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Supplementary Methods

Table S1. Canine cohort metadata Table S2. Canine library preparation, amplification, and complexity Table S3. COSMIC Cancer Gene Census genes Table S4. Summary of canine RNA-seq data Table S5. RNA-seq validation Table S6. RNA-seq survey Table S7. Enrichment of protein domains in hemangiosarcoma and angiosarcoma Table S8. Canine somatic copy number aberrations by oaCGH Table S9. Comparison of top SCNAs in human Angiosarcoma Project data with canine oaCGH data Figure S1 - Canine exome sequencing workflow Figure S2 - Canine SMGs called in normal vs. overamplified libraries Figure S3 - Mutational landscape of entire canine cohort Figure S4 - Mutational landscape: FFPE vs Frozen Figure S5 - Mutational landscape: Canine Tumor Location Figure S6 - FFPE vs Frozen exome SCNA data clustering

Supplemental Table 7

Table S1. Demographic and pathological features of human patients with angiosarcoma Table S2. Demographic and pathological features of dogs with HSA Table S3. Primary antibodies for immunohistochemical evaluation Table S4. Gene ontology and biological functions of fusion partner genes in human angiosarcomas Table S5. Gene ontology and biological functions of fusion partner genes in canine HSAs Table S6. Detection of fusion genes in canine HSAs by RT-PCR Table S7. Biological functions associated with 490 upregulated genes in human angiosarcomas Table S8. Association of frequency between fusion genes, TP53 and PIK3CA mutations Table S9. Association of fusion genes with dog signalment and tumor histology Table S10. Association of frequency between fusion genes, TP53 and PIK3CA mutations Table S11. 41 differentially expressed genes in PF (n = 4) compared to P (n = 9) group Table S12. 212 differentially expressed genes in PF (n = 4) compared to PK (n = 6) group Table S13. 377 differentially expressed genes in PF (n = 4) compared to PFK (n = 4) group Table S14. Immunohistochemical features of human angiosarcomas Table S15. Immunohistochemical features of canine HSAs

Angiosarcoma is a highly aggressive cancer of blood vessel–forming cells with few effective treatment options and high patient mortality. It is both rare and heterogenous, making large, well-powered genomic studies nearly impossible. Dogs commonly suffer from a similar cancer, called hemangiosarcoma, with breeds like the golden retriever carrying heritable genetic factors that put them at high risk. If the clinical similarity of canine hemangiosarcoma and human angiosarcoma reflects shared genomic etiology, dogs could be a critically needed model for advancing angiosarcoma research. We assessed the genomic landscape of canine hemangiosarcoma via whole-exome sequencing (47 golden retriever hemangiosarcomas) and RNA sequencing (74 hemangiosarcomas from multiple breeds). Somatic coding mutations occurred most frequently in the tumor suppressor TP53 (59.6% of cases) as well as two genes in the PI3K pathway: the oncogene PIK3CA (29.8%) and its regulatory subunit PIK3R1 (8.5%). The predominant mutational signature was the age-associated deamination of cytosine to thymine. As reported in human angiosarcoma, CDKN2A/B was recurrently deleted and VEGFA, KDR, and KIT recurrently gained. We compared the canine data to human data recently released by The Angiosarcoma Project, and found many of the same genes and pathways significantly enriched for somatic mutations, particularly in breast and visceral angiosarcomas. Canine hemangiosarcoma closely models the genomic landscape of human angiosarcoma of the breast and viscera, and is a powerful tool for investigating the pathogenesis of this devastating disease.Implications:We characterize the genomic landscape of canine hemangiosarcoma and demonstrate its similarity to human angiosarcoma.

Sporadic angiosarcomas are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic angiosarcomas. In this study, we leveraged RNA-sequencing data from 13 human angiosarcomas and 76 spontaneous canine hemangiosarcomas to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in seven of the 13 human tumors, with two tumors showing mutations of TP53. HRAS and NRAS mutations were found in angiosarcomas without fusions or TP53 mutations. We found 15 novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 in 11 of the 76 canine hemangiosarcomas; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS. In particular, fusion genes and mutations of TP53 cooccurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human angiosarcomas and canine hemangiosarcomas identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data suggest that genome 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 in this vascular malignancy.Implications:This study shows that, while drive events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphologic organization and biological behavior of these tumors in both species.

