Clinical, pathologic and molecular findings in 2 Rottweiler littermates with appendicular osteosarcoma

Appendicular osteosarcoma was diagnosed and treated in a pair of littermate Rottweiler dogs, resulting in distinctly different clinical outcomes despite similar therapy within the context of a prospective, randomized clinical trial (NCI-COTC021/022). Histopathology, immunohistochemistry, mRNA sequencing, and targeted DNA hotspot sequencing techniques were applied to both dogs’ tumors to define factors that could underpin their differential response to treatment. We describe the comparison of their clinical, histologic and molecular features, as well as those from a companion cohort of Rottweiler dogs, providing new insight into potential prognostic biomarkers for canine osteosarcoma.


Introduction
Osteosarcoma is the most common form of primary bone cancer found in both humans and canines. 1,2[11] Osteosarcoma is highly aggressive and tends to rapidly metastasize to the lungs, although disease progression to soft tissue, bone, and other internal organs, has been reported as well.7] However, canines diagnosed with osteosarcoma are typically between 7 and 10 years old and fall within the adult or geriatric age group. 112][23][24] While the literature con icts over which dog breeds are most predisposed to the disease, one study found that Rottweilers have a higher osteosarcoma odds ratio than any other breed. 24Rottweilers are also one of three breeds included in a large osteosarcoma Genome Wide Association Study (GWAS), making them an ideal candidate for uncovering genomic biomarkers in dogs with translational value for humans. 3,25In addition, Rottweilers may perform worse than other dog breeds despite receiving the same cancer treatment, though this evidence is observational. 21e Comparative Oncology Program (COP) within the intramural research program of the National Institutes of Health -National Cancer Institute launched the Comparative Oncology Trial Consortium (COTC) in 2003 to utilize the pet dog as a translational model for novel cancer treatments. 26Under the COTC infrastructure and with support of the Morris Animal Foundation, a 2-armed prospective, randomized trial was conducted in canine osteosarcoma patients, COTC021/22.Dogs enrolled in this trial received either Standard of Care (SOC) therapy, or SOC + adjuvant sirolimus (SOC + S) therapy. 27The trial enrolled 324 dogs, 18 of which were Rottweilers.Of this cohort, two Rottweiler dogs, patient ID numbers 1410 (spayed female) and 1411 (castrated male), were littermates and were both diagnosed with osteoblastic osteosarcomas within 3 months of each other.Both dogs, aged 6 at the time of osteosarcoma diagnosis, were raised in the same household and received treatment from the same DNA and RNA were isolated from canine frozen tumor and normal tissue in RNAlater using Qiagen Allprep DNA/RNA Mini Kit (Cat#80204).The total RNA quality and quantity was assessed using Nanodrop 8000 (Thermo sher) and Agilent 4200 Tapestation with RNA Screen Tape (Cat# 5067-5576) and RNA Screen Tape sample Buffer (Cat#5067-5577).All samples forwarded for mRNA sequencing had a RIN > 8 and a total RNA quantity > 100 ng.DNA quantity and quality were assessed using the Qubit Fluorometer 2.0 with the Qubit dsDNA BR assay (ThermoFisher Scienti c) and the TapeStation genomic DNA assay (Agilent Technologies).Samples with a DIN > 3 and with > 50 ng total DNA were utilized for DNA sequencing.

