Circulating let‐7g is down‐regulated in Bernese Mountain dogs with disseminated histiocytic sarcoma and carcinomas – a prospective study

Cancer is a prevalent cause of mortality in Bernese mountain dogs (BMDs). Circulating microRNAs (miRNAs) are found in blood and have been identified as promising biomarkers in various neoplastic diseases in humans. In the current study, the expression profile of different types of miRNAs was investigated in healthy BMDs and BMDs with cancer. Seven healthy and six non‐treated BMDs with cancer [four with disseminated histiocytic sarcomas (DHS)] were enrolled in this study. Clinical evaluations including physical examination, blood analysis, urinalysis and diagnostic imaging were performed on all dogs. Twenty‐four different miRNAs were profiled from RNA isolated from whole blood preserved in PAXgene^® tubes using quantitative real‐time PCR (qPCR). The miRNA let‐7g was significantly down‐regulated in dogs with cancer (P = 0.002) and dogs with DHS (P = 0.011) compared with healthy controls. This miRNA is a known tumour suppressor and further analyses are warranted to assess its value as a non‐invasive biomarker for early detection of different types of cancer in BMDs.     

Checkpoint molecule expression by B and T cell lymphomas in dogs

Immunotherapies targeting checkpoint molecule programmed cell death 1 (PD‐1) protein were shown to be effective for treatment of non‐Hodgkin lymphoma in people, but little is known about the expression of PD‐1 or its ligand PD‐L1 by canine lymphoma. Therefore, flow cytometry was used to analyse expression of PD‐1 and PD‐L1 in canine lymphoma, using fine‐needle aspirates of lymph nodes from 34 dogs with B cell lymphoma (BCL), 6 dogs with T cell lymphoma (TCL) and 11 dogs that had relapsed. Furthermore, fine‐needle aspirates were obtained from 17 healthy dogs for comparison. Lastly, the impact of chemotherapy resistance on expression of PD‐1 and PD‐L1 was assessed in vitro. These studies revealed increased expression of PD‐L1 by malignant B cells compared to normal B cells. In the case of TCL, tumour cells and normal T cells both showed low to negative expression of PD‐1 and PD‐L1. In addition, tumour infiltrating lymphocytes from both BCL and TCL had increased expression of both PD‐1 and PD‐L1 expression compared to B and T cells from lymph nodes of healthy animals. In vitro, chemotherapy‐resistant BCL and TCL cell lines exhibited increases in both PD‐1 and PD‐L1 expression, compared to non‐chemotherapy selected tumour cells. These findings indicate that canine lymphomas exhibit upregulated checkpoint molecule expression, though the impact of checkpoint molecule expression on tumour biological behaviour remains unclear.     

Ultrasound-guided core needle biopsy in dogs with thyroid carcinoma

Currently, a histological diagnosis of highly vascularized canine (c) thyroid carcinoma (TC) is primarily obtained following excisional biopsy (EB) through thyroidectomy. Non-EBs are contraindicated in unresectable invasive cTCs due to their highly vascularized nature, which subsequently, lack histological diagnosis. We hypothesised ultrasound-guided core needle biopsy (UGCNB) to be a safe biopsy technique to obtain an accurate histological diagnosis in unresectable TCs. Nine client-owned dogs with suspected naturally occurring TC, presented for surgical excision, were included. First, a UGCNB was taken from the cervical tumour, followed by EB. Haemorrhage following UGCNB was evaluated preoperatively and once the tumour was surgically exposed by visual inspection and ultrasonography. Histological analysis, including cell organisation, tumour capsular and vascular invasion, and immunohistochemistry were performed and compared between both biopsy specimens (i.e., UGCNB and EB) of the same dog. Pre- and peroperative visual inspection revealed minor, localised haemorrhage, subsequent to the UGCNB, in 7/9 dogs. Histology of the EBs confirmed TC in 8/9 dogs and was inconclusive in 1/9 dogs. Histology of the UGCNBs revealed neoplastic thyroid tissue in 7/9 UGCNBs and was inconclusive in 1/9 UGCNBs. The remaining UGCNB contained no mass related tissue and was, therefore, excluded. Histological parameters (i.e., cell organisation, tumour capsular and vascular invasion) were not concordant between 6/8 included UGCNBs and their respective EB. Immunolabelling for thyroglobulin and calcitonin was concordant between all eight included UGCNBs and their respective EB. The remaining evaluated immunohistochemical markers (i.e., cyclooxygenase-2 [COX-2], P-glycoprotein and vascular endothelial growth factor [VEGF]) were concordant between the included UGCNBs and the EBs in 6/8 dogs. To conclude, UGCNBs can be safely obtained in suspected cTCs and enable a reliable diagnosis of the thyroid origin, thyroid cell origin and potential therapeutic markers such as COX-2, P-glycoprotein and VEGF. Subsequently, UGCNB enables clinicians to establish an individually tailored treatment plan in dogs with unresectable TC.     