S1. Representative putative inter-chromosomal fusion transcripts in human AS and canine HSA. SCLT1-NIPBL fusion in human angiosarcoma. S2. Fusion genes and their association with tumor content and sequencing depth in canine HSA tissues. S3. A, Primers designed for amplification of SCLT1-NIPBL fusion transcripts identified in human angiosarcoma. B, SCLT1-NIPBL fusion gene amplification by quantitative RT-PCR. Expression value represents 2(-â^†Ct)(Fusion gene/GAPDH). C, Primers designed for amplification of four fusion transcripts identified in canine hemangiosarcomas. D, Fusion gene amplification was confirmed in canine tumors by quantitative RT-PCR. E, Electrophoresis (1% agarose gel) of DNA product by RTPCR was done. S4. Genetic alterations of fusion partner genes identified in human AS. S5. Cell type enrichment analysis in ASs and TCGA sarcomas. S6. Three distinct molecular groups of canine HSAs. S7. Kaplan-Meier probability of survival for dogs with angiogenic and inflammatory HSA. S8. Association of the fusion events with demographic and histopathologic features in canine HSA. S9. Gene pathways associated with commonly enriched genes in tumors with fusion genes and TP53 mutations in canine HSA. S10. Violin plots display IHC scores of p53, p-p53 (Ser15), and p-p53 (Ser20) in human AS and canine HSA. S11. Violin plots present IHC scores of AKT and p-AKT (Thr308) in human AS and canine HSA. S12. IHC scores of mTOR and p-mTOR (Ser2448) in human AS.

Although cancer is widely regarded as a major contributor to canine morbidity and mortality, its frequency in companion dogs has only infrequently been characterised. We analysed cross-sectional data from the baseline survey of owners of 27 541 living companion dogs enrolled in the Dog Aging Project as of 31 December 2020 to estimate the lifetime prevalence of malignant and benign tumours and several potentially-associated characteristics. Survey questions elicited information on history of ‘cancer or tumors’ including organ site and histologic type. Owners reported 819 malignant tumours (56% sited in the skin, muscle or other soft tissue) and 404 benign tumours (69% sited in the skin, muscle or other soft tissue). The lifetime prevalence of malignant tumours (29.7/1000 dogs) was approximately double the lifetime prevalence of benign tumours (14.7/1000 dogs). Lifetime prevalence of both malignant and benign tumours increased with dog age at survey completion. There were no statistically discernable differences in age-adjusted lifetime prevalence of malignant (prevalence ratio (PR) = 0.93 [95% confidence interval (CI) 0.82, 1.07] or benign tumours (PR = 1.10, 95% CI 0.91, 1.34) in mixed vs. purebred dogs. The lifetime prevalence of malignant tumours increased with increasing dog size class; compared to toy and small dogs, the age-adjusted PRs (95% CIs) for medium, standard, large, and giant dogs were 1.65 (1.28, 2.11), 2.92 (2.35, 3.64), 3.67 (2.92, 4.62) and 2.99 (1.23, 4.02), respectively. Similar though less pronounced patterns in relation to dog size class were observed for benign tumours. Ongoing prospective data collection will permit future studies on risk factors for canine tumour incidence.

BackgroundCanine hemangiosarcoma (HSA) is an aggressive cancer of endothelial cells associated with short survival times. Understanding the genomic landscape of HSA is critical to developing more effective therapeutic strategies.ObjectivesTo determine the relationships between genomic and clinical features including treatment and outcome in canine splenic HSA.Animals109 dogs with primary splenic HSA treated by splenectomy that had tumor sequencing via the FidoCure® Precision Medicine Platform targeted sequencing panel.MethodsPatient signalment, weight, metastasis at diagnosis, treatment, and survival time were retrospectively evaluated. The incidence of genomic alterations in individual genes and their relationship to patient variables and outcome were assessed.ResultsSomatic mutations inTP53(n = 45),NRAS(n = 20), andPIK3CA(n = 19) were most common. Survival was associated with metastases at diagnosis, germline variants inSETD2andNOTCH1, and nominally with breed. Age at diagnosis was associated withNRASmutations and breed.TP53andPIK3CAmutations were found in larger dogs, germlineSETD2variants in smaller dogs. Doxorubicin (DOX) treatment did not significantly improve survival time, while targeted therapies had a significant early survival benefit.Conclusions and clinical importanceDOX treatment may provide limited clinical benefit for dogs with splenic HSA, while targeted therapy may provide early survival benefit. Genetic signatures associated with splenic HSA may be useful in guiding targeted therapy to improve outcomes. Germline variants, age, size, and breed may be useful prognostic factors and provide insight into the genomic landscape of the tumor.

