Gene set enrichment in PTEN stratified samples.

Spontaneously occurring primary canine pulmonary adenocarcinoma (cPAC) exhibits clinicopathological and molecular similarities to never-smoker human lung cancers. Shared genomic alterations, including point mutations, indel mutations and copy number changes particularly in HER2 signalling, are significant therapeutic targets, especially for HER2 and tyrosine kinase inhibitors. Whilst progress has been made in identifying mutational drivers in canine cancers, the role of somatic gene fusions in cPAC remains poorly understood, despite their importance in other cancers as drivers and therapeutic targets. This study investigates the fusion landscape in cPAC by analysing RNA-seq data from a cohort of 36 primary tumour samples and reports oncogenic fusions with therapeutic potential. Notably, NRG1 fusions were identified in a subset of tumours, including recurrent SDC4::NRG1 events, potentially playing key roles in disease progression. NRG1 fusions, known to activate HER2 signalling, are mutually exclusive with HER2 gene alterations, indicating convergence on the same pathway. Tumours with SDC4::NRG1 fusions also overexpress HER2 pathway-related genes, reinforcing NRG1-driven activation. Similar fusions occur in never-smoker human non-small cell adenocarcinoma lacking other common drivers, underscoring their therapeutic importance. These findings highlight NRG1 fusions as critical contributors to cPAC tumorigenesis and warrant further clinical and comparative investigation. Additionally, novel fusions disrupting the PTEN axis were identified, leading to truncated PTEN and associated protein domains. These disruptions could impair tumour-suppressive pathways, presenting additional therapeutic targets. This research emphasises the broader relevance of fusion-driven mechanisms in cPAC tumorigenesis, advancing the understanding of both canine and human lung cancers for clinical and comparative studies.

Immune cell enrichment in MYC stratified samples.

Differential gene expression in MYC-amplified and MYC-unamplified samples.

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

Structural variation output using DELLY algorithm in discovery cohort (n = 55) sample.

List of hyper- and hypo-methylated marker genes and their associated promoter regions, along with their respective differential methylation profiles.

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.

Demographic and clinical outcome data linked to canine tumor samples used in WGS, WGBS, and bulk transcriptomics. 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.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive hematopoietic neoplasm that affects humans as well as dogs. While previous studies on canine DLBCL (cDLBCL) have significantly advanced our understanding of the disease, the majority of this research has relied on whole-exome sequencing, which is limited in its ability to detect copy number aberrations and other genomic changes beyond coding regions. Furthermore, many of these studies lack sufficient clinical follow-up data, making it difficult to draw meaningful associations between genetic variants and patient outcomes. Our study aimed to characterize the mutational landscape of cDLBCL using whole-genome sequencing of matched tumor-normal samples obtained from a cohort of 43 dogs previously enrolled in a clinical trial for which longitudinal follow-up was available. We focused on identifying genes that were significantly or recurrently mutated with coding point mutations, copy number aberrations, and their associations with patient outcomes. We identified 26 recurrently mutated genes, 18 copy number gains, and 8 copy number losses. Consistent with prior studies, the most commonly mutated genes included TRAF3, FBXW7, POT1, TP53, SETD2, DDX3X and TBL1XR1. The most prominent copy number gain occurred on chromosome 13, overlapping key oncogenes such as MYC and KIT, while the most frequent deletion was a focal loss on chromosome 26, encompassing IGL, PRAME, GNAZ, RAB36, RSPH14, and ZNF280B. Notably, our set of recurrently mutated genes was significantly enriched with genes involved in epigenetic regulation. In particular, we identified hotspot mutations in two histone genes, H3C8, and LOC119877878, resulting in H3K27M alterations predicted to dysregulate gene expression. Finally, a survival analysis revealed that H3K27M mutations in H3C8 were associated with increased hazard ratios for progression-free survival. No copy number aberrations were associated with survival. These findings underscore the critical role of epigenetic dysregulation in cDLBCL and affirm the dog as a relevant large animal model for interrogating the biological activity of novel histone-modifying treatment strategies.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a rare, deadly form of ovarian cancer that uniformly harbors mutations in SMARCA4, a member of the SWI/SNF chromatin remodeling complex. SWI/SNF impacts RNA splicing, and dysregulation of splicing can generate immunogenic tumor antigens. In this study, we explored the relationship between SMARCA4 loss and RNA splicing dysregulation. SCCOHT primary tumors harbored tumor-associated outlier splicing events compared with normal tissues. Many of the tumor events were retained introns encoding novel peptides predicted to bind to MHC-I complexes. Immune cells were observed in primary SCCOHT tumors, suggesting a potentially immune-reactive tumor microenvironment. Mutations in several switch/sucrose nonfermenting (SWI/SNF) subunits were associated with higher rates of outlier retained introns across tumor types in The Cancer Genome Atlas data. Interestingly, RNA sequencing of isogenic SCCOHT cell lines demonstrated a role for SMARCA4 in intron retention (IR). Distinct protein-protein interactions between splicing factors identified in SCCOHT cell lines supported a role for SMARCA4 in splicing regulation. Furthermore, SWI/SNF localized to genes, which were differentially spliced. Mass spectrometry analyses confirmed expression of some of these novel peptides, and a subset of these are predicted to bind to MHC-I complexes. A pool of these novel peptides derived from retained introns in SCCOHT triggered proliferation and expression of TNFα and INFγ in primary human T cells. Together, these data suggest that SMARCA4 loss in SCCOHT leads to IR. Furthermore, T-cell activation by novel peptides encoded by these tumor-specific splicing events suggests IR could be a source of tumor-associated antigens in SCCOHT. SIGNIFICANCE:SCCOHT, a rare ovarian cancer, features splicing dysregulation due to SMARCA4 loss that generates immunostimulatory peptides linked to potential immune responses and therapeutic avenues, challenging traditional views of the role of SMARCA4.