Library preparation and mRNA sequencing
Between 100ng to 1µg of total RNA was used as the input for the mRNA sequencing libraries.Libraries were generated using the TruSeq Stranded mRNA library kit (Illumina) according the to the manufacturers protocol.The libraries were pooled and sequenced on NovaSeq S1 using a 2x150 cycle kit.The HiSeq Real Time Analysis software (RTA v.3.4.4) was used for processing raw data les.The Illumina bcl2fastq2.17 was used to demultiplex and convert binary base calls and qualities to fastq format.The samples had 44 to 61 million pass lter reads with more than 91% of bases above the quality score of Q30.Reads of the samples were trimmed for adapters and low-quality bases using Cutadapt.The trimmed reads were mapped to the CanFam4 reference genome (GSD_1.0from NCBI) 29 using STAR aligner (version 2.7.0f) with two-pass alignment option.RSEM (version 1.3.1)was used for gene and transcript quanti cation based on the CanFam4 GTF le.The average mapping rate of all samples was 83% with unique alignment above 66%.There were 13.13-26.26%unmapped reads.The mapping statistics were calculated using Picard software.The samples had between 0.01-0.76%ribosomal bases.Percent coding bases were between 58-71%.Percent UTR bases were 10-16%, and mRNA bases were between 75-82% for all the samples.Library complexity was measured in terms of unique fragments in the mapped reads using Picard's MarkDuplicate utility.The samples had 48-78% nonduplicate reads.

mRNA sequencing data analysis
The COTC021/022 trials enrolled a total of 324 dogs with appendicular osteosarcoma.The DOG 2 cohort consists of a subset of 186 canine osteosarcoma patients for which mRNAseq data from their treatmentnaïve primary tumors is available.Eleven of the n = 186 dogs were of Rottweiler breed, including dogs 1410 (poor responder) and 1411 (elite responder).Of these 11 Rottweilers, 4 dogs were assigned to a group of "elite" responders (patient IDs 1411, 0608, 0511, 0301) with a median disease-free interval (DFI) of 453 days (range: 210-859 days) and a median overall survival (OS) of 826 days (range: 634-909 days).Six dogs were assigned to a group of "poor" responders (Patient IDs 0402, 0712, 1022, 1103, 1409, 1410) with a median DFI of 87 days (range: 55-126 days) and a median OS of 142 days (range: 75-194   days).One Rottweiler dog, 0518, was not included in this analysis because it was taken off study at 12 days post-operatively as the owner elected not to pursue further therapy.Differential expression analysis from the available mRNAseq datasets described above was performed using the R (version 4.03) package DESeq2 (version 1.30.1). 30Confounding covariates for batch effects and sex were accounted for in the DESeq2 model.A p-value cutoff was determined using Independent Hypothesis Weighting (IHW) with signi cance level of 0.05 using the IHW (version 1.18.0)R package. 31ierarchical clustering was performed with Ward's method using the function "clustermap" function from seaborn (version 0.10.1)Python (version 3.8.3)package using the Log 2 DESeq normalized counts per million (CPM) expression data.
Based on our previous work with the DOG 2 mRNAseq cohort, a 27 gene signature (GS-1) was shown to cluster canine primary tumors into two groups based on their relative expression of immune-related genes. 28Using this signature, the expression pro les were sorted by group (Immune-high, Immune-low, Rottweiler breed) and displayed in a heatmap composed using the seaborn (version 0.10.1)Python (version 3.8.3)package.

DNA sequencing and data analysis
A pan-cancer genomic sequencing panel was applied to DNA extracted from treatment-naïve tumor and matched normal tissue samples collected from both dogs at the time of limb amputation.[34] Data was analyzed using a custom tumor-only genomics pipeline for the identi cation of SNVs, CNVs, and ITDs.The rst step involved using Trimmomatic (v0.36) 35 to remove adapter sequences, low-quality bases, and other artifacts, and to generate FASTQ quality control metrics.Trimmed paired-end reads were then aligned to the canine reference genome, CanFam v3.1.99 36, using BWA-mem (v0.7.17) 37 .Consensus SNV/indel calls from Mutect2 (GATK-4.1.4.0) 38 and Pisces (v5.2.5.20) 39 were determined, and calls occurring at variant allele frequencies ≥ 3% were functionally annotated using SnpEff (v4.3) 40 to determine the effects of the variants on the encoded protein.The Ensembl Variant Effect Predictor (VEP) 41 was then utilized to determine the impacts of amino acid substitutions, incorporating SIFT annotation to assess the potential functional consequences.SIFT scores range from 0 to 1, with a lower score indicating a higher likelihood of being damaging to protein function.Substitutions with SIFT scores < 0.05 were considered high-impact ('HIGH'), while substitutions with scores ≥ 0.05 and < 0.5 were considered moderate impact ('MODERATE').Substitutions with SIFT scores ≥ 0.5 were considered tolerated and marked as 'BENIGN'.
