Multi-omics approach identifies germline regulatory variants associated with hematopoietic malignancies in retriever dog breeds.

Histiocytic sarcoma is an aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart affects flat-coated retrievers (FCRs) at unusually high frequency, with 20% developing the lethal disease. The similar clinical presentation combined with the closed population structure of dogs, leading to high genetic homogeneity, makes dogs an excellent model for genetic studies of cancer susceptibility. To determine the genetic risk factors underlying histiocytic sarcoma in FCRs, we conducted multiple genome-wide association studies (GWASs), identifying two loci that confer significant risk on canine chromosomes (CFA) 5 (Pwald = 4.83x10-9) and 19 (Pwald = 2.25x10-7). We subsequently undertook a multi-omics approach that has been largely unexplored in the canine model to interrogate these regions, generating whole genome, transcriptome, and chromatin immunoprecipitation sequencing. These data highlight the PI3K pathway gene PIK3R6 on CFA5, and proximal candidate regulatory variants that are strongly associated with histiocytic sarcoma and predicted to impact transcription factor binding. The CFA5 association colocalizes with susceptibility loci for two hematopoietic malignancies, hemangiosarcoma and B-cell lymphoma, in the closely related golden retriever breed, revealing the risk contribution this single locus makes to multiple hematological cancers. By comparison, the CFA19 locus is unique to the FCR and harbors risk alleles associated with upregulation of TNFAIP6, which itself affects cell migration and metastasis. Together, these loci explain ~35% of disease risk, an exceptionally high value that demonstrates the advantages of domestic dogs for complex trait mapping and genetic studies of cancer susceptibility.

A synonymous germline variant in a gene encoding a cell adhesion molecule is associated with cutaneous mast cell tumour development in Labrador and Golden Retrievers.

Mast cell tumours are the most common type of skin cancer in dogs, representing a significant concern in canine health. The molecular pathogenesis is largely unknown, but breed-predisposition for mast cell tumour development suggests the involvement of inherited genetic risk factors in some breeds. In this study, we aimed to identify germline risk factors associated with the development of mast cell tumours in Labrador Retrievers, a breed with an elevated risk of mast cell tumour development. Using a methodological approach that combined a genome-wide association study, targeted next generation sequencing, and TaqMan genotyping, we identified a synonymous variant in the DSCAM gene on canine chromosome 31 that is associated with mast cell tumours in Labrador Retrievers. DSCAM encodes a cell-adhesion molecule. We showed that the variant has no effect on the DSCAM mRNA level but is associated with a significant reduction in the level of the DSCAM protein, suggesting that the variant affects the dynamics of DSCAM mRNA translation. Furthermore, we showed that the variant is also associated with mast cell tumours in Golden Retrievers, a breed that is closely related to Labrador Retrievers and that also has a predilection for mast cell tumour development. The variant is common in both Labradors and Golden Retrievers and consequently is likely to be a significant genetic contributor to the increased susceptibility of both breeds to develop mast cell tumours. The results presented here not only represent an important contribution to the understanding of mast cell tumour development in dogs, as they highlight the role of cell adhesion in mast cell tumour tumourigenesis, but they also emphasise the potential importance of the effects of synonymous variants in complex diseases such as cancer.

Unclassified sarcomas: a study to improve classification in a cohort of Golden Retriever dogs.

Morphologically, canine soft-tissue sarcomas (STSs) resemble human STSs. In humans, proper classification of STSs is considered essential to improve insight in the biology of these tumors, and to optimize diagnosis and therapy. To date, there is a paucity of data published on the significance of detailed classification of STSs in the dog. We revised a cohort (n = 110) of proliferative lesions obtained from a study in Golden Retrievers that were considered "soft tissue sarcoma, not otherwise specified or of uncertain subtype" in order to optimize the diagnoses of these lesions. The criteria according to the veterinary WHO classification, recent veterinary literature, and the WHO classification for humans were applied. Revision was initially based on morphologic characteristics of hematoxylin and eosin-stained histologic sections of the neoplasms. If considered necessary (n = 76), additional immunohistochemistry was applied to aid characterization. The diagnosis of STS was confirmed in 75 neoplasms (68%). Of this group, diagnosis of a specific subtype of the STSs was possible in 58 neoplasms. Seven neoplasms had morphologic characteristics that were suggestive for sarcoma subtypes only described in the WHO classification for humans. Seventeen neoplasms remained "unclassified STSs." Thirty-one lesions (28%) were diagnosed "neoplasm, not being STS." Four lesions (4%) were considered nonneoplastic. Because incorrect classification of a tumor could lead to inappropriate therapeutic intervention and prognostication, the results of our study clearly illustrate the importance of revision and further diagnosis of "unclassified STSs" in dogs.