Comparison between stearoyl‐CoA desaturase expression in milk somatic cells and in mammary tissue of lactating dairy cows

Stearoyl‐CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, where it inserts a cis‐double bond at the Δ9 position in a wide range of fatty acids. Investigating SCD expression in the bovine mammary gland generally requires invasive biopsy to obtain mammary tissue. The aim of this study was to evaluate the use of milk somatic cells as a non‐invasive alternative to biopsy for measuring mammary SCD expression in dairy cows. Both milk somatic cells and mammary tissue were collected from 14 Holstein‐Friesian cows and used for analysis of SCD expression by real‐time PCR. The SCD5 mRNA levels in mammary tissue compared with SCD1 were low, and for several milk somatic cell samples, SCD5 expression was even below the limit of detection. A significant relationship was found between SCD1 expression in milk somatic cells and in mammary tissue. In addition, SCD1 expression in milk somatic cells was significantly related to Δ9‐desaturase indices in milk, which are commonly used as an indicator of SCD1 activity within the mammary gland. Our study showed that milk somatic cells can be used as a source of mRNA to study SCD1 expression in dairy cows, offering a non‐invasive alternative to mammary tissue samples obtained by biopsy.     

Assessment of the value of quantitative thyroid scintigraphy for determination of thyroid function in dogs

Objective : To assess the value of thyroid scintigraphy to determine thyroid status in dogs with hypothyroidism and various non‐thyroidal illnesses. Methods : Thyroid hormone concentrations were measured and quantitative thyroid scintigraphy performed in 21 dogs with clinical and/or clinicopathological features consistent with hypothyroidism. Results : In 14 dogs with technetium thyroidal uptake values consistent with euthyroidism, further investigations supported non‐thyroidal illness. In five dogs with technetium thyroidal uptake values within the hypothyroid range, primary hypothyroidism was confirmed as the only disease in four. The remaining dog had pituitary‐dependent hyperadrenocorticism. Two dogs had technetium thyroidal uptake values in the non‐diagnostic range. One dog had iodothyronine concentrations indicative of euthyroidism. In the other, a dog receiving glucocorticoid therapy, all iodothyronine concentrations were decreased. Markedly asymmetric technetium thyroidal uptake was present in two dogs. All iodothyronine concentrations were within reference interval but canine thyroid stimulating hormone concentration was elevated in one. Non‐thyroidal illness was identified in both cases. Clinical Significance : In dogs, technetium thyroidal uptake is a useful test to determine thyroid function. However, values may be non‐diagnostic, asymmetric uptake can occur and excess glucocorticoids may variably suppress technetium thyroidal uptake and/or thyroid hormone concentrations. Further studies are necessary to evaluate quantitative thyroid scintigraphy as a gold standard method for determining canine thyroid function.     

Outcome following treatment of soft tissue and visceral extraskeletal osteosarcoma in 33 dogs: 2008–2013

Extraskeletal osteosarcoma (EOS) is a rare, highly malignant mesenchymal neoplasm arising from viscera or soft tissues characterised by the formation of osteoid in the absence of bone involvement. Owing to the rarity of these neoplasms very little information exists on treatment outcomes. The purpose of this study was to describe the outcome following surgical treatment of non‐mammary and non‐thyroidal soft tissue and visceral EOS in dogs. Thirty‐three dogs were identified; the most common primary tumour site was the spleen. Dogs that had wide or radical tumour excision had longer survival times compared with dogs that had only marginal tumour excision performed [median survival time of 90 days (range: 0–458 days) versus median survival time of 13 days (range: 0–20 days)]. The use of surgery should be considered in the management of dogs with non‐mammary and non‐thyroidal soft tissue and visceral EOS.     

p62/SQSTM1 expression in canine mammary tumours: Evolutionary notes

Recent studies highlighted the role of autophagy as a cardinal regulatory system for homeostasis and cancer‐related signalling pathways. In this context, the deregulated expression of p62 – Sequestosome1 (p62/SQSTM1) – a protein acting both as an autophagy receptor and signalling hub, has been associated with tumour development and chronic inflammation. Multiple clinical studies test drugs targeting autophagy, and even more research is on the way to clinical trials. However, no comparative investigations have been carried out to identify adequate preclinical models to assess p62‐based medicine. In veterinary oncology the role of p62 in cancer‐related pathways has been largely ignored. We compared p62 sequences in multiple organisms and found that canine p62 significantly diverges from the humans and from other animals sequences. Then, we chart by immunohistochemistry the expression levels of p62 in canine mammary tumours. A total of 66 tumours and 10 non‐neoplastic mammary samples were examined. The expression of p62 was higher in normal tissue and adenomas than carcinomas, with lowest levels of p62 protein detected in high grade carcinomas. In all cases examined the tumour stroma appeared to be p62‐negative. Taken together our results would suggest that in dogs the association between p62 expression and cancer cells overturns that reported in human breast carcinoma, where p62 accumulates in malignant cells as compared to normal epithelium. Thus, at least in canine mammary tumours, p62 should be not considered a tumour‐rejection antigen for an anti‐cancer immunotherapy.     

