Establishment and characterization of two novel patient-derived lines from canine high-grade glioma.

High-grade glioma is an aggressive cancer that occurs naturally in pet dogs. Canine high-grade glioma (cHGG) is treated with radiation, chemotherapy or surgery, but has no curative treatment. Within the past eight years, there have been advances in our imaging and histopathology standards as well as genetic charactereization of cHGG. However, there are only three cHGG cell lines publicly available, all of which were derived from astrocytoma and established using methods involving expansion of tumour cells in vitro on plastic dishes. In order to provide more clinically relevant cell lines for studying cHGG in vitro, the goal of this study was to establish cHGG patient-derived lines, whereby cancer cells are expanded in vivo by injecting cells into immunocompromized laboratory mice. The cells are then harvested from mice and used for in vitro studies. This method is the standard in the human field and has been shown to minimize the acquisition of genetic alterations and gene expression changes from the original tumour. Through a multi-institutional collaboration, we describe our methods for establishing two novel cHGG patient-derived lines, Boo-HA and Mo-HO, from a high-grade astrocytoma and a high-grade oligodendroglioma, respectively. We compare our novel lines to G06-A, J3T-Bg, and SDT-3G (traditional cHGG cell lines) in terms of proliferation and sensitivity to radiation. We also perform whole genome sequencing and identify an NF1 truncating mutation in Mo-HO. We report the characterization and availability of these novel patient-derived lines for use by the veterinary community.

Canine Apocrine Gland Anal Sac Adenocarcinoma: A Review.

Apocrine gland anal sac adenocarcinoma (AGASAC) is a relatively uncommon tumor in the dog and comprises approximately 17% of perianal malignancies; however, it is one of the most common causes of paraneoplastic hypercalcemia. Clinical signs in affected dogs most commonly are associated with mechanical obstruction caused by the primary tumor or enlarged regional metastatic lymph nodes and the effects of paraneoplastic hypercalcemia when present. Surgical excision of the primary tumor and metastasectomy of affected locoregional lymph nodes is the preferred initial treatment option for most dogs, although radiation therapy and adjuvant chemotherapy are commonly incorporated into multi-modality treatment plans. A significant role for the use of adjuvant chemotherapy has not been clearly demonstrated. Prolonged survival times are possible, especially for dogs with smaller primary tumors and for dogs that undergo further treatments for recurrent disease. In this article, we review the clinical signs, diagnosis, staging, treatment, and prognosis of AGASAC in the dog.

Clinical validation of a next-generation sequencing-based multi-cancer early detection "liquid biopsy" blood test in over 1,000 dogs using an independent testing set: The CANcer Detection in Dogs (CANDiD) study.

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection "liquid biopsy" test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3-60.0%) sensitivity and a 98.5% (95% CI: 97.0-99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4-90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3-68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs.

A retrospective analysis of 11 dogs with surface osteosarcoma.

While the majority of canine osteosarcomas (OSA) arise from the medullary cavity, a subset arises from the surface of bone. In humans, surface OSA often has a more indolent disease course with better outcomes than medullary OSA. The aim of this retrospective case series was to evaluate the clinical outcome and potential prognostic factors of dogs with surface OSA. Medical records from 11 dogs previously diagnosed with surface OSA were included. Histopathology of cases was evaluated during case review by two veterinary anatomic pathologists. Median progression free interval (PFI) and overall median survival time (OST) were estimated using Kaplan-Meier methods. Intergroup comparisons were performed using log-rank tests. Six dogs were diagnosed with periosteal OSA, 4 dogs with parosteal OSA, and one dog with an unclassified surface OSA. Two dogs were found to have metastatic disease at the time of diagnosis and four developed metastatic lesions after treatment. The median PFI and median OST for all dogs with surface OSA was 425 and 555 days, respectively. The 6 dogs diagnosed with periosteal OSA had a median PFI of 461 days and median OST of 555 days, while the 4 dogs with parosteal OSA had a PFI of 350 days and the OST could not be calculated. Multiple prognostic factors (surgery, systemic adjunctive therapy, elevated alkaline phosphatase at diagnosis, appendicular vs axial location, mitotic count, and tumour grade) were evaluated and none were prognostic for PFI or OST. Dogs with surface OSA appear to have prolonged PFI and OST, consistent with humans with surface OSA.

Case Report: Cutaneous Pleomorphic Lymphangiosarcoma in a Dog Exhibiting Features of Human Composite Hemangioendothelioma.