Variants with a predicted impact of "HIGH" or "MODERATE" were subjected to additional ltering to exclude likely germline variants based on their presence in the European Variant Archive (EVA) 42 with a population allele frequency (AF) of ≥ 1% in studies comprising at least 10 dogs in each cohort.In addition to these ltering steps, we also annotated potential biomarker associations using our Precision Oncology Knowledgebase (Vidium Animal Health).This approach enables the identi cation of mutation biomarkers that have been described in human or canine cancers in published literature.Mutations identi ed in both the constitutional samples and the matched tumor samples were considered in downstream analysis.Mutations were considered somatic if present only in the tumor sample and germline if present in constitutional and tumor DNA.
Manta (v1.6) 43 was used for ITD calling of KIT and FLT3 genes, and CNVkit (v0.9.6) 44 for CNV calls.A two-copy loss in tumor suppressor genes and a six-copy gain in oncogenes were assumed to have a signi cant impact on function.For copy number events, gains in autosomal oncogenes were retained if the con dence interval (CI) lower bound > 0.368, and losses in autosomal tumor suppressors were retained if the CI upper bound < -0.238.For genes on the sex chromosomes in males, a true gain was considered to have a CI lower bound > -0.7, and a true loss was any event with a CI lower bound < -1.3.FASTQ les were generated and aligned to the canine reference genome, CanFam3.147.The primary analysis pipeline was automated to generate single-nucleotide variants (SNV), copy number variants (CNV), and internal tandem duplications (ITD), using the DNAnexus cloud-based computing platform (DNAnexus Inc.).Based on log2 fold change and tumor content, copy number gains or losses were inferred as single or multiple.For both 1410 and 1411, the Spearman correlation was calculated between the CNV variation reported from the SearchLight panel and scaled log 2 transformed DESeq normalized gene expression data.Additionally, expression values for genes exhibiting CNV events in either 1410 (poor) and 1411 (elite) were combined and the Spearman correlation between CNV variation and expression was calculated.

Clinical ndings
Dog 1410 (poor) presented with a 3x3x5 cm mass on the right distal femur and had a disease-free interval (DFI) of 62 days.However, her littermate, dog 1411(elite) presented with an 8x7x7 cm mass on the right distal tibia and had a DFI of 859 days, surviving nearly 13 times longer than their sibling.Both dogs were diagnosed with osteoblastic osteosarcomas (Fig. 1).Neither had evidence of lymphatic or vascular invasion in evaluated sections.A higher mitotic index was reported for 1411(elite) (20 vs. 8 in ten 400x elds).Serum Alkaline Phosphatase (ALP) levels for 1410 (poor) were normal but elevated for 1411; however, 1411 had a preexisting diagnosis of idiopathic epilepsy and history of phenobarbital treatment, which may have been responsible for the baseline elevation in liver-associated ALP.Radiographic ndings prior to surgical limb amputation for both dogs were consistent with appendicular osteosarcoma (Fig. 2).Metastatic progression was documented in both dogs during the study period.1411 (elite) had metastasis to the right distal femur, detected at 122 weeks (859 days) post-amputation, and 1410 (poor) to the lungs detected at 9 weeks (63 days) post-amputation.Neither patient underwent a post-mortem examination.
