Comparative Genomics Reveals Shared Mutational Landscape in Canine Hemangiosarcoma and Human Angiosarcoma.

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.

Genome-Wide Association Study of Golden Retrievers Identifies Germ-Line Risk Factors Predisposing to Mast Cell Tumours.

Canine mast cell tumours (CMCT) are one of the most common skin tumours in dogs with a major impact on canine health. Certain breeds have a higher risk of developing mast cell tumours, suggesting that underlying predisposing germ-line genetic factors play a role in the development of this disease. The genetic risk factors are largely unknown, although somatic mutations in the oncogene C-KIT have been detected in a proportion of CMCT, making CMCT a comparative model for mastocytosis in humans where C-KIT mutations are frequent. We have performed a genome wide association study in golden retrievers from two continents and identified separate regions in the genome associated with risk of CMCT in the two populations. Sequence capture of associated regions and subsequent fine mapping in a larger cohort of dogs identified a SNP associated with development of CMCT in the GNAI2 gene (p = 2.2x10-16), introducing an alternative splice form of this gene resulting in a truncated protein. In addition, disease associated haplotypes harbouring the hyaluronidase genes HYAL1, HYAL2 and HYAL3 on cfa20 and HYAL4, SPAM1 and HYALP1 on cfa14 were identified as separate risk factors in European and US golden retrievers, respectively, suggesting that turnover of hyaluronan plays an important role in the development of CMCT.

Genome-wide association study identifies shared risk loci common to two malignancies in golden retrievers.

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 the vesicle 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.

Genome-wide analyses implicate 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B.

BACKGROUND: Canine osteosarcoma is clinically nearly identical to the human disease, but is common and highly heritable, making genetic dissection feasible. RESULTS: Through genome-wide association analyses in three breeds (greyhounds, Rottweilers, and Irish wolfhounds), we identify 33 inherited risk loci explaining 55% to 85% of phenotype variance in each breed. The greyhound locus exhibiting the strongest association, located 150 kilobases upstream of the genes CDKN2A/B, is also the most rearranged locus in canine osteosarcoma tumors. The top germline candidate variant is found at a >90% frequency in Rottweilers and Irish wolfhounds, and alters an evolutionarily constrained element that we show has strong enhancer activity in human osteosarcoma cells. In all three breeds, osteosarcoma-associated loci and regions of reduced heterozygosity are enriched for genes in pathways connected to bone differentiation and growth. Several pathways, including one of genes regulated by miR124, are also enriched for somatic copy-number changes in tumors. CONCLUSIONS: Mapping a complex cancer in multiple dog breeds reveals a polygenic spectrum of germline risk factors pointing to specific pathways as drivers of disease.

Thorough investigation of a canine autoinflammatory disease (AID) confirms one main risk locus and suggests a modifier locus for amyloidosis.

Autoinflammatory disease (AID) manifests from the dysregulation of the innate immune system and is characterised by systemic and persistent inflammation. Clinical heterogeneity leads to patients presenting with one or a spectrum of phenotypic signs, leading to difficult diagnoses in the absence of a clear genetic cause. We used separate genome-wide SNP analyses to investigate five signs of AID (recurrent fever, arthritis, breed specific secondary dermatitis, otitis and systemic reactive amyloidosis) in a canine comparative model, the pure bred Chinese Shar-Pei. Analysis of 255 DNA samples revealed a shared locus on chromosome 13 spanning two peaks of association. A three-marker haplotype based on the most significant SNP (p<2.6×10(-8)) from each analysis showed that one haplotypic pair (H13-11) was present in the majority of AID individuals, implicating this as a shared risk factor for all phenotypes. We also noted that a genetic signature (F ST) distinguishing the phenotypic extremes of the breed specific Chinese Shar-Pei thick and wrinkled skin, flanked the chromosome 13 AID locus; suggesting that breed development and differentiation has played a parallel role in the genetics of breed fitness. Intriguingly, a potential modifier locus for amyloidosis was revealed on chromosome 14, and an investigation of candidate genes from both this and the chromosome 13 regions revealed significant (p<0.05) renal differential expression in four genes previously implicated in kidney or immune health (AOAH, ELMO1, HAS2 and IL6). These results illustrate that phenotypic heterogeneity need not be a reflection of genetic heterogeneity, and that genetic modifiers of disease could be masked if syndromes were not first considered as individual clinical signs and then as a sum of their component parts.

Antigen-specific human T-cell responses and T cell-dependent production of human antibodies in a humanized mouse model.

