Canine Hemangiosarcoma Research Articles

Liposome-encapsulated chemotherapy: Current evidence for its use in companion animals.

Cytotoxic drugs encapsulated into liposomes were originally designed to increase the anticancer response, while minimizing off-target adverse effects. The first liposomal chemotherapeutic drug was approved for use in humans more than 20 years ago, and the first publication regarding its use in a canine cancer patient was published shortly thereafter. Regardless, no general application for liposomal cytotoxic drugs has been established in veterinary oncology till now. Due to the popularity of canines as experimental models for pharmacokinetic analyses and toxicity studies, multiple publications exist describing various liposomal drugs in healthy dogs. Also, some evidence for its use in veterinary cancer patients exists, especially in canine lymphoma, canine splenic hemangiosarcoma and feline soft tissue sarcoma, however, the results have not been overwhelming. Reasons for this may be related to inherent issues with the enhanced permeability and retention effect, the tumour phenomenon which liposomal drugs exploit. This effect seems very heterogeneously distributed in the tumour. Also, it is potentially not as ubiquitously occurring as once thought, and it may prove important to select patients for liposomal therapy on an individual, non-histology-oriented, basis. Concurrently, new developments with active-release modified liposomes in experimental models and humans will likely be relevant for veterinary patients as well, and holds the potential to improve the therapeutic response. It, however, does not resolve the other challenges that liposomal chemotherapy faces, and more work still needs to be done to determine which veterinary patients may benefit the most from liposomal chemotherapy.

Adjuvant Doxorubicin with or without Metronomic Cyclophosphamide for Canine Splenic Hemangiosarcoma.

This retrospective study investigated the outcome of 33 dogs with splenic hemangiosarcoma treated with surgery followed by adjuvant dose-intensified doxorubicin (DOX) with or without low-dose metronomic cyclophosphamide (LDM-C) maintenance therapy. Among the 33 dogs, 18 dogs received LDM-C. Clinical stage was available for all dogs (5 stage I, 18 stage II, and 10 stage III). Nine dogs had macroscopic, and 24 dogs had microscopic disease at the start of DOX treatment. Median progression-free survival (PFS) and overall survival were 125 and 133 days, respectively. Clinical stage and tumor burden (microscopic versus macroscopic) at the start of chemotherapy was prognostic for PFS. No significant difference was observed in PFS or overall survival for the addition of LDM-C after a completed DOX protocol (P = .563 and P = .148, respectively). Based on the results of this retrospective study, the addition of LDM-C therapy as a maintenance regimen following a completed protocol of DOX adjuvant treatment of canine hemangiosarcoma may not improve outcome.

