Hemangiosarcoma Research Articles

Abstract PO007: Role of PI3K pathway in reprogramming the tumor niche of angiosarcoma

Abstract Angiosarcoma (AS) is a highly aggressive, rare soft-tissue sarcoma that forms malignant blood vessels. Approximately half of patients have metastatic or unresectable disease with a median overall survival of less than 6 months, and tumor-related mortality is high. Inflamed tissue is frequently seen in angiosarcomas, and immune cells are key components that contribute to the landscape of the tumor microenvironment. Hemangiosarcoma (HSA) occurs commonly in companion dogs, and it shares clinical and morphological features with human AS. Recurrent mutations in TP53 and genes involved in PI3K pathway such as PIK3CA, and PIK3R1 were observed in both human AS and canine HSA, and the mutational landscape was associated with distinct molecular tumor subtypes; in particular, differing immunophenotypes. Recently, we have shown that human AS and canine HSA establish convergent transcriptional programs driven by angiogenic fusion genes and TP53 mutation. However, these vascular tumors are genomically complex, and molecular mechanisms that establish the tumor microenvironment are incompletely known. In this study, we analyzed RNA-seq transcriptomic data generated from human AS tissues (n=13) and canine HSA tissues (n=76) and HSA cell lines (n=11). The data showed that the tumors of both species enriched gene signatures associated with maintenance of hematopoietic stem cells. Then, we found that canine HSA cells supported expansion and differentiation of human CD34+ umbilical cord blood cells using long-term culture-initiating cell and colony-forming unit assays. We also developed xenograft models of canine HSA that facilitate myelopoiesis and macrophage infiltration in the tumor tissue. Our data showed that gene signatures of monocytes and naïve macrophages were detectable in canine HSA cells. In human ASs, macrophage-associated genes, in particular M2 macrophage signatures were significantly enriched, compared to normal tissues. Collectively, our data suggest that human AS and canine HSA have cell-autonomous capacity to govern hematopoietic progenitors and immune cells, potentially establishing the tumor immune niche. We engineered HSA cells to induce homozygous H1047R mutations in PIK3CA gene using CRISPR-Cas9 system. Our ongoing work is to determine if regulating PI3K pathway in the tumor cells contributes to the molecular programs that create the tumor niche of the vascular malignancy. Citation Format: Sophia Wenthe, Ashley J. Schulte, Mathew G. Angelos, Dan S. Kaufman, Jaime F. Modiano, Jong Hyuk Kim. Role of PI3K pathway in reprogramming the tumor niche of angiosarcoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO007.

Candidate Glycoprotein Biomarkers for Canine Visceral Hemangiosarcoma and Validation Using Semi-Quantitative Lectin/Immunohistochemical Assays.

Visceral hemangiosarcoma (HSA) is one of the more frequent cancers in dogs and has a high metastatic rate and poor prognosis, as clinical signs only become apparent in advanced stages of tumor development. In order to improve early and differential diagnostic capabilities and hence, prognosis for dogs with HSA, two types of biomarker are needed: a point-of-care diagnostic biomarker and a prognostic biomarker—preferentially based on samples obtained with minimally invasive methods. In this study, we applied a lectin magnetic bead array-coupled tandem mass spectrometry (LeMBA-MS/MS) workflow through discovery and validation phases to discover serum glycoprotein biomarker candidates for canine HSA. By this approach, we found that Datura stramonium (DSA), wheat germ agglutinin (WGA), Sambucus nigra (SNA), and Pisum sativum (PSA) lectins captured the highest number of validated candidate glycoproteins. Secondly, we independently validated serum LeMBA-MS/MS results by demonstrating the in situ relationship of lectin-binding with tumor cells. Using lectin-histochemistry and immunohistochemistry (IHC) for key proteins on tissues with HSA and semi-quantitation of the signals, we demonstrate that a combination of DSA histochemistry and IHC for complement C7 greatly increases the prospect of a more specific diagnosis of canine HSA.

Evaluation of an autologous cancer vaccine for the treatment of metastatic canine hemangiosarcoma: a preliminary study