Serum Galectin-3 Levels in Dogs with Metastatic and Non-metastatic Mammary Tumors.

Galectin-3 is implicated in tumor progression and metastasis. High levels of galectin-3 have been reported in intravasated cells in primary and metastatic tumor sites of canine malignant mammary tumors (CMMT). Nevertheless, it is still unknown whether this increase is limited to the site of the lesion or if it is a systemic feature. To better understand the pattern of the expression of galectin-3 and to investigate the possibility of using serum galectin-3 levels as a relevant biomarker in this disease, galectin-3 concentrations were determined in a series of sera from CMMT-bearing female dogs. None of the dogs included in the study had detectable metastases at the time of presentation. Animals were retrospectively divided into two groups dependent on whether or not they developed metastatic lesions during a 25-month follow-up period. Samples were collected from all dogs before surgery, 1 month after resection of the primary tumor and every 3 months during the postoperative period. Galectin-3 levels were significantly higher 1 month after than at the time of surgery (p=0.0058). Higher galectin-3 was found in samples collected 7 (p=0.0007), 10 (p=0.0061) and 13 months (p=0.0052) after surgery from dogs of the metastatic group when compared to those remaining free of development of detectable metastases. In conclusion, increased serum galectin-3 levels seem to be present in both metastatic and non-metastatic cases during the postoperative period, however, while in non-metastatic cases the values tend to return to baseline levels after surgery, in metastatic cases, levels remain persistently elevated.

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.

Hypoxia Up-Regulates Galectin-3 in Mammary Tumor Progression and Metastasis.

The tumor microenvironment encompasses several stressful conditions for cancer cells such as hypoxia, oxidative stress and pH alterations. Galectin-3, a well-studied member of the beta-galactoside-binding animal family of lectins has been implicated in multiple steps of metastasis as cell-cell and cell-ECM adhesion, promotion of angiogenesis, cell proliferation and resistance to apoptosis. However, both its aberrantly up- and down-regulated expression was observed in several types of cancer. Thus, the mechanisms that regulate galectin-3 expression in neoplastic settings are not clear. In order to demonstrate the putative role of hypoxia in regulating galectin-3 expression in canine mammary tumors (CMT), in vitro and in vivo studies were performed. In malignant CMT cells, hypoxia was observed to induce expression of galectin-3, a phenomenon that was almost completely prevented by catalase treatment of CMT-U27 cells. Increased galectin-3 expression was confirmed at the mRNA level. Under hypoxic conditions the expression of galectin-3 shifts from a predominant nuclear location to cytoplasmic and membrane expressions. In in vivo studies, galectin-3 was overexpressed in hypoxic areas of primary tumors and well-established metastases. Tumor hypoxia thus up-regulates the expression of galectin-3, which may in turn increase tumor aggressiveness.

The two main forms of histiocytic sarcoma in the predisposed Flatcoated retriever dog display variation in gene expression.

Examination of gene functions in specific tumor types improves insight in tumorigenesis and helps design better treatments. Due to the rarity of histiocytic/dendritic cell sarcoma in humans, it is difficult to accrue such knowledge. Therefore, comparative research of these cancers in predisposed dog breeds, such as the Flatcoated retriever, can be of value. Histiocytic sarcoma in the dog can be grouped into a soft tissue- and visceral form. The soft tissue form at first is localized, while the visceral form progresses more quickly to a terminal state, which might be related to variations in gene expression. Microarray analyses were performed on fresh-frozen tissue from Flatcoated retrievers with either soft tissue- or visceral histiocytic sarcoma. Expression differences of ten most significantly differentially expressed genes were validated with quantitative real-time PCR (q PCR) analyses. Q PCR analyses confirmed the significantly aberrant expression of three of the selected genes: C6 was up-regulated; CLEC12A and CCL5 were down-regulated in the visceral histiocytic sarcoma compared to the soft tissue form. The findings of our study indicate that these two forms of histiocytic sarcoma in the dog display a variation in gene expression and warrant analysis of functional changes in the expression of those genes in these rare sarcomas in man.

