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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.

Osteosarcoma is a debilitating bone cancer that affects humans, especially children and adolescents. A homologous form of osteosarcoma spontaneously occurs in dogs, and its differential incidence observed across breeds allows for the investigation of tumor mutations in the context of multiple genetic backgrounds. Using whole-exome sequencing and dogs from three susceptible breeds (22 golden retrievers, 21 Rottweilers, and 23 greyhounds), we found that osteosarcoma tumors show a high frequency of somatic copy-number alterations (SCNA), affecting key oncogenes and tumor-suppressor genes. The across-breed results are similar to what has been observed for human osteosarcoma, but the disease frequency and somatic mutation counts vary in the three breeds. For all breeds, three mutational signatures (one of which has not been previously reported) and 11 significantly mutated genes were identified. TP53 was the most frequently altered gene (83% of dogs have either mutations or SCNA in TP53), recapitulating observations in human osteosarcoma. The second most frequently mutated gene, histone methyltransferase SETD2, has known roles in multiple cancers, but has not previously been strongly implicated in osteosarcoma. This study points to the likely importance of histone modifications in osteosarcoma and highlights the strong genetic similarities between human and dog osteosarcoma, suggesting that canine osteosarcoma may serve as an excellent model for developing treatment strategies in both species.Significance: Canine osteosarcoma genomics identify SETD2 as a possible oncogenic driver of osteosarcoma, and findings establish the canine model as a useful comparative model for the corresponding human disease. Cancer Res; 78(13); 3421–31. ©2018 AACR.

File contains list of GISTIC Defined Recurrent SCNA genes that overlap between Humans and Dogs.

Supplementary Figure 1 - Workflow for Somatic Point and Indel Mutation detection. Supplementary Figure 2 - Heatmap counts of samples with recurrent focal SCNAs per chromosome. Supplementary Figure 3 - oaCGH profiles penetrance plots and GISTIC analysis. Supplementary Figure 4 - Venn diagrams of shared and unique somatic point mutations and somatic indel mutations across the three breeds. Supplementary Figure 5 - Scatter plot shows age of diagnosis is positively correlated to the mutation counts across all breeds. Supplementary Figure 6 - Mutational signature distribution per breed shows enrichment for the novel signature in Golden Retriever. Supplementary Figure 7 - Overview and functional classes for the mutations in histone modifier genes across the three breeds. Supplementary Figure 8 - Overview of germ-line candidate mutation across three breeds and their functional impacts.

File contains genes that have focal somatic copy number deletions.

File contains supplementary tables and figures for the main article. Supplementary Table 1 - Source for tools and databases used in the methods for analysis. Supplementary Table 2 - Summary of Somatic Point and Indel Mutation across the three breeds. Supplementary Table 3 - Summary of GISTIC-defined recurrent large-scale SCNAs and gene frequency. Supplementary Table 4 - List of GISTIC classified amplification genes. See attached excel spreadsheet. Supplementary Table 5 - List of GISTIC classified deletion genes. See attached excel spreadsheet. Supplementary Table 6 - List of GISTIC genes and their predicted roles in cancer. Supplementary Table 7 - Significantly over-represented pathways for GISTIC- and MuSiC-defined genes. Supplementary Table 8 - Significantly mutated genes as identified by MuSiC and the proportion of samples per breed that show mutation in them. Supplementary Table 9 - Putative driver genes as detected by MutSigCV algorithm Supplementary Table 10 - Putative driver genes assessed by VEP high-impact mutations and their variant allele frequency. Supplementary Table 11 - Mutations and their effects on p53 protein. Supplementary Table 12 - Mutations and their effects on SETD2 protein. Supplementary Table 13 - Count of non-silent mutations per breed/per germline for OSA susceptibility genes Supplementary Table 14 - Overlap of GISTIC-defined recurrent SCNA genes between humans and dogs. See attached excel spreadsheet.

File contains genes that have focal somatic copy number amplifications

File contains additional information regarding methods for identifying putative driver genes with MutSigCV and high-impact mutations based analysis. Also methods for predicting impacts of TP53 and SETD2 genes. And further information about the oaCGH analysis for somatic copy number aberrations across a subset of 22 dogs.

PDF file - 843K, Unsupervised hierarchical clustering of cBCLs.

PDF file - 881K, Western blot comparing phospho-NF-κB (p65) levels in "ABC-like" and "GCB-like" canine samples.