Abstract LB-201: Durable remission of lethal canine hemangiosarcoma

Abstract Background: Angiosarcoma is an uncommon but deadly malignant neoplasm of the vascular endothelium. Primary disease may present at any vascularized site but often occurs in the dermis on the neck and scalp or in the radiation field of women previously-treated for breast cancer. Metastases are often present at first diagnosis, and five-year survival is an abysmal 10-30%. In contrast to the relative paucity of human diagnoses, angiosarcoma is a major killer in the companion canine population, responsible for an estimated 120,000 deaths per year in the United States. The canine disease (termed HSA) closely mimics the human disease in both pathology and genetics. It frequently presents in the dog as acute hemoabdomen secondary to splenic rupture. Even if life-saving splenectomy is performed, median OS is a scant 48 days, and one-year OS hovers at 0%. Given the lack of an analogous rodent model and the accelerated timeline of the canine disease, the outbred companion canine is an ideal system in which to translate novel therapeutic approaches. Here we report an interim analysis of a phase I multi-site, open-label veterinary trial of chemo-immunotherapy performed on consecutively-presenting splenectomized companion canines with histopathologically-verified HSA. Methods: In addition to two cycles of 20 mg/m2 (low-dose) doxorubicin, enrolled study subjects received an autologous cell-therapy reported to generate durable CD8+ memory similar to that of physiologic viral infection. Vaccine was generated from mobilized peripheral blood and cryopreserved tumor antigen and was administered in four doses given in conjunction with type I interferon. Disease burden was monitored monthly by abdominal ultrasound, chest x-ray, and echocardiogram. Results: At the time of abstract submission, median OS had not yet been reached in the per protocol population, with 75% of animals alive and healthy (NED or ongoing PR) at up to one year post-splenectomy (p<0.0002). Abdominal ultrasound documented the resolution of local mesenteric disease in one animal, sternal lymphadenopathy in another, and hepatic metastases in a third. In the intent-to-treat population, median OS was 109 days with 43% of animals alive and healthy at up to one year post-splenectomy. A single incidence of grade 1/2 uveitis resolved promptly with administration of ophthalmic corticosteroids. Conclusions: Administration of autologous cell therapy in conjunction with low-dose doxorubicin is feasible, safe, and highly efficacious in the companion canine population. Generally, if subjects survived long enough to begin treatment, the prognosis became quite favorable. The results suggest that additional clinical studies in both canines and humans are warranted. Citation Format: Matthew M. Halpert, Vanaja Konduri, Yunyu Chen, Dan Liang, Jonathan M. Levitt, Brittany Neal, Caleb Hudson, Heidi Hottinger, Sindy Piscoya, Nicola Wilson, Lisa DiBernardi, Shonda Stallings, Zharkyn Omarbekov, Lori Seelhof, Oscar Ramirez, Qizhi C. Yao, Ratan D. Bhardwaj, Vinod Ravi, Laurel Douglass, William K. Decker. Durable remission of lethal canine hemangiosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-201. doi:10.1158/1538-7445.AM2017-LB-201

Active specific immunotherapy for lethal canine hemangiosarcoma: A model system for the treatment of angiosarcoma with curative intent.

e14539 Background: Angiosarcoma is a deadly malignant neoplasm of the vascular endothelium. Metastases are often present at diagnosis, and 5-year survival is only 10-30%. Though it comprises only 1% all soft tissue sarcoma diagnoses, it is a major killer among companion canines, responsible for an estimated 120,000 deaths per year in the US. The canine disease (termed HSA) closely mimics the human disease in both pathology and genetics. It frequently presents in the dog as acute hemoabdomen secondary to splenic rupture. Even if life-saving splenectomy is performed, median OS is only 48 days, and 1 year OS hovers at 0%. Given the accelerated timeline of the canine disease, the outbred dog is an ideal system in which to translate novel therapeutic approaches. Here we report an analysis of a phase I multi-site, open-label veterinary trial of chemo-immunotherapy performed on consecutively-presenting splenectomized companion canines with histopathologically-verified HSA. Methods: Subjects were administered two cycles of 20 mg/m2 (low-dose) doxorubicinand an autologous cell-therapy reported to generate durable CD8+ memory similar to that of physiologic viral infection. Vaccine was generated from mobilized peripheral blood and cryopreserved tumor antigen and was administered with type I interferon. Disease burden was monitored monthly by abdominal ultrasound, chest x-ray, and echocardiogram. Results: At the time of submission, median OS had not been reached in the per protocol population, with 75% of animals alive and healthy (NED or ongoing PR) at up to 1 year post-splenectomy (p < 0.0002). Ultrasound documented resolution of local mesenteric disease in one animal, sternal lymphadenopathy in another, and hepatic metastases in a third. In the intent-to-treat population, median OS was 109 days with 43% of animals alive and healthy at up to 1 year post-splenectomy. Conclusions: Administration of autologous cell therapy with low-dose doxorubicin is feasible, safe, and highly efficacious in the companion canines. If subjects survived long enough to begin treatment, prognosis became favorable. The results suggest that additional clinical studies in both canines and humans are warranted.

Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR.