Differential expression of galectin-1 and galectin-3 in canine non-malignant and malignant mammary tissues and in progression to metastases in mammary tumors.

BACKGROUND: Galectin-1 and galectin-3 are carbohydrate-binding proteins that have been implicated in the pathobiology of several types of cancer. The aim of the present study was to investigate the expression pattern of both these galectins in canine non-neoplastic mammary tissues and mammary tumors (CMT). MATERIALS AND METHODS: Protein and mRNA expression of galectin-1 and -3 were assessed in 12 benign and 41 malignant CMT. RESULTS: Galectin-1 was overexpressed in the majority of malignant CMT cases in tumor cells and stroma. Its expression in malignant tumor cells was associated with smaller-sized tumours. Distant metastases presented a strong intensity of galectin-1 and reduced galectin-3 expression, while the opposite was observed in circulating tumor cells. Interestingly intravascular tumor cells presented galectin-3 up-regulation at the mRNA level. Double-labelling further made it clear that galectin-3 and galectin-1 expression did not overlap in normal-adjacent mammary and CMT cells. CONCLUSION: Taken together, our data suggest that malignant CMT cell sub-populations have alternating expression of galectin-1 or -3. This might confer survival advantage to tumour cells in different phases of tumour progression.

Estimated incidence rate and distribution of tumours in 4,653 cases of archival submissions derived from the Dutch golden retriever population.

BACKGROUND: A genetic predisposition for certain tumour types has been proven for some dog breeds. Some studies have suggested that this may also be true for the Golden retriever breed. The present study aimed to examine a possible existence of a tumour (type) predisposition in the Dutch population of Golden retrievers by evaluating annual estimated incidence rates compared to incidence rates from previous publications. A second aim was to evaluate whether incidences of various tumours differed as related to the diagnostic method chosen, being either cytology or histology. RESULTS: Tumours submitted to Utrecht University during the period 1998-2004 diagnosed either by means of cytology (n = 2,529) or histology (n = 2,124), were related to an average annual Dutch kennel club population of 29,304 Golden retrievers.Combining individual tumours from both the cytological and the histopathological data-set resulted in an annual estimated incidence rate of 2,242 for 100,000 dog-years at risk regarding tumour development in general.The most common cytological tumor diagnoses were 'fat, possibly lipoma' (35%), mast cell tumour (21%) and non-Hodgkin lymphoma (10%). The most commonly diagnosed tumours by histology were mast cell tumour (26%), soft tissue sarcomas (11%) and melanoma (8%). Both the cytological and histopathological data-sets, showed variation; in patient age distribution, age of onset and incidence of various tumours. CONCLUSION: Comparing our data with previous reports in non-breed-specified dog populations, the Golden retriever breed shows an increased risk for the development of tumours in general, as well as an increased risk for the development of specific tumour types, including the group of soft tissue sarcomas. Variations in age, location and incidence of various tumours were observed between the two data-sets, indicating a selection bias for diagnostic procedure.

Causes of death and the impact of histiocytic sarcoma on the life expectancy of the Dutch population of Bernese mountain dogs and Flat-coated retrievers.

Bernese mountain dogs and Flat-coated retrievers are predisposed to hereditary oncological diseases. Since 1986 several authors have reported a high prevalence of tumours in both breeds, especially malignant histiocytosis/histiocytic sarcoma, which has a negative influence on life expectancy. However, many earlier reports included relatively low numbers of dogs, distributed over a small number of broad categories, often using outdated disease criteria. The aim of this study was to provide new data on causes of death, and the relative role of tumours, especially histiocytic sarcoma, collected and verified in a large number of dogs of both breeds in co-operation with dog owners and veterinarians. The study demonstrates that the death of at least 55.1% of Bernese mountain dogs and 63.8% of Flat-coated retrievers is associated with malignant tumours. In addition, it appears that over 1/7 of all Bernese mountain dogs and Flat-coated retrievers die because of histiocytic sarcoma. This emphasises the need for further research on tumours, especially histiocytic sarcoma.