Small cell carcinoma of the ovary-hypercalcemic type (SCCOHT) is a rare ovarian cancer affecting young females and is driven by the loss of both SWI/SNF ATPases SMARCA4 and SMARCA2. As loss of SWI/SNF alters enhancers, we hypothesized that super-enhancers, which regulate oncogene expression in cancer, are disparately impacted by SWI/SNF loss. We discovered differences between SWI/SNF occupancy at enhancers vs. super-enhancers. SCCOHT super-enhancer target genes were enriched in developmental processes, most notably nervous system development. This may further support neuronal cell-of-origin previously proposed. We found high sensitivity of SCCOHT cell lines to triptolide. Triptolide inhibits expression of many super-enhancer-associated genes, including oncogenes. SALL4 expression is decreased by triptolide and is highly expressed in SCCOHT tumors. In patient-derived xenograft models, triptolide and prodrug minnelide effectively inhibit tumor growth. These results reveal unique features of super-enhancers in SCCOHT, which may be one mechanism through which triptolide has high activity in these tumors.

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 characteristics, as well as those from a companion cohort of Rottweiler dogs. A pan-cancer genomic sequencing panel conducted in the sibling dogs demonstrated both shared and distinct alterations in several genes implicated in osteosarcoma, including CDKN2B, SETD2, MYC, and PDGFRA, while transcriptional profiling of primary tumor tissue indicated under-expression of key immunological response genes. This report provides new insight into molecular features and potential prognostic biomarkers for canine osteosarcoma.

