (DFI). Survivin attenuation in canine osteosarcoma cells inhibited cell-cycle progression, increased apoptosis, mitotic arrest, and chemosensitivity, and cooperated with chemotherapy to significantly improve in vivo tumor control. Our findings illustrate the utility of a canine system to more accurately model human osteosarcoma and strongly suggest that survivin-directed therapies might be highly effective in its treatment.

Preclinical Investigation of PEGylated Tumor Necrosis Factor α in Dogs with Spontaneous Tumors: Phase I Evaluation

Purpose: Tumor necrosis factor-α (TNF) is a cytokine with potent antitumor activity; however, toxicity and short half-life have limited its utility. Polyethylene glycol (PEG) conjugation of biotherapeutics can decrease immunogenicity while improving bioactivity and half-life. PEGylation of TNF (PEG-TNF) significantly improved half-life and toxicity in mice, resulting in enhanced antitumor activity. This study characterized toxicity, biological effect, and antitumor activity of PEG-TNF in pet dogs with spontaneous cancer. Experimental Design: A phase I clinical trial enrolled dogs with measurable tumors in which standard therapy had failed or been declined. Physiologic, hematologic, and biochemical parameters were evaluated and tumor biopsies obtained serially. A subset of patients underwent serial dynamic contrast-enhanced magnetic resonance imaging. Results: Fifteen dogs were enrolled at doses from 20.0 to 30.0 μg/kg. Dose-limiting toxicity at 30.0 μg/kg consisted of vascular leak in one and hypotension/coagulopathy in one, establishing 26.7 μg/kg as the maximum tolerated dose. Mean elimination half-life was 15.3 ± 4.9 hours. Biological activity (transient fever and leukopenia, increased tumor inflammation, and necrosis) was observed at all dosages. A significant increase in tumor blood flow was observed with dynamic contrast-enhanced magnetic resonance imaging. Minor/transient antitumor responses were observed in dogs with melanoma, squamous cell carcinoma, and mammary carcinoma, and a partial response was observed in a dog with angiosarcoma. Conclusions: Using a clinically relevant, spontaneous large animal model of neoplasia, we have shown that biologically effective doses of PEG-TNF can be administered safely, and that PEG-TNF administration is associated with encouraging biological activity. These results justify the clinical evaluation of PEG-TNF in human cancer. Clin Cancer Res; 16(5); 1498–508

Pigmented Epidermal Plaques in Three Dogs

Papillomavirus was identified in pigmented epidermal plaques (PEP) from three dogs: a miniature schnauzer with hyperadrenocorticism and hypoglobulinemia, an American Staffordshire terrier with hypoglobulinemia, and a Pomeranian with unconfirmed hypothyroidism. Squamous cell carcinoma (SCC) arose within several plaques in the Pomeranian. Clinical improvement coincided in the first two cases with treatment of the concurrent disease and the administration of low-dose oral interferon-alpha. This is the first report of PEP in an American Staffordshire terrier and a Pomeranian. The potential for malignant transformation of PEP to SCC emphasizes the need for recognition and clinical management of PEP.

Evaluation of risk factors for outcome associated with adrenal gland tumors with or without invasion of the caudal vena cava and treated via adrenalectomy in dogs: 86 cases (1993–2009)

OBJECTIVE To evaluate risk factors for outcome for dogs with adrenal gland tumors with or without invasion of the caudal vena cava treated via adrenalectomy. DESIGN Retrospective study. ANIMALS 86 dogs that underwent adrenalectomy for treatment of adrenal gland tumors. PROCEDURES Medical records of dogs that underwent adrenalectomy for treatment of an adrenal gland tumor from 1993 to 2009 were reviewed; data collected including signalment, clinical signs, diagnostic test findings, treatments prior to surgery, findings at surgery including additional procedures performed and extent of caudal vena caval invasion (local invasion [caudal to the hepatic portion of the vena cava] or extensive invasion [cranial to the hepatic portion of the vena cava]), procedures performed during surgery, histopathologic diagnosis, perioperative complications, follow-up data, and necropsy findings. RESULTS Of the 86 dogs, 14 had adenomas, 45 had adrenocortical carcinomas, and 27 had pheochromocytomas. Fourteen dogs had invasion of the caudal vena cava; of these tumors, 7 were locally invasive and 7 were extensively invasive. Risk factors for poor short-term survival (death within 14 days following surgery) were vena caval invasion, extent of invasion, pheochromocytoma, intraoperative transfusion, and postoperative factors including disseminated intravascular coagulation, pancreatitis, hypotension, hypoxemia, and renal failure. Multivariate analysis of risk factors for poor short-term survival revealed that extensive invasion was the most important factor. Regardless of extent of invasion or tumor type, long-term survival was possible. CONCLUSIONS AND CLINICAL RELEVANCE Invasion of the caudal vena cava, particularly tumor thrombus extension beyond the hepatic hilus, was associated with a higher postoperative mortality rate, but did not affect long-term prognosis in dogs undergoing adrenalectomy because of an adrenal gland tumor.

