Editorial: can urine‐based metabolomics improve diagnosis of advanced fibrosis in NAFLD? Authors' reply

2. Taylor RS, Taylor RJ, Bayliss S, et al. Association between fibrosis stage and outcomes of patients with nonalcoholic fatty liver disease: a systematic review and meta-analysis. Gastroenterology. 2020. https://doi.org/10.1053/j.gastro.2020.01.043 [Epub ahead of print] 3. Hagstrom H, Thiele M, Roelstraete B, Soderling J, Ludvigsson JF. Mortality in biopsy-proven alcohol-related liver disease: a population-based nationwide cohort study of 3453 patients. Gut. 2020. https://doi.org/10.1136/gutjn l-2019-320446 [Epub ahead of print]. 4. Parker R, Aithal GP, Becker U, et al. Natural history of histologically proven alcohol-related liver disease: a systematic review. J Hepatol. 2019;71:586-593. 5. Younossi ZM, Loomba R, Rinella ME, et al. Current and future therapeutic regimens for nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Hepatology (Baltimore, MD). 2018;68:361-371. 6. Younossi ZM, Ratziu V, Loomba R, et al. Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial. Lancet. 2019;394:2184-2196. 7. Moolla A, de Boer J, Pavlov D, et al. Accurate non-invasive diagnosis and staging of non-alcoholic fatty liver disease using the urinary steroid metabolome. Aliment Pharmacol Ther. 2020;51:1188-1197 8. Vilar-Gomez E, Chalasani N. Non-invasive assessment of non-alcoholic fatty liver disease: clinical prediction rules and blood-based biomarkers. J Hepatol. 2018;68:305-315. 9. Hagstrom H, Talback M, Andreasson A, Walldius G, Hammar N. Ability of noninvasive scoring systems to identify individuals in the population at risk for severe liver disease. Gastroenterology. 2019;158:200-214


Editorial: can urine-based metabolomics improve diagnosis of advanced fibrosis in NAFLD? Authors' reply
We are pleased by the enthusiasm with which our paper on machine learning GMLVQ analysis of the urinary steroid metabolome has been received, and the recognition of its potential as a novel, non-invasive test to accurately stage NAFLD fibrosis. 1 In addition, with developing pharmacotherapies, the reliable tracking of fibrosis progression or regression will be critical, and the development of non-invasive tools to be able to achieve this is a clinical priority. 2 We do recognise that in our cohort, the FIB-4 test was highly predictive of advanced fibrosis and this almost certainly is a reflection of the high prevalence of advanced disease in our secondary care cohort. However, within our analysis, we also tested the ability of the urinary steroid metabolome to identify advanced NALFD fibrosis from healthy control subjects, who are perhaps more representative of a primary care population. 3 In this context, the GMLVQ analysis performed very well (AUC ROC = 0.98 [0.98-0.99]) and therefore we would anticipate that this test may have significant clinical potential to identify advanced NAFLD in the community.
Whilst we accept that the analysis of the urine sample requires specialist equipment and expertise, the methodology that we describe is widely used, 4 and in that respect, it is no different from a serum-based assay. In addition, the use of liquid chromatography tandem mass spectrometry is also becoming more common. 5 As a simple urine test, there is even the potential for it to be collected in the patients' own home; it requires no specialist equipment, no expertise in collection and no complex storage conditions. Indeed, with suitable containerisation, it could even be mailed directly, by the patient, to the analysing laboratory.
A further important consideration is that whilst the FIB-4 test may be perceived as a 'cheap' test as it incorporates routine blood tests (ALT, AST, platelets), cost estimates from the UK are in excess of £60 (~$75) per sample. 6 This takes into account, not only the physical measurement of the analytes, but also the personnel, time, equipment and processing prior to sample analysis. We have already proposed moving to a liquid chromatography tandem mass spectrometry platform 2,5 that has the benefit of not only dramatically increasing throughput, but in parallel, significantly reducing costs to the level of (or below) that for the routine FIB-4 analysis. We believe that this, combined with the very high degree of patient acceptability and ease of sampling, makes the urine steroid metabolome analysis a highly attractive proposition both for the accurate staging of NAFLD fibrosis as well as potentially screening for advanced disease in primary and secondary care.
There is still a great deal of work to be done and we certainly agree that the future development and direction for this test will be reliant upon a detailed validation and confirmation of findings in a larger cohort of patients with biopsy-proven NAFLD as well as healthy controls. In addition, it will also be important to examine the ability of the test to track changes in liver histology and crucially also to predict meaningful clinical outcomes.

ACK N OWLED G EM ENT
The authors' declarations of personal and financial interests are unchanged from those in the original article. 3 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. This study 9 emphasises that EPI is not investigated in most PC and CP patients. Furthermore, only a minority of patients received