Altered expression of G proteins in thyroid gland adenomas obtained from hyperthyroid cats

OBJECTIVE: To determine whether expression of G proteins (G(i) and G(s)) is altered in thyroid gland adenomas obtained from hyperthyroid cats. SAMPLE POPULATION: Adenomatous thyroid glands obtained from 8 hyperthyroid cats and thyroid glands obtained from 4 age-matched euthyroid cats. PROCEDURE: Expression of G(i) and G(s) was quantified in enriched membrane preparations of thyroid gland tissue, using immunoblotting with G(i) and G(s) antibodies and toxin-catalyzed ADP-ribosylation. RESULTS: Expression of G(i) was significantly reduced in thyroid gland adenomas from hyperthyroid cats, compared with normal thyroid gland tissue from euthyroid cats. Expression of G(s) was similar between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: A decrease in expression of G in adenomatous thyroid glands of cats may reduce the negative inhibition of the cAMP cascade in thyroid cells, leading to autonomous growth and hypersecretion of thyroxine. Understanding the molecular mechanisms for hyperthyroidism in cats may lead to better treatment or, ultimately, prevention of the disease.     

MicroRNAs as tumour suppressors in canine and human melanoma cells and as a prognostic factor in canine melanomas

Malignant melanoma (MM) is one of the most aggressive cancers in dogs and in humans. However, the molecular mechanisms of its development and progression remain unclear. Presently, we examined the expression profile of microRNAs (miRs) in canine oral MM tissues and paired normal oral mucosa tissues by using the microRNA‐microarray assay and quantitative RT‐PCR. Importantly, a decreased expression of miR‐203 was significantly associated with a shorter survival time. Also, miR‐203 and ‐205 were markedly down‐regulated in canine and human MM cell lines tested. Furthermore, the ectopic expression of miR‐205 had a significant inhibitory effect on the cell growth of canine and human melanoma cells tested by targeting erbb3. Our data suggest that miR‐203 is a new prognostic factor in canine oral MMs and that miR‐205 functions as a tumour suppressor by targeting erbb3 in both canine and human MM cells.     

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.     

Photodynamic detection of canine mammary gland tumours after oral administration of 5‐aminolevulinic acid

5‐Aminolevulinic acid (5‐ALA) is widely used in photodynamic detection (PDD) and therapy. We evaluated the pharmacokinetics of 5‐ALA‐induced porphyrins and its effectiveness in PDD in dogs with mammary gland tumours (MGTs) following oral administration. Healthy dogs and those with MGTs (nine each) were orally administered 4 mg kg^?1 5‐ALA. Protoporphyrin IX (PpIX) was not detected in the plasma of healthy dogs but it peaked in dogs with MGT at 2 h after 5‐ALA administration. In the PDD study, 16 dogs with MGT were orally administered 40 mg kg^?1 5‐ALA, and MGT but not normal tissue showed red fluorescence after 2–4 h. Photon counts were 6635–63 890 and 59–4011 (median, 19 943 and 919) for MGT and non‐tumour tissues, respectively. Cell density strongly correlated with PpIX photon counts of MGT tissue of the dogs (R = 0.743, P = 0.0009). We suggest that 5‐ALA‐PDD might be an effective diagnostic tool for MGTs.     

Ultrasonographic evaluation of the thyroid gland in healthy, hypothyroid, and euthyroid Golden Retrievers with nonthyroidal illness

Ultrasound evaluation of the thyroid gland was performed in healthy, hypothyroid, and euthyroid Golden Retriever dogs with nonthyroidal illness (NTI) to determine the diagnostic usefulness of ultrasound for differentiating between euthyroid and hypothyroid dogs. Thirty-six healthy, 11 hypothyroid, and 35 euthyroid dogs with NTI were evaluated. Each thyroid lobe was examined ultrasonographically for size, shape, echogenicity, and homogeneity. Thyroid lobe volume was estimated by using the equation for an ellipsoid: pi/6(length X height x width). No differences were found between healthy dogs and euthyroid dogs with NTI. In the majority of euthyroid dogs, the thyroid lobes were fusiform and triangular in shape in longitudinal and transverse planes, respectively. The thyroid capsule appeared smooth. The thyroid parenchyma had a homogeneous echogenic pattern and usually was hyperechoic or isoechoic compared with the surrounding musculature. Ultrasound findings in hypothyroid dogs were more variable, including a greater frequency of round to oval-shaped thyroid lobes in the transverse imaging plane (P < .05), hypoechogenicity of the thyroid parenchyma compared with surrounding musculature (P < .001), and a decrease in the size and volume of the thyroid lobes and total volume of the thyroid gland (P < .05) compared with euthyroid dogs. Other findings in hypothyroid dogs included an irregular surface to the thyroid capsule, a heterogeneous pattern to the thyroid parenchyma, and differences in the echogenic pattern between the left and right thyroid lobes. Results suggest that determination of thyroid size and volume by ultrasound may be a useful adjunctive test for differentiating between hypothyroid and euthyroid dogs with NTI.