Gene expression profiling of histiocytic sarcomas in a canine model: the predisposed flatcoated retriever dog.

BACKGROUND: The determination of altered expression of genes in specific tumor types and their effect upon cellular processes may create insight in tumorigenesis and help to design better treatments. The Flatcoated retriever is a dog breed with an exceptionally high incidence of histiocytic sarcomas. The breed develops two distinct entities of histiocytic neoplasia, a soft tissue form and a visceral form. Gene expression studies of these tumors have value for comparable human diseases such as histiocytic/dendritic cell sarcoma for which knowledge is difficult to accrue due to their rare occurrence. In addition, such studies may help in the search for genetic aberrations underlying the genetic predisposition in this dog breed. METHODS: Microarray analysis and pathway analyses were performed on fresh-frozen tissues obtained from Flatcoated retrievers with localized, soft tissue histiocytic sarcomas (STHS) and disseminated, visceral histiocytic sarcomas (VHS) and on normal canine spleens from various breeds. Expression differences of nine genes were validated with quantitative real-time PCR (qPCR) analyses. RESULTS: QPCR analyses identified the significantly altered expression of nine genes; PPBP, SpiC, VCAM1, ENPEP, ITGAD (down-regulated), and GTSF1, Col3a1, CD90 and LUM (up-regulated) in the comparison of both the soft tissue and the visceral form with healthy spleen. DAVID pathway analyses revealed 24 pathways that were significantly involved in the development of HS in general, most of which were involved in the DNA repair and replication process. CONCLUSIONS: This study identified altered expression of nine genes not yet implicated in histiocytic sarcoma manifestations in the dog nor in comparable human histiocytic/dendritic sarcomas. Exploration of the downside effect of canine inbreeding strategies for the study of similar sarcomas in humans might also lead to the identification of genes related to these rare malignancies in the human.

Safety and efficacy field study of artesunate for dogs with non-resectable tumours.

The anti-malarial drug artesunate has shown anticancer activity in vitro and in preliminary animal experiments, but experience in patients with cancer is very limited. Pre-clinical studies in dogs indicated morbidity at high dosage levels. This study evaluated the effects of artesunate in canine cancer cell lines and in canine cancer patients. Four canine cell lines were tested in vitro for sensitivity towards artesunate and dihydroartemisinin (DHA; active metabolite of artesunate). The half-maximal inhibitory concentration (IC50) values for artesunate or DHA were 2-60 µM in three cell lines, while one cell line was much less sensitive to artesunate (IC50 337 µM) than to DHA (IC50 50 µM). A safety/efficacy field study with artesunate was conducted in 23 dogs with non-resectable tumours. Artesunate was administered for 7-385 days at a dosage of 651-1178 (median 922) mg/m(2). No neurological or cardiac toxicity was observed and seven dogs exhibited no adverse effects at all. Fever and haematological/gastrointestinal toxicity, mostly transient, occurred in 16 dogs. One dog died from pneumonia. Plasma artesunate and DHA levels fell below the limit of detection within 8-12 h after artesunate administration, while levels after two hours were close to 1 µM. Artesunate produced a long-lasting complete remission in one case of cancer and short-term stabilization of another seven cases.

The MTAP-CDKN2A locus confers susceptibility to a naturally occurring canine cancer.

