Short communication Serum miR-181b-5p predicts ascites onset in patients with compensated cirrhosis Authors Ana Garcia Garcia de Paredes, Càndid Villanueva, Carolina Blanco, Joan Genescà, Nicolo Manicardi, Juan Carlos Garcia-Pagan, Jose Luis Calleja, Carlos Aracil, Rosa M. Morillas, Maria Poca, Beatriz Peñas, Salvador Augustin, Juan G. Abraldes, Eldimar Alvarado, Félix Royo, Maria Laura Garcia-Bermejo, Juan Manuel Falcon-Perez, Rafael Bañares, Jaime Bosch, Jordi Gracia-Sancho, Agustin Albillos Correspondence agustin.albillos@uah.es (A. Albillos). Graphical abstract miR-429 predicts the acute HVPG response to NSBB miR-181b-5p predicts ascites onset miR-452-5p miR-452-5p miR-429 miR-429 miR-885-5p miR-885-5p miR-181b-5p miR-181b-5p miR-122-5p miR-122-5p BASELINE 1 YEAR NSBB n=2 n = 17 Ascites onset n = 52 Patients with compensated cirrhosis and n = 19 clinically significant portal hypertension Placebo n = 105 n = 53 HVPG after intravenous propranolol HVPG (acute HVPG response to NSBB) (HVPG response) Serum samples Serum samples Highlights Lay summary  miR-181b-5p appears to be a useful serum Ascites marks the transition from the compensated to biomarker to anticipate ascites onset. decompensated stage in cirrhosis and indicates a worsening in prognosis. There are currently no easily  Low serum miR-181b-5p indicates low risk of as- accessible tools to identify patients with compensated cites in compensated cirrhosis. cirrhosis at risk of developing ascites. We evaluated  Low serum miR-429 reflects acute hemodynamic the levels of novel molecules termed microRNAs in the response to non-selective beta-blockers. blood of patients with compensated cirrhosis and observed that miR-181b-5p can predict which patients are going to develop ascites. https://doi.org/10.1016/j.jhepr.2021.100368 Short communication Serum miR-181b-5p predicts ascites onset in patients with compensated cirrhosis Ana Garcia Garcia de Paredes,1 Càndid Villanueva,2,3 Carolina Blanco,4 Joan Genescà,3,5 Nicolo Manicardi,6 Juan Carlos Garcia-Pagan,3,7 Jose Luis Calleja,8 Carlos Aracil,9 Rosa M. Morillas,3,10,11 Maria Poca,2 Beatriz Peñas,1 Salvador Augustin,5 Juan G. Abraldes,7,14 Eldimar Alvarado,2 Félix Royo,3,12 Maria Laura Garcia-Bermejo,4 Juan Manuel Falcon-Perez,3,12 Rafael Bañares,3,13 Jaime Bosch,3,7,15 Jordi Gracia-Sancho,3,6,# Agustin Albillos1,3,*,# 1 Gastroenterology and Hepatology Department, Hospital Universitario Ramon y Cajal, Instituto Ramon y Cajal de Investigacion Sanitaria (IRYCIS), Universidad de Alcala, Madrid, Spain; 2Hospital of Santa Creu and Sant Pau, Autonomous University of Barcelona, Hospital Sant Pau Biomedical Research Institute (IIB Sant Pau) Barcelona, Spain; 3Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Spain; 4Biomarkers and Therapeutic Targets Group, Instituto Ramon y Cajal de Investigacion Sanitaria (IRYCIS), Madrid, Spain; 5Liver Unit, Vall d’Hebron University Hospital, Vall d’Hebron Institute of Research (VHIR), Vall d’Hebron Barcelona Hospital campus, Autonomous University of Barcelona, Barcelona, Spain; 6Liver Vascular Biology Research Group, IDIBAPS Biomedical Research Institute, Barcelona, Spain; 7Barcelona Hepatic Haemodynamic Laboratory, Liver Unit, Institute of Digestive and Metabolic Diseases, August Pi i Sunyer Institute of Biomedical Research, Hospital Clínic, Barcelona, Spain; 8Gastroenterology and Hepatology Department, Hospital Universitario Puerta de Hierro, Puerta de Hierro Hospital Research Institute, Autonomous University of Madrid, Madrid, Spain; 9Institute of Biomedical Research, Arnau de Vilanova University Hospital (IRB Lleida), Lleida, Spain; 10 Liver Section, Hospital Universitari Germans Trias i Pujol, IGTP. Badalona, Spain; 11Universitat Autònoma de Barcelona, Spain; 12Exosomes Laboratory, Center for Cooperative Research in Biosciencies (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Bizkaia, 48160, Spain; 