Background: Angiosarcomas are a broad category of vascular origin neoplasms that are poorly characterized in veterinary species. Lymphangiosarcoma (LAS) is an uncommon type of angiosarcoma reported in humans and canines arising from lymphatic endothelium. LAS can be differentiated from other angiosarcomas in dogs based on expression of Prospero-related homeobox gene-1 (PROX-1) or lymphatic vessel endothelial receptor-1 (LYVE-1). Composite hemangioendothelioma (CHE) is a rare angiosarcoma subtype described in people and characterized by a variable biologic behavior and infrequent metastasis. This variant of angiosarcoma histologically combines features of retiform hemangioendothelioma and epithelioid hemangioendothelioma. Information regarding the cytologic and histopathologic appearance and clinical course of dogs with vascular tumors that exhibit features of CHE are unknown. Here, we report a case of pleomorphic LAS with features of CHE arising in a dog and treated with surgery and adjuvant chemotherapy. Case presentation: A 10-year-old intact male Labrador retriever presented with an approximately 6-cm-diameter cutaneous mass caudal to the left elbow that was progressively growing over 1.5 years. On physical examination, palpable extensions were identified coursing proximally over the triceps with concurrent loco-regional peripheral lymphadenopathy. Fine needle aspirates (FNA) and cytologic assessment of the cutaneous mass, left prescapular, and accessory axillary lymph nodes reported that this appeared to be a metastatic epithelial neoplasm, although a mixed carcinoma or collision tumor could not be excluded. An incisional biopsy of the mass was submitted for histopathology and was consistent with a well-differentiated angiosarcoma with features of CHE. The neoplasm expressed vimentin, CD31, von Willebrand factor (vWf), and PROX-1, supporting the diagnosis of LAS. Complete staging was performed, and no additional metastatic lesions were identified. Left forelimb amputation and lymph node removal were performed. Based on the diagnosis of metastatic LAS, doxorubicin chemotherapy was administered. 7 months post-amputation, the tumor recurred at the amputation site without evidence of metastatic disease. Conclusion: This report describes a malignant, locally aggressive lymphatic origin vascular tumor in a dog, with features consistent with descriptions of CHE in humans. Cytologic features in this case were discordant with its true mesenchymal etiology, obfuscating diagnosis. The morphologic features of the mesenchymal neoplastic population and immunohistochemistry (IHC) labeling ultimately supported a diagnosis of pleomorphic LAS with features of CHE.

Development of an exosomal gene signature to detect residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning.

Osteosarcoma has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, their clinical application is precluded by challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma in mouse xenografts to distinguish tumor-derived from host-response exosomal messenger RNAs (mRNAs). The model allows for the identification of canine osteosarcoma-specific gene signatures by RNA sequencing and a species-differentiating bioinformatics pipeline. An osteosarcoma-associated signature consisting of five gene transcripts (SKA2, NEU1, PAF1, PSMG2, and NOB1) was validated in dogs with spontaneous osteosarcoma by real-time quantitative reverse transcription PCR (qRT-PCR), while a machine learning model assigned dogs into healthy or disease groups. Serum/plasma exosomes were isolated from 53 dogs in distinct clinical groups ("healthy", "osteosarcoma", "other bone tumor", or "non-neoplastic disease"). Pre-treatment samples from osteosarcoma cases were used as the training set, and a validation set from post-treatment samples was used for testing, classifying as "osteosarcoma detected" or "osteosarcoma-NOT detected". Dogs in a validation set whose post-treatment samples were classified as "osteosarcoma-NOT detected" had longer remissions, up to 15 months after treatment. In conclusion, we identified a gene signature predictive of molecular remissions with potential applications in the early detection and minimal residual disease settings. These results provide proof of concept for our discovery platform and its utilization in future studies to inform cancer risk, diagnosis, prognosis, and therapeutic response.

Case Report: Metastatic Parosteal Osteosarcoma in a Dog.

This case report describes a rare form of malignant bone tumor in an 8-year-old Labrador retriever. This dog initially presented for evaluation of a right distal humeral mass. Radiographs of the right elbow and thorax were performed, revealing a smooth mineralized mass adjacent to the lateral aspect of the distal humerus and a 5mm pulmonary nodule. Computed tomography (CT) of the humerus and thorax showed a smooth mineralized lesion adjacent to the lateral humeral epicondyle, and a right cranial lung lobe nodule with a thin mineral rim. Surgical biopsies of both lesions were diagnostic for parosteal osteosarcoma (POSA). The dog was then treated with stereotactic body radiation therapy (SBRT) which controlled the dog's discomfort for 14 months until he became progressively painful and subsequently had his right forelimb amputated. This case report is the first to document the CT imaging characteristics of a metastatic appendicular POSA in a dog and the first dog described with POSA treated with SBRT. The dog lived for 623 days after histopathologic diagnosis and 849 days after initial presentation with pulmonary metastatic disease.