There were 16 other Rottweilers with osteosarcoma enrolled in COTC021/022.These dogs were not rstdegree relatives with 1410 and 1411 and were included in this case report as a basis of comparison to the sibling Rottweilers and to the overall COTC021/022 cohort.The non-sibling Rottweiler cohort comprised 5 females and 11 males with a mean age of 7.1 years.The average Rottweiler weight was 42.4 kg for females and 50.8 kg for males.Serum ALP values varied equally within the non-sibling Rottweilers with 50% reporting elevated levels and 50% reporting normal levels.The majority of the nonsibling cohort (69%) was treated with SOC consisting of surgical limb amputation and carboplatin chemotherapy, while 31% also received adjuvant sirolimus (SOC + S).Most Rottweilers (75%) received 4 doses of carboplatin; the remaining dogs received fewer doses before exiting the study.The most common reason dogs were taken off study was disease progression (94%) while the most common method of death was euthanasia (81%).At the trial's conclusion, all but one Rottweiler had documented metastatic disease and 44% had metastases in multiple locations.The most common sites of metastases were lung (81%), bone (31%), and kidney (31%).The median disease-free interval (DFI) for all non-sibling Rottweilers was 143 days and did not vary signi cantly from 1411 and 1410 (p = 0.6) or the overall median DFI for the COTC 021/022 trial (Standard of Care median DFI, 180 days; Standard of Care + sirolimus median DFI, 204 days). 27rolimus pharmacokinetics 1410 and 1411 were randomly assigned to one of two treatment arms as part of the COTC021/22 trial: SOC or SOC + S. Dog 1411 (elite) completed 4 doses of carboplatin and 4 doses of sirolimus after limb amputation, whereas dog 1410 (poor) received the SOC treatment after amputation and only 2 doses of carboplatin before removal from study due to disease progression.Ultimately, the results of COTC021/22 found that there was no signi cant difference between SOC + S and SOC with respect to DFI and overall survival time. 27This could be due to the high variability in oral drug absorption and bioavailability of sirolimus and is further supported by 1411's pharmacokinetic summary (Supplemental Table 1), which demonstrated an estimated trough level of sirolimus far below 10 ng/ml, which is the exposure threshold for the drug thought to exert therapeutic e cacy in sarcoma. 45Thus, although its contribution cannot be ruled out, the treatment type 1411 received was thought not to be the primary determinant of their extended survival time compared to 1410.
No shared mutations were detected between siblings and no pathogenic germline SNV was detected in either dog.Although an NF2 mutation was observed in the germline and tumor of 1411, this variant is likely benign.The NF2 variant is a known SNP that, though rare in the general canine population (0.09% frequency among 1,172 measured dogs 46 ), has not previously been reported in the human or canine cancer literature.The mutation also occurs at a 44-50% VAF, consistent with a heterozygous state with no sign of a second hit in the tumor.
When considering copy number variations (CNVs) that were detected with a log2 fold-change equivalent to at least a single copy gain or loss (≤ -0.35 for a loss and ≥ 0.35 for a gain), 38 genes were found to be impacted by CNVs in both tumors.No CNVs were detected at signi cant levels in germline samples.Thus, no obvious shared germline pathogenic CNV was observed in SearchLight DNA regions.CNVs detected in the tumors were mostly unique to each sample and demonstrate notable differences in copy number alterations between the two dogs involving genes implicated in osteosarcoma.Both dogs' tumors exhibited a homozygous CDKN2B loss and partial loss of IKZF1, MSH3, NF1, NOTCH1 and TSC1, 1411 (elite) demonstrated partial losses of BAP1, BRCA2, MEN1, SETD2, SMARCA4, STK11, and VHL, with gains of CCNE1 and MYCN.In contrast, 1410 (poor) demonstrated partial losses in TP53, APC, ATM, ATR, ATRX, BRCA1, CDK12, FLCN, and MLH1, with small gains in RICTOR, AKT1, CCND1, and FGF3, and a more signi cant gain of chr13 that spanned MYC, KIT, KDR, and PDGFRA.
