Biomarkers and genetics of canine visceral haemangiosarcoma

Visceral haemangiosarcoma (HSA) is a vascular endothelial cell cancer that arises in internal organs, especially in the spleen. HSA carries a very poor prognosis but is also difficult to diagnose. Therefore, dogs with HSA symptoms are often euthanized without full investigation. The goal of the thesis was to improve the diagnosis and prevention of this cancer by exploring several HSA biomarkers.The thesis comprises four independent biomarker studies regarding glycoproteins, genetics and Infrared spectral markers. The aim of the first study (chapter 2) was to validate the candidates for canine visceral HSA serum biomarkers, by performing tissue immuno- and lectin-labelling of HSA (n = 32) and HSA-like (n=26) formalin-fixed paraffin-embedded (FFPE) tissues with C7 (component complement 7), MGAM (maltase-glucoamylase), VTN (vitronectin) antibodies and DSA (Datura stramonium), WGA (Wheat germ agglutinin), SNA (Sambucus nigra) and PSA (Pisum sativum)lectins. The results showed lectin/immuno-positive signal on HSA cells, endothelial cell of the veins and arteries. IHC and LHC signal intensities were heterogeneous across the tissue types, diagnosis groups (HSA vs HSA-like) and tissue markers. A semi-quantitative assay was applied to assess and compare the levels of IHC/LHC signal intensities of each marker. Among the candidates tested, complement component 7 (C7) and DSA binding glycoproteins were considered the most promising tissue markers as they demonstrated the ability to distinguish HSA tissue from HSA-like tissues (e.g. hematoma), by the comparison of glycoprotein expression level.HSA is overrepresented in Golden Retrievers, German Shepherds and Labrador Retrievers which suggested that genetic background plays an important role in this cancer. Inherited germline mutation was hypothesized to be the major factor. The aim of chapter 3 was to identify germline variants/polymorphisms that were related to HSA, allowing for development of HSA predisposing genetic markers in the future. Whole exome sequence data analysis was performed in 28 dogs with HSA. The sources of data included novel whole exome sequencing from the samples collected in this study (n = 11) and database archival data (n = 17).  Control data (n = 247) were obtained from sequence read archive databases. Exome variants association scan was performed in parallel with functional annotation analysis. The results showed genetic variants/polymorphisms in four genes, including PLSCR3, FANCM, MRPL40 and ZNF704, were strongly associated with HSA. The allele frequencies were detected in over 80% of HSA cases and were mostly homozygous. While the allele frequencies in the control were around 50%, none was homozygous.  Gene set enrichment analysis showed five molecular pathways where genes with HSA-related variants resided, including Interferon gamma, Complement, Adipogenesis, Genes down-regulated in CD4 T-cells, and Genes down-regulated in marginal zone B-lymphocytes.In chapter 4, somatic mutations were investigated further in five samples, where matched blood-tumour samples were available, by performing whole exome sequencing and bioinformatics data analysis. Gene mutation candidates were manually selected based on; 1) their annotated functions, 2) co-mutated candidates which most HSA tumours have in common, and 3) genes that had high numbers of mutations.  Co-mutated (more than 60% of samples) somatic mutations were found in ADCY1, CNN2, ARHGAP45, ATXN2L, CYP1A2, KRT3, SBNO2, TUFM and U6 genes.  When considering the severity of molecular functions that were disrupted by gene mutations, genes containing one or more binding sites for MZF1 (myeloid zinc finger 1) in their promoter regions (DUSP6, NFKBID, COPS3, KPNB1, KIF21A, SERF2, PABPC1L and COL24A1) were found to have undergone mutations in canine HSA tumours.  Genes with high numbers of mutations included GLUL, RIDA, ESRRA, COL11A2, ZNF296, RSAD2, RIN2, XRCC1, POLRMT, MTFR2 and PSMD4, with high numbers of the mutations occurring in the 3'-UTR.  Combining all the mutations, the mitotic spindle pathway was found to be disrupted by mutations in NF1, SASS6, CLASP1, ARHGEF3 and STAU1 genes. The proportion of samples having mutations on gene TP53 and PIK3CA in this study cohort was 20%.The study in chapter 5 explored the capability of Fourier Transform Infrared (FTIR) imaging plus Partial Least Squares-Discriminant analysis (PLS-DA) in discriminating FFPE spleen tissue sections containing HSA (cancer), which could lead to the development of rapid, label-free tissue diagnostic technologies to aid in canine HSA differential diagnosis. FTIR images of 26 splenic HSA and 32 non-HSA splenic lesions were blind tested. The prediction model correctly identified 24 out of 26 HSA samples while 21 non-HSA samples were incorrectly predicted as cancer, which yielded a sensitivity of 92% and 34% specificity. Potential improvement for this technology is suggested.The studies in this thesis contributed to further development of canine visceral HSA biomarkers such as differential diagnostic markers, disease predisposing genetic markers and genomic markers for personalized treatments.

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