Treatment of indolent cutaneous T-cell lymphoma with hypofractionated radiation therapy in three cats.

BACKGROUND: Feline indolent cutaneous T-cell lymphoma (ICL) is an uncommon neoplastic disease. There is currently no consensus on treatment recommendations for ICL. OBJECTIVE: To report the clinical outcome of three cats with ICL treated with hypofractionated electron-beam radiotherapy (RT). ANIMALS: Three privately owned cats with ICL. MATERIALS AND METHODS: Medical records and client surveys were reviewed. A diagnosis of probable ICL was based on history, clinical presentation and histopathological findings, and confirmed using CD3 immunohistochemical analysis and PCR for antigen receptor gene rearrangement (PARR). All cats were treated with hypofractionated RT (four fractions of 8 Gy). RESULTS: All cats presented with skin lesions characterised by erythema and alopecia that were refractory to previous treatment with systemic glucocorticoids. Before hypofractionated RT treatment, lesions were histologically described as having diffuse infiltration of the dermis with CD3+ T cells. Molecular clonality analysis revealed clonal T-cell receptor gamma gene rearrangement. After RT, two cats showed histological improvement defined by decreased infiltration of lymphocytes, with cellular infiltrate present only in the deeper dermis; one cat had near complete histological resolution of lesions with only minimal residual lymphocytes. One cat was determined to have a complete clinical response while the other showed partial responses. No acute adverse effects of radiation were observed; chronic effects included leukotrichia, partial alopecia and mild fibrosis. All clients reported improvement in quality of life for their cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical and histological improvement in these cats suggests that hypofractionated RT can be a useful treatment modality for cats with ICL.

Adoptive Natural Killer Cell Immunotherapy for Canine Osteosarcoma.

Osteosarcoma is the most common primary bone tumor in both humans and dogs. It is a highly metastatic cancer and therapy has not improved significantly since the inclusion of adjuvant chemotherapy into disease treatment strategies. Osteosarcoma is an immunogenic tumor, and thus development of immunotherapies for its treatment, especially treatment of microscopic pulmonary metastases might improve outcomes. NK cells are lymphocytes of the innate immune system and can recognize a variety of stressed cells, including cancer cells, in the absence of major histocompatibility complex (MHC)-restricted receptor ligand interactions. NK cells have a role in controlling tumor progression and metastasis and are important mediators of different therapeutic interventions. The core hypothesis of adoptive natural killer (NK) cell therapy is there exists a natural defect in innate immunity (a combination of cancer-induced reduction in NK cell numbers and immunosuppressive mechanisms resulting in suppressed function) that can be restored by adoptive transfer of NK cells. Here, we review the rationale for adoptive NK cell immunotherapy, NK cell biology, TGFß and the immunosuppressive microenvironment in osteosarcoma, manufacturing of ex vivo expanded NK cells for the dog and provide perspective on the present and future clinical applications of adoptive NK cell immunotherapy in spontaneous osteosarcoma and other cancers in the dog.

Validation of protein arginine methyltransferase 5 (PRMT5) as a candidate therapeutic target in the spontaneous canine model of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) is a heterogeneous group of blood cancers arising in lymphoid tissues that commonly effects both humans and dogs. Protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes the symmetric di-methylation of arginine residues, is frequently overexpressed and dysregulated in both human solid and hematologic malignancies. In human lymphoma, PRMT5 is a known driver of malignant transformation and oncogenesis, however, the expression and role of PRMT5 in canine lymphoma has not been explored. To explore canine lymphoma as a useful comparison to human lymphoma while validating PRMT5 as a rational therapeutic target in both, we characterized expression patterns of PRMT5 in canine lymphoma tissue microarrays, primary lymphoid biopsies, and canine lymphoma-derived cell lines. The inhibition of PRMT5 led to growth suppression and induction of apoptosis, while selectively decreasing global marks of symmetric dimethylarginine (SDMA) and histone H4 arginine 3 symmetric dimethylation. We performed ATAC-sequencing and gene expression microarrays with pathway enrichment analysis to characterize genome-wide changes in chromatin accessibility and whole-transcriptome changes in canine lymphoma cells lines upon PRMT5 inhibition. This work validates PRMT5 as a promising therapeutic target for canine lymphoma and supports the continued use of the spontaneously occurring canine lymphoma model for the preclinical development of PRMT5 inhibitors for the treatment of human NHL.