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I-II study of 23 dogs with spontaneous, stage I-II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 μg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956-65. ©2017 AACR.

The Anti‐cancer Effects of Resveratrol on Canine Hemangiosarcoma Cells

Hemangiosarcoma (HSA) is a highly malignant tumor of vascular endothelial origin that exhibits aggressive biological behavior. HSAs are more common in dogs than other species including cats, sheep, horses, and cattle. Generally, canine HSAs have poor prognosis. After the complete surgical excision of the primary tumor, the survival time of dogs with HSA is typically short, less than 3 months. Adjuvant treatment with chemotherapy, especially anthracycline‐based regimens such as doxorubicin, can result in modest improvement of survival rates. However, the median survival time of dogs with HSA is still short, even after chemotherapy and surgery. Therefore, there is an urgent need to improve the adjuvant chemotherapeutic regimens in order to reach better clinical outcomes in dogs with HSA. Metformin and resveratrol have been shown to have strong anti‐proliferative and/or pro‐apoptotic properties in several human cancer cell lines. However, the potential anticancer effects of metformin and resveratrol have not been determined in canine HSAs. Therefore, the objective of this study is to investigate the potential growth inhibitory effects of metformin and resveratrol on HSA cell lines when used alone or in combination with doxorubicin. FROG and DD‐1 canine HSA cell lines were treated with varying concentrations of metformin or resveratrol with and without doxorubicin. Cell viability was measured by the MTT assay, and the expression of apoptotic proteins was assessed by western blotting. In contrast to human cell line studies, metformin had a minimal inhibitory effect on the growth of HSA cells. However, resveratrol markedly inhibited the growth of HSA cells, which is in agreement with human cell line studies. In combination, resveratrol and doxorubicin inhibited the growth of both HSA cell lines more than doxorubicin alone. Mechanistically, resveratrol induced apoptosis in HSA cells, and augmented doxorubicin‐induced apoptosis in these cells. Additionally, MTT cell viability of the cardiac‐derived H9c2 cells showed that resveratrol does not augment doxorubicin‐induced cell death in cardiac cells. These findings suggest that resveratrol may have anticancer effects against canine HSA and could be a potential adjunct therapy to the standard‐of‐care anticancer drugs used in canine HSA patients. Since resveratrol has been shown to have both anti‐cancer and cardioprotective effects in rodent models, canine cancer patients can be a perfect translational platform to move the findings of these rodent studies forward to the clinic.Support or Funding InformationResearch reported in this abstract was supported by a Pre‐K Career Development Award from the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Role of monocyte recruitment in hemangiosarcoma metastasis in dogs.

Canine hemangiosarcoma (HSA) is a highly malignant tumour associated with short survival times because of early and widespread metastasis. In humans and rodents, monocytes play key roles in promoting tumour metastasis through stimulating tumour cell extravasation, seeding, growth and angiogenesis. Therefore, we investigated the potential association between monocyte infiltration and tumour metastasis in HSA and other common canine tumours. Immunohistochemistry was used to quantify CD18+ monocytes within metastases. We found that HSA metastases had significantly greater numbers of CD18+ monocytes compared with metastases from other tumour types. HSA cells were the highest producers of the monocyte chemokine CCL2, and stimulated canine monocyte migration in a CCL2 dependent manner. These results are consistent with the hypothesis that overexpression of CCL2 and recruitment of large numbers of monocytes may explain in part the aggressive metastatic nature of canine HSA. Thus, therapies designed to block monocyte recruitment may be an effective adjuvant strategy for suppressing HSA metastasis in dogs.

Use of routine histopathology and factor VIII-related antigen/von Willebrand factor immunohistochemistry to differentiate primary hemangiosarcoma of bone from telangiectatic osteosarcoma in 54 dogs.