BackgroundCanine hemangiosarcoma (HSA) is an aggressive cancer arising from multipotential bone marrow-derived stem cells. Anthracycline chemotherapy drugs have been the mainstay adjuvant chemotherapy following surgery with only modest improvement in survival and an attendant risk for adverse events. Immunotherapy, using a whole cell autologous cancer vaccine adjuvanted with MIM-SIS, may improve outcomes for dogs with HSA with a lower risk for adverse events compared with chemotherapy.ResultsIn cultured DH82 canine monocyte-like cells, autologous cancer vaccines prepared from 13 dogs with HSA increased MHC-II surface expression ranging from 20.0-60.4% on single-stained cells, CD80 surface expression ranging from 23.7–45.9% on single-stained cells, and MHC-II/CD80 surface expression ranging from 7.2–20.1% on double-stained cells. Autologous cancer vaccines were able to, on average, stimulate an up-regulation of MHC-II and CD80 by 48-fold as compared to media only (MHC-II + CD80 + cells: 12.19 ± 3.70% vs. 0.25 ± 0.06%; p < 0.001). The overall median survival time for dogs treated with the autologous cancer vaccine was 142 days (range, 61 to 373 days). Dogs treated with the autologous cancer vaccine or maximum tolerated dose (MTD) chemotherapy had significantly (P < 0.001) longer survival than dogs treated with surgery alone. The 1-year survival rate was 12.5% for dogs treated with the autologous cancer vaccine, and 0% for dogs treated with surgery alone or MTD chemotherapy. No adverse events were observed in the dogs treated with the autologous cancer vaccine.ConclusionsThe adjuvanted autologous cancer vaccine is capable of up-regulating MHC-II and CD80 in cultured canine monocyte-derived cells, which are important stimulatory molecules in generating an immune response and improves survival time in dogs with metastatic (stage III) HSA when compared to surgical treatment alone. Autologous cancer vaccine-treated dogs had survival similar to those dogs treated with MTD chemotherapy without any observed adverse events. This autologous cancer vaccine represents an effective form of individualized immunotherapy that is an appealing option for dog owners not wanting to pursue adjuvant chemotherapy for HSA.

Effect of simvastatin on cell proliferation and Ras activation in canine tumour cells.

Statins are inhibitors of the mevalonate cascade that is responsible for cholesterol biosynthesis and the formation of intermediate metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) used in the prenylation of proteins. Although statins are widely used in the treatment of hypercholesterolemia, recent studies suggest that they also inhibit proliferation of tumour cells by reducing prenylation of small GTP-binding proteins, such as, Ras. This study aimed to evaluate the effect of simvastatin on cell proliferation and Ras activation in various canine tumour cell lines, including hemangiosarcoma (HSA), melanoma, and lymphoma cell lines. Simvastatin inhibited cell proliferation of all cell lines tested in a concentration- and time-dependent manner, but the susceptibilities were different amongst the cell lines. Simvastatin induced apoptotic cell death via activation of caspase-3 and cell cycle arrest. The cytotoxic effects of simvastatin were attenuated by GGPP and FPP. Simvastatin decreased the amount of prenylated Ras and GTP-bound Ras in HSA and melanoma cell lines, but not in lymphoma cell lines. These results indicate that simvastatin induces cytotoxic effects through the depletion of GGPP and FPP in a variety of canine tumour cells, whereas multiple mechanisms are involved in the effects. Further study is required to elucidate the underlying mechanisms of simvastatin-induced cytotoxic effects in a variety of canine tumour cells.

Abstract 195: Molecular mechanisms that activate convergent oncogenic pathway in genomically complex angiosarcoma

Abstract Angiosarcoma is an aggressive, albeit rare cancer in humans. Angiosarcomas are vascular malignancies that can occur anywhere in the body, and their metastatic propensity is high. The cause of the vast majority of sporadic angiosarcomas is unknown, and no therapeutic targets have been identified to improve outcomes. Angiosarcomas are genomically complex, with chaotic karyotypes and massive chromosomal abnormalities. Despite the genomic complexity, angiosarcomas share a histological morphology that consists of disorganized, malignant vessel-forming cells. Hemangiosarcoma (HSA) is a common cancer of dogs; it shares clinical and morphological features, as well as aspects of its mutational landscape with human angiosarcoma. Our previous work has revealed that canine HSAs and human angiosarcomas share transcriptional signatures that establish angiogenic and inflammatory molecular subtypes. A comparative genomics approach is useful to apply knowledge from appropriately powered canine studies to inform research into human angiosarcomas. In this study, we leveraged next generation RNA-Seq data from a cohort of 76 spontaneous canine HSAs and from thirteen human angiosarcomas to identify fusion genes. Fifteen novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 were identified in 11 of the 76 canine HSAs; these fusion genes were exclusively seen in tumors of the angiogenic molecular subtype. Mutations of TP53 and fusion genes co-occurred in tumors with higher frequency than expected by random chance. Pathway analysis revealed that co-occurring mutations of TP53 and PIK3CA were associated with gene expression signatures of chromatin remodeling and immunosuppression, and co-occurrence of fusion genes and TP53 mutation enriched a gene signature predicting activation of angiogenic pathways. In human angiosarcomas, ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in 7 of 13 tumors, with two showing mutations of TP53. Immunohistochemical assays showed that canine HSA and human angiosarcoma activated p53, AKT, and mTOR signaling pathways independent of fusion genes and mutational conditions, suggesting that both tumors activate convergent signaling pathways to retain the ontogenetical properties. In summary, our comparative analysis identified shared molecular signatures between canine HSA and human angiosarcoma. Specifically, the data suggests that genomic instability induced by TP53 mutations might create a predisposition for fusion events that may contribute to tumor progression by promoting selection and/or enhancing fitness through activation of convergent angiogenic pathways. Our ongoing work seeks to define the key molecular programs that establish the mutational landscape, which consequently activates convergent pathways that contribute to angiosarcoma development. Citation Format: Jong Hyuk Kim, Kate Megquier, Aaron L. Sarver, Rachael Thomas, Ashley J. Schulte, Chao Wang, Ingegerd Elvers, Elinor Karlsson, Matthew Breen, Kerstin Lindblad-Toh, Jaime F. Modiano. Molecular mechanisms that activate convergent oncogenic pathway in genomically complex angiosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 195.