Humanized mice with a functional human immune system would be very useful for in vivo studies of human immunobiology. We have previously shown that cotransplantation of human fetal thymus/liver tissues and CD34(+) fetal liver cells into immunodeficient nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice leads to the development of multiple lineages of human lymphohematopoietic cells and formation of secondary lymphoid organs with normal architecture. Here, we evaluated the ability of these humanized mice to develop antigen-specific, T cell-dependent antibody responses after in vivo immunization with T-dependent antigen, 2,4-dinitrophenyl hapten-keyhole limpet hemocyanin (DNP(23)-KLH). Human T cells from DNP(23)-KLH-immunized mice showed strong proliferation in response to KLH in vitro. Furthermore, T cell-dependent production of DNP-specific human antibodies (mainly IgG1 and IgG2) was detected in all immunized mice. These results confirm that a functional human immune system can be established in immunodeficient mice through cotransplantation of human fetal thymus/liver tissues and CD34(+) hematopoietic stem/progenitor cells.

Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation.

Studies of the human immune system have been limited by the lack of an appropriate in vivo model. For this reason, efforts have been made to develop murine models with a functional human immune system. We report here that cotransplantation of human fetal thymus/liver tissues and CD34(+) hematopoietic stem/progenitor cells led to the development of sustained human hematopoiesis and a functional human immune system in immunodeficient NOD/SCID mice. The humanized mice showed systemic repopulation with a comprehensive array of human lymphohematopoietic cells, including T cells, B cells, and dendritic cells, and the formation of secondary lymphoid organs. Furthermore, these mice produce high levels of human IgM and IgG antibodies and mediate strong immune responses in vivo as demonstrated by skin xenograft rejection. Thus, the humanized NOD/SCID mice described in this paper provide a powerful model system to study human immune function.

Hyperbaric oxygen inhibits benign and malignant human mammary epithelial cell proliferation.

BACKGROUND: Hyperbaric oxygenation (HBO) therapy is the administration of 100%-inhaled oxygen to patients at increased atmospheric pressure. MATERIALS AND METHODS: We used an in vitro model to examine the effects of HBO on mammary cell proliferation. Normal mammary epithelia, primary tumor and metastatic tumor cells derived from the same patient and immortalized by transfection with the human papilloma virus E6 oncogene, as well as the MCF7 human mammary adenocarcinoma cell line, were studied. RESULTS: HBO (97.9% O2, 2.1% CO2, 2.4 atmospheres absolute) inhibited the proliferation of all 4 cell types as measured by light microscopy, [3H]thymidine uptake, a tetrazolium-based colorimetric assay and a clonogenicity assay. The anti-proliferative effect of HBO was time-dependent (p < 0.01 for all 4 cell types). Hyperoxia alone (95% O2, 5% CO2, 1 atmosphere absolute) and increased atmospheric pressure alone (8.75% O2, 2.1% CO2, 2.4 atmospheres absolute) also inhibited proliferation, but their effects were not as profound as HBO (p < 0.01 when either hyperoxia or increased pressure was compared to HBO for all 4 cell types). HBO enhanced the anti-proliferative effects of melphalan (p < 0.05), gemcitabine (p < 0.001) and paclitaxel (p < 0.001). The clonogenicity assay demonstrated that the effects of HBO were still evident 2 weeks after the exposure (p < 0.01 for all 4 cell types). Experiments using Hoechst-propidium iodide or annexin V-propidium iodide staining showed no HBO-induced increases in necrosis or apoptosis. CONCLUSION: HBO inhibits benign and malignant mammary epithelial cell proliferation, but does not enhance cell death.

Glucocorticoid-induced apoptosis of thymocytes: requirement of proteasome-dependent mitochondrial activity.

Thymocytes undergo negative and positive selection during development in the thymus. During this selection process, the majority of thymocytes are eliminated by apoptosis through signaling via TCR or die by neglect, possibly mediated through glucocorticoids. In this study, we report that thymocytes require molecular oxygen to undergo apoptosis induced by dexamethasone (DEX), a synthetic glucocorticoid, and treatment with N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibits thymocyte apoptosis in vivo as well as ex vivo. We detected elevated intracellular levels of hydrogen peroxide (H(2)O(2)) during DEX-induced apoptosis, which is reduced by NAC treatment, indicating that the elevated levels of intracellular H(2)O(2) are proapoptotic. We also show that loss of mitochondrial membrane potential, cytochrome c release, as well as caspase-3 activation induced by DEX are attenuated by NAC treatment. We identified the production site for H(2)O(2) as the ubiquinone cycle at complex III of mitochondria by using various inhibitors of the mitochondrial electron transport chain, and we show that the cell death events mediated by mitochondria are also significantly reduced when the inhibitors were used. Through inhibition of the proteasome, we also show that the production of H(2)O(2) and the cell death events mediated by mitochondria are regulated by proteosomal activities in DEX-induced thymocyte apoptosis. We conclude that in DEX-treated thymocytes, the increased production of H(2)O(2) originates from mitochondria and is proapoptotic for cell death mediated by mitochondria. We also conclude that all the apoptotic events mediated by mitochondria are regulated by proteasomes.