Development of a diagnostic assay for liver disease – a ‘One Health, One Medicine’ case study

Richard Mellanby0 commentsBlog

Liver disease is a major cause of morbidity and mortality in dogs and cats, but non-invasive tests such as ALT lack sensitivity and specificity. This article reports on a veterinary/human medicine collaboration to identify a better alternative.

Chronic liver disease occurs commonly in dogs and can cause severe clinical signs such as hepatic encephalopathy, ascites, loss of interest in food, weight loss and jaundice (1). Acute hepatitis is also well recognised in dogs and can occur secondary to numerous disease processes including drug reactions and infections (2, 3). Definitive treatment of both acute and chronic liver diseases in dogs is often challenging and is frequently associated with poor outcomes (4). For example, we recently reported that dogs with chronic hepatitis had a median survival time of just 38 days (4). A significant cause of the poor outcomes in dogs with liver disorders is the delay in disease detection; due to the limitations of current tests, liver pathology is often well advanced before it is biochemically detected, making successful treatment outcomes harder to achieve.

A definitive diagnosis of liver disease in companion animals requires a liver biopsy. This procedure typically requires an anaesthetic and even minimally invasive approaches such as tru-cut biopsy carry a significant risk of complications (5). These issues have driven the need for non-invasive tests of liver damage. The most commonly used biomarker to non-invasively assess hepatocellular damage in cats and dogs is the measurement of serum or plasma concentrations of alanine aminotransferase (ALT) (6, 7). In dogs and cats with liver pathology, the normal cellular architecture of the liver is damaged and ALT leaks into the circulation where it is detected in higher than normal concentrations.

However, there are two key limitations to the use of ALT as marker of liver damage. Firstly, it has suboptimal specificity; numerous other tissues, notably skeletal and cardiac muscle, also express ALT. Consequently, non-hepatic pathology can lead to a rise in circulating ALT concentrations. Secondly, ALT also has suboptimal sensitivity for detection of liver damage. It is becoming increasingly apparent that ALT assays have a poor sensitivity to detect histologically confirmed liver disease in many dogs. This was dramatically demonstrated in a recent study which found that the ALT assay had a sensitivity of only 45% for the detection of histologically confirmed acute hepatitis in Labrador dogs (8). Therefore, there is a clear need for a more sensitive and specific test to detect hepatocellular damage in companion animals.

MicroRNAs (miRNA) are small (~22 nucleotide-long) non-protein coding RNAs that regulate post-transcriptional gene expression which includes RNA silencing. The properties of circulating miRNAs that support their utility as biomarkers include organ specificity (for certain microRNAs), relativestability in blood and their ability to be amplified and measured by polymerase chain reaction (PCR). miR-122 is highly expressed in hepatocytes, accounting for up to 70% of the total liver miRNA content (9). Studies in human patients have demonstrated that miR-122 is a highly sensitive and specific marker of liver disease including drug-induced liver injury and ethanol-induced liver disease (10-13). Crucially, miR-122 has enhanced sensitivity and specificity when compared with the present gold standard marker of liver damage, ALT (11). For example, miR-122 accurately reports drug-induced liver injury after paracetamol overdose at first presentation when ALT is still within its reference range (11, 13).

The need for better diagnostic tests for canine liver disease and the growing evidence that miR-122 is a valuable biomarker of liver damage in human diseases catalysed a medicine-veterinary medicine collaboration which measured miR-122 concentration in a cohort of healthy dogs, dogs with non-hepatic disorders and histology confirmed liver disease. We discovered that miR-122 is significantly increased in dogs with liver disease compared to healthy dogs and ill dogs with non-hepatic illnesses (14). The upper limit of miR-122 concentrations in healthy dogs had a sensitivity of 79% and specificity of 95% for identifying liver disease. The diagnostic performance of miR-122 in this pilot study was significantly better than ALT in many previously reported studies (8).