Chromatin run-on sequencing analysis finds that ECM remodeling plays an important role in canine hemangiosarcoma pathogenesis

BackgroundCanine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression.ResultsChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECM-associated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions.ConclusionOutcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA.

Abstract B22: A method for early detection of hemangiosarcoma in dogs

Abstract Aims: We developed a test to detect hemangiosarcoma (HSA)-associated cells in the circulation of dogs prior to the onset of grossly detectable disease. Our objective is to assign dogs into risk categories for development of HSA, allowing for the rational deployment of eBAT, a drug to target the cancer-stem cell compartment and the tumor niche in these dogs, as an effective means for cancer prophylaxis. Methods: We used samples from 125 dogs (28 with hemangiosarcoma, 29 with other cancers, 27 with benign vascular pathology, and 41 for healthy) as a training set for machine learning. We used 10-fold cross validation, “leave-one-out” analyses to establish parameters of the test for detection of HSA-associated cells, and to determine sensitivity and specificity. We then applied the algorithms to a prospective cohort of 209 clinically healthy golden retrievers, boxers, and Portuguese Water Dogs older than 6 years of age as a validation set to assess the performance of the test in the early detection setting. Results: Using 10-fold cross validation, the test achieved a classification accuracy of 85% for healthy dogs and up to 89% for dogs with HSA, with 89% sensitivity and 95% specificity for detection of HSA. In the prospective cohort, more than 50% of clinically healthy dogs were categorized into one of the three conditions indicative of pathology (HSA, other cancer, benign splenic pathology). The test seems to have outstanding specificity and acceptable sensitivity for use in early detection: the false negative rate at 6-months after testing was &amp;lt;1% (1 of 99 dogs). Twenty-one dogs in the prospective cohort have developed cancer or another chronic condition. Nineteen that had a prediction of cancer-associated pathology were diagnosed with cancer (90%). Only two of these 21 dogs (10%) were misclassified. One had a prediction of cancer-associated pathology but died from a nonmalignant condition, and one had a prediction of “healthy” but died of cancer. Conclusions: We developed a blood test that can successfully detect the presence of HSA-associated cells in dogs with active disease, and that can be used in the early-detection setting to categorize risk of developing HSA. Our results from a prospective cohort of 200 dogs over 6 years of age suggest as many as 50% of these dogs have inapparent disease. The performance of the test is acceptable for use as an actionable component of a cancer prevention platform. Clinical Significance: To our knowledge, this is the first test that can accurately assign risk for development of hemangiosarcoma to clinically healthy dogs, providing a rationale for cancer chemoprophylaxis. Our study also provides proof of concept for prospective, large-scale trials for early cancer detection in companion dogs. Citation Format: Taylor A. DePauw, Ali Khammanivong, Jaime F. Modiano. A method for early detection of hemangiosarcoma in dogs [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B22.

Development of synthetic microRNA-214 showing enhanced cytotoxicity and RNase resistance for treatment of canine hemangiosarcoma.

