Heather Gardner

Copy number variation in each expansion cohort (n = 209) tumor samples. Abbreviations: COTC = Comparative Oncology Trials Consortium, WGS = Whole Genome Sequencing, ALP = Alkaline Phosphatase, DFI = Disease-Free Interval, TME = Tumor Microenvironment.

The list of TP53 copy number, somatic (consensus of muTect2, Strelka and Seurat), structural variations, along with somatic and germline mutations identified by GATK haplotype caller were shown.

Purpose:Osteosarcoma is a heterogeneous and aggressive primary bone malignancy that affects both canines and humans. Standardized treatment regimens prescribed to both species do not address the complexity of the disease and thus have resulted in stagnant patient outcomes for more than 30 years. Experimental Design:In this study, we present the first multiomic dataset created from a large outcome-linked biobank of canine osteosarcoma treatment-naïve primary tumors, utilizing a computational framework designed to interrogate each dataset individually and to compare and integrate findings. Results:This exploratory work suggests that the presence of MYC amplification is a poor prognostic indicator in canines and highlights alterations in DNA damage repair, metabolism, and cell-cycle genes that are shared with humans. Furthermore, we show relationships between the local tumor immune microenvironment, TP53 mutations, MYC and PTEN status, and global gene methylation patterns. Conclusions:This work highlights the complexity of the disease and provides new insight into the utility of prognostic biomarkers and potential druggable targets for future study.

The list of marker genes used in cell deconvolution using MCPcounter.

Gene set enrichment in MYC stratified samples.

Germline mutations of the Discovery cohort tumor/normal paired samples: a: Normal samples b: tumor samples. HGVS.c: Variant using HGVS notation (DNA level) and HGVS.c: Variant using HGVS notation (Protein level).

GISTIC output of discovery cohort (n = 55) sample. Cytoband annotations are based on the CanFam4 UCSC cytoBandIdeo file. Classical cytogenetic band subdivisions (p/q arms, numbered bands) are not included, as these are not provided in the CanFam4 annotation.

Copy-number variation (CNV), single-nucleotide variation (SNV), and structural variation (SV) for each discovery cohort sample (n = 55). Combined information for CNVs, SNVs, and SVs for each gene is provided in Sheet 2. Each column in Sheet 2 represents an individual dog, indicated by the case ID. Abbreviations: COTC = Comparative Oncology Trials Consortium, WGS = Whole Genome Sequencing, ALP = Alkaline Phosphatase, DFI = Disease-Free Interval, TME = Tumor Microenvironment, SOC = Standard of Care, PH = proximal humerus, NPH = non-proximal to humerus.

Supplementary Figure 1 | The total sample size for each clinical trial, the treatments received, and the number of multiomic samples associated with each COTC021/022 /030 cohort. Supplementary Figure 2 | The detailed parameter and steps used in copy number variation, single nucleotide and structural variation. Supplementary Figure 3 | Genetic profiling of Discovery cohort patients (n = 55). Supplementary Figure 4 | Forest plots of adjusted hazard ratios for TP53 stratification and clinical features derived from multivariable Cox proportional hazards regression analyses. (a) Disease-free interval (DFI). (b) Overall survival (OS). *P < 0.05 Supplementary Figure 5 | Genetic profiling of Expansion cohort patients (n = 209). Supplementary Figure 6 | Consensus matrix of control and primary OS WGBS samples using K value 2 to 10. Supplementary Figure 7 | Forest plots of adjusted hazard ratios for methylation stratification and clinical features derived from multivariable Cox proportional hazards regression analyses. (a) Disease-free interval (DFI). (b) Overall survival (OS). *P < 0.05. Supplementary Figure 8 | Forest plots of adjusted hazard ratios for MYC stratification and clinical features derived from multivariable Cox proportional hazards regression analyses. (a) Disease-free interval (DFI). (b) Overall survival (OS). *P < 0.05 Supplementary Figure 9 | Box plots represent distribution of SBS in MYC-amplified (n = 31) and MYC-unamplified (n = 24) tumors. Supplementary Figure 10 | Heatmap depicting the fraction of MTAP stratified patients (with MTAP deletion: n = 31 and without MTAP deletion n = 24) grouped into TME subtype (IE, IE-ECM, and ID). IE = Immune Enriched, IE-ECM = Immune Extracellular Matrix, ID = Immune Dessert. Supplementary Figure 11 | Forest plots of adjusted hazard ratios for PTEN stratification and clinical features derived from multivariable Cox proportional hazards regression analyses. (a) Disease-free interval (DFI). (b) Overall survival (OS). *P < 0.05.

Differential gene expression in samples with and without PTEN copy number loss.