Biomarkers of acute-on-chronic liver failure (ACLF) progression

Abstract

The invention relates to the field of liver diseases and, more in particular, to methods for determining the presence of acute-on chronic liver-failure or methods for determining the risk of a patient suffering from cirrhosis or delayed pre-ACLF of developing ACLF. The invention also relates to methods for distinguishing patients suffering from compensated cirrhosis, acutely decompensated cirrhosis (ADC) or delayed ACLF from patients suffering from early ACLF or ACLF 1, 2, 3, methods for determining the degree of systemic inflammation and methods for determining the presence of a bacterial infection with sepsis.

Description

[0001] BIOMARKERS OF ACUTE-ON-CHRONIC LIVER FAILURE (ACLF) PROGRESSION

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the field of liver diseases and, more in particular, to methods for determining the presence of acute-on chronic liver-failure or methods for determining the risk of a patient suffering from cirrhosis or delayed pre-acute-on-chronic liver failure (ACLF) of developing ACLF. The invention also relates to methods for distinguishing patients suffering from compensated cirrhosis, acutely decompensated cirrhosis (ADC) or delayed ACLF from patients suffering from early ACLF or ACLF 1 , 2, 3, methods for determining the degree of systemic inflammation and methods for determining the presence of a bacterial infection with sepsis.

[0004] BACKGROUND OF THE INVENTION

[0005] The appearance of liver failure in a patient with cirrhosis represents a decisive time point both in terms of medical management and prognosis since this condition is frequently associated with rapidly evolving multi-organ dysfunction. Among the liver diseases, cirrhosis is by far the most frequent reason for hospital admission or liver transplantation. Only 20% of patients with advanced cirrhosis globally can be treated with liver transplantation owing to the great imbalance between donation and potential recipients. Cirrhosis progresses over several years. It is first compensated when the disease has not functional symptoms or complications. The occurrence of complications indicated the transition to the so-called decompensated wherein complications such ascites, gastrointestinal bleeding, renal failure, bacterial infections or hepatic encephalopathy appears.

[0006] The pathophysiology of Acutely Decompensated Cirrhosis (ADC), a syndrome characterized by the development of ascites, encephalopathy and / or gastrointestinal hemorrhage secondary to portal hypertension and liver failure, is being challenged by recent large-scale observational investigations combined with omics technology. The identification of a new syndrome, the Acute-on-Chronic Liver Failure (ACLF), characterized by single or multiple organ failure and high risk of 28-day mortality, of other phenotypes in patients with ADC without ACLF, and of systemic inflammation as a central pathophysiological mechanism, and the observation in Latin American patients that severity of systemic inflammation is influenced by genetic ancestry, place ADC into a new scenario that requires further investigation. Although the exact pathophysiology of the development of ACLF remains to be elucidated, unregulated inflammation is thought to be a major contributing factor. A characteristic feature of ACLF is its rapid progression, the requirement for multiple organ supports and high short and medium-term mortality, of 50-90%. Unfortunately, available therapeutic options for ACLF are limited. Liver transplantation remains the only definitive therapy for patients with ACLF, however, as has been mentioned above, limited availability of the donor organs limits its usefulness in the management of patients with ACLF.

[0007] Although in theory the currently used working definition of ACLF seems straightforward to use, medical practice is hampered by an often complex clinical picture, the absence of a clear differentiation from a patient with end-stage liver disease and problems in determining the ideal moment and / or necessity or usefulness of certain therapeutic interventions. With regard to prognosis, ACLF also poses problems since it coalesces both an acute potentially life- threatening insult and a severe chronic underlying disease. The few available prognostic models for decision making-ACLF for conventional management or liver transplantation are usually far for being satisfactory. The Model for End Stage Liver Disease (MELD) is the scoring system for assessing the severity of chronic liver diseases useful in determining prognosis and prioritizing for receipt of a liver transplant. However, MELD score has several limitations including interlaboratory variations for measurement of creatinine and international normalized ratio of prothrombin time and systematic adverse female gender bias.

[0008] Patients with ACLF are stratified into three stages of severity (ACLF-1 , ACLF 2, and ACLF-3) according to the number of organ failures (1 , 2, or 3 or more), which correlate with severity of systemic inflammation. Systemic inflammation, as estimated by C- reactive protein (CRP), cytokines, or white blood cell count (WBC), also correlates to patient’s clinical course. Among patients hospitalized with ADC without ACLF, who show lower systemic inflammation than patients with ACLF, there is a group known as pre- ACLF because they develop ACLF during hospitalization (“early” pre-ACLF) or later, during a follow-up period of 90 days (“delayed” pre-ACLF). The rest of the patients with ADC are stratified into “unstable” or “stable” decompensated cirrhosis depending on whether they require or not further hospitalization(s) during the 90-day follow-up. Patients with unstable cirrhosis present similar grade of systemic inflammation as do patients with stable decompensated cirrhosis, but present higher prevalence of events associated with severe portal hypertension, and shorter 1 -year survival. Current research in patients with ADC and ACLF is limited by the low accuracy of standard inflammatory biomarkers in cirrhosis. Well designated predictive models are necessary to establish an optimal assessment and management for ACLF, as well as to more accurately discriminate high and low severity ACLF phenotypes.