Whilst this test is currently based around qPCR technologies, the development of next generation assays which can measure MicroRNAs in a quick, cost effective and accurate manner are well advanced which should allow for rapid expansion of point-of-care miR-122 quantification (15). This case study demonstrates the merits of veterinarians and medics working together on important, unmet needs of both patient groups with the aim of improving the welfare of humans and animals.

 

 

References

  1. Watson PJ, Roulois AJ, Scase TJ, Irvine R, Herrtage ME. Prevalence of hepatic lesions at post-mortem examination in dogs and association with pancreatitis. The Journal of small animal practice. 2010;51(11):566-72.
  2. MacPhail CM, Lappin MR, Meyer DJ, Smith SG, Webster CR, Armstrong PJ. Hepatocellular toxicosis associated with administration of carprofen in 21 dogs. Journal of the American Veterinary Medical Association. 1998;212(12):1895-901.
  3. Hutchins RG, Messenger KM, Vaden SL. Suspected carprofen toxicosis caused by coprophagia in a dog. Journal of the American Veterinary Medical Association. 2013;243(5):709-11.
  4. Kilpatrick S, Dreistadt M, Frowde P, Powell R, Milne E, Smith S, et al. Presence of Systemic Inflammatory Response Syndrome Predicts a Poor Clinical Outcome in Dogs with a Primary Hepatitis. PloS one. 2016;11(1):e0146560.
  5. Bigge LA, Brown DJ, Penninck DG. Correlation between coagulation profile findings and bleeding complications after ultrasound-guided biopsies: 434 cases (1993-1996). Journal of the American Animal Hospital Association. 2001;37(3):228-33.
  6. Lawrence YA, Steiner JM. Laboratory Evaluation of the Liver. The Veterinary clinics of North America Small animal practice. 2017;47(3):539-53.
  7. Chapman SE, Hostutler RA. A laboratory diagnostic approach to hepatobiliary disease in small animals. The Veterinary clinics of North America Small animal practice. 2013;43(6):1209-25, v.
  8. Dirksen K, Burgener IA, Rothuizen J, van den Ingh T, Penning LC, Spee B, et al. Sensitivity and Specificity of Plasma ALT, ALP, and Bile Acids for Hepatitis in Labrador Retrievers. Journal of veterinary internal medicine / American College of Veterinary Internal Medicine. 2017;31(4):1017-27.
  9. Bandiera S, Pfeffer S, Baumert TF, Zeisel MB. miR-122–a key factor and therapeutic target in liver disease. Journal of hepatology. 2015;62(2):448-57.
  10. Dear JW, Antoine DJ, Starkey-Lewis P, Goldring CE, Park BK. Early detection of paracetamol toxicity using circulating liver microRNA and markers of cell necrosis. British journal of clinical pharmacology. 2014;77(5):904-5.
  11. Dear JW, Clarke JI, Francis B, Allen L, Wraight J, Shen J, et al. Risk stratification after paracetamol overdose using mechanistic biomarkers: results from two prospective cohort studies. The lancet Gastroenterology & hepatology. 2018;3(2):104-13.
  12. McCrae JC, Sharkey N, Webb DJ, Vliegenthart AD, Dear JW. Ethanol consumption produces a small increase in circulating miR-122 in healthy individuals. Clinical toxicology. 2016;54(1):53-5.
  13. Antoine DJ, Dear JW, Lewis PS, Platt V, Coyle J, Masson M, et al. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. Hepatology. 2013;58(2):777-87.
  14. Oosthuyzen W, Ten Berg PWL, Francis B, Campbell S, Macklin V, Milne E, et al. Sensitivity and specificity of microRNA-122 for liver disease in dogs. Journal of veterinary internal medicine / American College of Veterinary Internal Medicine. 2018.
  15. Rissin DM, Lopez-Longarela B, Pernagallo S, Ilyine H, Vliegenthart ADB, Dear JW, et al. Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury. PloS one. 2017;12(7):e0179669.

 

 

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