BACKGROUND: Advantages offered by canine population substructure, combined with clinical presentations similar to human disorders, makes the dog an attractive system for studies of cancer genetics. Cancers that have been difficult to study in human families or populations are of particular interest. Histiocytic sarcoma is a rare and poorly understood neoplasm in humans that occurs in 15% to 25% of Bernese Mountain Dogs (BMD). METHODS: Genomic DNA was collected from affected and unaffected BMD in North America and Europe. Both independent and combined genome-wide association studies (GWAS) were used to identify cancer-associated loci. Fine mapping and sequencing narrowed the primary locus to a single gene region. RESULTS: Both populations shared the same primary locus, which features a single haplotype spanning MTAP and part of CDKN2A and is present in 96% of affected BMD. The haplotype is within the region homologous to human chromosome 9p21, which has been implicated in several types of cancer. CONCLUSIONS: We present the first GWAS for histiocytic sarcoma in any species. The data identify an associated haplotype in the highly cited tumor suppressor locus near CDKN2A. These data show the power of studying distinctive malignancies in highly predisposed dog breeds. IMPACT: Here, we establish a naturally occurring model of cancer susceptibility due to CDKN2 dysregulation, thus providing insight about this cancer-associated, complex, and poorly understood genomic region.

Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability.

DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development.

Sialylation regulates galectin-3/ligand interplay during mammary tumour progression--a case of targeted uncloaking.

Galectin-3 is involved both in facilitating detachment of cells from primary tumour sites and favouring cancer cell adhesion and survival to anoikis in the blood stream. The mechanisms behind these apparently contradictory roles of the lectin have not yet been resolved. In order to investigate possible interplays between galectin-3 and its ligands underlying their role in the metastatic process, we examined mucin-1 (MUC1) and epidermal growth factor receptor (EGFR), well-known galectin-3 ligands, as well as galectin-3-binding site expression in a series of spontaneous canine malignant mammary tumours (CMMT) and a metastatic CMMT cell line. Despite the fact that CMMT cells expressed MUC1 and EGFR homogeneously over their plasma membrane, intravascular tumour cells, positive for galectin-3, expressed MUC1 and EGFR in a more focal membrane localization. Moreover, MUC1 overexpression in primary CMMT was present in parallel with down-regulation of galectin-3. Furthermore, in the CMT-U27 cell line, galectin-3 knock-down led to increased MUC1 expression, while MUC1 knock-down led to down-regulation of the lectin. Finally, removal of sialic acid from both CMMT and CMT-U27 xenograft samples exposed galectin-3-ligands throughout the tumour tissue, whereas these ligands were only present in galectin-3-positive invading cells in untreated samples. Interestingly indeed, we show that in vessel-invading cells, there is interaction between galectin-3 and the T antigen in vivo. We therefore hypothesized that loss of galectin-3 and sialylation-related masking of its ligands, in conjunction with their overexpression in specific tumour cell subpopulations, are crucial in regulating adhesive/de-adhesive events in the progression and invasive capacity of metastatic cells.

Coordinated expression of galectin-3 and galectin-3-binding sites in malignant mammary tumors: implications for tumor metastasis.

Galectin-3 is a glycan-binding protein that mediates cell-cell and/or cell-extracellular matrix (ECM) interactions. Although galectin-3 is implicated in the progression of various types of cancers, the mechanisms by which galectin-3 enhances metastasis remain unclear. In order to elucidate the role of galectin-3 in the complex multistage process of cancer metastasis, we examined galectin-3 and galectin-3-binding site expression in a series of 82 spontaneous canine mammary tumors (CMT) and two CMT cell lines. Benign CMT tumors exhibited strong nuclear/cytoplasmic galectin-3 immunostaining, whereas malignant CMT tumors and metastases exhibited dramatically decreased galectin-3 expression with the majority of the immunostaining confined to the cytoplasm. Interestingly, intravascular tumor cells overexpressed galectin-3 regardless of their location. CMT-U27 xenografts displayed the same pattern of galectin-3 expression found in spontaneous malignant CMT. In parallel with the downregulation of galectin-3, malignant CMT displayed an overall loss of galectin-3-binding sites in the ECM and focal expression of galectin-3-binding sites mainly detected in intravascular tumor cells and endothelium. Furthermore, loss of galectin-3-binding sites was correlated with the downregulation of GLT25D1, a ß (1-O) galactosyltransferase that modifies collagen, and upregulation of stromal galectin-1. Finally, GLT25D1 mRNA expression was strikingly downregulated in malignant CMT-U27 compared with the benign cell line, and its expression was further decreased in a galectin-3 knockdown CMT-U27 cell line. We therefore hypothesized that the loss of galectin-3-binding sites in the ECM in conjunction with the overexpression of galectin-3 in specific tumor cell subpopulations are crucial events for the development of mammary tumor metastases.

Extensive expression of craniofacial related homeobox genes in canine mammary sarcomas.

The global gene expression in three types of canine mammary tumors: carcinoma, fibrosarcoma and osteosarcoma were investigated by Affymetrix gene array technology. Unsupervised clustering analysis revealed a close clustering of the respective tumor types, with fibrosarcomas clustering close to the osteosarcomas and the carcinomas clustering closer to non-malignant mammary tissues (NMTs). A number of epithelial markers were expressed in both carcinomas and NMTs, whereas the sarcomas expressed genes related to mesenchymal differentiation. A comparison of the gene expression profile of the sarcomas versus carcinoma/NMTs revealed that the sarcomas, in particular the osteosarcomas, showed a striking upregulation of a panel of homeobox genes previously linked to craniofacial bone formation. In line with this finding, osteosarcomas showed an upregulation of bone morphogenetic proteins (BMPs) and of genes associated with retinoic acid signaling. Increased homeobox gene expression in sarcomas was also confirmed at the protein level by immunohistochemical analysis of tumor tissue, and in an osteosarcoma cell line after stimulation by BMP-2. These findings suggest that the development of mammary sarcomas specifically involves triggering of a set of homeobox genes related to neural crest and craniofacial bone development.

TP53 gene mutations in canine osteosarcoma.

OBJECTIVE: To investigate mutations of the TP53 gene in canine osteosarcoma (OS). STUDY DESIGN: Clinical historic cohort study. ANIMALS: Client-owned dogs. METHODS: OS (n=59) were screened for mutations of the complete TP53 gene using polymerase chain reaction and the mutation was analyzed by single-strand conformational polymorphism. Clinical outcome of dogs with TP53-mutated OS were compared with dogs with OS without a mutation after complete surgical excision of the primary tumor. RESULTS: TP53 gene mutations were observed in 24 of 59 (40.7%) OS; 3 mutated OS had 2 mutations. The alterations consisted mainly of point mutations (74%). Dogs with mutated OS had a significantly shorter survival time (ST) after surgery than dogs with normal tumor TP53 gene expression (P=.03). Other significant prognosticators for ST and disease-free interval included elevated serum alkaline phosphatase (P<.01) and tumor grade (P=.01). CONCLUSION: TP53 genetic mutations are common in canine OS and may have a prognostic value. CLINICAL RELEVANCE: Mutations of the TP53 gene may influence survival and should be considered when evaluating canine OS.

A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues.

For a proper determination of relative mRNA expression levels with real-time quantitative PCR (Q-PCR) internal standards, such as the expression of reference genes, are of utmost importance. For cats, in contrast to dogs, no validation of reference genes has been published. Our goal was to evaluate frequently used reference genes for the analysis of relative mRNA levels from feline tissues in a SYBR Green-based Q-PCR protocol. First, primers were optimized on mRNA-derived cDNA from liver and kidney tissues of randomly chosen (healthy and diseased) cats. Then, the expression variation and stability of each reference gene within a specific tissue was determined. Dental roots and crowns, heart (left ventricle), renal, liver, lung, and mammary gland tissues from 3 to 11 cats of different breeds, sexes, ages, and disease status were included in this study. Averaging relative stabilities over these six tissues revealed the usefulness of each tested gene as reference gene. In order to compensate for the expression variation of a reference gene within a specific tissue, as much as six reference genes (e.g. RPL17, RPL30, RPS7, YWHAZ, and HPRT) were required to obtain highly reliable data in cat tissues. The optimal set of reference genes depended on the tissue analyzed and should, ideally, be selected and evaluated at the start of each experimental condition. A comparison with a similar evaluation in dogs revealed three issues: (i) most ribosomal genes are suitable in both species; (ii) good non-ribosomal reference genes differ; (iii) more feline than canine reference genes are required for proper analysis.