J. B. Charles

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.

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.

Immunohistochemical characterization of feline oral squamous cell carcinoma.

OBJECTIVE To evaluate the expression of Ki67 and epidermal growth factor receptor (EGFR), mitotic index (MI), and microvascular density (MVD) in feline oral squamous cell carcinoma (SCC) via immunohistochemical staining on archival tumor tissues and to seek a correlation between these markers and clinical variables. SAMPLE 22 archived tumor samples of feline oral SCC. PROCEDURES Immunohistochemical staining for Ki67, MVD, and EGFR was performed and scored. Patient survival information was obtained from the medical records. These molecular markers as well as MI were correlated with tumor locations and patient survival time. RESULTS The 22 tumors had wide variation in Ki67 expression, MI, MVD, and EGFR expression. Tongue SCC had higher MVD than did mandibular and maxillary SCC. Tumor expression of EGFR was inversely proportional to survival time. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that EGFR expression might be a valuable prognostic factor for treatment outcome in feline oral SCC. It also identified higher angiogenesis in tongue SCC, compared with mandibular and maxillary SCC, which may account for a different clinical outcome. Further prospective characterization of feline oral SCC may provide a better understanding of the underlying molecular factors that drive its behavior and offer the possibility for future patient-specific treatment plans.

Assessment of predictive molecular variables in feline oral squamous cell carcinoma treated with stereotactic radiation therapy

This study evaluated molecular characteristics that are potentially prognostic in cats with oral squamous cell carcinoma (SCC) that underwent stereotactic radiation therapy (SRT). Survival time (ST) and progression-free interval (PFI) were correlated with mitotic index, histopathological grades, Ki67 and epidermal growth factor receptor expressions, tumour microvascular density (MVD), and tumour oxygen tension (pO(2)). Median ST and PFI were 106 and 87 days, respectively (n = 20). Overall response rate was 38.5% with rapid improvement of clinical symptoms in many cases. Patients with higher MVD or more keratinized SCC had significantly shorter ST or PFI than patients with lower MVD or less keratinized SCC (P = 0.041 and 0.049, respectively). Females had significantly longer PFI and ST than males (P ≤ 0.016). Acute toxicities were minimal. However, treatment-related complications such as fractured mandible impacted quality of life. In conclusion, SRT alone should be considered as a palliative treatment. MVD and degree of keratinization may be useful prognostic markers.

Glucose transporter 1 expression in canine osteosarcoma

Hypoxia in tumours has been associated with an increased resistance to radiation and chemotherapy, and increased metastatic potential. Hypoxia-inducible factor 1-alpha (HIF-1alpha) is a transcription factor induced by hypoxia. Glucose transporter 1 (GLUT-1), a downstream product of HIF-1alpha pathway activation, is over-expressed in a variety of human tumours. The purpose of this study was to determine if GLUT-1 is expressed in canine osteosarcomas (OSAs) and if the expression is related to tumour necrosis and outcome. Immunohistochemistry was performed on 44 histologically confirmed OSA tissue samples to assess expression of GLUT-1. Of 44 cases, 27 (61%) expressed GLUT-1. There was no statistical correlation between GLUT-1 and disease-free interval, survival time or percentage of necrosis. As hypothesized, GLUT-1 is present in canine appendicular OSAs. A more objective evaluation of GLUT-1 and other proteins in the HIF-1alpha pathway may be warranted.

The use of novel lymphatic endothelial cell-specific immunohistochemical markers to differentiate cutaneous angiosarcomas in dogs.

Lymphangiosarcomas are uncommon vascular neoplasms that arise from lymphatic endothelial cells (LECs). They efface and replace normal subcutaneous tissue and are characterised by arborising, vascular channels lined by a single layer of pleomorphic endothelial cells and a paucity of erythrocytes. Lymphangiosarcomas are architecturally similar to hemangiosarcomas, a common malignancy of vascular origin arising from blood vascular endothelial cells. Common immunohistochemical markers for vascular endothelium, such as Factor VIII-related antigen (F8RA) and CD31, have traditionally been used to confirm the diagnosis of tumours of vascular origin. However, these markers fail to differentiate between lymphangiosarcoma and hemangiosarcoma, which often show overlapping morphologic features, disparate clinical behaviour and require different treatment modalities. Here we describe the use of two novel LEC-specific markers, lymphatic vessel endothelial receptor-1 (LYVE-1) and prospero-related homeobox gene-1 (PROX-1), to further differentiate between vascular tumours of lymphatic (lymphangiosarcoma) and blood (hemangiosarcoma) endothelial cell origin in the dog.