MicroRNA-214 (miR-214), a pivotal tumour-suppressive miRNA, is downregulated in canine hemangiosarcoma (HSA) cells. Although these tumour-suppressive miRNAs are potential therapeutic agents, their clinical efficacy may be limited because of their vulnerability to RNase-rich microenvironments and low in vivo transfection rates. We developed synthetic miR-214s with enhanced cytotoxicity, RNase resistance and quantity of miR-214 in/on cells. These synthetic miR-214s were synthesized by various chemical modifications (such as 4'-aminoethyl-2'-fluoro, 2'-fluoro, 2'-O-methyl, phosphorothioate and oligospermine modifications) of the wild-type mature miR-214 sequences. Transfection of HSA cells with synthetic miR-214 (miR-214 5AE) demonstrated significant growth suppressive effect and induced the strongest apoptotic response. Synthetic miR-214s (miR-214 5AE, miR-214 10AE and miR-214 OS) were much more stable than mature miR-214s in foetal bovine serum. Similar to mature miR-214, 5AE and OS suppressed the expression level of COP1 in HSA cells. The quantity of synthetic miR-214s in/on cells was higher than that of mature miR-214. In conclusion, we developed a clinically applicable, synthetic miR-214 5AE that regulates the COP1 protein expression similar to that mediated by mature miR-214. Additionally, miR-214 5AE confers better cytotoxicity, nuclease resistance and transfection rate than mature miR-214. Thus, miR-214 5AE could potentially be a novel miRNA-based chemotherapeutic agent that could improve the prognosis of HSA. Its in vivo effects on canine HSA need to be examined in future.

Molecular subtypes in canine hemangiosarcoma reveal similarities with human angiosarcoma.

Angiosarcoma (AS) is a rare neoplasm with limited treatment options and a poor survival rate. Development of effective therapies is hindered by the rarity of this disease. Dogs spontaneously develop hemangiosarcoma (HSA), a common, histologically similar neoplasm. Metastatic disease occurs rapidly and despite chemotherapy, most dogs die several months after diagnosis. These features suggest that HSA might provide a tractable model to test experimental therapies in clinical trials. We previously reported whole exome sequencing of 20 HSA cases. Here we report development of a NGS targeted resequencing panel to detect driver mutations in HSA and other canine tumors. We validated the panel by resequencing the original 20 cases and sequenced 30 additional cases. Overall, we identified potential driver mutations in over 90% of the cases, including well-documented (in human cancers) oncogenic mutations in PIK3CA (46%), PTEN (6%), PLCG1(4%), and TP53 (66%), as well as previously undetected recurrent activating mutations in NRAS (24%). The driver role of these mutations is further demonstrated by augmented downstream signaling crucial to tumor growth. The recurrent, mutually exclusive mutation patterns suggest distinct molecular subtypes of HSA. Driver mutations in some subtypes closely resemble those seen in some AS cases, including NRAS, PLCG1, PIK3CA and TP53. Furthermore, activation of the MAPK and PI3K pathways appear to be key oncogenic mechanisms in both species. Together, these observations suggest that dogs with spontaneous HSA could serve as a useful model for testing the efficacy of targeted therapies, some of which could potentially be of therapeutic value in AS.

Abscessation associated with an intramuscular haemangiosarcoma in a dog

Haemangiosarcoma (HSA) is a neoplasm of the endothelial cells. Intramuscular HSA is an uncommon presentation of this endothelial cell neoplasm. This report describes an unusual case of an intramuscular HSA...

Primary Hemangiosarcoma of the Falciform Fat in Seven Dogs (2007-2015).

Canine hemangiosarcoma (HSA), a malignant neoplasm of vascular endothelial or bone marrow progenitor cell origin, most often affects the spleen, heart, and liver and typically has an aggressive biologic behavior. Canine HSA arising from the falciform fat/ligament represents a rare anatomic variant, with only two reports in the veterinary literature. In this study, we describe the clinical presentation, treatment, and outcome of seven dogs with primary HSA of the falciform ligament. Histologic grade and mitotic score were not significantly associated with outcome. All dogs had the primary tumor surgically excised except for one diagnosed at necropsy. Median overall survival for all dogs diagnosed prior to necropsy was 339 days, and the 1 yr survival rate was 50%. Four dogs were treated with adjuvant chemotherapy and had a significantly longer median overall survival (394 versus 83 days) than those that did not (P = .018). Dogs with HSA of the falciform ligament may have improved 1 yr survival rates and longer median survival time compared with dogs with HSA in more common visceral locations.

Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma from across the United States.