Sporadic angiosarcomas are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic angiosarcomas. In this study, we leveraged RNA-sequencing data from 13 human angiosarcomas and 76 spontaneous canine hemangiosarcomas to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in seven of the 13 human tumors, with two tumors showing mutations of TP53. HRAS and NRAS mutations were found in angiosarcomas without fusions or TP53 mutations. We found 15 novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 in 11 of the 76 canine hemangiosarcomas; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS. In particular, fusion genes and mutations of TP53 cooccurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human angiosarcomas and canine hemangiosarcomas identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data suggest that genome 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 in this vascular malignancy. Implications: This study shows that, while drive events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphologic organization and biological behavior of these tumors in both species.

INTRODUCTION: Before the 1800s, dogs were probably primarily selected for functional roles such as hunting, guarding, and herding. Modern dog breeds are a recent invention defined by conformation to a physical ideal and purity of lineage. Breeds are commonly ascribed temperaments and behavioral proclivities based on the purported function of the ancestral source population. By extension, the breed ancestry of individual dogs is assumed to be predictive of temperament and behavior. Through our community science project Darwin’s Ark (darwinsark.org), we enrolled a diverse cohort of pet dogs to explore how genetics shapes complex behavioral traits in this exceptional natural model. RATIONALE: Dogs are a natural system for investigating the genetics of complex traits. Millions of pet dogs live in human homes, sharing our environment, and receive sophisticated medical care. Behavioral disorders are treated with human psychiatric drugs, achieving similar response rates, and genetic studies suggest shared etiology with some human psychiatric conditions. We developed Darwin’s Ark as an open data resource for collecting owner-reported phenotypes and genetic data and invited any dog owner to enroll their dog. We paired this with low-pass sequencing to capture nearly all common variation in this outbred population. Our inclusive approach achieved the large samples needed to investigate complex traits. RESULTS: We surveyed owners of 18,385 dogs (49% purebred) and sequenced the DNA of 2155 dogs. Most behavioral traits are heritable [heritability (h(2)) > 25%], but behavior only subtly differentiates breeds. Breed offers little predictive value for individuals, explaining just 9% of variation in behavior. For more heritable, more breed-differentiated traits, like biddability (responsiveness to direction and commands), knowing breed ancestry can make behavioral predictions somewhat more accurate (see the figure). For less heritable, less breed-differentiated traits, like agonistic threshold (how easily a dog is provoked by frightening or uncomfortable stimuli), breed is almost uninformative. We used dogs of mixed breed ancestry to test the genetic effect of breed ancestry on behavior and compared that to survey responses from purebred dog owners. For some traits, like biddability and border collie ancestry, we confirm a genetic effect of breed that aligns with survey responses. For others, like human sociability and Labrador retriever ancestry, we found no significant effect. Through genome-wide association, we found 11 regions that are significantly associated with behavior, including howling frequency and human sociability, and 136 suggestive regions. Regions associated with aesthetic traits are unusually differentiated in breeds, consistent with a history of selection, but those associated with behavior are not. CONCLUSION: In our ancestrally diverse cohort, we show that behavioral characteristics ascribed to modern breeds are polygenic, environmentally influenced, and found, at varying prevalence, in all breeds. We propose that behaviors perceived as characteristic of modern breeds derive from thousands of years of polygenic adaptation that predates breed formation, with modern breeds distinguished primarily by aesthetic traits. By embracing the full diversity of dogs—including purebred dogs, mixed-breed dogs, purpose-bred working dogs, and village dogs—we can fully realize dogs’ long-recognized potential as a natural model for genetic discovery.

Dogs represent a unique spontaneous cancer model. Osteosarcoma (OSA) is the most common primary bone tumor in dogs (OMIA 001441-9615), and strongly resembles human forms of OSA. Several large- to giant-sized dog breeds, including the Leonberger, have a greatly increased risk of developing OSA. We performed genome-wide association analysis with high-density imputed SNP genotype data from 273 Leonberger cases with a median age of 8.1 [3.1–13.5] years and 365 controls older than eight years. This analysis revealed significant associations at the CDKN2A/B gene locus on canine chromosome 11, mirroring previous findings in other dog breeds, such as the greyhound, that also show an elevated risk for OSA. Heritability (h2SNP) was determined to be 20.6% (SE = 0.08; p-value = 5.7 × 10−4) based on a breed prevalence of 20%. The 2563 SNPs across the genome accounted for nearly all the h2SNP of OSA, with 2183 SNPs of small effect, 316 SNPs of moderate effect, and 64 SNPs of large effect. As with many other cancers it is likely that regulatory, non-coding variants underlie the increased risk for cancer development. Our findings confirm a complex genetic basis of OSA, moderate heritability, and the crucial role of the CDKN2A/B locus leading to strong cancer predisposition in dogs. It will ultimately be interesting to study and compare the known genetic loci associated with canine OSA in human OSA.

Naturally occurring canine cancers have remarkable similarities to their human counterparts. In order to determine whether these similarities occur at the molecular level, we investigated hotspot mutations in a variety of spontaneously arising canine cancers and found high concordance in oncogenic drivers between cancers in both species. These findings suggest that canines may present a powerful and complementary model for preclinical investigations for targeted cancer therapeutics. Through analysis of 708 client-owned dogs from 96 breeds (plus mixed breeds) with 23 common tumor types, we discovered mutations in 50 well-established oncogenes and tumor suppressors, and compared them to those reported in human cancers. TP53 is the most commonly mutated gene, detected in 30.81% of canine tumors overall and >40% in hemangiosarcoma and osteosarcoma. Canine tumors share mutational hotspots with human tumors in oncogenes including PIK3CA, KRAS, NRAS, BRAF, KIT and EGFR. Hotspot mutations with significant (P

Despite a considerable expenditure of time and resources and significant advances in experimental models of disease, cancer research continues to suffer from extremely low success rates in translating preclinical discoveries into clinical practice. The continued failure of cancer drug development, particularly late in the course of human testing, not only impacts patient outcomes, but also drives up the cost for those therapies that do succeed. It is clear that a paradigm shift is necessary if improvements in this process are to occur. One promising direction for increasing translational success is comparative oncology—the study of cancer across species, often involving veterinary patients that develop naturally-occurring cancers. Comparative oncology leverages the power of cross-species analyses to understand the fundamental drivers of cancer protective mechanisms, as well as factors contributing to cancer initiation and progression. Clinical trials in veterinary patients with cancer provide an opportunity to evaluate novel therapeutics in a setting that recapitulates many of the key features of human cancers, including genomic aberrations that underly tumor development, response and resistance to treatment, and the presence of comorbidities that can affect outcomes. With a concerted effort from basic scientists, human physicians and veterinarians, comparative oncology has the potential to enhance the cost-effectiveness and efficiency of pipelines for cancer drug discovery and other cancer treatments.

Cancer progression is an evolutionary process. During this process, evolving cancer cell populations encounter restrictive ecological niches within the body, such as the primary tumor, circulatory system, and diverse metastatic sites. Efforts to prevent or delay cancer evolution—and progression—require a deep understanding of the underlying molecular evolutionary processes. Herein we discuss a suite of concepts and tools from evolutionary and ecological theory that can inform cancer biology in new and meaningful ways. We also highlight current challenges to applying these concepts, and propose ways in which incorporating these concepts could identify new therapeutic modes and vulnerabilities in cancer.