Detection of calprotectin and apoptotic activity within the equine colon from horses with black walnut extract-induced laminitis

The black walnut extract (BWE) model of equine laminitis is associated with a systemic inflammatory response manifest by increased expression of inflammatory cytokines in the lungs and liver as well as the laminae. The specific role of the gastrointestinal tract in development of this response is unclear and is of utmost importance, as gastrointestinal disease and laminitis are intimately related. We investigated calprotectin expression and epithelial and endothelial apoptosis in the colon of horses exposed to orally administered BWE. Sections of colon from 19 horses including 7 controls not exposed to BWE, 6 horses at the developmental time-point of leukopenia (DTP) and 6 at the onset of Obel grade 1 laminitis (LAM) after BWE-administration were histologically examined. Immunohistochemical evaluation for calprotectin expression with MAC 387 antibody was performed along with assessment of epithelial and endothelial apoptosis with caspase-3 active antibody. Calprotectin expression and percentage of apoptotic cells were compared between controls and the two treatment groups and presence of a correlation between calprotectin expression and apoptosis was evaluated. Histological findings from BWE-treated horses included eosinophil and lymphocyte epitheliotropism. The DTP group had a higher (p<0.01) calprotectin score with respect to the control group, while there was no significant difference in percentage of epithelial and endothelial apoptotic cells between groups (p=0.08 and p=0.48 respectively). No significant correlation was found between calprotectin score and epithelial or endothelial apoptosis (p=0.69 and p=0.29 respectively). There is preliminary evidence that exposure of horses to BWE results in an early inflammatory response in the colon. Further studies are needed to characterize the nature of the colonic injury in BWE-exposed horses and the link to the development of laminitis.

Survivin inhibition via EZN-3042 in canine lymphoma and osteosarcoma

Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Survivin, an inhibitor of apoptosis (IAP) family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in dogs with LSA and OS, and canine LSA and OS cell lines express high levels of survivin. In this study, we demonstrate that survivin downregulation in canine LSA and OS cells using a clinically applicable locked nucleic acid antisense oligonucleotide (EZN-3042, Enzon Pharmaceuticals, Piscataway Township, NJ, USA) inhibits growth, induces apoptosis and enhances chemosensitivity in vitro, and inhibits survivin transcription and protein production in orthotopic canine OS xenografts. Our findings strongly suggest that survivin-directed therapies might be effective in treatment of canine LSA and OS and support evaluation of EZN-3042 in dogs with cancer.

Abstract B35: Survivin inhibition via EZN-3042 in canine models of lymphoma and osteosarcoma

Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Due to the strong similarities between canine and human LSA and OS, canine LSA and OS are excellent models for the human disease. Survivin, an IAP family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in dogs and humans with LSA and OS. The objective of our research was to determine the effects of survivin inhibition using a locked nucleic acid antisense oligonucleotide (EZN-3042) on canine LSA and canine OS cell lines, with respect to cell proliferation, apoptosis, and chemosensitivity in vitro. Furthermore, we sought to determine the efficacy of EZN-3042 on inhibition of survivin transcription, survivin protein production, and tumor growth in subcutaneous and orthotopic canine OS xenografts. We inhibited survivin using EZN-3042 in two canine LSA and two canine OS cell lines. Survivin inhibition was confirmed by qRT-PCR and western blot. Percent dead and total cell number was assessed by manual cell counting with trypan blue. Growth inhibition was confirmed with a bioreductive fluorometric assay. A caspase-3/7 assay was used to determine levels of apoptosis in EZN-3042 treated cells compared to controls. Cells were then treated with antineoplastic drug doxorubicin (DOX) +/- EZN-3042 and assays repeated. In vivo, nude mice with subcutaneous and orthotopic OS xenografts were given 100 mg/kg EZN3042 intraperitoneally. Survivin inhibition was confirmed with Immunohistochemistry and qRT-PCR analysis. Survivin inhibition in canine LSA and OS cells via EZN-3042 resulted in 34-72% decrease in survivin protein expression and a 1.3-3.4 fold decrease in endogenous survivin mRNA expression. When EZN-3042 treated cells were compared to controls, total and viable cell numbers were decreased, and apoptosis was increased. Survivin inhibition via EZN-3042 enhanced canine LSA and OS cell lines sensitivity to DOX. IHC and qRT-PCR analysis of subcutaneous and orthotopic canine OS xenografts confirmed decreased tumor survivin expression in EZN-3042 treated mice. Mice treated with EZN-3042 in combination with DOX had significantly decreased tumor growth when compared to single agent treatment and control groups. These results demonstrate that survivin inhibition via EZN-3042 decreased LSA and OS cell proliferation, and increased cellular apoptosis and chemosensitivity to DOX, and that EZN-3042 treatment inhibited tumor survivin expression in vivo and significantly decreased tumor growth when combined with DOX. Survivin-directed therapies may be highly effective in treatment of both canine and human LSA and OS, and spontaneous canine cancer may be a valuable model for the evaluation of survivin-targeted treatment. Citation Format: Jenette K. Shoeneman, Eugene J. Ehrhart, III, Joseph B. Charles, Douglas H. Thamm. Survivin inhibition via EZN-3042 in canine models of lymphoma and osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B35.