Adjuvant Sirolimus Does Not Improve Outcome in Pet Dogs Receiving Standard-of-Care Therapy for Appendicular Osteosarcoma: A Prospective, Randomized Trial of 324 Dogs.

PURPOSE: The mTOR pathway has been identified as a key nutrient signaling hub that participates in metastatic progression of high-grade osteosarcoma. Inhibition of mTOR signaling is biologically achievable with sirolimus, and might slow the outgrowth of distant metastases. In this study, pet dogs with appendicular osteosarcoma were leveraged as high-value biologic models for pediatric osteosarcoma, to assess mTOR inhibition as a therapeutic strategy for attenuating metastatic disease progression. PATIENTS AND METHODS: A total of 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a two-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb, followed by adjuvant carboplatin chemotherapy ± oral sirolimus therapy. The primary outcome measure was disease-free interval (DFI), as assessed by serial physical and radiologic detection of emergent macroscopic metastases; secondary outcomes included overall 1- and 2-year survival rates, and sirolimus pharmacokinetic variables and their correlative relationship to adverse events and clinical outcomes. RESULTS: There was no significant difference in the median DFI or overall survival between the two arms of this trial; the median DFI and survival for standard-of-care (SOC; defined as amputation and carboplatin therapy) dogs was 180 days [95% confidence interval (CI), 144-237] and 282 days (95% CI, 224-383) and for SOC + sirolimus dogs, it was 204 days (95% CI, 157-217) and 280 days (95% CI, 252-332), respectively. CONCLUSIONS: In a population of pet dogs nongenomically segmented for predicted mTOR inhibition response, sequentially administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend DFI or survival in dogs with appendicular osteosarcoma.

Pharmacokinetics and Tolerability of the Novel Non-immunosuppressive Fingolimod Derivative, OSU-2S, in Dogs and Comparisons with Data in Mice and Rats.

In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 µM, and Caco-2 data suggested low permeability with active efflux at 2 µM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5-20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUClast (26.1 µM*h), and Cmax (0.899 µM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.

Proliferative and Invasive Colorectal Tumors in Pet Dogs Provide Unique Insights into Human Colorectal Cancer.

Spontaneous tumors in pet dogs represent a valuable but undercharacterized cancer model. To better use this resource, we performed an initial global comparison between proliferative and invasive colorectal tumors from 20 canine cases, and evaluated their molecular homology to human colorectal cancer (CRC). First, proliferative canine tumors harbor overactivated WNT/ß-catenin pathways and recurrent CTNNB1 (ß-catenin) mutations S45F/P, D32Y and G34E. Invasive canine tumors harbor prominent fibroblast proliferation and overactivated stroma. Both groups have recurrent TP53 mutations. We observed three invasion patterns in canine tumors: collective, crypt-like and epithelial⁻mesenchymal transition (EMT). We detected enriched Helicobacter bilis and Alistipes finegoldii in proliferative and crypt-like tumors, but depleted mucosa-microbes in the EMT tumor. Second, guided by our canine findings, we classified 79% of 478 human colon cancers from The Cancer Genome Atlas into four subtypes: primarily proliferative, or with collective, crypt-like or EMT invasion features. Their molecular characteristics match those of canine tumors. We showed that consensus molecular subtype 4 (mesenchymal) of human CRC should be further divided into EMT and crypt-like subtypes, which differ in TGF-ß activation and mucosa-microbe content. Our canine tumors share the same pathogenic pathway as human CRCs. Dog-human integration identifies three CRC invasion patterns and improves CRC subtyping.

NCI Comparative Oncology Program Testing of Non-Camptothecin Indenoisoquinoline Topoisomerase I Inhibitors in Naturally Occurring Canine Lymphoma.

PURPOSE: Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics. EXPERIMENTAL DESIGN: Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined. RESULTS: The MTDs were 17.5 mg/m2 for LMP 776 and 100 mg/m2 for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m2 LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744. CONCLUSIONS: These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.

SETD2 Is Recurrently Mutated in Whole-Exome Sequenced Canine Osteosarcoma.