Hemangiosarcoma (HSA), a locally invasive and highly metastatic endothelial cell neoplasm, accounts for two-thirds of all cardiac and splenic neoplasms in dogs. Bartonella spp. infection has been reported in association with neoplastic and non-neoplastic vasoproliferative lesions in animals and humans. The objective of this study was to determine the prevalence of Bartonella spp. in conjunction with two other hemotropic pathogens, Babesia spp. and hemotropic Mycoplasma spp., in tissues and blood samples from 110 dogs with histopathologically diagnosed HSA from throughout the United States. This was a retrospective, observational study using clinical specimens from 110 dogs with HSA banked by the biospecimen repository of the Canine Comparative Oncology and Genomics Consortium. Samples provided for this study from each dog included: fresh frozen HSA tumor tissue (available from n = 100 of the 110 dogs), fresh frozen non-tumor tissue (n = 104), and whole blood and serum samples (n = 108 and 107 respectively). Blood and tissues were tested by qPCR for Bartonella, hemotropic Mycoplasma, and Babesia spp. DNA; serum was tested for Bartonella spp. antibodies. Bartonella spp. DNA was amplified and sequenced from 73% of dogs with HSA (80/110). In contrast, hemotropic Mycoplasma spp. DNA was amplified from a significantly smaller proportion (5%, p<0.0001) and Babesia spp. DNA was not amplified from any dog. Of the 100 HSA tumor samples submitted, 34% were Bartonella PCR positive (32% of splenic tumors, 57% of cardiac tumors, and 17% of other tumor locations). Of 104 non-tumor tissues, 63% were Bartonella PCR positive (56% of spleen samples, 93% of cardiac samples, and 63% of skin/subcutaneous samples). Of dogs with Bartonella positive HSA tumor, 76% were also positive in non-tumor tissue. Bartonella spp. DNA was not PCR amplified from whole blood. This study documented a high prevalence of Bartonella spp. DNA in dogs with HSA from geographically diverse regions of the United States. While 73% of all tissue samples from these dogs were PCR positive for Bartonella DNA, none of the blood samples were, indicating that whole blood samples do not reflect tissue presence of this pathogen. Future studies are needed to further investigate the role of Bartonella spp. in the development of HSA.

Clinical features and treatment of ten patients with primary hepatic angiosarcoma

Objective To investigate the clinical characteristics, treatment and curative effect of primary hepatic angiosarcoma. Methods A retrospective study was conducted on 10 patients with primary hepatic angiosarcoma who were treated in the Department of Hepatobiliary Surgery, the First Affiliated Hospital of Zhengzhou University from April 2012 to July 2018. There were 6 males and 4 females, with an average age of 55.1 (range 39.0 to 71.0) years. The clinical features, imaging findings, treatment methods and survival time of the patients were analyzed. Results The clinical manifestations of 10 patients with primary hepatic angiosarcoma were non-specific and all were confirmed by pathology. Three cases had diffuse liver echo changes on ultrasound, and 7 cases had hypoechoic lesions with blurred borders. Space occupying lesions were found in 10 cases by CT. A total of five patients underwent partial hepatectomy, 1 case received chemotherapy, 1 case received transcatheter arterial chemoembolization (TACE), 2 case received molecular targeted therapy, and 1 case was untreated. From diagnosis to the last follow-up, the survival time of the 5 patients receiving surgical resection were 9 months, 15 months, 19 months, 21 months and 26 months. The survival time of the patient receiving chemotherapy was 7 months. The survival time of the patient receiving TACE was 3 months. Two patients were targeted for treatment, one survived for 32 months, another had been targeted for 15 months, and treatment was continuing. One patient survived for 1 month without treatment. Conclusions Primary hepatic angiosarcoma has a high degree of malignancy and a poor prognosis. No specific clinical manifestations and imaging findings, confirmed by pathological examination. Patients with partial hepatectomy have a better survival. Targeted therapy may be the most effective treatment for primary hepatic angiosarcoma in the future. Key words: Hemangiosarcoma; Liver; Diagnosis; Treatment outcome

Tenovin-6 induces the SIRT-independent cell growth suppression and blocks autophagy flux in canine hemangiosarcoma cell lines

Canine hemangiosarcoma (HSA) is a commonly occurring aggressive tumor stemming from the vascular endothelial cells and is considered to be a good model for a similar disease in humans, called angiosarcoma. In this study, we reviewed drug libraries to identify new signal transduction inhibitors that can suppress the cell growth of canine HSA in vitro. We observed that tenovin-6, a sirtuin (SIRT) inhibitor, inhibited cell proliferation and induced cell death in three canine HSA cell lines (JuB4, Re12, and Ud6). These effects were induced through G1 cell cycle arrest and caspase-3 activation. Although tenovin-6 is known as an inhibitor of SIRT1 and SIRT2, knockout (KO) of genes encoding SIRT1 and/or SIRT2 had no apparent impact on cell proliferation in canine HSA. In addition, tenovin-6 showed cell growth inhibition in SIRT KO cells, as well as parental cells. These results indicated the cytotoxicity of tenovin-6 was a SIRT-independent event. Instead, we found that tenovin-6 inhibited autophagy flux in canine HSA cells, as evidenced by the suppression of lysosomal proteolysis. These results suggested that tenovin-6 induces cell growth suppression in canine HSA cells by impairing the lysosomal function. Therefore, tenovin-6 could be used in a new therapeutic strategy to treat canine HSA.