Hemangiosarcoma (HSA) of bone and telangiectatic osteosarcoma (tOSA) can appear similar histologically, but differ in histogenesis (malignant endothelial cells versus osteoblasts), and may warrant different treatments. Immunohistochemistry (IHC) for endothelial cell marker factor VIII-related antigen/von Willebrand factor (FVIII-RAg/vWF) is a well-documented ancillary test to confirm HSA diagnoses in soft tissues, but its use in osseous HSA is rarely described. Archived samples of 54 primary appendicular bone tumours previously diagnosed as HSA or tOSA were evaluated using combination routine histopathology (RHP) and IHC. Approximately 20% of tumours were reclassified on the basis of FVIII-RAg/vWF immunoreactivity, typically from an original diagnosis of tOSA to a reclassified diagnosis of HSA. No sample with tumour osteoid clearly identified on RHP was immunopositive for FVIII-RAg/vWF. RHP alone was specific but not sensitive for diagnosis of HSA, compared with combination RHP and IHC. Routine histopathological evaluation in combination with FVIII-RAg/vWF IHC can help differentiate canine primary appendicular HSA from tOSA.

Identification of the two KIT isoforms and their expression status in canine hemangiosarcomas.

BackgroundKIT is a tyrosine kinase growth factor receptor. High expression of KIT has been found in several tumors including canine hemangiosarcoma (HSA). This study investigated the correlation of KIT expression and c-kit sequence mutations in canine HSAs and benign hemangiomas (HAs).ResultsImmunohistochemistry (IHC) staining confirmed KIT expression in 94.4 % (34/36) of HSAs that was significantly higher than 0 % in HAs (0/16). Sequencing the entire c-kit coding region of HSAs and normal canine cerebellums (NCCs) revealed GNSK-deletion in exon 9. As for exon 9 genotyping by TA-cloning strategy, GNSK-deletion c-kit accounted for 48.6 % (68/140) colonies amplified from12 KIT-positive HSAs, a significantly higher frequency than 14.1 % (9/64) of colonies amplified from six NCCs.ConclusionsDue to the distinct expression pattern revealed by IHC, KIT might be used to distinguish benign or malignant vascular endothelial tumors. Moreover, the high incidence of GNSK-deletion c-kit in canine HSAs implicates KIT isoforms as possibly participating in the tumorigenesis of canine HSAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0772-y) contains supplementary material, which is available to authorized users.

Abstract 383: In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma

Abstract While extremely rare in humans, hemangiosarcoma (HSA) accounts for nearly 2% of canine neoplasia, and is characterized by both aggressive local growth/invasion and a high rate of metastasis. Both canine and human HSA exhibit sustained aberrant PI3K/Akt/mTOR pathway signaling. The purpose of this study was to examine the in vitro effects of a novel dual PI3K/mTOR inhibitor, VDC-597, in canine HSA cells. Three canine HSA cell lines (DEN-HSA, CIN-HSA, and SB-HSA) were employed in multiple in vitro assays. Western analysis evaluated activation (phosphorylation) of key downstream pro-survival proteins in the PI3K/mTOR pathway. Changes in tumor cell growth/apoptosis were assessed using both bioreductive assays (Alamar Blue) and in vitro live-cell imaging (IncuCyte), in the presence and absence of doxorubicin, a standard-of-care cytotoxic drug for canine and human sarcomas, as well as in the presence and absence of U-0126, an inhibitor of the MEK pathway. Migration was assessed using both Boyden chamber and scratch assays, via live-cell imaging (IncuCyte). Matrigel invasion was assessed using traditional Boyden chambers. Finally, ELISA was utilized to quantify relative expression of vascular endothelial growth factor (VEGF). VDC-597 suppressed activation of both Akt and 4eBP1 in canine HSA cells in a dose- and time-dependent fashion, with an IC50 of approximately 0.3 uM, a concentration predicted to be clinically achievable based on preliminary early-phase canine and human studies. VDC-597 dose-dependently reduced proliferation, migration, invasion, and VEGF expression in HSA cells, while promoting tumor cell apoptosis. VDC-597 demonstrated additive antiproliferative effects when combined with doxorubicin and U-0126. Together, these results suggest that inhibitors of the PI3K/Akt/mTOR pathway may act against multiple components of the neoplastic process, including proliferation/apoptosis, chemosensitivity, invasion/migration and angiogenesis, and justify the evaluation of PI3K/mTOR inhibitors in canine, and eventually human, HSA. Experiments to examine the effect of VDC-597 in reducing tumor burden and metastasis in a rodent model are ongoing. Citation Format: Alex A. Pyuen, Douglas H. Thamm. In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 383.