13 Gastroenterology and Hepatology Department, Hospital Universitario Gregorio Marañon, Instituto de Investigacion Sanitaria Gregorio Marañon (IiSGM), Universidad Complutense de Madrid, Madrid, Spain; 14Liver Unit, Division of Gastroenterology, University of Alberta, Edmonton, Canada; 15 Department of Biomedical Research and University Clinic for Visceral Medicine and Surgery, Inselspital, Bern, Switzerland JHEP Reports 2021. https://doi.org/10.1016/j.jhepr.2021.100368 Background & Aims: Treatment with non-selective beta-blockers (NSBBs) reduces the risk of ascites, which is the most common decompensating event in cirrhosis. This study aimed to assess the ability of a serum microRNA (miRNA) signature to predict ascites formation and the hemodynamic response to NSBBs in compensated cirrhosis. Methods: Serum levels of miR-452-5p, miR-429, miR-885-5p, miR-181b-5p, and miR-122-5p were analyzed in patients with compensated cirrhosis (N = 105). Hepatic venous pressure gradient (HVPG) was measured at baseline, after intravenous propranolol, and 1 year after randomization to NSBBs (n = 52) or placebo (n = 53) (PREDESCI trial). miRNAs were analyzed at baseline and at 1 year. Results: Nineteen patients (18%) developed ascites, of whom 17 developed ascites after 1 year. miR-181b-5p levels at 1 year, but not at baseline, were higher in patients that developed ascites. The AUC of miR-181b-5p at 1 year to predict ascites was 0.7 (95% CI 0.59–0.78). miR-429 levels were lower at baseline in acute HVPG responders to NSBBs (AUC 0.65; 95% CI, 0.53–0.76), but levels at baseline and at 1 year were not associated with the HVPG response to NSBBs at 1 year. Conclusions: Serum miR-181b-5p is a promising non-invasive biomarker to identify patients with compensated cirrhosis at risk of ascites development. Lay summary: Ascites marks the transition from the compensated to decompensated stage in cirrhosis and indicates a worsening in prognosis. There are currently no easily accessible tools to identify patients with compensated cirrhosis at risk of developing ascites. We evaluated the levels of novel molecules termed microRNAs in the blood of patients with compensated cirrhosis and observed that miR-181b-5p can predict which patients are going to develop ascites. © 2021 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction decompensation.1,2 Ascites results from an increase in portal Ascites develops in 5%–10% of patients with compensated pressure – estimated by the hepatic venous pressure gradient cirrhosis per year and represents the most frequent cause of (HVPG) which must increase above 10 mmHg, i.e. clinically sig- nificant portal hypertension (CSPH) – and the development of circulatory dysfunction and renal sodium retention. The devel- Keywords: Ascites; beta-blockers; cirrhosis; microRNA; portal hypertension. opment of CSPH is a hallmark in the natural history of cirrhosis, Received 5 July 2021; received in revised form 19 August 2021; accepted 8 September 2021; available online 27 September 2021 since it places patients at risk of ascites and other complications # Authors share senior authorship of portal hypertension.3 We currently lack other predictors of * Corresponding author. Address: Gastroenterology and Hepatology Department, ascites formation and, in this regard, there is a clinical need for Hospital Universitario Ramon y Cajal, M-607, km. 9.100, 28034 Madrid, Spain. E-mail address: agustin.albillos@uah.es (A. Albillos). non-invasive biomarkers. A recent study has shown that non- Short communication selective beta-blockers (NSBBs) reduce the risk of decompensa- collected at baseline and 1 year.2 A multivariate Cox propor- tion and death in patients with compensated cirrhosis and CSPH tional hazards regression was used with adjustment for treat- by approximately half.2 The benefit of NSBBs was predominant in ment arm and first-order interaction