Abstract C098: Preclinical evaluation of eribulin in a veterinary clinical trial of metastatic spontaneous hemangiosarcoma in pet dogs

Abstract Eribulin is a microtubule dynamics inhibitor that is currently in clinical use for treatment of certain patients with advanced breast cancer or liposarcoma. FDA approval for both indications was based on extension of overall survival. Based on eribulin’s known vascular remodeling effects, its activity against at least one sarcoma type, and literature reports of taxane activity in angiosarcoma, we hypothesized that eribulin may show activity against angiosarcoma. Due to a paucity of preclinical cell-based, xenograft, and PDX models for human angiosarcoma, we sought other alternatives for preclinical testing in this disease. While rare in humans, angiosarcoma is common in some breeds of domestic dogs, where it is known in veterinary parlance as hemangiosarcoma (HSA). Accordingly, we undertook a small veterinary clinical trial to evaluate eribulin against spontaneous HSA in pet dogs. Canine HSA most frequently occurs in the spleen, so splenectomy is a common first-line treatment; however, non-splenic sites also occur and these are often unresectable. In keeping with eribulin’s clinical use in the metastatic setting, we selected metastatic canine HSA, which we defined as dogs with histologically confirmed HSA with either non-resectable or post-splenectomy residual disease confirmed by imaging (PET/CT, CT). Of 19 pets initially screened, 12 dogs were imaged; 6 of these had imageable disease and thus began eribulin treatment. Eribulin was administered using the human clinical schedule (d1,8Q21), with the first cycle at 0.3 mg/m2 followed by intra-patient dose escalation to 0.6 mg/m2 after determination of tolerability. Of the 6 dogs entering treatment, 5 completed cycle 1, 3 completed cycle 2, and 2 completed the full per-protocol 3 cycles of treatment. These 2 dogs survived 121 and 179 days after initial diagnosis, contrasting with the 0.9 month (~27 days) median survival time reported by Wendelburg et al. (JAVMA, 2015) for 52 post-splenectomy canine HSA patients with residual metastases. Although limited in scope, our results with spontaneous metastatic canine HSA in pet dogs provides a preclinical foundation for further exploration of eribulin in malignancies of vascular origin in both the veterinary and human settings. Citation Format: Christina Kelly Lopes, Gerald Post, Lindsay Lambert, Benjamin Lewis, Thaddeus David Allen, Angel Patel, Bruce A Littlefield. Preclinical evaluation of eribulin in a veterinary clinical trial of metastatic spontaneous hemangiosarcoma in pet dogs [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C098. doi:10.1158/1535-7163.TARG-19-C098

Contrast-enhanced computed tomography findings of canine primary renal tumors including renal cell carcinoma, lymphoma, and hemangiosarcoma.

In veterinary medicine, abdominal ultrasonography is used to rank the differential diagnosis of renal lesions. However, a conventional sonographic examination may show nonspecific findings. The purpose of this study was to assess the computed tomography (CT) findings of canine renal tumors, including renal cell carcinoma (RCC), lymphoma, and hemangiosarcoma (HSA). In this retrospective study, the following CT parameters were recorded for each dog: 1) extent of renal involvement of tumors, 2) enhancement pattern, 3) number of renal tumors, 4) renal tumor vessel enhancement in the corticomedullary phase, 5) presence of lymphadenopathy and lung metastasis, and 6) attenuation values of the renal tumors on the pre- and post-contrast corticomedullary, nephrographic, and excretory phase images. Fifteen dogs met the inclusion criteria, of which nine had RCCs, four had lymphomas, and two had HSAs. RCCs tended to show heterogeneous enhancement and unilateral renal involvement, and vessel enhancement was detected in the corticomedullary phase in dogs with RCC. Conversely, renal lymphomas showed homogeneous enhancement, bilateral renal involvement, and multiple masses; in these dogs, no vessel enhancement was detected in the corticomedullary phase, and the incidence of lymphadenopathy was low. However, in dogs with lymphadenopathy, the renal lymphoma was associated with regionally severe lymphadenopathy. Finally, renal HSAs tended to show heterogeneous enhancement with a non-enhanced area and unilateral renal involvement; in these dogs, vessel enhancement was detected in the nephrographic phase, with the enhancement expanding around the vessel. These findings had no significant differences. Further studies with a larger sample size are required to examine the association between CT and histopathological findings.