Altres ajuts: A comprehensive canine genetics resource including gene and variation annotation (NIH /OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH) NIH/OD/5R24OD018250 ; NCI/5R37CA218570 ; NIMH/R21MH109938 ; NHGRI/1R01HG008742 Dogs are an unparalleled natural model for investigating the genetics of health and disease, particularly for complex diseases like cancer. Comprehensive genomic annotation of regulatory elements active in healthy canine tissues is crucial both for identifying candidate causal variants and for designing functional studies needed to translate genetic associations into disease insight. Currently, canine geneticists rely primarily on annotations of the human or mouse genome that have been remapped to dog, an approach that misses dog-specific features. Here, we describe BarkBase, a canine epigenomic resource available at barkbase.org. BarkBase hosts data for 27 adult tissue types, with biological replicates, and for one sample of up to five tissues sampled at each of four carefully staged embryonic time points. RNA sequencing is complemented with whole genome sequencing and with assay for transposase-accessible chromatin using sequencing (ATAC-seq), which identifies open chromatin regions. By including replicates, we can more confidently discern tissue-specific transcripts and assess differential gene expression between tissues and timepoints. By offering data in easy-to-use file formats, through a visual browser modeled on similar genomic resources for human, BarkBase introduces a powerful new resource to support comparative studies in dogs and humans.

Angiosarcoma is a highly aggressive cancer of blood vessel-forming cells with high fatality and few effective treatment options. It is both rare and heterogenous, making large, well powered genomic studies nearly impossible. In dogs, angiosarcoma is common, with breeds like the golden retriever carrying heritable genetic factors that put them at very high risk. If the clinical similarity of canine and human angiosarcoma reflects shared genomic etiology, dogs could be a critically needed model for advancing angiosarcoma research. We assessed the genomic landscape of canine angiosarcoma via whole exome sequencing (47 golden retriever angiosarcomas) and RNA sequencing (74 angiosarcomas from multiple breeds). The predominant mutational signature was the age-associated deamination of cytosine to thymine, and somatic coding mutations occurred most frequently in the tumor suppressorTP53(59.6% of cases) as well as two genes in the PI3K pathway: the oncogenePIK3CA(29.8%) and its regulatory subunitPIK3R1(8.5%). We compared the canine data to human data recently released by The Angiosarcoma Project, and found the same genes and many of the same pathways significantly enriched for somatic mutations, most notably protein kinases, glycoproteins, fibronectin Type III domains, EGF-like domains, and cell adhesion proteins such as cadherins. As in human angiosarcoma,CDKN2A/Bwas recurrently deleted andVEGFA, KDR, and KITrecurrently gained. Canine angiosarcoma closely models human angiosarcoma on a genomic level, and is a powerful tool for investigating the pathogenesis of this devastating disease.

Angiosarcoma is a highly aggressive cancer of blood vessel-forming cells with few effective treatment options and high patient mortality. It is both rare and heterogenous, making large, well powered genomic studies nearly impossible. Dogs commonly suffer from a similar cancer, called hemangiosarcoma, with breeds like the golden retriever carrying heritable genetic factors that put them at high risk. If the clinical similarity of canine hemangiosarcoma and human angiosarcoma reflects shared genomic etiology, dogs could be a critically needed model for advancing angiosarcoma research. We assessed the genomic landscape of canine hemangiosarcoma via whole exome sequencing (47 golden retriever hemangiosarcomas) and RNA sequencing (74 hemangiosarcomas from multiple breeds). Somatic coding mutations occurred most frequently in the tumor suppressor TP53 (59.6% of cases) as well as two genes in the PI3K pathway: the oncogene PIK3CA (29.8%) and its regulatory subunit PIK3R1 (8.5%). The predominant mutational signature was the age-associated deamination of cytosine to thymine. As reported in human angiosarcoma, CDKN2A/B was recurrently deleted and VEGFA, KDR, and KIT recurrently gained. We compared the canine data to human data recently released by The Angiosarcoma Project, and found many of the same genes and pathways significantly enriched for somatic mutations, particularly in breast and visceral angiosarcomas. Canine hemangiosarcoma closely models the genomic landscape of human angiosarcoma of the breast and viscera, and is a powerful tool for investigating the pathogenesis of this devastating disease.

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.

Pet dogs are a powerful spontaneous model for human cancers. Dogs develop the same cancers that humans do, with striking similarities at the clinical, histopathologic, and genomic levels. These cancers have an accelerated clinical course compared with their human counterparts, and canine patients undergo many of the same interventions and treatments as human patients. The dog model thus offers the opportunity to implement rapid clinical trials informative for human medicine. We are developing liquid biopsy in dogs to further refine its clinical use by addressing questions that are difficult to assess in human patients. We have four ongoing pilot studies examining several different aspects of blood biopsy in canine patients. (1) We characterized the yield and tumor fraction of cell-free DNA (cfDNA) in nine canine cancer histologies using prospectively collected and banked plasma samples. (2) We tested the correlation between large-scale somatic copy number aberrations (SCNAs) in 24 osteosarcoma tumor samples and matched plasma samples and are now preparing to perform whole-exome sequencing (WES) of these samples to assess the concordance of SNVs and INDELs. (3) We investigated the use of cfDNA as a biomarker for response to therapy in four dogs with multicentric lymphoma treated with L-asparaginase via longitudinal blood biopsy. (4) We are optimizing phlebotomy techniques and characterizing short-term cfDNA dynamics by testing the effect of blood draw site and time of day on yield and tumor fraction. Samples from three sites (jugular, saphenous, and cephalic veins) were collected in a cohort of ten patients, and collection of samples at three time points throughout the day in a separate cohort is ongoing. (1) The majority (93%) of plasma samples yielded or were projected to yield sufficient DNA (>2ng) for ultra-low-pass whole-genome sequencing, and 37% had a tumor fraction ≥10%, allowing for WES given sufficient input DNA. (2) We found a strong correlation (Spearman coefficient of 0.74) between SCNAs in 13 tumor/plasma pairs with tumor fraction ≥10%. We are moving forward with WES of these cfDNA samples to examine simple somatic mutations. (3) Longitudinal cfDNA yield and tumor fraction correlated with response to therapy in three of four lymphoma patients. (4) Sample collection and sequencing are under way for the studies on the effect of blood draw site and time of day. Our studies demonstrated the feasibility of blood biopsy in dogs, the high concordance between cfDNA and tumor samples, and suggested that blood biopsy is a promising method for monitoring response to therapy. Emerging results will help to optimize phlebotomy protocols and characterize cfDNA dynamics over short time scales, as well as assessing in more detail the concordance between cfDNA and matched tumor samples. Further development of blood biopsy in dogs has the potential to improve and accelerate clinical application of this technology in human medicine and to facilitate further high-impact comparative translational studies in the dog model. Citation Format: Kate Megquier, Kan Xiong, Heather L. Gardner, Justin Rhoades, Viktor Adalsteinsson, Cheryl A. London, Elinor K. Karlsson. Advancing blood biopsy through the canine comparative model [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 A58.

Canine degenerative myelopathy (DM) is a naturally occurring neurodegenerative disease with similarities to some forms of amyotrophic lateral sclerosis (ALS). Most dogs that develop DM are homozygous for a common superoxide dismutase 1 gene (SOD1) mutation. However, not all dogs homozygous for this mutation develop disease. We performed a genome-wide association analysis in the Pembroke Welsh Corgi (PWC) breed comparing DM-affected and -unaffected dogs homozygous for the SOD1 mutation. The analysis revealed a modifier locus on canine chromosome 25. A haplotype within the SP110 nuclear body protein (SP110) was present in 40% of affected compared with 4% of unaffected dogs (P = 1.5 × 10(-5)), and was associated with increased probability of developing DM (P = 4.8 × 10(-6)) and earlier onset of disease (P = 1.7 × 10(-5)). SP110 is a nuclear body protein involved in the regulation of gene transcription. Our findings suggest that variations in SP110-mediated gene transcription may underlie, at least in part, the variability in risk for developing DM among PWCs that are homozygous for the disease-related SOD1 mutation. Further studies are warranted to clarify the effect of this modifier across dog breeds.

Angiosarcoma is an aggressive, albeit rare cancer in humans. The cause of the vast majority of sporadic angiosarcomas is unknown, mortality is high, and no therapeutic targets have been identified to improve outcomes. Hemangiosarcoma (HSA) is a common cancer of dogs, and it shares histopathologic features with human angiosarcoma. In our previous work, canine HSAs were classified into angiogenic, inflammatory, and adipogenic subtypes based on transcriptional profiles. However, the genetic and molecular events that regulate transcriptional subtypes in angiosarcoma are not currently understood. Our goal was to use a comparative genomics approach to apply knowledge from appropriately powered canine studies to inform our research into human sarcomas. In this study, we identified recurrent mutations in RNASeq data from 93 HSAs and 16 nonmalignant controls, based on mutations first identified in exomes from 42 paired tumor and normal samples. In addition to identifying recurrent somatic mutations we also identified translocation fusions, allowing elucidation of oncogenic mechanisms for vascular endothelial growth factor receptors (VEGFR), phosphoinositide-3 kinase (PIK3) signaling pathways, and the p53 DNA damage repair pathway in canine HSA. Significantly, mutational signatures were associated with distinct molecular subtypes of canine hemangiosarcomas, and both the angiogenic and the inflammatory subtypes were apparent in RNASeq data from human angiosarcomas (n=14), suggesting that comparable etiologic mechanisms are operative in the canine and human disease. Our ongoing work seeks to understand how the molecular mechanisms give rise to molecular subtypes of angiosarcoma by defining the association between driver mutations, signaling pathway alterations and transcriptional patterns, which should allow us to identify rational therapeutic targets. Citation Format: Jong Hyuk Kim, Kate Megquier, Aaron L. Sarver, Rachael Thomas, Chao Wang, Ingegerd Elvers, Elinor Karlsson, Matthew Breen, Kerstin Lindblad-Toh, Jaime F. Modiano. Mutational and transcriptomic profiling identify distinct angiogenic and inflammatory subtypes of angiosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5357.

Dogs, with their breed-determined limited genetic background, are great models of human disease including cancer. Canine B-cell lymphoma and hemangiosarcoma are both malignancies of the hematologic system that are clinically and histologically similar to human B-cell non-Hodgkin lymphoma and angiosarcoma, respectively. Golden retrievers in the US show significantly elevated lifetime risk for both B-cell lymphoma (6%) and hemangiosarcoma (20%). We conducted genome-wide association studies for hemangiosarcoma and B-cell lymphoma, identifying two shared predisposing loci. The two associated loci are located on chromosome 5, and together contribute ~20% of the risk of developing these cancers. Genome-wide p-values for the top SNP of each locus are 4.6×10-7 and 2.7×10-6, respectively. Whole genome resequencing of nine cases and controls followed by genotyping and detailed analysis identified three shared and one B-cell lymphoma specific risk haplotypes within the two loci, but no coding changes were associated with the risk haplotypes. Gene expression analysis of B-cell lymphoma tumors revealed that carrying the risk haplotypes at the first locus is associated with down-regulation of several nearby genes including the proximal gene TRPC6, a transient receptor Ca2+-channel involved in T-cell activation, among other functions. The shared risk haplotype in the second locus overlaps thevesicle transport and release gene STX8. Carrying the shared risk haplotype is associated with gene expression changes of 100 genes enriched for pathways involved in immune cell activation. Thus, the predisposing germ-line mutations in B-cell lymphoma and hemangiosarcoma appear to be regulatory, and affect pathways involved in T-cell mediated immune response in the tumor. This suggests that the interaction between the immune system and malignant cells plays a common role in the tumorigenesis of these relatively different cancers., Golden Retriever Foundation, Morris Animal Foundation (Grant D10CA-501), National Institutes of Health (U.S.) (P30CA077598 (MCC Core)), Land of PureGold Foundation, Inc., Uppsala University, American Kennel Club Canine Health Foundation, Inc. (Grant 422), American Kennel Club Canine Health Foundation, Inc. (Grant 615), American Kennel Club Canine Health Foundation, Inc. (Grant 2254), American Kennel Club Canine Health Foundation, Inc. (Grant 1131), American Kennel Club Canine Health Foundation, Inc. (Grant 593), American Kennel Club Canine Health Foundation, Inc. (Grant 1168), American Kennel Club Canine Health Foundation, Inc. (Grant 1169), National Institutes of Health (U.S.) (RO1CA112211), American Kennel Club Canine Health Foundation, Inc. (Grant 1147), American Kennel Club Canine Health Foundation, Inc. (Starlight Fund), National Institutes of Health (U.S.) (NIH Short-term Training Grant (T32 RR18267)), Swedish Medical Research Council, University of Minnesota, United States. Army Medical Research and Materiel Command (Training Grant (W81XWH-06-1-064)), European Research Council (ERC Young Investigator Award), European Science Foundation (European Young Investigator Award Program)