[0009] SUMMARY OF THE INVENTION

[0010] The authors of the present invention have developed a new scoring system, based on genes related to innate immune and B cells, which can discriminate high- and low- severity ADC phenotypes more accurately than current biomarkers and identified new relevant aspects on pathophysiology. In particular, the authors of the present invention have found that the expression levels of the different marker genes allows distinguishing patients suffering from compensated cirrhosis (phenotype 1), ADC (phenotype 2) or delayed ACLF (phenotype 3) from patients suffering from early ACLF (phenotype 4) or ACLF 1 , 2, 3 (phenotypes 5, 6 and 7, respectively). They have also shown that the levels of the marker genes change according to the progression of ACLF.

[0011] The authors of the present invention have also found that the same scoring system can be used in the determination of the degree of systemic inflammation in a subject and in the determination of the presence of a bacterial infection with sepsis in a subject.

[0012] Thus, in a first aspect, the present invention relates to an in vitro method for determining the presence of acute-on chronic liver-failure (ACLF) in a subject suffering from cirrhosis or delayed pre-ACLF which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has ACLF.

[0013] In another aspect, the present invention relates to an in vitro method for determining the risk of developing ACLF in a subject suffering from cirrhosis or delayed pre-ACLF which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has high risk of developing ACLF.

[0014] In another aspect, the present invention relates to an in vitro method for monitoring the progression of an ACLF in a subject which comprises: a) determining the expression levels of the HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject, b) comparing the expression levels of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point after the onset of ACLF, wherein if the expression level of HMGB2, RETN, ZNF608 and PYCARD genes are decreased with respect to said reference value, then it is indicative of ACLF resolution.

[0015] In another aspect, the present invention relates to an in vitro method for monitoring the effect of a therapy in a patient suffering from ACLF and being treated with said therapy which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point, wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject are decreased with respect to said reference value, there is indicative of the therapy is being effective.

[0016] In another aspect, the present invention relates to an in vitro method for determining the degree of systemic inflammation in a subject, which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has a severe systemic inflammation.

[0017] In another aspect, the present invention relates to an in vitro method for determining the presence of a bacterial infection with sepsis in a subject which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has a bacterial infection with sepsis.

[0018] In another aspect, the present invention relates to a kit or assay device, hereinafter the first kit or assay device of the invention, comprising reagents adequate for the determination of the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes.

[0019] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes; or the first kit of the invention for determining the presence of ACLF, for determining the risk of developing ACLF, for monitoring the progression of ACLF, for monitoring the effect of a therapy, for selecting a patient suffering from ACLF for a therapy or for determining the degree of systemic inflammation in a subject.

[0020] In another aspect, the present invention relates to a kit or assay device, hereinafter the second kit or assay device of the invention, comprising reagents adequate for the determination of the expression level of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28.

[0021] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28; or the second kit of the invention for determining the presence of ACLF, for determining the risk of developing ACLF, for monitoring the progression of ACLF, for monitoring the effect of a therapy, for selecting a patient suffering from ACLF for a therapy or for determining the degree of systemic inflammation in a subject.

[0022] In another aspect, the present invention relates to a method for determining the presence of acute-on chronic liver-failure (ACLF) in a subject suffering from cirrhosis or delayed pre-ACLF and treating the patient comprising: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said patient; and b) comparing the expression level of said genes with reference values for each gene, wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the patient is greater than the respective reference values, said patient is treated with an adequate therapy including antibiotics, diuretics, albumin, lactulose, beta-adrenergic blocker agents, vasoconstrictors, sclerosing agents, intrahepatic portosystemic shunting, plasma exchange, artificial respiratory support, renal hemodialysis, albumin dialysis, liver transplantation and / or combinations thereof.

[0023] In another aspect, the present invention relates to a method for determining the risk of developing ACLF in a subject suffering from cirrhosis or delayed pre-ACLF and treating the patient which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein if expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes, then the patient is treated with a therapy adequate for the prevention of ACLF including antibiotics, diuretics, albumin, lactulose, beta-adrenergic blocker agents, terlipressin, sclerosing agents, intrahepatic portosystemic shunting and / or combinations thereof. In another aspect, the present invention relates to a method for determining the degree of systemic inflammation in a subject and treating the subject which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject is greater than the reference value for said genes, then the patient is treated with a therapy adequate for severe systemic inflammation including antibiotics, corticosteroids, albumin, plasma exchange and / or combinations thereof.

[0024] In another aspect, the present invention relates to a method for determining the presence of a bacterial infection with sepsis in a subject and treating the subject which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject is greater than the reference value for said genes, then the patient is treated with a therapy adequate for bacterial infection and / or sepsis including antibiotics, vasoconstrictors, organ support including renal hemodialysis and / or artificial respiratory support.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1. A. Relative contribution of each of the 28 selected genes to the CLIF-SIG score; B. Comparison of the CLIF-SIG score at T1 between healthy subjects (HS) and patients from the low- and high-severity groups. Of note the high-severity group is split into “early” pre-ACLF and ACLF to illustrate the different CLIF-SIG score presented by these subgroups; C. Comparison of the CLIF-SIC score at T1 between healthy subjects (HS), and patients from the low- and high-severity groups; D. ROC curves differentiating low-severity from high-severity groups by CLIF-SIG (black line) and CLIF-SIC (gray line) scores at T1 in the train (solid line) and test (dashed line) sets with complete records for CLIF-SIC score; asterisks represent p<0.001 between scores in each set; E. Correlation between individual CLIF-SIG and CLIF-SIC scores at T1. Figure 2. Comparisons of CLIF-SIG score (A), CLIF-SIC score (B), CRP (C), and WBC (D) at T1 across the different phenotypes of ADC in Predict study patients from Cohort 1 ; HS: healthy subjects; SDC: stable decompensated cirrhosis; UDC: unstable decompensated cirrhosis.