Utilizing the bulk mRNAseq and paired clinical data from n = 10 Rottweiler dogs within the DOG 2 cohort, which included both 1411 and 1410, correlations between log2 fold changes in CNV and gene expression were explored for speci c genes to establish a gene dose-gene expression relationship.In dog 1410 (poor), a signi cant correlation was seen but not for dog 1411 (elite), likely due to the higher incidence of CNV in 1410 (Fig. 4).We then sought to determine if transcriptionally-de ned clusters and/or differentially expressed genes (DEGs) could be identi ed to de ne differences between 1410 and 1411, and how they relate to other Rottweilers with osteosarcoma based on known clinical outcomes with equivalent standardized therapy.Our a priori de nition of elite (DFI > 200 days) and poor (DFI < 200 days) responder groups allowed segregation of dogs and a supervised analysis of DEGs between these two groups (Fig. 5).Although the sample size is small, 97 DEGs (Supplemental Table 2), were identi ed that de ne these two outcome-linked groups of Rottweilers.We then went on to apply a transcriptional signature originally derived from an external canine osteosarcoma dataset, GS-1, that consists mainly of genes involved in immune responses. 5,28This analysis indicates that Rottweilers in the DOG 2 cohort, including both siblings, appear to have under-expression of GS-1 genes (Supplemental Fig. 1A), consistent with an 'immune low' environment, which has been previously shown to correlate with immune cell in ltration as demonstrated by labeling of immune cells including macrophages (Supplemental Fig. 1B).Although decreased GS-1 enrichment has been associated with poor prognosis 28 , the DFI and survival of the Rottweiler cohort was not signi cantly different than the remainder of the COTC021/022 cohort (Supplemental Fig. 2).

Discussion
Osteosarcoma is uncommon among humans, and even rarer between siblings, however, studying such a phenomenon can provide key insight into the genetic behavior and pathogenesis of the disease. 479][50] Instances of osteosarcoma between children and parents or between cousins have been recorded as well. 51,524][55][56] In human osteosarcoma patients, this includes Paget's disease, Li-Fraumeni syndrome, hereditary retinoblastoma, ATR-X syndrome, Rothmund-Thomson syndrome, and Werner and Bloom syndromes.In dogs, germline variants in APC2, BLM, BRCA2, TP53, RB1, WRN, and CDKN2B have also been observed. 5Although not evident in the sibling dogs described herein, genomic sequencing studies of related dogs may provide insight into germline variants in genes not previously linked to OS, as described by Mirabello et al in a study of over 1200 patients with osteosarcoma. 56e goals of this case report were to describe the clinical, histologic and molecular/genomic features Rottweilers within the DOG 2 cohort and speci cally, a sibling pair of affected Rottweiler dogs with disparate outcomes with similar therapy.Review of medical records and associated clinical trial data did not identify obvious differences within their home environment, histologic or clinical features, or therapeutic management.This directed us to examine the molecular features of their tumors.What we describe here is the clinical and transcriptomic landscape of Rottweiler dogs from a prospective, randomized clinical trial with evidence of a differential genomic and transcriptional program between 2 sibling Rottweiler dogs with contemporaneously occurring osteosarcoma but vastly different clinical outcomes.
In this study, we used SearchLight to investigate CNV changes that relate to genes with known and/or suspected association with osteosarcoma in prior canine and human literature.SearchLight DNA is a commercially-available pan-cancer tumor genomic sequencing panel that reports on multiple mutation types, including single-nucleotide variants, copy number variants, and internal tandem duplications in 120 pre-selected cancer genes.][59] Although the data from these sibling Rottweiler dogs did not uncover a shared germline pathogenic variant to explain their contemporaneous osteosarcoma development, we were able to describe genomic changes in their respective osteosarcomas that may have a role in progression or resistance to therapy.