tested with a global patients with HVPG decreasing >10% from baseline at 1 year. likelihood ratio test. We plotted receiver-operating character- MicroRNAs (miRNAs) are non-coding RNAs that regulate istic (ROC) curves and calculated the AUC and corresponding cellular processes by repressing the translation of mRNA.4 miR- 95% CI to evaluate the diagnostic performances of the signifi- NAs are secreted by cells and retrieved in body fluids which, in cant miRNAs. We performed a sensitivity analysis calculating conjunction with their high stability, makes them attractive and time-to-event ROC curves in a survival data framework.9 All accessible biomarkers.5 Our group has recently identified a analyses were 2-tailed and significance was set at p <0.01 after serum signature of 5 miRNAs – miR-452-5p, miR-429, miR-885- adjustment for multiple comparisons (supplementary materials 5p, miR-181b-5p, and miR-122-5p – in decompensated cirrhosis and methods). that differentiates patients with diuretic-sensitive from those with refractory ascites (through miR-181b-5p) and identifies those achieving an HVPG response to NSBBs (by means of miR- Results 452-5p and miR-429).6 A recent study has also characterized a Patient characteristics set of circulating miRNAs that are altered in patients who We included 105 out of the 201 patients enrolled in the PRE- progress to acute-on-chronic liver failure.7 The aim of the pre- DESCI trial. The clinical characteristics of the study cohort and sent study was to evaluate our recently described serum miRNA that of the patients excluded for lacking paired archived samples signature as a biomarker to predict ascites formation and the were similar (Table 1). Fifty-two (49.5%) and 53 (50.5%) patients HVPG response to NSBBs in a large cohort of patients with belonged to the NSBB and placebo groups, respectively. The compensated cirrhosis and CSPH. median follow-up was 36.2 months (IQR 26.3–47.6). Patients and methods Serum miRNA levels and ascites formation Patients and study design Of the 105 patients, 19 (18%) developed ascites during follow-up, Serum samples were obtained from a registered collection 6 (11.5%) and 13 (24.5%) in the NSBB and placebo group, (C.0005353, Collections Section, Biobank Registry of Instituto de respectively. The median time to ascites formation was 26.3 Salud Carlos III, Madrid, Spain) of patients with compensated cirrhosis included in a randomized trial to evaluate the ability of NSBBs to prevent decompensation (PREDESCI trial; Table 1. Baseline characteristics of the study population and of patients from the PREDESCI trial with no archived samples. NCT01059396).2 Cirrhosis was diagnosed on histological or compatible clinical, biochemical, and ultrasonographic findings. Study population No archived All patients’ HVPG was measured at baseline and 1 year after N = 105 samples n = 96 being randomized to NSBBs or placebo. Only patients with baseline HVPG > Age (years) 65 (55–73) 60 (52–68) −10 mmHg were included. Patients were followed Male sex 63 (60%) 60 (62.5%) up to clinical decompensation (ascites, gastrointestinal bleeding, Cirrhosis etiology or overt hepatic encephalopathy) or death. Alcohol 16 (15.2%) 17 (17.7%) The novel miRNA signature – miR-452-5p, miR-429, miR-885- HCV 67 (63.8%) 46 (48%) 5p, miR-181b-5p, and miR-122-5p – was analyzed in serum Alcohol and HCV 10 (9.5%) 7 (7.3%) samples collected and archived at baseline and at 1 year after Other 12 (11.5%) 26 (27%) Child-Pugh A/B/C 92/13/0 69/27/0 randomization.8 The study protocol adhered to the ethical MELD score 6 (4–8) 5 (4-7) guidelines of the Declaration of Helsinki and was approved by Baseline HVPG (mmHg) 15 (12-18) 13 (11–16) the Ethics Committee for Clinical Research (IRB number: 362/19). Esophageal varices at baseline All participants granted written informed consent authorizing None 40 (38%) 49 (51%) the storage and research use of