Supplementary Tables 1-2 from Distinct B-Cell and T-Cell Lymphoproliferative Disease Prevalence among Dog Breeds Indicates Heritable Risk

XLSX file - 40K, Canine genes identified by Ingenuity Pathway Analysis as part of the NF-kB or B-cell receptor signaling pathways (Fig. 5).

PDF file - 60K

Immunophenotypes in lymphoproliferative diseases (LPD) are prognostically significant, yet causative factors for these conditions, and specifically those associated with heritable risk, remain elusive. The full spectrum of LPD seen in humans occurs in dogs, but the incidence and lifetime risk of naturally occurring LPD differs among dog breeds. Taking advantage of the limited genetic heterogeneity that exists within dog breeds, we tested the hypothesis that the prevalence of LPD immunophenotypes would differ among different breeds. The sample population included 1,263 dogs representing 87 breeds. Immunophenotype was determined by the presence of clonal rearrangements of immunoglobulin heavy chain or T-cell receptor γ chain. The probability of observing the number of B-cell or T-cell tumors in a particular breed or breed group was compared with three reference populations. Significance was computed using χ2 test, and logistic regression was used to confirm binomial predictions. The data show that, among 87 breeds tested, 15 showed significant differences from the prevalence of LPD immunophenotypes seen across the dog population as a whole. More significantly, elevated risk for T-cell LPD seems to have arisen ancestrally and is retained in related breed groups, whereas increased risk for B-cell disease may stem from different risk factors, or combinations of risk factors, arising during the process of breed derivation and selection. The data show that domestic dogs provide a unique and valuable resource to define factors that mediate risk as well as genes involved in the initiation of B-cell and T-cell LPD.

PDF file - 41K, Distribution of lymphoma samples into "Ongoing" or "Static" categories based on the proportion of IGHV subclones that are identical at the CDR3 region for each sample.

XLSX file - 116K, Canine genes that distinguish "ABC-like" and "GCB-like" subtypes (Fig. 5).

Background Patients presenting with head and neck squamous cell carcinoma of unknown primary (HNSCCUP) remain challenging clinical scenarios as large variation exists in practices used to locate the primary. Objective To perform a systematic review of the literature and offer recommendations for oropharyngeal biopsies in HNSCCUP. Method Pubmed, Medline and Embase were searched to identify studies from inception to October 2021. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines were followed. Results 483 articles were included and screened, 40 studies met the inclusion criteria, including over 3400 patients from the original articles and 1575 patients from 3 meta-analyses. The primary site identification rate following random biopsies or deep tissue biopsies is less than 5% in most studies. The mean detection rate following ipsilateral tonsillectomy is 34%; two pooled analyses indicate that the mean detection rate following tongue base mucosectomy is 64%, with this figure rising when the tonsils are negative. Conclusions High level evidence is lacking, with heterogeneity in the reported studies. Published meta analyses are based on retrospective data. There is little evidence supporting the practice of random/non-directed oropharyngeal biopsies. Available evidence supports palatine tonsillectomy and tongue base mucosectomy compared to deep tissue biopsies.

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.

Despite the strikingly high worldwide prevalence of vulvovaginal candidiasis (VVC), treatment options for recurrent VVC (RVVC) remain limited, with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown, with few studies identifying potential mechanisms of treatment failure. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, the vaginal microbiome, and how their interaction influences disease pathology. 16S rRNA sequencing characterized disease by a reduction in specific health-associated Lactobacillus species, such as Lactobacillus crispatus, coupled with an increase in Lactobacillus iners. In vitro analysis showed that Candida albicans clinical isolates are capable of heterogeneous biofilm formation, and we found the presence of hyphae and C. albicans aggregates in vaginal lavage fluid. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to identify a possible mechanism by which L. crispatus may contribute to re-establishing a healthy vaginal environment through amino acid acquisition from C. albicans. This study highlights the potential formation and impact of Candida biofilms in RVVC. Additionally, it suggests that RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and that understanding interactions between this yeast and vaginal Lactobacillus species may be crucial to elucidating the cause of RVVC and developing appropriate therapies. IMPORTANCE RVVC is a significant burden, both economically and for women's health, but its prevalence is poorly documented globally due to the levels of self-treatment. Identifying triggers for development and recurrence of VVC and the pathogenesis of the microbes involved could considerably improve prevention and treatment options for women with recurrent, azole-resistant cases. This study therefore aimed to examine the interkingdom dynamics from healthy women and those with RVVC using next-generation sequencing techniques and to further investigate the molecular interactions between C. albicans and L. crispatus in a relevant biofilm coculture system.