[0027] Figure 3. A. Density curves of patients capturing upregulation, minor changes, and downregulation of CLIF-SIG score in Sub-cohort 1a. Stratification was based on symmetric cut-offs selected to keep a minimum proportion >20% in patients showing up- or downregulation. T1 values were weighted to increase sensitivity in patients placed in the extremes of the distribution; B. Individual changes of the CLIF-SIG score in Subcohort 1a. C. ROC curve of the CLIF-SIG score differentiating patients from the low- and high-severity groups, and critical score level (CSL, 0.386) that better discriminates these groups in Cohort 1 ; D. Course of CLIF-SIG score in Sub-cohort 1b;. E. The three phases of the systemic inflammatory reaction were totally or partially detected within the first 3 weeks after hospital admission in only 4 out of the 59 patients included in Sub-cohort 1b; F and G. Representative examples of the systemic inflammation curves in patients lacking the A-D phase alone, and the ITSI plus A-D phases; H. The figure illustrates CLIF-SIG score curves corresponding to three patients developing recurrent bursts of systemic inflammation (gray). Following the descending-recovery phase of a previous burst of systemic inflammation, patients developed an intense reactivation of the systemic inflammatory reaction. The figure also illustrates the three patients with normal CLIF-SIG score at T1 in whom ADC developed in the absence of a detectable systemic inflammatory reaction. Of note, estimated portions of each curve are represented by dashed lines. The shadowed area represents days prior T 1 .

[0028] Figure 4. A. Individual changes of CLIF-SIG score in the low- and high-severity group of patients included in Sub-cohort 1a. The upper discontinued horizontal line represents the CSL limiting patients with moderate (gray) and severe (black) systemic inflammation at T2; the lower discontinued horizontal line represents the median level CLIF-SIG score in healthy subjects (HS). The main results are expressed below the figure; B. Clinical course of most patients developing ACLF during hospital admission in Sub-cohort 1b took place above a CSL > 0.386. Of note, the horizontal axis is broken to reduce the impact of 4 patients with prolonged hospitalizations on the length of the figure. The early course of these 4 patients is also not represented in the left part of the figure to reduce the density of lines within this period. The vertical discontinued lines correspond to the first three weeks after T1 ; the diamonds represent time-points of ACLF detection; black and gray lines represent the course of patients who did and did not develop ACLF, respectively.

[0029] Figure 5. External validation of CLIF-SIG score genes. Results of analyses using a publicly available RNA-seq dataset (Monaco et al. Cell Rep 2019; 26: 1627-1640). The data set was composed of three groups: healthy subjects (HS) (n=40), non-cirrhotic patients with bacterial infections (n=32, including 20 with sepsis, and 12 with septic shock). (A) Heatmap showing expression of CLIF-SIG score genes across the 3 groups. (B) Principal component analysis (PCA) plot across the 3 study groups. (C) Box plots discriminating HS and non-cirrhotic patients with and without septic shock by the first principal component score.

[0030] Figure 6. Results derived from the score composed of 4 genes. A. Comparison of the 4- genes CLIF-SIG score at T1 between healthy subjects (HS) and patients from the low- and high-severity groups. Of note the high-severity group is split into “early” pre-ACLF and ACLF to illustrate the different 4-genes CLIF-SIG score presented by these subgroups; B. ROC curves differentiating low-severity from high-severity groups by CLIF-SIG (black lines) and CLIF-SIC (gray lines) scores at T1 in the train (solid lines) and test (dashed lines) sets with complete records for CLIF-SIC score; asterisks represent p<0.001 between scores in each set; C. Correlation between individual CLIF- SIG and CLIF-SIC scores at T1 .

[0031] Figure 7. Correlation between the gene expression of the 28 genes composing the CLIF- SIG score as determined by RNA-seq and their gene expression as determined by Nanostring and Fluidigm technologies.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] The authors of the present invention have found a new scoring system, based on genes related to innate immune and B cells, which discriminated high- and low-severity ADC phenotypes more accurately than current biomarkers and identified new relevant aspects on pathophysiology.

[0034] First method of the invention

[0035] In a first aspect, the present invention relates to an in vitro method, hereinafter the first method of the invention, for determining the presence of acute-on chronic liver-failure (ACLF) in a subject suffering from cirrhosis or delayed pre-ACLF which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has ACLF.

[0036] In a particular embodiment, the first method of the invention further comprises: a) determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11 , CRISPLD2, OSCAR, C3AR1 ,IGKV1 D-33.FPR1 ,SAMD3, MS4A4A, IGKV2-28; and b) comparing the expression level of said genes with their reference values; wherein an altered expression level of said genes when compared to the reference value for said genes, wherein said altered expression level is: an increased expression level of the genes IGFBP7, ORM1 ,ACSL1 ,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or a decreased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes, is indicative that said subject has ACLF.

[0037] Thus, in a particular embodiment, the first method of the invention comprises determining the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes and the expression level of at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes or at least 34 genes selected from the group consisting of the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28 genes.

[0038] The term “acute-on-chronic liver failure” or “ACLF” as used herein, refers to a syndrome characterized by acute deterioration of liver function in patients with compensated or decompensated cirrhosis who have failure of one or several organs, as explained below, and a poor short-term survival. According to the CANONIC study, said syndrome is classified in three grades of severity depending on the organ failure and mortality data:

[0039] - ACLF grade 1 : this grade includes 3 subgroups of patients: (1) patients with single kidney failure, (2) patients with single failure of the liver, coagulation, circulation or respiration who have a serum creatinine level ranging from 1 .5 to 1.9 mg / dL and / or mild moderate hepatic encephalopathy, and (3) patients with single cerebral failure who had a serum creatinine level ranging from 1.5 and 1.9 mg / dL. The 28-day and 90-day mortality rates are about 22.1% and 40.7%, respectively.