Background Children with relapsed central nervous system (CNS tumors), neuroblastoma, sarcomas, and other rare solid tumors face poor outcomes. This prospective clinical trial examined the feasibility of combining genomic and transcriptomic profiling of tumor samples with a molecular tumor board (MTB) approach to make real‑time treatment decisions for children with relapsed/refractory solid tumors. Methods Subjects were divided into three strata: stratum 1—relapsed/refractory neuroblastoma; stratum 2—relapsed/refractory CNS tumors; and stratum 3—relapsed/refractory rare solid tumors. Tumor samples were sent for tumor/normal whole-exome (WES) and tumor whole-transcriptome (WTS) sequencing, and the genomic data were used in a multi-institutional MTB to make real‑time treatment decisions. The MTB recommended plan allowed for a combination of up to 4 agents. Feasibility was measured by time to completion of genomic sequencing, MTB review and initiation of treatment. Response was assessed after every two cycles using Response Evaluation Criteria in Solid Tumors (RECIST). Patient clinical benefit was calculated by the sum of the CR, PR, SD, and NED subjects divided by the sum of complete response (CR), partial response (PR), stable disease (SD), no evidence of disease (NED), and progressive disease (PD) subjects. Grade 3 and higher related and unexpected adverse events (AEs) were tabulated for safety evaluation. Results A total of 186 eligible patients were enrolled with 144 evaluable for safety and 124 evaluable for response. The average number of days from biopsy to initiation of the MTB-recommended combination therapy was 38 days. Patient benefit was exhibited in 65% of all subjects, 67% of neuroblastoma subjects, 73% of CNS tumor subjects, and 60% of rare tumor subjects. There was little associated toxicity above that expected for the MGT drugs used during this trial, suggestive of the safety of utilizing this method of selecting combination targeted therapy. Conclusions This trial demonstrated the feasibility, safety, and efficacy of a comprehensive sequencing model to guide personalized therapy for patients with any relapsed/refractory solid malignancy. Personalized therapy was well tolerated, and the clinical benefit rate of 65% in these heavily pretreated populations suggests that this treatment strategy could be an effective option for relapsed and refractory pediatric cancers. Trial registration ClinicalTrials.gov, NCT02162732. Prospectively registered on June 11, 2014.

Precision medicine focuses on the clinical management of the individual patient, not on population-based findings. Successes from human precision medicine inform veterinary oncology. Early evidence of success for canines shows how precision medicine can be integrated into practice. Decreasing genomic profiling costs will allow increased utilization and subsequent improvement of knowledge base from which to make better informed decisions. Utility of precision medicine in canine oncology will only increase for improved cancer characterization, enhanced therapy selection, and overall more successful management of canine cancer. As such, practitioners are called to interpret and leverage precision medicine reports for their patients.

A genomic understanding of the oncogenic processes and individual variability of human cancer has steadily fueled improvement in patient outcomes over the past 20 years. Mutations within tumour tissues are routinely assessed through clinical genomic diagnostic assays by academic and commercial laboratories to facilitate diagnosis, prognosis and effective treatment stratification. The application of genomics has unveiled a wealth of mutation-based biomarkers in canine cancers, suggesting that the transformative principles that have revolutionized human cancer medicine can be brought to bear in veterinary oncology. To advance clinical genomics and genomics-guided medicine in canine oncology, we have developed and validated a canine cancer next-generation sequencing gene panel for the identification of multiple mutation types in clinical specimens. With this panel, we examined the genomic landscapes of 828 tumours from 813 dogs, spanning 53 cancer types. We identified 7856 alterations, encompassing copy number variants, single nucleotide variants, indels and internal tandem duplications. Additionally, we evaluated the clinical utility of these alterations by incorporating a biomarker framework from comprehensive curation of primary canine literature and inferences from human cancer genomic biomarker literature and clinical diagnostics. Remarkably, nearly 90% of the cases exhibited mutations with diagnostic, prognostic or therapeutic implications. Our work represents a thorough assessment of genomic landscapes in a large cohort of canine cancers, the first of its kind for its comprehensive inclusion of multiple mutation types and structured annotation of biomarkers, demonstrating the clinical potential of leveraging mutation-based biomarkers in veterinary oncology.

List of validated siRNA screen hits.

List of high-throughput drug screen hits.

Canine primary lung cancer cell line sensitivity to erlotinib. Five canine cell lines (three HER2WT and two HER2V659E) and one human cell line BT474 (HER2amp) were treated with 10 erlotinib doses ranging from 5x10-8 to 50 μM for 72 hours with CellTiterGlo viability endpoints measured and shown as percent growth inhibition relative to DMSO vehicle control.