Therapeutic potential of endothelin inhibitors in canine hemangiosarcoma

AimsHemangiosarcoma (HSA) that originates from vascular endothelial cells is the most common splenic malignant neoplasm in dogs, as it accounts for approximately 20% of all canine soft tissue sarcomas. In this study, inhibitory effects of endothelin receptor antagonists on the growth of HSA cells were examined using cell lines established from canine HSA. Main methodsThe preproendothelin-1 (PPET-1), endothelin type A receptor (ETA) and endothelin type B receptor (ETB) mRNA expression levels in HSA cell lines (n=5) were analyzed quantitatively by real-time RT-PCR. These levels were compared with those in HSA tissues (n=11) and those in normal splenic tissues (n=6). ETA and ETB protein expression was examined by western blot. The production and secretion of endothelin-1 (ET-1) and big ET-1 by cell lines were analyzed by measuring the levels in the culture medium by ELISA. The inhibitory effects of endothelin receptor antagonists (ambrisentan, BQ788 and bosentan) on cell growth were evaluated by WST-8 assay. Key findingsThe PPET1 and ETA mRNA expression levels were elevated in HSA tissues and HSA cell lines compared with normal tissues. In cell lines, the production of ET-1 and big ET-1 peptide as well as the expression of ETA protein were detected, but the levels of ETB were not measured. Ambrisentan and bosentan inhibited growth activity in cell lines. Ambrisentan was more effective than bosentan. SignificanceThese findings demonstrate the importance of the ETA axis in canine HSA as well as the potential of ETA inhibitors in the treatment of canine HSA.

Clinical, Pathological and Immunohistochemical Evaluation of a Primary Hemangiosarcoma in a Pinscher Dog