[0040] - ACLF grade 2: this group includes patients with 2 organ failures. The 28-day and 90-day mortality rates are about 32% and 52.3%, respectively.

[0041] - ACLF grade 3: this group includes patients with 3 organ failure or more. The 28- day and 90-day mortality rates are about 76.7% and 79.1 %, respectively.

[0042] The term “cirrhosis” as used herein, refers to a condition characterized by replacement of liver tissue by fibrosis and regenerative nodules which lead to loss of liver function. Ascites (fluid retention in the abdominal cavity) is the most common complication of cirrhosis. It is associated with a poor quality of life, increased risk of infection and poor long-term outcome. Other potentially life-threatening complications are hepatic encephalopathy (confusion and coma) and bleeding from esophageal varices. Cirrhosis has many possible manifestations. These signs and symptoms may be either as a direct result of the failure of liver cells or secondary to the resultant portal hypertension. Effects of portal hypertension include splenomegaly, gastroesophageal varices, and portocollateral circulation as a result of formation of venous collateral veins between portal system and the periumbilical veins as a result of portal hypertension.

[0043] Cirrhosis is divided in two clinical categories: compensated and decompensated cirrhosis. The term “compensated cirrhosis” as used herein, means that the liver is heavily scarred but can still perform many important bodily functions. Patients suffering from compensated cirrhosis experience few or no symptoms and can live without serious clinical complications. Patients at early stages of compensated cirrhosis are characterized by low levels of portal hypertension and lack of esophageal varices. Patients at advanced stages of compensated cirrhosis are characterized by higher levels of portal hypertension and presence of esophageal varices but without ascites and without bleeding. The term “decompensated cirrhosis” or “acute decompensated cirrhosis” as used herein, means that the liver is extensively scarred and unable to function properly. Patients suffering from decompensated cirrhosis develop a variety of symptoms such as fatigue, loss of appetite, jaundice, weight loss, ascites and / or edema, hepatic encephalopathy and / or bleeding. Patients at early stages of decompensated cirrhosis are characterized by the presence of ascites with or without esophageal varices in a patient that has never bled. Patients at advanced stages of compensated cirrhosis are characterized by more severe ascites alone or in association with bleeding, bacterial infections and / or hepatic encephalopathy.

[0044] The term “subject” or “patient” as used herein, refers to any animal classified as a mammal and includes but is not restricted to domestic and farm animals, primates and humans, for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. The subject is preferably a male or female human being of any age or race.

[0045] In a particular embodiment, the subject suffers unstable or stable acute decompensated cirrhosis.

[0046] The term “pre-ACLF” as used herein, refers to subjects that develop ACLF during hospitalization (“early” pre-ACLF) or later, during a follow-up period of 90 days (“delayed” pre-ACLF).

[0047] As it is used herein, the term “marker” or “marker gene” refers to a gene which is differentially expressed in populations showing different phenotypes and the differential expression of which, alone or in combination with other genes is correlated with a specific phenotype to a greater extent than what would be expected randomly.

[0048] The term “sample”, as used herein refers to biological material isolated from a subject and therefore includes biological samples. Said sample can contain any biological material suitable for detecting the desired marker and can comprise cells and / or non- cellular material from the subject. In general, a sample can be isolated from any suitable biological tissue or fluid; nevertheless, said sample is preferably a biofluid from the subject under study for putting the present invention into practice. Said biofluid sample can be a urine sample, a blood sample, a serum sample, etc., and can be obtained by means of any conventional method. In a particular embodiment, the sample is a biofluid, and in a more particular embodiment, said biofluid is blood.

[0049] In a first step, the first method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0050] As mentioned above, in a particular embodiment, the first method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1,IGKV1 D-33,FPR1,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0051] As it is used herein, the term “expression level” refers to the value of a parameter that measures the degree of expression of a specific gene. In a particular embodiment, said value can be determined by measuring the mRNA level of the gene of interest or a fragment thereof or by measuring the amount of protein encoded by said gene of interest or a variant thereof.

[0052] Virtually any conventional method for detecting and quantifying the expression level of a gene can be used within the framework of the present invention for detecting and quantifying the expression level of a specific gene. By way of non-limiting illustration, the expression level of a gene can be determined by means of quantifying the mRNA level of said gene or by means of quantifying the level of protein encoded by said gene.

[0053] Methods for determining the amount of mRNA are well-known in the state of the art. For example, the nucleic acid contained in the sample, such as the biofluid sample from the subject under study, is extracted according to conventional methods, for example, by means of using lytic enzymes, chemical solutions or fixing resins. The extracted mRNA can be detected by hybridization (for example by means of Northern blot analysis or DNA or RNA arrays (microarrays) after converting mRNA into labeled cDNA) and / or amplification by means of an enzymatic chain reaction. In general, quantitative or semi- quantitative enzymatic amplification methods are preferred. The polymerase chain reaction (PCR) or quantitative real-time RT-PCR or semi-quantitative RT-PCR technique is particularly advantageous. Primer pairs are preferably designed for the purpose of superimposing an intron to distinguish cDNA amplification from the contamination from genomic DNA (gDNA). Additional primers or probes, which are preferably labeled, for example with fluorescence, which hybridize specifically in regions located between two exons, are optionally designed for the purpose of distinguishing cDNA amplification from the contamination from gDNA. If desired, said primers can be designed such that approximately the nucleotides comprised from the 5’ end to half the total length of the primer hybridize with one of the exons of interest, and approximately the nucleotides comprised from the 3’ end to half the total length of said primer hybridize with the other exon of interest. Suitable primers can be readily designed by a person skilled in the art. Other amplification methods include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). The amount of mRNA is preferably measured quantitatively or semi-quantitatively. Relevant information about conventional methods for quantifying the expression level of a gene can be found, for example, in Sambrook et al., 2001 [Sambrook, J., et al., “Molecular cloning: a Laboratory Manual”, 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3],

