Association of serum and fecal microRNA profiles in cats with gastrointestinal cancer and chronic inflammatory enteropathy.

BACKGROUND: Differentiation of gastrointestinal cancer (GIC) from chronic inflammatory enteropathies (CIE) in cats can be challenging and often requires extensive diagnostic testing. MicroRNAs (miRNAs) have promise as non-invasive biomarkers in serum and feces for diagnosis of GIC. HYPOTHESIS/OBJECTIVES: Cats with GIC will have serum and fecal miRNA profiles that differ significantly from healthy cats and cats with CIE. Identify serum and fecal miRNAs with diagnostic potential for differentiation between cats with GIC and CIE as compared to healthy cats. ANIMALS: Ten healthy cats, 9 cats with CIE, and 10 cats with GIC; all client-owned. METHODS: Cats were recruited for an international multicenter observational prospective case-control study. Serum and feces were screened using small RNA sequencing for miRNAs that differed in abundance between cats with GIC and CIE, and healthy cats. Diagnostic biomarker potential of relevant miRNAs from small RNA sequencing and the literature was confirmed using reverse transcription quantitative real-time PCR (RT-qPCR). RESULTS: Serum miR-223-3p was found to distinguish between cats with GIC and CIE with an area under the curve (AUC) of 0.9 (95% confidence interval [CI], 0.760-1.0), sensitivity of 90% (95% CI, 59.6-99.5%), and specificity of 77.8% (95% CI, 45.3-96.1%). Serum miR-223-3p likewise showed promise in differentiating a subgroup of cats with small cell lymphoma (SCL) from those with CIE. No fecal miRNAs could distinguish between cats with GIC and CIE. CONCLUSION AND CLINICAL IMPORTANCE: Serum miR-223-3p potentially may serve as a noninvasive diagnostic biomarker of GIC in cats, in addition to providing a much needed tool for the differentiation of CIE and SCL.

Association of fecal and serum microRNA profiles with gastrointestinal cancer and chronic inflammatory enteropathy in dogs.

BACKGROUND: Reliable biomarkers to differentiate gastrointestinal cancer (GIC) from chronic inflammatory enteropathy (CIE) in dogs are needed. Fecal and serum microRNAs (miRNAs) have been proposed as diagnostic and prognostic markers of GI disease in humans and dogs. HYPOTHESIS/OBJECTIVES: Dogs with GIC have fecal and serum miRNA profiles that differ from those of dogs with CIE. AIMS: (a) identify miRNAs that differentiate GIC from CIE, (b) use high-throughput reverse transcription quantitative real-time PCR (RT-qPCR) to establish fecal and serum miRNA panels to distinguish GIC from CIE in dogs. ANIMALS: Twenty-four dogs with GIC, 10 dogs with CIE, and 10 healthy dogs, all client-owned. METHODS: An international multicenter observational prospective case-control study. Small RNA sequencing was used to identify fecal and serum miRNAs, and RT-qPCR was used to establish fecal and serum miRNA panels with the potential to distinguish GIC from CIE. RESULTS: The best diagnostic performance for distinguishing GIC from CIE was fecal miR-451 (AUC: 0.955, sensitivity: 86.4%, specificity: 100%), miR-223 (AUC: 0.918, sensitivity: 90.9%, specificity: 80%), and miR-27a (AUC: 0.868, sensitivity: 81.8%, specificity: 90%) and serum miR-20b (AUC: 0.905, sensitivity: 90.5%, specificity: 90%), miR-148a-3p (AUC: 0.924, sensitivity: 85.7%, specificity: 90%), and miR-652 (AUC: 0.943, sensitivity: 90.5%, specificity: 90%). Slightly improved diagnostic performance was achieved when combining fecal miR-451 and miR-223 (AUC: 0.973, sensitivity: 95.5%, specificity: 90%). CONCLUSIONS AND CLINICAL IMPORTANCE: When used as part of a diagnostic RT-qPCR panel, the abovementioned miRNAs have the potential to function as noninvasive biomarkers for the differentiation of GIC and CIE in dogs.

Use of a videofluoroscopic feeding evaluation to guide management of dogs with congenital idiopathic megaoesophagus.

BACKGROUND: Evidence-based guidelines for determining dietary management in dogs with megaoesophagus are lacking. OBJECTIVES: This study looked to compare oesophageal clearance times (ECT) of liquid and two food consistencies using a contrast videofluoroscopy feeding evaluation, and to assess if recommendations made based on findings could improve regurgitation and quality of life in dogs with congenital megaoesophagus. METHODS: Twenty-one dogs with congenital megaoesophagus and nine healthy dogs received liquid, slurry, and meatball diets containing barium while in an upright position. Follow-up was performed to determine response to recommendations. RESULTS: Healthy dogs had significantly shorter median ECT for all consistencies (p < 0.001). In the megaoesophagus group, ECT varied by consistency and individual. The number of dogs in the megaoesophagus group with complete clearance was four (median ECT 10 min) for liquid, five (median ECT 5 min) for slurry, and two (median ECT 5 and 30 min, respectively) for meatballs. Partial clearance was seen in 11 dogs (median clearance 25%) with liquid, seven with slurry (median clearance 50%), and five with meatballs (median clearance 60%). Recommendations included altering current medications (13/21 dogs), diet consistency (6/21), time upright (12/21), water delivery (21/21), and adding activity (7/21). Regurgitation episodes/week decreased significantly from 5.5 to 2.5 (p < 0.001) at follow-up 3-5 weeks post-evaluation, with 95% of owners reporting improvement in quality of life. Seventy percent were alive 46-777 days after last recheck. Three dogs died from megaoesophagus associated complications (median survival 461 days after diagnosis). CONCLUSIONS: The findings of this study suggest that a videofluoroscopic feeding evaluation may help guide management of dogs with congenital megaoesophagus.