Background: Hemangiosarcoma (HSA) is a malignant tumor that arises from the vascular endothelium affecting more often dogs than other species as cats, cows and horses. It comprises approximately 2% of all tumors in dogs. The most common primary site for the HSA in dogs is the spleen, and other locations include the right atrium, pericardium, liver andprostate. Other authors have reported this tumor in lungs, kidney, oral cavity, muscle, bone, urinary bladder, left ventricle, tongue and retroperitoneum. Due to the importance of the HSA in canine species, the aim of this study was to describe the clinical and pathological fndings, besides therapeutic protocol in an unusual case of HSA.Case:A six-year-old male pinscher was referred to the veterinary hospital with a history of cyanosis and choking. The animal was submitted to radiographic examination in lateral cervical view, which identifed the presence of a mass of 1.2 cm in diameter near the pharynx. In order to evaluate the oral cavity, general anesthesia was performed, and it was possible to see a soft, rosy, circumscribed and vascularized lump in pharyngeal region. Due to suspicion of neoplasm, excisional biopsywithout surgical margin was performed. The histopathological exam diagnosed hemangiosarcoma. Immunohistochemistry against vimentin, factor VIII, VEGF and Ki67 was performed and confrmed diagnosis of low grade hemangiosarcoma. Antineoplastic chemotherapy protocol was initiated with doxorubicin and cyclophosphamide every 21 days totaling sixsessions. However, the animal died after the cyclophosphamide intoxication with a three-fold recommended dose (660 mg/m² total in the last session), showing a median survival rate of 220 days.Discussion: The most common primary site for HSA in dogs is the spleen. The pharyngeal location is rare, with only a few reports in literature. In the present case, solitary tumor was observed in pharynx with no involvement of other organs, evidenced by radiographic examination, abdominal ultrasound and echocardiogram, suggesting that pharynx was theprimary location of the tumor. Main features of HSA comprise a solitary nodule or multifocal lesions within the organ or widely disseminated. Histologically, they consist of pleomorphic immature endothelial cells with formation of vascular spaces with variable amount of blood and/or thrombi. In some cases, HSA shows a polymorphic subtype and immunohistochemistry is necessary to provide a defnitive diagnosis. The sample was submitted to histopathological examination which revealed proliferation of endothelial cells with pronounced pleomorphism ranging from polygonals to ovoid, sparse cytoplasm, round to oval nucleus with visible nucleolus, few mitotic fgures, some of them, aberrant, which confrmed diagnosis of HAS. Due to the unusual location, we performed immunohistochemical staining for vimentin, factor VIII,VEGF and Ki67 antibodies to confrm mesenchymal origin of the tumor. In IHC, it was possible to identify positive reac tion for vimentin protein, factor VIII, VEGF and few Ki67 positive cells, confrming histopathological diagnosis. Despite literature describes an aggressive biological behavior of canine HSA, with common occurrence of metastasis, recurrencewas not observed at the site of the removal of the tumor. In histopathological evaluation, it was observed low number ofmitoses, besides the low Ki67 expression on IHC, featuring a low grade tumor with minor ability to metastasize. To the author’s knowledge, this case describes an unusual presentation of HSA, with low metastatic potential, in which chemo therapy protocol achieved survival time of 220 days.Keywords: angiosarcoma, dogs, immunohistochemistry, pharynx.

Immunohistochemical detection of a potential molecular therapeutic target for canine hemangiosarcoma

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm of dogs for which there is currently no effective treatment. A recent study suggested that receptor tyrosine kinases (RTKs), the PI3K/Akt/m-TOR and MAPK pathways are all activated in canine and human HSA. The aim of the present study was to investigate the overexpression of these proteins by immunohistochemistry in canine splenic HSA to identify potential molecular therapeutic targets. A total of 10 splenic HSAs and two normal splenic samples surgically resected from dogs were sectioned and stained with hematoxylin and eosin for histological diagnosis or analyzed using immunohistochemistry. The expression of RTKs, c-kit, VEGFR-2 and PDGFR-2, as well as PI3K/Akt/m-TOR and MEK was higher in canine splenic HSAs compared to normal spleens. These proteins may therefore be potential therapeutic targets in canine splenic HSA.

Pathobiology of Hemangiosarcoma in Dogs: Research Advances and Future Perspectives.

Hemangiosarcoma (HSA) is an aggressive and common cancer in dogs. While cutaneous masses are often treatable by tumor excision, visceral tumors are almost always incurable. Treatment advances for this disease have been limited due to a poor understanding of the overall tumor biology. Based upon its histological appearance, HSA has been presumed to originate from transformed endothelial cells; however, accumulating data now suggest a pluripotent bone marrow progenitor as the cell of origin for this disease. More recently, the identification of a novel subclassification of HSAs has provided a foundation to further our understanding of the cellular characteristics of HSA tumor cells, along with those of the cells comprising the tumor microenvironment. These discoveries hold promise for the development of new approaches to improve treatments for canine HSA, as well as to establish the utility of this disease as a spontaneous model to understand the pathogenesis and develop new treatments for vascular tumors of humans. In this review, we will provide a brief historical perspective and pathobiology of canine HSA, along with a focus on the recent advances in the molecular and cellular understanding of these tumors. In addition, future directions that should continue to improve our understanding of HSA pathogenesis will be discussed.

MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases

Malignant endothelial proliferative diseases including human angiosarcoma (AS) and canine hemangiosarcoma (HSA) are serious diseases with a grave prognosis. Establishing liquid biopsy-based biomarkers for screening has definite clinical utility; however, plasma miRNAs up- or down-regulated in these sarcomas have been unclear. For identifying possible diagnostic plasma miRNAs for these sarcomas, we investigated whether plasma miR-214 and miR-126, which miRNAs play important roles in angiogenesis and tumorigenesis, were elevated in malignant endothelial proliferative diseases. For this investigation, human angiosarcoma and canine hemangiosarcoma cell lines and clinical plasma samples of canine hemangiosarcoma were examined by performing miRNA qRT-PCR. We report here that human angiosarcoma and canine hemangiosarcoma cell lines over-secreted miR-214 and miR-126 via microvesicles; in addition, their levels in the plasma samples from canines with hemangiosarcoma were increased. Moreover, the surgical resection of primary tumors decreased the levels of plasma miR-214 and miR-126. Our findings suggest that these malignant endothelial proliferative diseases over-secreted miR-214 and miR-126, thus suggesting that these miRNAs have potential as diagnostic biomarkers for malignant endothelial proliferative diseases in canine and possible in human angiosarcoma.

Increased Survival after Treatment of Canine Hemangiosarcoma with Fenofibrate

Hemangiosarcoma (HSA) is a common and highly metastatic tumor that affects predominantly large-breed dogs. The median survival time for dogs diagnosed with splenic HSA without adjuvant chemotherapy after surgery is 86 days.

MicroRNA-214 Promotes Apoptosis in Canine Hemangiosarcoma by Targeting the COP1-p53 Axis.

MicroRNA-214 regulates both angiogenic function in endothelial cells and apoptosis in various cancers. However, the regulation and function of miR-214 is unclear in canine hemangiosarcoma, which is a spontaneous model of human angiosarcoma. The expression and functional roles of miR-214 in canine hemangiosarcoma were presently explored by performing miRNA TaqMan qRT-PCR and transfecting cells with synthetic microRNA. Here, we report that miR-214 was significantly down-regulated in the cell lines used and in clinical samples of canine hemangiosarcoma. Restoration of miR-214 expression reduced cell growth and induced apoptosis in canine hemangiosarcoma cell lines through transcriptional activation of p53-regulated genes although miR-214 had a slight effect of growth inhibition on normal endothelial cells. We identified COP1, which is a critical negative regulator of p53, as a novel direct target of miR-214. COP1 was overexpressed and the specific COP1 knockdown induced apoptosis through transcriptional activation of p53-regulated genes as well as did miR-214-transfection in HSA cell lines. Furthermore, p53 knockdown abolished the miR-214-COP1-mediated apoptosis; thus, miR-214 and COP1 regulated apoptosis through controlling p53 in HSA. In conclusion, miR-214 functioned as a tumor suppressor in canine hemangiosarcoma by inducing apoptosis through recovering the function of p53. miR-214 down-regulation and COP1 overexpression is likely to contribute to tumorigenesis of HSA. Therefore, targeting miR-214-COP1-p53 axis would possibly be a novel effective strategy for treatment of canine hemangiosarcoma and capable of being applied to the development of novel therapeutics for human angiosarcoma.

Interactions between CXCR4 and CXCL12 promote cell migration and invasion of canine hemangiosarcoma.

The CXCR4/CXCL12 axis plays an important role in cell locomotion and metastasis in many cancers. In this study, we hypothesized that the CXCR4/CXCL12 axis promotes migration and invasion of canine hemangiosarcoma (HSA) cells. Transcriptomic analysis across 12 HSA cell lines and 58 HSA whole tumour tissues identified heterogeneous expression of CXCR4 and CXCL12, which was associated with cell movement. In vitro, CXCL12 promoted calcium mobilization, cell migration and invasion that were directly proportional to surface expression of CXCR4; furthermore, these responses proved sensitive to the CXCR4 antagonist, AMD3100, in HSA cell lines. These results indicate that CXCL12 potentiates migration and invasion of canine HSA cells through CXCR4 signalling. The direct relationship between these responses in HSA cells suggests that the CXCR4/CXCL12 axis contributes to HSA progression.