[0054] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0055] In another particular embodiment, the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes and of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is determined by means of determining the expression level of the protein encoded by these genes, because increased expression of a gene is usually accompanied by an increase in the amount of corresponding protein, is also possible. The determination of the amount of a protein corresponding to the expression of a specific gene can be performed using any conventional method for protein detection and quantification, for example by means of an immunoassay, etc. By way of non-limiting illustration, said determination can be performed using antibodies with the capability to bind specifically to the protein to be determined (or fragments thereof with the antigenic determinants) and subsequent quantification of the antigen-antibody complex derivatives. The antibodies can be, for example, polyclonal sera, hybridoma supernatants or monoclonal antibodies, fragments of antibodies, Fv, Fab, Fab' and F(ab')2, scFv, diabodies, triabodies, tetrabodies, humanized antibodies, etc. Said antibodies may (or may not) be labeled with a marker. Illustrative, non-limiting examples of markers that can be used in the present invention include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme cofactors, enzyme substrates, enzyme inhibitors, etc. There is a wide range of well-known assays that can be used in the present invention, such as, for example, assays based on Western-blot or immunoblot techniques, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical or immunohistochemical techniques, etc. Other ways of detecting and quantifying the protein include affinity chromatogaphy, ligand binding assay techniques, particle-enhanced turbidimetric immunoassay (PETIA), etc.

[0056] The second step of the first method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value.

[0057] In a particular embodiment, the first method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 with a reference value for each of the genes.

[0058] In some embodiments, the expression levels of the genes under consideration are normalized. In order to normalize the values of mRNA expression among the different samples, it is possible to compare the expression levels of the mRNA of interest in the test samples with the expression of a control RNA. A “control RNA” as used herein, relates to RNA whose expression levels do not change or change only in limited amounts in tumor cells with respect to non-tumorigenic cells. Preferably, the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions. Preferred housekeeping genes for use in the present invention include p-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin, IPO8, HPRT, GAPDH, PSMB4, tubulin and p-actin.

[0059] The term “reference value” as used herein, refers to a laboratory value used as a reference for the values / data obtained from samples obtained from the subjects. The reference value (or reference level) can be an absolute value, a relative value, a value which has an upper and / or lower limit, a series of values, an average value, a median, a mean value, or a value expressed by reference to a control or reference value. A reference value can be based on the value obtained from an individual sample, such as, for example, a value obtained from a sample from the subject object of study but obtained at a previous point in time. The reference value can be based on a high number of samples, such as the values obtained in a population of the subjects of the chronological age group coinciding with that of the subject object of study or based on a set of inclusion or exclusion samples of the sample to be analyzed. For example, the reference value can be based on the expression level of the marker to be analyzed obtained from subjects who have cirrhosis or delayed pre-ACLF. In another embodiment, the reference value for the expression level of the gene or genes of interest is the mean level of expression of said gene or genes in a pool of samples from the same type as the sample that is being analyzed obtained from a plurality of subjects suffering from cirrhosis or delayed pre-ACLF.

[0060] Once the reference value has been established, the level of HMGB2, RETN, ZNF608 and PYCARD genes, and optionally IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A or IGKV2-28 expressed in samples from subjects can be compared with this reference value, and thus be assigned a level of “increased”, “decreased” or “equal”.

[0061] In the context of the present invention, it is considered that an expression level of HMGB2, RETN, ZNF608, PYCARD, IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 genes in the sample from the subject is “increased” or “greater than” the reference value for said markers when the expression level of these genes in the sample from the subject increases, for example, 5%, 10%, 25%, 50%, 100% or even more when compared with the reference value for said genes, or when it increases, for example, at least 1.1 -fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more when compared with the reference value for said markers.

[0062] In the context of the present invention, it is also considered that an expression level of the markers of interest, (i.e. HMGB2, RETN, ZNF608, PYCARD, IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 genes) in the sample from the subject is “decreased” or “less than” the reference value for said markers when the expression level of these genes in the sample from the subject decreases, for example, 5%, 10%, 25%, 50%, 75%, or even 100% when compared with the reference value for said markers.

[0063] In the context of the present invention, it is also considered that an expression level of the markers of interest (i.e. HMGB2, RETN, ZNF608, PYCARD, IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 genes) in the sample from the subject is “equal to” the reference value for said markers when the expression level of these genes is substantially unchanged with respect to the reference value; for example, it is considered that the expression level of HMGB2, RETN, ZNF608, PYCARD, IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33.FPR1 ,SAMD3, MS4A4A, IGKV2-28 genes in the sample from the subject under study is “equal to” the reference value when the levels differ by not more than 0.1%, not more than 0.2%, not more than 0.3%, not more than 0.4%, not more than 0.5%, not more than 0.6%, not more than 0.7%, not more than 0.8%, not more than 0.9%, not more than 1 %, not more than 2%, not more than 3%, not more than 4%, not more than 5%, or not more than the percentage value which is the same as the error associated with the experimental method used in the determination. Once the comparison is made between the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject and the reference value for said markers, the first method of the invention allows determining if a subject has ACLF based on if the expression level of HMGB2, RETN, ZNF608 and PYCARD genes is increased with respect to the reference value.

[0064] In a particular embodiment, the first method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33.FPR1 ,SAMD3, MS4A4A, IGKV2-28, and: if the expression level of GFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33 genes is increased with respect to the reference value, or

[0065] - the expression level of XCL1 , GZMH, GNLY, FASLG or SAMD3 genes is decreased with respect to the reference value it is indicative that the subject has ACLF.

[0066] Second method of the invention

[0067] The scoring system developed by the authors of the present invention allows distinguishing patients suffering from compensated cirrhosis (phenotype 1), ADC (phenotype 2) or delayed ACLF (phenotype 3) from patients suffering from early ACLF (phenotype 4) or ACLF 1 , 2, 3 (phenotypes 5, 6 and 7, respectively). Since the presence of delayed ACLF or of cirrhosis may result in the development of ACLF, then the method developed by the inventors also allows predicting whether a patient suffering from cirrhosis or delayed pre-ACLF is at high risk of developing ACLF. Accordingly, in another aspect, the present invention relates to an in vitro method, hereinafter the second method of the invention, for determining the risk of developing ACLF in a subject suffering from cirrhosis or delayed pre-ACLF which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has high risk of developing ACLF.

[0068] In a particular embodiment, the second method of the invention further comprises: a) determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1,IGKV1D-33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 ; and b) comparing the expression level of said genes with their reference values; wherein an altered expression level of said genes when compared to the reference value for said genes, wherein said altered expression level is: an increased expression level of the genes IGFBP7, ORM1 ,ACSL1 ,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or

[0069] - a decreased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes, is indicative that said subject has high risk of developing ACLF.

[0070] Thus, in a particular embodiment, the second method of the invention comprises determining the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes and the expression level of at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes or at least 34 genes selected from the group consisting of the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28 genes.

[0071] The expression “determining the risk” or “prediction of the risk”, or similar, as used herein, is synonymous of the expression “assessing the risk” or “assessment of the risk”, means that the present invention makes it possible to predict, estimate or evaluate the risk of a patient with cirrhosis or delayed pre-ACLF to develop ACLF. The prediction of risk generally implies that the risk is either increased or reduced. As it will be understood by those skilled in the art, the prediction (or the risk), although preferred to be, need not be correct for 100% of the cirrhosis or delayed pre-ACLF patients to be evaluated. The term, however, requires that a statistically significant portion of cirrhosis or delayed pre-ACLF patients can be identified as having an increased probability of having ACLF. Whether a subject is statistically significant can be determined without further ado by the person skilled in the art by using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%. The p-values are, preferably 0.05, 0.025, 0.001 , 0.0001 or lower.

[0072] The terms “ACLF”, “cirrhosis”, “compensated cirrhosis”, “decompensated cirrhosis”, “pre- ACLF”, “marker”, “subject” and “sample” have been defined or explained above, and these definitions are applicable to the second method of the invention.

[0073] In a particular embodiment, the subject suffers unstable or stable acute decompensated cirrhosis.

[0074] In another particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood.

[0075] In a first step, the second method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0076] In a particular embodiment, the second method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0077] The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the second method of the invention.

[0078] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11, CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0079] The second step of the second method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value.

[0080] In a particular embodiment, the second method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0081] The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the second method of the invention.

[0082] Once the comparison is made between the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject and the reference value for said markers, the second method of the invention allows determining if a subject has high risk of developing ACLF based on if the expression level of HMGB2, RETN, ZNF608 and PYCARD genes is increased with respect to the reference value.

[0083] In a particular embodiment, the second method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33.FPR1.SAMD3, MS4A4A, IGKV2-28, and: if the expression level of IGFBP7, ORM1 ,ACSL1,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11, OSCAR, IGKV2-28, IGKV2-33 genes is increased with respect to the reference value, or the expression level of XCL1 , GZMH, GNLY, FASLG or SAMD3 genes is decreased with respect to the reference value it is indicative that the subject has a high risk of developing ACLF.

[0084] Third method of the invention

[0085] The authors of the present invention have also observed that the score also acts a marker of the progression of the diseases and thus, it can be used for monitoring the clinical course of the patient. Accordingly, in another aspect, the present invention relates to an in vitro method, hereinafter the third method of the invention, for monitoring the progression of an ACLF in a subject which comprises: a) determining the expression levels of the HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject, b) comparing the expression levels of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point after the onset of ACLF, wherein if the expression level of HMGB2, RETN, ZNF608 and PYCARD genes are decreased with respect to said reference value, then it is indicative of ACLF resolution.

[0086] In a particular embodiment, the third method of the invention further comprises: a) determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33.FPR1.SAMD3, MS4A4A, IGKV2-28 ; and b) comparing the expression level of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point after the onset of ACLF, wherein an altered expression level of said genes when compared to reference value of said genes in a sample is indicative of ACLF resolution, wherein said altered expression level is: a decreased expression level of the genes IGFBP7, ORM1 ,ACSL1,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or an increased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes.

[0087] Thus, in a particular embodiment, the third method of the invention comprises determining the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes and the expression level of at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes or at least 34 genes selected from the group consisting of the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28 genes.

[0088] The terms “ACLF”, “marker”, “subject” and “sample” have been defined or explained above, and these definitions are applicable to the third method of the invention.

[0089] In a particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood. The term “ACLF resolution”, as used herein, refers to a situation in which a subject previously diagnosed with ACLF recovers from the disease. The determination of whether a subject is recovering from the disease can be carried out using different parameters that are commonly used to measure the severity, such as the variation in international normalized ratio (I NR), total bilirubin (TB) and total creatinine (TC) among others, at different times. Resolution of coagulation failure is considered when INR decreases to < 2.5, liver failure resolution is defined as a TB < 12 mg / dl and renal failure resolution as a TC < 2 mg / dl. Criteria that define vascular, respiratory and brain failure resolution are withdrawal of vasopressors, PaCh / F h > 200 and hepatic encephalopathy <3, respectively.

[0090] In a first step, the third method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0091] In a particular embodiment, the third method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0092] The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the third method of the invention.

[0093] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0094] The second step of the third method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value. In a particular embodiment, the third method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM 1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0095] The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the third method of the invention. In the third method of the invention reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point after the onset of ACLF. In a particular embodiment, the reference values are obtained at diagnosis of the syndrome and daily in the first 1 to 2 weeks in patients admitted to the ICU. Those admitted to the regular ward require less controls, typically, three times per week.

[0096] Thus according to the third method of the invention, if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject are decreased with respect to the expression level of these genes determined in a sample from said patient at earlier after the onset of ACLF, then it is indicative of ACLF resolution.

[0097] In a particular embodiment, if the third method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33.FPR1 ,SAMD3, MS4A4A, IGKV2-28, then: a decreased expression level of the genes IGFBP7, ORM1 ,ACSL1 ,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33 with respect to the expression level of these genes determined in a sample from said patient at earlier after the onset of ACLF, is indicative of ACLF resolution or an increased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes with respect to the expression level of these genes determined in a sample from said patient at earlier after the onset of ACLF, is indicative of ACLF resolution.

[0098] Fourth method of the invention

[0099] In another aspect, the present invention relates to an in vitro method, hereinafter the fourth method of the invention, for monitoring the effect of a therapy in a patient suffering from ACLF which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point, wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject are decreased with respect to said reference value, there is indicative of the therapy is being effective.

[0100] In a particular embodiment, the fourth method of the invention further comprises: a) determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11 , CRISPLD2, OSCAR, C3AR1 ,IGKV1 D-33.FPR1 ,SAMD3, MS4A4A, IGKV2-28 ; and b) comparing the expression level of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point, wherein an altered expression level of said genes when compared to reference value of said genes in a sample is indicative that the therapy is being effective, wherein said altered expression level is: a decreased expression level of the genes IGFBP7, ORM1 ,ACSL1 ,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or an increased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes. Thus, in a particular embodiment, the fourth method of the invention comprises determining the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes and the expression level of at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes or at least 34 genes selected from the group consisting of the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28 genes.

[0101] The term “therapy”, as used herein, refers to the attempted remediation of a health problem, usually following a diagnosis, or to prevention or the appearance of a health problem. As such, it is not necessarily a cure, i.e. a complete reversion of a disease. Said therapy may or may not be known to have a positive effect on a particular disease. This term includes both therapeutic treatment and prophylactic or preventative measures, in which the object is to prevent or stop (reduce) an undesired physiological change or disorder, such as, ACLF. For the purpose of this invention, beneficial or desired clinical results include, without limitation, relieving symptoms, stabilizing pathological state (specifically not worsening), slowing down or stopping the progression of the disease, improving or mitigating the pathological impairment. Particularly, for the purpose of the present invention, therapy is directed to slow the progression of liver damage and reduce the risk of further complications. It can also involve prolonging survival in comparison with the expected survival if treatment is not received. Those subjects needing treatment include those subjects already suffering the condition or disorder, as well as those with the tendency to suffer the condition or disorder or those in which the condition or disorder must be prevented.

[0102] The term “treatment”, as used herein, relates to both therapeutic measures and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as ACLF. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, stabilizing pathological state (specifically not worsening), slowing down or stopping the progression of the disease, improving or mitigating the pathological. Particularly, for the purpose of the present invention, treatment is directed to slow the progression of liver damage and reduce the risk of further complications.

[0103] The terms “ACLF”, “marker”, “subject” and “sample” have been defined or explained above, and these definitions are applicable to the fourth method of the invention.

[0104] In a particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood.

[0105] In a first step, the fourth method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0106] In a particular embodiment, the fourth method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0107] The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the fourth method of the invention.

[0108] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0109] The second step of the fourth method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value. In a particular embodiment, the fourth method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM 1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0110] The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the fourth method of the invention. In the fourth method of the invention reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point after the onset of ACLF.

[0111] According to this inventive aspect, the expression level of HMGB2, RETN, ZNF608 and PYCARD genes (and optionally one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28) are determined in a sample from a subject having ACLF obtained at a first time (first sample) and the expression level of these genes determined in the sample from said subject at second period of time within the course of the therapy (second sample). Both values can then be e compared allowing the monitorization of the effect of a therapy in said subject having ACLF.