Osteosarcoma is a debilitating bone cancer that affects humans, especially children and adolescents. A homologous form of osteosarcoma spontaneously occurs in dogs, and its differential incidence observed across breeds allows for the investigation of tumor mutations in the context of multiple genetic backgrounds. Using whole-exome sequencing and dogs from three susceptible breeds (22 golden retrievers, 21 Rottweilers, and 23 greyhounds), we found that osteosarcoma tumors show a high frequency of somatic copy-number alterations (SCNA), affecting key oncogenes and tumor-suppressor genes. The across-breed results are similar to what has been observed for human osteosarcoma, but the disease frequency and somatic mutation counts vary in the three breeds. For all breeds, three mutational signatures (one of which has not been previously reported) and 11 significantly mutated genes were identified. TP53 was the most frequently altered gene (83% of dogs have either mutations or SCNA in TP53), recapitulating observations in human osteosarcoma. The second most frequently mutated gene, histone methyltransferase SETD2, has known roles in multiple cancers, but has not previously been strongly implicated in osteosarcoma. This study points to the likely importance of histone modifications in osteosarcoma and highlights the strong genetic similarities between human and dog osteosarcoma, suggesting that canine osteosarcoma may serve as an excellent model for developing treatment strategies in both species.Significance: Canine osteosarcoma genomics identify SETD2 as a possible oncogenic driver of osteosarcoma, and findings establish the canine model as a useful comparative model for the corresponding human disease. Cancer Res; 78(13); 3421-31. ©2018 AACR.

Immunohistochemical detection of p53, PTEN, Rb, and p16 in canine osteosarcoma using tissue microarray.

Although inactivating mutations of tumor suppressor genes are well described in cell lines of canine osteosarcoma (OS), expression of tumor suppressor proteins in spontaneous disease is poorly characterized. We determined the immunohistochemical expression of p53, PTEN, Rb, and p16 in a large cohort of dogs with OS. Formalin-fixed, paraffin-embedded samples of canine OS were analyzed retrospectively. Primary tumor samples from 145 dogs, collected between 2003 and 2008, were evaluated by tissue microarray. Streptavidin-biotin complex immunohistochemistry was performed using monoclonal antibodies for Rb and PTEN and polyclonal antibodies for p16 and p53. The average age of dogs was 7.6 y, and 118 of 145 (81%) were purebred. Most commonly represented purebreds were Greyhound (23%), Rottweiler (11%), and Labrador Retriever (10%). Immunohistochemical detection of p53, PTEN, Rb, and p16 was 81%, 61%, 66%, and 66%, respectively. The staining pattern for p16 was primarily cytoplasmic; the predominant pattern for PTEN, Rb, and p53 was cytoplasmic and nuclear. Exclusively cytoplasmic staining was noted in 19% of samples positive for p53 and 8% of samples positive for Rb. Kaplan-Meier curves showed that protein expression was not associated with significant differences in overall survival ( p > 0.191). We documented heterogeneity in both immunostaining and subcellular localization of tumor suppressor proteins, providing further characterization of canine OS.

Sensitivity of osteosarcoma cells to HDAC inhibitor AR-42 mediated apoptosis.

BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumor in both humans and dogs and is the second leading cause of cancer related deaths in children and young adults. Limb sparing surgery along with chemotherapy has been the mainstay of treatment for OS. Many patients are not cured with current therapies, presenting a real need for developing new treatments. Histone deacetylase (HDAC) inhibitors are a promising new class of anticancer agents. In this study, we investigated the activity of the novel HDAC inhibitor AR-42 in a panel of human and canine OS cell lines. METHODS: The effect of AR-42 and suberoylanilide hydroxamic acid (SAHA) alone or in combination with doxorubicin on OS cell viability was assessed. Induction of histone acetylation after HDAC inhibitor treatment was confirmed by Western blotting. Drug-induced apoptosis was analyzed by FACS. Apoptosis was assessed further by measuring caspase 3/7 enzymatic activity, nucleosome fragmentation, and caspase cleavage. Effects on Akt signaling were demonstrated by assessing phosphorylation of Akt and downstream signaling molecules. RESULTS: AR-42 was a potent inhibitor of cell viability and induced a greater apoptotic response compared to SAHA when used at the same concentrations. Normal osteoblasts were much less sensitive. The combination of AR-42 with doxorubicin resulted in a potent inhibition of cell viability and apparent synergistic effect. Furthermore, we showed that AR-42 and SAHA induced cell death via the activation of the intrinsic mitochondrial pathway through activation of caspase 3/7. This potent apoptotic activity was associated with the greater ability of AR-42 to downregulate survival signaling through Akt. CONCLUSIONS: These results confirm that AR-42 is a potent inhibitor of HDAC activity and demonstrates its ability to significantly inhibit cell survival through its pleiotropic effects in both canine and human OS cells and suggests that spontaneous OS in pet dogs may be a useful large animal model for preclinical evaluation of HDAC inhibitors. HDAC inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in both canine and human OS.