High drug efflux pump capacity and low DNA damage response induce doxorubicin resistance in canine hemangiosarcoma cell lines

Canine hemangiosarcoma (HSA) is an aggressive malignant endothelial tumor in dogs and characterized by poor prognosis because of its high invasiveness, high metastatic potential, and poor responsiveness to anti-cancer drugs. Although doxorubicin-based chemotherapy is regularly conducted after surgical treatment, its effects on survival rates are limited. Acquisition of drug resistance is one of the causes of this problem, but the underlying mechanisms remain unclear. In the present study, we aimed to identify the drug-resistance mechanism in canine HSA by establishing doxorubicin-resistant (DR) HSA cell lines. HSA cell lines were exposed to doxorubicin at gradually increasing concentrations. When the cells were able to grow in the presence of a 16-fold higher doxorubicin concentration compared with the initial culture, they were designated DR-HSA cell lines. Characterization of these DR-HSA cell lines revealed higher drug efflux pump capacity compared with the parental cell lines. Furthermore, the DR-HSA cell lines did not show activation of the DNA damage response despite carrying high DNA damage burdens, meaning that apoptosis was not strongly induced. In conclusion, canine HSA cell lines acquired doxorubicin resistance by increasing their drug efflux pump capacity and decreasing the DNA damage response. This study provides useful findings to promote further research on the drug-resistance mechanisms in canine HSA.

Abstract 3421: Comparison of mutations in different subtypes of sarcomas and its clinical implications

Abstract Background: Sarcoma is a malignant cancer that arises from transformed cells of mesenchymal origin. Sarcoma has the characteristics of low incidence and wide distribution of onset age. Sarcoma patients have poor prognosis and very limited selection of approved targeted drugs. Till now, the genomic features of sarcoma patients in China have not been well understood. Here we present the comprehensive genomic features of 202 sarcoma patients and compared mutations in different subtypes of sarcomas. Methods: Deep sequencing targeting 450 cancer genes was performed on FFPE and matched blood samples collected from 202 sarcoma patients. Based on the histology patients included 33 osteosarcoma (OS), 24 rhabdomyosarcoma (RMS), 20 liposarcoma (LPS), 19 fibrosarcoma (FBS), 16 leiomyosarcoma (LMS), 14 Ewing’s sarcoma (ES), 11 hemangiosarcoma (HMS), 10 synovial sarcoma (SS), 9 chondrosarcoma (CDS), and 46 others in this study. Genomic alterations (GAs) including single nucleotide variants (SNVs), insertions and deletions, copy number variations (CNV) and fusions were assessed. Results: Based on the deep sequencing results, there are 1219 somatic mutations detected in all 202 sarcomas, average 6.0 mutations per sample. The most common GAs of LPS, ES and SS’s are consistent with those mentioned in NCCN guidelines (MDM2 55%, EWSR1 78.6% and SS18 80%, respectively). OS, RMS and LMS shared the same most common GAs of TP53 (25%-50%). The second ranked common GAs were NCOR1 (24%), FRS2 (20.8%), ATRX (25%) genes, and the third ranked GAs were RB1 (24%), CDK4 (16.7%) and MAP2K4 (25%) genes. 161/202 patients (79.7%) harbored at least one actionable mutations which might benefit from FDA approved drugs or drugs were under investigation in clinical trials. All FBS, 93.8% LMS and 90.9% HMS have actionable mutations, however, only 10% SS and 35.7% ES have actionable mutations. 8 samples have CNV (including MDM2/4, EGFR and CCND1) which was related to hyper-progression of immunotherapy. All these 8 samples were bone tumor including 4 OS, 1 CDS, 2 ES and 1 fibrosarcoma of bone. Conclusions: Through the comprehensive genomic profiling, different types of sarcomas showed different characteristics of genomic alterations, and potential therapeutic targets may be identified for the patients. Further, NGS provides information on whether or not patients may have the potential hyper-progression of immunotherapy. Citation Format: Xinghua Song, Zuping Lian, YanBin Xiao, Kunpeng Bu, Ye Qiu, Tao Ji, Dan Liu, Angen Liu, Kai Wang. Comparison of mutations in different subtypes of sarcomas and its clinical implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3421.

Simultaneous Occurrence of Hemangiosarcoma and Grade II Mast Cell Tumor in a Pit Bull Terrier

Background: The incidence of cutaneous neoplasms in dogs is high and quite variable. Hemangiosarcoma (HSA) and mast cell tumor (MCT) are commonly diagnosed neoplasms in isolation; however, reports of concomitant occurrence in a single patient are rare. HSA is a malignant mesenchymal neoplasm of endothelial origin; the spleen is the most commonly affected organ. MCT is a common neoplasm that may affect any region of the body, without predilection for sex, although some breeds have shown higher incidence. This report describes a case of HSA and MCT in a Pit bull terrier.Case: A 5-year-old white male Pit bull was presented to the Small Animal Clinic of the Federal Rural University of Pernambuco with nodules in preputial region measuring 5,4 x 3,7 cm and an ulcerated nodule in the right lateral thoracic region measuring 23,0 x 19,0 x 5,5 cm. The owner reported surgical excision of two previous nodules one year before the consultation, but neither were submitted for cytopathological or post-surgical histopathological examination. Two months after the procedure, the nodules recurred. Cytopathological examinations of preputial and lateral thoracic nodules were performed, with a suggestive diagnosis for HSA and MCT, respectively. Due to the unfavorable prognosis and the weakness of the animal, euthanasia was elected. Necroscopic examination revealed an ideal body condition score (4/9), hypertrophy of right pre-scapular and axillary lymph nodes, red hepatization in the apical lobe of the right lung, with multiple, soft and pigmented nodules in the spleen, liver, pancreas and testis. Several tissue samples were collected, conditioned in 10% buffered formaldehyde solution, routinely processed for histology, and stained with hematoxylin-eosin and toluidine blue. Microscopically, the lateral thoracic nodule consisted of round cells in cordonal arrangement, with sparse basophilic and discretely granular cytoplasm, and a round, central nucleus with a single evident nucleolus. Some cells were positively marked by toluidine blue, and the neoplasm was classified as grade II MCT. The preputial and digital nodules were confirmed as well-differentiated HSA, with fusiform cells forming vascular structures containing large numbers of erythrocytes and irregular fibrovascular stroma. Metastases of HSA were observed in the lung, liver, spleen, pancreas, and testis. Invasion of mast cells was observed in lymph node.Discussion: Although the etiology of HSA has not yet been clarified, its occurrence in dogs with poorly pigmented skin exposed to ultraviolet light is well established, consistent with observations in this case. MCTs often occur on the pelvic and thoracic limbs, abdomen, and chest. Primary site of HSA may be in any tissue, but it is commonly seen in highly vascular organs such as the spleen. When it occurs on the skin, HSA has more frequently been reported in the preputial and scrotal regions in dogs. For both neoplasms, the locations in this Pit bull corresponded to those commonly reported. MCTs with ulceration, edema, pruritus, and flushing are associated with worse prognosis. Metastasis is a major concern when evaluating oncologic patients. While it is difficult to determine the primary site of HSAs, metastasis often occurs in the lungs and liver. In this case, metastatic lesions were identified in the lungs, liver, spleen, pancreas, and testis by necropsy and histopathology. Fine needle aspiration of neoplasm-suggestive lesions should be performed prior to surgical procedures, especially because of its high sensitivity for MCTs. MCT and HSA are tumors of great significance, due to their high occurrence and variable prognosis. Multidisciplinary diagnostics involving clinical and pathological aspects are essential to guide the prognosis and treatment of these tumors.Mariana Lumack do Monte Barretto, Rômulo Freitas Francelino Dias, Saulo Romero Felix Gonçalves, Órion Pedro da Silva, Roseane Oliveira Feitosa, Stephanie Caroline Gueiros Silva, Valdemiro Amaro da Silva Junior &amp; Andrea Alice da Fonseca Oliveira

Development and validation of a hemangiosarcoma likelihood prediction model in dogs presenting with spontaneous hemoabdomen: The HeLP score.

To calculate a risk prediction model for hemangiosarcoma (HSA) diagnosis in dogs presenting with nontraumatic hemoabdomen. Retrospective multicenter observational cohort study enrolling dogs presented 2003-2016. Five academic veterinary medical centers. A total of 406 dogs with nontraumatic hemoabdomen as the presenting complaint that underwent surgical exploration or necropsy and received a histological diagnosis. Overall, 219 dogs from 3 centers provided the data for model construction, and 187 dogs from 2 centers provided the population for external validation. None. The risk score was modeled on 4 predictors: bodyweight (P = 0.01), total plasma protein (P < 0.01), platelet count (P < 0.01), and thoracic radiograph findings (P = 0.02). The incidence of HSA diagnosis was 36%, 76%, and 96% in the low risk (≤40), medium risk (41-55), and high risk (>55) score groups, respectively. The risk score AUROC was 0.85 (95% CI 0.79-0.90) on the construction population, and 0.77 (95% CI 0.70-0.84) on the validation population. The risk of HSA diagnosis in dogs presenting with nontraumatic hemoabdomen could be predicted using a simple risk score, which could aid in identification and treatment of dogs at lower risk for this diagnosis.