their samples. Small 65 (62%) 47 (49%) Treatment arm Placebo 53 (50.5%) 48 (50%) miRNA signature determination in serum samples NSBB 52 (49.5%) 48 (50%) Extraction of total RNA and subsequent analysis of individual Propranolol/carvedilol 33/19 34/14 miRNAs by quantitative real-time PCR was performed following Decompensation previously reported technical steps, and miR-103a-3p was also Before 12 months follow-up used as reference gene for data normalization.6 The relative Ascites 2 4 levels of each miRNA were calculated with the comparative Ct Hepatic encephalopathy 0 0 Variceal bleeding 0 2 (cycle threshold) method: DCt, with DCt = Average Cttested miRNA − Spontaneous bacterial peritonitis 0 1 Average CtmiR-103a-3p (average Ct = mean of the technical tripli- Hepatorenal syndrome 0 0 cate). DCt is inversely correlated with the amount of miRNA in After 12 months follow-up the serum (i.e., the higher the quantity or number of copies of Ascites 17 6 miRNA, the lower the DCt). Detailed information is displayed in Hepatic encephalopathy 4 5 the supplementary materials and methods. Variceal bleeding 4 1 Spontaneous bacterial peritonitis 4 1 Hepatorenal syndrome 0 2 Statistical analysis HVPG, hepatic venous pressure gradient; MELD, model for end-stage liver disease; Sample size was not prespecified. Instead, we included all pa- NSBBs, non-selective beta-blockers. Qualitative variables are provided as absolute tients from the PREDESCI trial with archived serum samples values and percentages. Quantitative variables are provided as median (IQR). JHEP Reports 2021 vol. 3 j 100368 2 months (IQR 21.2–38.5). Of the 19 patients, 17 (89.5%) developed Discussion ascites after 1 year. Our study shows that circulating miR-181b-5p is altered in pa- The serum levels of all 5 miRNAs were reduced at 1 year tients with compensated cirrhosis and that its elevated serum (Fig. S1). Baseline miRNA levels did not associate with ascites levels are associated with ascites development. These findings development (Fig. S2). miR-181b-5p serum levels at 1 year were extend the results of our previous study to compensated significantly higher in patients who eventually developed ascites cirrhosis. Thus, miR-181b-5p is emerging as a novel non-invasive than in those who did not (p = 0.001), with no differences in biomarker of ascites formation and progression through the other miRNAs levels (Fig. 1). stages of cirrhosis. miR-181b-5p did not correlate with the HVPG at 1 year (q = The goal of therapy in patients with compensated cirrhosis is −0.09; p = 0.34). In survival analysis adjusted by treatment, miR- to prevent decompensation. Accordingly, predictors of disease 181b-5p remained significantly associated with ascites formation progression, specifically ascites formation, are hugely important. (hazard ratio 0.73; 95% CI 0.53–0.99; p = 0.04). No interaction Circulating miRNAs have been extensively investigated in with NSBBs was found (p value for first-order interaction = 0.27). chronic liver disease10 and, to a lesser extent, in decompensated The AUC of miR-181b-5p in a binary data framework to identify cirrhosis7,11 to determine their association with cirrhosis etiology patients at risk of ascites was 0.7 (95% CI 0.59–0.78). When we and prognosis. However, we are unaware of studies addressing performed a time-to-event analysis, the AUCs of ascites onset at the value of circulating miRNAs in the transition from compen- 3, 6, 12, and 24 months after 1-year analysis were 0.92, 0.75, 0.70, sated to decompensated cirrhosis. and 0.41, respectively. The serum levels of the 5 miRNAs comprising the signature significantly changed after 1 year of follow-up, illustrating their dynamic levels as cirrhosis evolves. However, only miR-181b-5p Serum miRNAs levels and the hemodynamic response to NSBB was associated with progression from compensated to decom- miRNA levels showed no correlation