Abstract 1694: Preclinical evaluation of a novel bispecific targeted toxin for the treatment of sarcomas

Abstract Introduction: EGFuPA is a bispecific ligand-targeted toxin consisting of epidermal growth factor (EGF) and urokinase (uPA) on the same molecule with truncated Pseudomonas exotoxin. The toxin was mutated to remove amino acids recognized by B-cells. EGFuPA is uniquely designed to simultaneously target EGF receptors (EGFR) and/or urokinase receptors (uPAR) on tumor cells and uPAR in the microenvironment. EGFuPA is safe and effective in laboratory animals and has excellent in vitro activity against sarcomas. Because EGFuPA crosses species platforms, an adaptive phase 1/2 study was undertaken using canine hemangiosarcoma (HSA) as a model of a highly aggressive, metastatic vascular sarcoma. We hypothesized that six-month survival would exceed 50% for dogs enrolled. Canine HSA is a tumor derived from blood vessel forming cells, which can overexpress both uPAR and EGFR rendering it an excellent target for the drug. It bears similarity to human angiosarcoma. Methods: Enrollment began in November 2012. Dogs with histologically confirmed, non-metastatic stage-I or II splenic HSA were eligible after splenectomy. EGFuPA was administered intravenously for one cycle on Days 1, 3, and 5. Adjuvant doxorubicin was given according to standard of care (SOC) protocols starting seven days after the last dose of EGFuPA. Results: EGFuPA was detectable in the systemic circulation 5 minutes post-infusion with clearance by 15 minutes. The drug was well tolerated with self-limiting, reversible hypovolemia at the highest dose. Thus far, 50 μg/kg appears to be the optimal dose. When compared to a historical control group of 19 dogs treated with SOC between 2008 and 2011, median survival is significantly longer for all 16 dogs treated with EGFuPA (SRCBST group, 240 vs. 129 days, p = 0.03) and for the 10 dogs in the Optimal Dose group (MS not reached, p = 0.01). Six-month survival for the Optimal Dose group was 65%. Overall survival at 400 days was 7% for the SOC group, 32.7% for the SRCBST group, and 56% for the Optimal Dose group. Outcomes were not affected by the presence of neutralizing antibodies. Conclusions: A biologically active dose of EGFuPA was established and found to be safe in companion dogs with HSA in the minimal residual disease setting. The unique nature of its bispecific targeting and the observed significant, biologic activity warrants consideration of EGFuPA for a phase 1 study for drug-refractory sarcomas in human patients. Citation Format: Antonella Borgatti, Amber Winter, Kathleen Stuebner, Joseph Koopmeiners, Jaime F. Modiano, Daniel Vallera. Preclinical evaluation of a novel bispecific targeted toxin for the treatment of sarcomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1694. doi:10.1158/1538-7445.AM2015-1694

Association of Sphingosine-1-phosphate (S1P)/S1P Receptor-1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma.

BackgroundSphingosine‐1‐phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs.Hypothesis/ObjectivesS1P/S1P1 signaling will contribute to the progression of hemangiosarcoma (HSA).AnimalsThirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used.MethodsThis was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT‐PCR, immunohistochemistry, and immunoblotting were performed to examine S1P1 expression. S1P concentrations were measured by high‐performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca2+ mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining.ResultsCanine HSA cells expressed higher levels of S1P1 mRNA than nonmalignant endothelial cells. S1P1 protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P1, decreased S1P1 protein expression and induced apoptosis of HSA cells.Conclusions and clinical importanceS1P/S1P1 signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P1 or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA.