Circulating MicroRNAs as Non-invasive Biomarkers for Canine Cushing's Syndrome.

Canine Cushing's syndrome (hypercortisolism) can be caused by a pituitary tumor (pituitary-dependent hypercortisolism; PDH) or a cortisol-secreting adrenocortical tumor (csACT). For both cases, non-invasive biomarkers that could pre-operatively predict the risk of recurrence after surgery would greatly impact clinical decision making. The aim of this study was to determine whether circulating microRNAs (miRNAs) can be used as diagnostic (presence of PDH or csACT) and/or prognostic (disease recurrence, histological grade) non-invasive biomarkers for canine Cushing's syndrome. After a pilot study with 40 miRNAs in blood samples of healthy dogs (n = 3), dogs with PDH (n = 3) and dogs with a csACT (n = 4), we selected a total of 20 miRNAs for the definitive study. In the definitive study, these 20 miRNAs were analyzed in blood samples of healthy dogs (n = 6), dogs with PDH (n = 19, pre- and post-operative samples) and dogs with a csACT (n = 26, pre-operative samples). In dogs with PDH, six miRNAs (miR-122-5p, miR-126-5p, miR-141-3p, miR-222-3p, miR-375-3p and miR-483-3p) were differentially expressed compared to healthy dogs. Of one miRNA, miR-122-5p, the expression levels did not overlap between healthy dogs and dogs with PDH (p = 2.9x10-4), significantly decreased after hypophysectomy (p = 0.013), and were significantly higher (p = 0.017) in dogs with recurrence (n = 3) than in dogs without recurrence for at least one year after hypophysectomy (n = 7). In dogs with csACTs, two miRNAs (miR-483-3p and miR-223-3p) were differentially expressed compared to healthy dogs. Additionally, miR-141-3p was expressed significantly lower (p = 0.009) in dogs with csACTs that had a histopathological Utrecht score of ≥ 11 compared to those with a score of <11. These results indicate that circulating miRNAs have the potential to be non-invasive biomarkers in dogs with Cushing's syndrome that may contribute to clinical decision making.

Molecular markers of prognosis in canine cortisol-secreting adrenocortical tumours.

Hypercortisolism is caused by a cortisol-secreting adrenocortical tumour (ACT) in approximately 15%-20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol-secreting ACTs were included from 40 dogs, of which follow-up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate-high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT-PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour-transforming gene-1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor-1 (SF-1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets.

Heat shock protein expression analysis in canine osteosarcoma reveals HSP60 as a potentially relevant therapeutic target.

Heat shock proteins (HSP) are highly conserved across eukaryotic and prokaryotic species. These proteins play a role in response to cellular stressors, protecting cells from damage and facilitating recovery. In tumor cells, HSPs can have cytoprotective effects and interfere with apoptotic cascades. This study was performed to assess the prognostic and predictive values of the gene expression of HSP family members in canine osteosarcoma (OS) and their potential for targeted therapy. Gene expressions for HSP were assessed using quantitative PCR (qPCR) on 58 snap-frozen primary canine OS tumors and related to clinic-pathological parameters. A significant increased expression of HSP60 was found in relation to shorter overall survival and an osteoblastic phenotype. Therefore, the function of HSP60 was investigated in more detail. Immunohistochemical analysis revealed heterogeneous staining for HSP60 in tumors. The highest immunoreactivity was found in tumors of short surviving dogs. Next HSP expression was shown in a variety of canine and human OS cell lines by qPCR and Western blot. In two highly metastatic cell lines HSP60 expression was silenced using siRNA resulting in decreased cell proliferation and induction of apoptosis in both cell lines. It is concluded that overexpression of HSP60 is associated with a poor prognosis of OS and should be evaluated as a new target for therapy.

Expression of epidermal growth factor receptor in canine osteosarcoma: association with clinicopathological parameters and prognosis.

Expression of epidermal growth factor receptor (EGFR) is associated with aggressive growth and metastasis of a range of tumours, including osteosarcomas (OS), although some studies have reported no relevance to clinicopathological events or prognosis. The present study evaluated EGFR mRNA and protein expression in a panel of OS cell lines, normal bones, frozen primary OS and tissue microarrays. EGFR expression was significantly elevated in primary OS compared to normal bones and in metastases of OS to the lungs in comparison with extrapulmonary sites. However, there were no clinical or pathological associations with mRNA expression levels in frozen tumours. Tissue microarray analysis demonstrated that a subset of canine OS with high EGFR expression was associated with significantly shorter survival times and disease-free intervals. Cytoplasmic expression of EGFR was present in 75% of metastases and was similar to expression in primary tumours. EGFR expression alone is not a reliable predictor of outcome and other markers are necessary for further prognostic stratification of dogs with OS. However, these findings suggest that a subset of dogs may benefit from anti-EGFR adjuvant therapies.