The SearchLight results demonstrated CNV changes occurring in both dogs that relate to genes with known and/or suspected association with osteosarcoma in prior canine and human literature.Both dogs had shared copy number losses in a subset of genes, such as CDKN2B and NOTCH1, but the remainder of alterations appeared mutually exclusive to each dog's tumor.For example, in dog 1411, a segmental gain in CFA13 was observed that includes KDR, KIT, and PDGFRA.An analogous segmental ampli cation of human chromosome 4q11-12 involving KIT, KDR and PDGFRA has been identi ed in 6-20% of osteosarcoma patients. 60This gain has been implicated as a both druggable event and a negative prognostic factor in some human cancers. 61Dog 1411 also demonstrated a gain in MYC, also located on CFA 13.MYC ampli cation has garnered much attention recently as a potentially prognostic biomarker for human OS. 62Larger studies of this gene-dense region are needed to de ne an association between segmental and/or single-gene ampli cation and prognosis in canine osteosarcoma.
The SearchLight assay provided data on TP53, a known driver of osteosarcoma in both humans and dogs.Homologous loss-of-function mutations in TP53 were observed, with dog 1410 exhibiting both a copy number loss and truncating mutation, and dog 1411 exhibiting a missense mutation.Alterations in TP53 are the most common genomic lesions observed in osteosarcomas of both dogs and humans, but the nature of the alterations varies.In dogs, point mutations appear to dominate while in humans, both point mutations and structural variations, particularly translocations involving intron 1, are seen. 63,64As computational tools become more widely available for assessment of structural variations in canine genomes, more data will emerge to characterize these alterations more fully.
Losses of CDKN2A, BRCA2, SETD2, ATRX and others have also been identi ed in cohorts of human osteosarcoma patients. 65,66Both dogs' tumors exhibit a homozygous CDKN2B loss but no evidence of a germline event at this locus.Both CDKN2A and CDKN2B act as tumor suppressors through encoding proteins p16 INK4a , p14 ARF and p15 INK4b , which regulate G 1 cell cycle arrest. 66To this point, genome-wide association studies (GWAS) carried out in 3 high-risk breeds (Greyhounds, Rottweilers, and Irish Wolfhounds) as well as the Leonberger dog, implicated regulatory elements upstream of the CDKN2A/B locus as highly associated with osteosarcoma development and possibly responsible for disruption of enhancer elements and thus altered expression of genes responsible for cell cycle control in this region. 25,67It is possible that identi cation of alterations upstream of the CDKN2A/B locus can be identi ed through whole-genome sequencing of both tumor and normal tissues from dogs 1410 and 1410, as well as other Rottweilers in the DOG 2 cohort.A recent GWAS study in Bernese Mountain Dogs, Rottweilers, golden retrievers and at-coated retrievers across 3 hematopoietic cancers (histiocytic sarcoma, lymphoma and mast cell tumor) also implicates the CDKN2A locus as well as other loci on canine chromosomes 5 and 20. 68e histone methyltransferase SETD2 is a tumor-suppressor gene that has been documented to harbor mutations in a small subset of human osteosarcomas. 65In a study of whole-exome sequencing in 66 dogs with osteosarcoma including 21 Rottweilers, SETD2 was the second most frequently mutated gene after TP53; this was further supported by a second sequencing study which reported SETD2 mutations in 42% of 24 canine patients. 5,69Dog 1411 had copy number loss of this gene in their tumor based on the SearchLight assay.SETD2 has been implicated as a potential driver in both canine and human osteosarcoma, which is consistent with data that implicates epigenetic modulation in tumor progression and differential outcomes. 70Additional work is needed to de ne the speci c role of SETD2 in gene regulation and the DNA damage response (DDR), which would be highly relevant in a cancer type that is characterized by treatment with DNA damaging agents such as platinum chemotherapy.This could be further exacerbated by loss of or mutations in DDR genes such as ATM, ATR, ATRX, BRCA1/2 and BAP1. 71As compared with the genomes of other pediatric cancers, human osteosarcoma genomes have a relatively high-level, homologous recombination-de cient signature (typically characteristic of BRCA1/2-de cient cancers). 71,72It has been suggested that alterations in these genes in osteosarcoma may be best identi ed through copy number variation and not through whole-genome or whole-exome sequencing 10,72 , which is a current focus of work within the larger DOG 2 cohort.SETD2 loss in dog 1411 may also be associated with sirolimus exposure and response to mTOR inhibition.Although the pharmacokinetic pro le for this dog suggests inadequate exposure to the drug, it is possible that some measure of mTOR inhibition may have occurred within tumor tissue, which may have been augmented by the SETD2 de cient nature of 1411's primary tumor.SETD2 loss or inactivation has been associated with enhanced response to mTOR inhibition through alterations in oxidative metabolism and protein synthesis pathways. 73,74ared loss of NOTCH1 in both dogs also carries relevance to osteosarcoma, as the NOTCH signaling pathway plays an important role in osteogenic differentiation. 75Dysregulation of this pathway is linked to occurrence and progression of defects involving this process. 768][79] This may be due to the time-sensitive expression of NOTCH receptors during osteogenic differentiation as some members (NOTCH1, NOTCH3) maintain the undifferentiated state of osteoprogenitor cells, while others (NOTCH2, NOTCH4) promote osteoblastic differentiation. 80 both dogs, analysis of the bulk mRNAseq data and its relationship to copy number data for the genes contained within the SearchLight panel provided the opportunity to link gene expression to gene dose.
Although many factors can in uence gene expression aside from loss or gain of copies of individual genes, this data demonstrates a signi cant relationship between CNV and mRNAseq of selected genes from dog 1411 (elite).Whole-genome sequencing data could be assessed alongside the bulk mRNAseq data from these and other dogs for which paired data are available within the DOG 2 cohort, to make additional observations on the relationship between these two complementary datasets.
The dataset and case reports presented herein provides interesting insight into differential genomic lesions that may in uence outcomes and allowed comparison of these two sibling dogs to other nonrelated Rottweilers within the larger DOG 2 cohort.Our study did not uncover a shared germline pathogenic variant which could help explain the development of osteosarcoma in related dogs.The exploration of cancer susceptibility is best performed in the context of a genome-wide association (GWAS) study.In contrast to humans, osteosarcoma in dogs is generally thought to be highly heritable with some large and giant breed dogs, including Rottweilers, at > 10x fold risk of developing the disease.However, given the high incidence across companion dogs in general and those of mixed breeding, the incidence of canine osteosarcoma cannot be purely explained by heritable risk.Future genomic sequencing studies of both related and unrelated dogs should be prioritized to provide insight into germline variants in genes not previously linked to osteosarcoma in canine and human patients.A better understanding of affected genes and their respective pathways will facilitate the development of diagnostic and therapeutic biomarkers and identi cation of novel therapeutic targets for osteosarcoma patients.

Declarations
Ethics approval and consent to participate: Dogs described herein were enrolled in a previously published clinical trial (NCI-COTC021/022), for which a signed informed consent was obtained from each dog owner prior to enrollment.Each enrolling site maintained their own approved Institutional Animal Care and Use protocol for the trial.
Consent for publication: Not applicable Figures Figure 1 In the distal metaphysis of the right femur, there is a mild moth-eaten bone lysis and marked sclerosis extending into the distal diaphysis and epiphysis.Radiographic images from dog 1411 (elite outcome), also from the right hindlimb but highlighting the tarsal joint (panel C: anterior-posterior projection, panel D: lateral projection).At the distal metaphysis and epiphysis of the tibia there is moth eaten lysis.The cranial and caudal margins of the cortex are smooth but thinned.Within the mid diaphysis, the medullary cavity has a mottled appearance.Circumferentially to the tarsus and within the tibiotarsal joint, there is a severe (more severe dorsomedially) amount of soft tissue swelling.2.

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Figure 4