Prospective molecular profiling of canine cancers provides a clinically relevant comparative model for evaluating personalized medicine (PMed) trials

Background Molecularly-guided trials (i.e. PMed) now seek to aid clinical decision-making by matching cancer targets with therapeutic options. Progress has been hampered by the lack of cancer models that account for individual-to-individual heterogeneity within and across cancer types. Naturally occurring cancers in pet animals are heterogeneous and thus provide an opportunity to answer questions about these PMed strategies and optimize translation to human patients. In order to realize this opportunity, it is now necessary to demonstrate the feasibility of conducting molecularly-guided analysis of tumors from dogs with naturally occurring cancer in a clinically relevant setting. Methodology A proof-of-concept study was conducted by the Comparative Oncology Trials Consortium (COTC) to determine if tumor collection, prospective molecular profiling, and PMed report generation within 1 week was feasible in dogs. Thirty-one dogs with cancers of varying histologies were enrolled. Twenty-four of 31 samples (77%) successfully met all predefined QA/QC criteria and were analyzed via Affymetrix gene expression profiling. A subsequent bioinformatics workflow transformed genomic data into a personalized drug report. Average turnaround from biopsy to report generation was 116 hours (4.8 days). Unsupervised clustering of canine tumor expression data clustered by cancer type, but supervised clustering of tumors based on the personalized drug report clustered by drug class rather than cancer type. Conclusions Collection and turnaround of high quality canine tumor samples, centralized pathology, analyte generation, array hybridization, and bioinformatic analyses matching gene expression to therapeutic options is achievable in a practical clinical window (<1 week). Clustering data show robust signatures by cancer type but also showed patient-to-patient heterogeneity in drug predictions. This lends further support to the inclusion of a heterogeneous population of dogs with cancer into the preclinical modeling of personalized medicine. Future comparative oncology studies optimizing the delivery of PMed strategies may aid cancer drug development.

Anti-angiogenic effects of interleukin-12 delivered by a novel hyperthermia induced gene construct

Purpose: Interleukin-12 (IL-12) is a pro-inflammatory cytokine possessing anti-cancer and anti-angiogenic properties. This study quantitatively assessed the anti-angiogenic effect of IL-12 delivered using an adenoviral vector with murine IL-12 placed under control of a heat shock promoter. This approach limits systemic toxicity by restricting IL-12 delivery locally to the tumour. The kinetics of the downstream cytokines interferon-γ (IFN-γ) and interferon inducible protein-10 (IP-10) and other molecules affecting angiogenesis, vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) were also studied. Materials and methods: 4T1 tumours were grown in Balb/C mice and the AdhspmIL-12 construct was injected intra-tumourally. The tumours were heated after 24 h using a water bath. At various time points post-heating the tumours were collected and quantitatively assessed for cytokine production and vascularity. Results: A significant reduction was seen in the tumour vasculature of the treated group vs. the control group mice. Systemic effects of IL-12 were limited to generalized immunostimulation. No hepatoxicity was noted. Conclusions: This study suggests that IL-12 can be effectively delivered using a gene-based approach with a heat shock promoter. This results in quantitatively measurable anti-angiogenesis and general immunostimulation. The complex inter-play of other pro- and anti-angiogenic factors (IFN-γ, IP-10, VEGF and PAI-1) was also studied.

Comparative analysis of survivin expression in untreated and relapsed canine lymphoma.

BACKGROUND Survivin, a member of the inhibitor of apoptosis protein family, has a dual role in tumor cell proliferative and antiapoptotic pathways. Survivin expression has been shown to be a negative prognostic factor in several cancers of humans, including B-cell non-Hodgkins lymphoma. HYPOTHESES High survivin expression will be a negative prognostic factor in dogs with lymphoma (LSA) treated with chemotherapy. In addition, survivin expression will be upregulated in relapsed canine LSA when compared with patient-matched, pretreatment biopsies. ANIMALS Thirty-one client-owned dogs with stage IIIa or IVa LSA. METHODS Retrospective evaluation of survivin immunoreactivity was performed on pretreatment lymph node biopsies and patient-matched samples obtained from dogs at relapse after being treated with an abbreviated CHOP-based protocol. RESULTS In this population of dogs presenting with stage IIIa or IVa B-cell LSA, those dogs that had high survivin immunoreactivity scores had a significantly (P < .01, hazard ratio = 0.30) shorter median disease-free interval than did dogs with low survivin immunoreactivity scores (171 days versus 321 days, respectively). Survivin immunoreactivity was not significantly different in relapsed canine LSA when compared with patient-matched, pretreatment biopsies. CONCLUSIONS AND CLINICAL IMPORTANCE Survivin expression is a negative prognostic factor that can predict early treatment failure of dogs that present with stage IIIa or IVa, B-cell LSA when treated with a CHOP-based protocol.