Radiation resistance is a significant clinical problem in rectal cancer treatment, the mechanisms of which are poorly understood. NRF2 signalling is known to contribute to chemo/radioresistance in some cancers, but its role in therapeutic resistance in colorectal cancer (CRC) is unexplored. Using siRNA and CRiSPR/Cas9 isogenic CRC cell lines, we investigated the effect of the knockdown and upregulation of the NRF2 pathway on chemo-radiosensitivity. Poly (A) enriched RNA sequencing and geneset enrichment analysis (GSEA) were carried out on both sensitive and resistant cell models for mechanistic insights. Finally, a cohort of rectal patient samples was profiled to understand the clinical relevance of NRF2 signalling. Radioresistant cell lines were significantly radiosensitised by siRNA knockdown (SW1463, SER10 1.22, ANOVA p &lt, 0.0001, HT55, SER10 1.17, ANOVA p &lt, 0.01), but not the (already) radiosensitive HCT116. The constitutive activation of NRF2 via a CRISPR Cas9 NFE2L2 mutation, E79K, induced radioresistance in HCT116 (SER10 0.71, ANOVA, p &lt, 0.0001). GSEA demonstrated significant opposing metabolic dependencies in NRF2 signalling, specifically, the downregulation of amino acid and protein synthesis with low levels of NRF2 and upregulation with over expression. In a clinical cohort of 127 rectal patients, using a validated mRNA signature, higher baseline NRF2 signalling was associated with incomplete responses to radiation higher final neoadjuvant rectal (NAR) score (OR 1.34, 95% C.I. 1.01–1.80, LRT p-value = 0.023), where high NAR indicates poor radiation response and poor long-term prognosis. This is the first demonstration of NRF2-mediated radiation resistance in colorectal cancer. NRF2 appears to regulate crucial metabolic pathways, which could be exploited for therapeutic interventions.

An amendment to this paper has been published and can be accessed via the original article.

Introduction Iatrogenic facial nerve palsy is distressing to the patient and clinician. The deformity is aesthetically displeasing, and can be functionality problematic for oral competence, dental lip trauma and speech. Furthermore such injuries have litigation implications. Marginal mandibular nerve (MMN) palsy causes an obvious asymmetrical smile. MMN is at particular risk during procedures such as rhytidoplasties, mandibular fracture, tumour resection and neck dissections. Cited causes for the high incidence are large anatomical variations, unreliable landmarks, an exposed course and tumour grade or nodal involvement dictating requisite nerve sacrifice. An alternative cause for post-operative asymmetry is damage to the cervical branch of the facial nerve or platysmal dysfunction. This tends to have a transient course and recovers. Distinction between MMN palsy and palsy of the cervical branch of the facial nerve should therefore be made. In 1979 Ellenbogen differentiated between MMN palsy and “Pseudo-paralysis of the mandibular branch of the facial nerve”. Despite this, there is paucity in the literature & confusion amongst clinicians in distinguishing between these palsies, and there is little regarding these post-operative sequelae and neck dissections. Method This article reflects on the surgical anatomy of the MMN and cervical nerve in relation to danger zones during lymphadenectomy. The authors review the anatomy of the smile. Finally, we utilise case studies to evaluate the differences between MMN palsy and its pseudo-palsy to allow clinical differentiation. Conclusion Here we present a simple method for clinical differentiation between these two prognostically different injuries, allowing appropriate reassurance, therapy & management.

Vulvovaginal Candidiasis (VVC) is the most prevalent Candida infection in humans affecting 75% of women at least once throughout their lifetime. In its debilitating recurrent form, RVVC is estimated to affect 140 million women annually. Despite this strikingly high prevalence, treatment options for RVVC remain limited with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown with few studies identifying potential mechanisms of failed treatment. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, vaginal microbiome and antagonism between Candida and Lactobacillus on disease pathology. 16S rRNA sequencing characterised disease by a reduction in specific health-associated Lactobacillus such as L. crispatus, coupled with an increase in L. iners. In vitro analysis showed Candida albicans clinical isolates are capable of heterogeneous biofilm formation and show the presence of hyphae and C. albicans aggregates in vaginal lavage. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to exploit a possible mechanism by which L. crispatus may aim to re-establish a healthy vaginal environment through amino-acid acquisition from C. albicans. This study suggests RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and understanding interactions between the yeast and vaginal Lactobacillus species may be more crucial to elucidating the cause of RVVC and developing appropriate therapies.