with the HVPG at baseline pensated cirrhosis. Indeed, miR-181b-5p levels predicted ascites (Table S2). An acute hemodynamic response after propranolol occurrence since they were higher in individuals that developed was achieved in 72 of the 105 patients (68.5%). Serum miR-429 ascites in subsequent months. As reinforced by the time-to-event baseline levels were lower in responders than in non- analysis at different points, lower serum miR-181b-5p levels responders (p = 0.01) with no differences for the other miRNAs were associated with a low risk of ascites formation in the short- (Fig. S3). The AUC of miR-429 to identify acute response to NSBBs term. Notably, the predictive ability of miR-181b-5p in serum was 0.65 (95% CI 0.53–0.76). declined in parallel with the time elapsed to ascites onset, which miRNA levels at 1 year were similar in patients in the NSBB is the most likely explanation of the absence of an association and placebo groups (Table S3). HVPG was reduced > −10% from between baseline levels and ascites occurrence. We hypothesize baseline at 1 year in 53% and 28% of the patients in the NSBB and that the greater ability to predict ascites appearance of miR- placebo group, respectively (p = 0.015). In patients on NSBB, 181b-5p levels at 1 year compared to those at baseline was miRNAs levels at baseline and 1 year were not associated with due to the closer temporal relationship to the event of the the HVPG response at 1 year (Table S4). p = 0.08 p = 0.65 p = 0.39 15 15 10 hsa-miR-452-5p (∆Ct) hsa-miR-885-5p (∆Ct) 8 hsa-miR-429 (∆Ct) 10 10 6 4 5 5 2 0 0 0 -2 No ascites Will No ascites Will No ascites Will develop ascites develop ascites develop ascites p = 0.001 4 p = 0.22 10 hsa-miR-122-5p (∆Ct) hsa-miR-181b-5p (∆Ct) 2 8 6 0 4 -2 2 -4 0 -6 No ascites Will No ascites Will develop ascites develop ascites Fig. 1. MicroRNA serum levels at 1-year follow-up in patients who will and will not develop ascites. The middle horizontal line represents the median while the horizontal boundaries of the boxes represent the first and third quartiles. Levels of significance were assessed with the Mann-Whitney U test. DCt (cycle threshold) is inversely correlated with the amount of miRNA in the serum (i.e., the higher the quantity or number of copies of miRNA in the sample, the lower the DCt). JHEP Reports 2021 vol. 3 j 100368 3 Short communication former. Furthermore, NSBBs did not appear to influence the miR-429 was not associated with the hemodynamic response to predictive ability of miR-181b-5p. NSBBs in the long-term, a finding similar to the low correlation Previous evidence could provide some clues to understand observed between baseline miR-181b-5p levels and ascites the association between serum miR-181b-5p and ascites onset. development. Taken together, these results suggest that the Ascites in cirrhosis results from interrelated mechanisms, more distant the occurrence of the event of interest, the lower including progressive portal hypertension, endothelial dysfunc- the predictive ability of the miRNA. tion, and functional renal impairment. In this regard, miR-181b- The results of this study represent a step forward in the 5p seems involved in endothelial dysfunction and cardiovascular search for innovative non-invasive biomarkers of cirrhosis pro- remodeling in different settings, such as vascular stiffness gression and the response to NSBBs. In this study, we measured signaling in arterial hypertension,12 diabetic cardiomyopathy miRNA by the same robust and affordable technique described in development in mice,13 and progression of abdominal aortic decompensated cirrhosis.6 We identified the same miRNA as aneurysms.14 In cirrhosis with ascites, we observed a direct predictors of disease progression (i.e., miR-181b-5p) and correlation between circulating levels of miR-181b-5p and response to NSBBs (i.e., miR-429) in the compensated and