Evaluation of fecal microRNA stability in healthy cats.

BACKGROUND: Gastrointestinal (GI) cancer accounts for 14% of feline malignancies. There is a great need for reliable noninvasive diagnostic biomarkers to reach a timely diagnosis and initiate treatment. Fecal microRNAs (miRNAs) could be such a biomarker and have shown great potential in colorectal screening in people but have yet to be investigated in cats. OBJECTIVES: We aimed to evaluate the presence and stability of feline fecal miRNA under different storage conditions (room temperature [RT], 4, and -20°C) and to evaluate the expression levels of specific fecal miRNAs collected on three separate days (days 1, 4, and 7) in healthy cats. METHODS: Healthy cats were prospectively recruited. Fecal samples were collected, aliquoted, and stored for 24 hours at RT and then transferred to -20°C, stored for 24 hours at 4°C and then transferred to -20°C, or were immediately placed at -20°C on day 1 or at -20°C on days 4 and 7 postcollection. Expression of 22 miRNAs was investigated using quantitative real-time PCR. RESULTS: Ten miRNA assays worked well, and one, let-7b, was used for normalization. No differences in miRNA expression were seen between the three storage temperatures for the nine miRNAs investigated. Only miR-26a showed a significant increase in expression between samples of days 1 and 7. The rest of the miRNAs levels were stable over time. CONCLUSIONS: Fecal miRNA can be isolated from healthy cats. The expression was stable at different temperatures and for most of the miRNAs over time. Prospective studies evaluating fecal miRNA as biomarkers in cats with GI neoplasia are warranted.

Validation of a method for quantitation of the clopidogrel active metabolite, clopidogrel, clopidogrel carboxylic acid, and 2-oxo-clopidogrel in feline plasma.

INTRODUCTION: The clopidogrel active metabolite (CAM) is unstable and challenging to quantitate. The objective was to validate a new method for stabilization and quantitation of CAM, clopidogrel, and the inactive metabolites clopidogrel carboxylic acid and 2-oxo-clopiodgrel in feline plasma. ANIMALS: Two healthy cats administered clopidogrel to demonstrate assay in vivo utility. MATERIALS AND METHODS: Stabilization of CAM was achieved by adding 2-bromo-3'methoxyacetophenone to blood tubes to form a derivatized CAM (CAM-D). Method validation included evaluation of calibration curve linearity, accuracy, and precision; within and between assay precision and accuracy; and compound stability using spiked blank feline plasma. Analytes were measured by high performance liquid chromatography with tandem mass spectrometry. In vivo utility was demonstrated by a pharmacokinetic study of cats given a single oral dose of 18.75mg clopidogrel. RESULTS: The 2-oxo-clopidogrel metabolite was unstable. Clopidogrel, CAM-D, and clopidogrel carboxylic acid appear stable for 1 week at room temperature and 9 months at -80°C. Standard curves showed linearity for CAM-D, clopidogrel, and clopidogrel carboxylic acid (r > 0.99). Between assay accuracy and precision was ≤2.6% and ≤7.1% for CAM-D and ≤17.9% and ≤11.3% for clopidogrel and clopidogrel carboxylic acid. Within assay precision for all three compounds was ≤7%. All three compounds were detected in plasma from healthy cats receiving clopidogrel. DISCUSSION: This methodology is accurate and precise for simultaneous quantitation of CAM-D, clopidogrel, and clopidogrel carboxylic acid in feline plasma but not 2-oxo-clopidogrel. CONCLUSIONS: Validation of this assay is the first step to more fully understanding the use of clopidogrel in cats.

Hypoadrenocorticism mimicking protein-losing enteropathy in 4 dogs.

Four dogs referred for suspected protein-losing enteropathy based on clinical signs, severe hypoalbuminemia, and hypocholesterolemia, and in 2 dogs, abdominal effusion or peripheral edema, were diagnosed with hypoadrenocorticism. Dogs with hypoadrenocorticism may have features of protein-losing enteropathy, including ascites or peripheral edema, which have not been described in dogs with hypoadrenocorticism.

Hypoadrénocorticisme imitant l'entéropathie avec perte de protéines chez 4 chiens. Quatre chiens recommandés pour une entéropathie suspectée avec perte de protéines fondée sur les signes cliniques, accompagnée d'une hypoalbuméniémie grave et de l'hypocholestérolémie et, chez 2 chiens, d'une effusion abdominale ou d'un œdème périphérique, ont reçu un diagnostic d'hypoadrénocorticisme. Les chiens atteints d'hypoadrénocorticisme peuvent présenter des signes d'entéropathie avec perte de protéines, y compris de l'ascite ou de l'œdème périphérique, qui n'ont pas été décrits chez les chiens atteints d'hypoadrénocorticisme.(Traduit par Isabelle Vallières).