Both colorectal (CRC, 15%) and endometrial cancers (EC, 30%) exhibit microsatellite instability (MSI) due to MLH1 hypermethylation and silencing. The MLH1 promoter polymorphism, rs1800734 is associated with MSI CRC risk, increased methylation and reduced MLH1 expression. In EC samples, we investigated rs1800734 risk using MSI and MSS cases and controls. We found no evidence that rs1800734 or other MLH1 SNPs were associated with the risk of MSI EC. We found the rs1800734 risk allele had no effect on MLH1 methylation or expression in ECs. We propose that MLH1 hypermethylation occurs by different mechanisms in CRC and EC.

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

Mast cell tumor (MCT) is the most common skin malignancy of domestic dogs and presents with a widely variable clinical behavior. Although activating KIT mutations are present in approximately 20% of canine MCTs, molecular etiology is largely unknown for the majority of this cancer. Characterization of genomic alterations in canine MCTs may identify genomic regions and/or genes responsible for their development and progression, facilitating the discovery of new therapeutic targets and improved clinical management of this heterogeneous cancer. We performed genome-wide DNA copy number analysis of 109 primary MCTs derived from three popular canine breeds (the Boxer, Labrador Retriever, and Pug) as well as nontarget breeds using oligonucleotide array comparative genomic hybridization (oaCGH). We demonstrated a stepwise accumulation of numerical DNA copy number aberrations (CNAs) as tumor grade increases. DNA sequencing analysis revealed that KIT mutations were found less frequently in the Pug tumors and were strongly associated with high histological grade. Tumors with KIT mutations showed genome-wide aberrant copy number profiles, with frequent CNAs involving genes in the p53 and RB pathways, whereas CNAs were very limited in tumors with wild-type KIT. We evaluated the presence of four CNAs to predict aggressive tumor phenotypes. This approach predicted aggressive tumors with a sensitivity of 78-94% and specificity of 88-93%, when using oaCGH and droplet digital PCR platforms. Further investigation of genome regions identified in this study may lead to the development of a molecular tool for classification and prognosis, as well as identification of therapeutic target molecules.

The raw data is available on Mendeley at https://data.mendeley.com/datasets/hfpbctm7tg/draft?a=1c91e494-cadc-4be0-a8ff-91d8736a28e7 © The Author(s) 2019. Expression of the mismatch repair gene MutL homolog 1 (MLH1) is silenced in a clinically important subgroup of sporadic colorectal cancers. These cancers exhibit hypermutability with microsatellite instability (MSI) and differ from microsatellite-stable (MSS) colorectal cancers in both prognosis and response to therapies. Loss of MLH1 is usually due to epigenetic silencing with associated promoter methylation; coding somatic mutations rarely occur. Here we use the presence of a colorectal cancer (CRC) risk variant (rs1800734) within the MLH1 promoter to investigate the poorly understood mechanisms of MLH1 promoter methylation and loss of expression. We confirm the association of rs1800734 with MSI+ but not MSS cancer risk in our own data and by meta-analysis. Using sensitive allele-specific detection methods, we demonstrate that MLH1 is the target gene for rs1800734 mediated cancer risk. In normal colon tissue, small allele-specific differences exist only in MLH1 promoter methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in MSI+ cancers. We show that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression. The transcription factor TFAP4 binds to the rs1800734 region but with much weaker binding to the risk than the protective allele. TFAP4 binding is absent on both alleles when promoter methylation is present. Thus we propose that TFAP4 binding shields the protective rs1800734 allele of the MLH1 promoter from BRAF induced DNA methylation more effectively than the risk allele. Funding was provided by a Medical Research Council New Investigator Research Grant (MR/P000738/1). Core funding to the Wellcome Trust Centre for Human Genetics was provided by the Wellcome Trust (090532/Z/09/Z). D.T., N.S. and D.F. were supported by the EPIGENOMICS FLAGSHIP PROJECT- EPIGEN (project number 08934412) and by University of Insubria. M.T. and S.K. were funded by Ludwig Cancer Research.

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.