creatinine.6 In the same line, miR-181b-5p, along with other decompensated settings, reinforcing the reproducibility of our circulating miRNAs, could be used to identify progression to results. We acknowledge some limitations. First, the analysis acute-on-chronic liver failure in decompensated cirrhosis.7 The included only those patients from the original trial with archived value of circulating miR-181b-5p to predict cirrhosis progression samples at baseline and at 1 year suitable for analysis as well as is independent of etiology, since this property of miR-181b-5p with HVPG assessment at 1 year. However, a related bias seems has been identified in both alcohol- and virus-related unlikely since we did not find significant differences between the cirrhosis.6,7,15,16 cohorts. Second, the number of patients that developed ascites NSBBs reduce the risk of portal hypertension-related com- was low. However, our study cohort was representative of the plications in compensated and decompensated cirrhosis.2,17-19 original trial in terms of treatment groups, ascites development, The hemodynamic response to NSBBs is heterogeneous and we and hemodynamic response. Finally, our findings require lack optimal non-invasive alternatives to assess it. In the present external validation. study, miR-429 levels were lower in patients who achieved an In summary, this study supports the usefulness of serum acute HVPG response to NSBBs, a finding in agreement with levels of miR-181b-5p as a non-invasive biomarker to anticipate results in the decompensated setting, where NSBB responders at ascites development in patients with compensated cirrhosis and 1 month also showed lower levels of this miRNA.6 Interestingly, CSPH who could benefit from a personalized follow-up. Abbreviations AA. Formal analysis: AGGP, LGB, JGS, AA. Funding acquisition: AA, JGS, RB. CSPH, clinically significant portal hypertension; Ct, cycle threshold; Investigation: AGGP, LGB, JGS, AA. Methodology: AGGP, LGB, JGS, AA. HVPG, hepatic venous pressure gradient; miRNAs, microRNAs; NSBBs, Supervision: AA, JGS. Drafting of the manuscript: AGGP, LGB, JGS, AA. non-selective beta-blockers; ROC, receiver-operating characteristic. Critical revision of the manuscript for important intellectual content: AGGP, CV, CB, JG, NM, JCGP, JLC, CA, RM, MP, BP, SA, JGA, EA, FR, LGB, JMFP, Financial support RB, JB, JGS, AA. Supported by grants from the Ministerio de Ciencia e Innovación and Instituto de Salud Carlos III (SAF 2017-86343-R awarded to A.A., PI20/ Data availability statement 01302 to A.A., PI18/01901 to R.B., CIBEREHD-16PI03 and PI20/00220 to The data shown in this article are available from the corresponding au- J.G.S.). A.G.G.P is the recipient of a grant from Ministerio de Ciencia e thors upon request. Innovación and Instituto de Salud Carlos III (Contrato Rio Hortega CM18/ 00091). Centro de Investigación Biomédica en Red en Enfermedades Supplementary data Hepáticas y Digestivas (CIBEREHD) is funded by the Instituto de Salud Supplementary data to this article can be found online at https://doi.org/1 Carlos III with grants cofinanced by the European Development Regional 0.1016/j.jhepr.2021.100368. Fund “A way to achieve Europe” (EDRF). Supported in part by a grant from Gilead Sciences (GLD19/00045). References Conflicts of interest Author names in bold designate shared co-first authorship The authors have declared that no personal or financial competing in- terests exist. [1] D’Amico G, Garcia-Tsao G, Pagliaro L. Natural history and prognostic in- Please refer to the accompanying ICMJE disclosure forms for further dicators of survival in cirrhosis: a systematic review of 118 studies. details. J Hepatol 2006;44(1):217–231. https://doi.org/10.1016/j.jhep.2005.10.013. [2] Villanueva C, Albillos A, Genesca J, Garcia-Pagan JC, Calleja JL, Aracil C, et al. 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