MiR-9 is overexpressed in spontaneous canine osteosarcoma and promotes a metastatic phenotype including invasion and migration in osteoblasts and osteosarcoma cell lines.

BACKGROUND: MicroRNAs (miRNAs) regulate the expression of networks of genes and their dysregulation is well documented in human malignancies; however, limited information exists regarding the impact of miRNAs on the development and progression of osteosarcoma (OS). Canine OS exhibits clinical and molecular features that closely resemble the corresponding human disease and it is considered a well-established spontaneous animal model to study OS biology. The purpose of this study was to investigate miRNA dysregulation in canine OS. METHODS: We evaluated miRNA expression in primary canine OS tumors and normal canine osteoblast cells using the nanoString nCounter system. Quantitative PCR was used to validate the nanoString findings and to assess miR-9 expression in canine OS tumors, OS cell lines, and normal osteoblasts. Canine osteoblasts and OS cell lines were stably transduced with pre-miR-9 or anti-miR-9 lentiviral constructs to determine the consequences of miR-9 on cell proliferation, apoptosis, invasion and migration. Proteomic and gene expression profiling of normal canine osteoblasts with enforced miR-9 expression was performed using 2D-DIGE/tandem mass spectrometry and RNA sequencing and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR. OS cell lines were transduced with gelsolin (GSN) shRNAs to investigate the impact of GSN knockdown on OS cell invasion. RESULTS: We identified a unique miRNA signature associated with primary canine OS and identified miR-9 as being significantly overexpressed in canine OS tumors and cell lines compared to normal osteoblasts. Additionally, high miR-9 expression was demonstrated in tumor-specific tissue obtained from primary OS tumors. In normal osteoblasts and OS cell lines transduced with miR-9 lentivirus, enhanced invasion and migration were observed, but miR-9 did not affect cell proliferation or apoptosis. Proteomic and transcriptional profiling of normal canine osteoblasts overexpressing miR-9 identified alterations in numerous genes, including upregulation of GSN, an actin filament-severing protein involved in cytoskeletal remodeling. Lastly, stable downregulation of miR-9 in OS cell lines reduced GSN expression with a concomitant decrease in cell invasion and migration; concordantly, cells transduced with GSN shRNA demonstrated decreased invasive properties. CONCLUSIONS: Our findings demonstrate that miR-9 promotes a metastatic phenotype in normal canine osteoblasts and malignant OS cell lines, and that this is mediated in part by enhanced GSN expression. As such, miR-9 represents a novel target for therapeutic intervention in OS.

Preclinical Evaluation of the Novel BTK Inhibitor Acalabrutinib in Canine Models of B-Cell Non-Hodgkin Lymphoma.

Acalabrutinib (ACP-196) is a second-generation inhibitor of Bruton agammaglobulinemia tyrosine kinase (BTK) with increased target selectivity and potency compared to ibrutinib. In this study, we evaluated acalabrutinib in spontaneously occurring canine lymphoma, a model of B-cell malignancy similar to human diffuse large B-cell lymphoma (DLBCL). First, we demonstrated that acalabrutinib potently inhibited BTK activity and downstream effectors in CLBL1, a canine B-cell lymphoma cell line, and primary canine lymphoma cells. Acalabrutinib also inhibited proliferation in CLBL1 cells. Twenty dogs were enrolled in the clinical trial and treated with acalabrutinib at dosages of 2.5 to 20mg/kg every 12 or 24 hours. Acalabrutinib was generally well tolerated, with adverse events consisting primarily of grade 1 or 2 anorexia, weight loss, vomiting, diarrhea and lethargy. Overall response rate (ORR) was 25% (5/20) with a median progression free survival (PFS) of 22.5 days. Clinical benefit was observed in 30% (6/20) of dogs. These findings suggest that acalabrutinib is safe and exhibits activity in canine B-cell lymphoma patients and support the use of canine lymphoma as a relevant model for human non-Hodgkin lymphoma (NHL).