[0112] The second sample can be taken at any time after the first sample, e.g., one day, one week, two weeks, three weeks, one month, two month, three months or more after the first subject sample. In a particular embodiment, the first sample is taken after the subject has started received treatment for said clinical condition. Therapies which can be used to treat a patient suffering from ACLF are focused to treat the liver failure and the endorgan dysfunction associated to said condition. Examples of said therapies include but are not limited to therapies directed to attenuate the immune response, such as pentoxifylline, therapies to prevent translocation of aerobic bacteria from the gut, such as norfloxacin or rifaximin, therapies to treat portal hypertension and portal hypertensin related complications (e.g. variceal bleeding) such as somatostain or terlipressin, therapies to treat bacterial infection, including systemic administration of antibiotics, therapies to treat circulatory dysfunction, including the administration of vasopressor agents, therapies to treat hepatic encephalopathy including lactulose, lactitol, and cleansing enemas, therapies to treat respiratory failure, including oxygen administration or assisted ventilation, therapies to treat kidney failure, including hemodyalisis or filtration therapies, therapies to treat liver failure including the so-called liver-assisted devices such as MARS or Prometheus or plasma exchange or any other therapy aimed at improving the effects of liver failure . The effectiveness of the therapy after the treatment can be easily followed according to the teachings of this invention.

[0113] Thus according to the fourth method of the invention, if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject are decreased with respect to the expression level of these genes determined in a sample from said patient at earlier point of time, the therapy is being effective.

[0114] In a particular embodiment, if the fourth method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33.FPR1 ,SAMD3, MS4A4A, IGKV2-28, then: a decreased expression level of the genes IGFBP7, ORM1 ,ACSL1 ,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33 with respect to the expression level of these genes determined in a sample from said patient at earlier point of time the therapy, is indicative that the therapy is being effective, or an increased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes with respect to the expression level of these genes determined in a sample from said patient at earlier point of time the therapy, is indicative that the therapy is being effective.

[0115] Fifth method of the invention

[0116] In another aspect, the invention relates to an in vitro method, hereinafter fifth method of the invention, for selecting a therapy for a patient suffering from ACLF which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point, wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject are increased with respect to said reference value, then it is indicative that patient is to be treated with a therapy aimed at treating the effects of liver failure and / or for liver transplantation.

[0117] The term “selecting a patient for a therapy”, as used herein, relates to the identification of a patient for a therapy designed to cure a disease or palliate the symptoms associated with one or more diseases or conditions. In the particular case of ACLF therapy, it is understood any therapy which abolishes, retards or reduces the symptoms associated with liver failure. Details of adequate therapies which can be used according to the invention to treat ACLF are antibiotics, diuretics, albumin, lactulose, beta-adrenergic blocker agents, vasoconstrictors, sclerosing agents, intrahepatic portosystemic shunting, plasma exchange, artificial respiratory support, renal hemodialysis, albumin dialysis, liver transplantation and / or combinations thereof.

[0118] The terms “ACLF”, “marker”, “subject” and “sample” have been defined or explained above, and these definitions are applicable to the fourth method of the invention.

[0119] In a particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood.

[0120] In a first step, the fifth method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0121] In a particular embodiment, the fifth method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0122] The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the fourth method of the invention. In a particular embodiment, the determination of the expression level of at least the HMGB2, RETN, ZNF608, PYCARD genes and at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes or at least 34 genes selected from the group consisting of the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0123] The second step of the fifth method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value.

[0124] In a particular embodiment, the fifth method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0125] The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the fourth method of the invention. In the fourth method of the invention reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point after the onset of ACLF.

[0126] Thus according to the fifth method of the invention, if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject are decreased with respect to the expression level of these genes determined in a sample from said patient at earlier point of time, the therapy is being effective. In another particular embodiment, if the fourth method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG. XCL1, RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D- 33.FPR1 ,SAMD3, MS4A4A, IGKV2-28; then an increased expression level of the genes IGFBP7, ORM1 ,ACSL1 ,BCL6, OLFM4,SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or a decreased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes, is indicative that the therapy is not being effective and that the patient is candidate for receiving a therapy aimed at treating the effects of liver failure and / or for liver transplantation.

[0127] As will be understood by those skilled in the art, the selection of the therapy patient, although preferred to be, need not be adequate for 100% of the subjects selected according to the fourth method of the invention. The term, however, requires that a statistically significant portion of subjects were correctly selected. Whether the selection of a patient in a population of subjects is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The p-values are, preferably, 0.2, 0.1 or 0.05.

[0128] The term “therapy aimed at treating the effects of liver failure” include but are not limited to therapies directed to attenuate the immune response, such as pentoxifylline, therapies to prevent translocation of aerobic bacteria from the gut, such as norfloxacin or rifaximin, therapies to treat portal hypertension and portal hypertensin related complications (e.g. variceal bleeding) such as somatostain or terlipressin, therapies to treat bacterial infection, including systemic administration of antibiotics, therapies to treat circulatory dysfunction, including the administration of vasopressor agents, therapies to treat hepatic encephalopathy including lactulose, lactitol, and cleansing enemas, therapies to treat respiratory failure, including oxygen administration or assisted ventilation, therapies to treat kidney failure, including hemodyalisis or filtration therapies, therapies to treat liver failure including the so-called liver-assisted devices such as MARS or Prometheus or plasma exchange or any other therapy aimed at improving the effects of liver failure . The effectiveness of the therapy after the treatment can be easily followed according to the teachings of this invention.

[0129] The term “liver transplantation” as used herein, refers to the replacement of a diseased liver with some or all of a healthy liver from another person. The most commonly used technique is orthotopic transplantation, in which the native liver is removed and replaced by a liver from an organ donor in the same anatomic location as the original liver.

[0130] Sixth method of the invention

[0131] In another aspect, the present invention relates to an in vitro method, hereinafter the sixth method of the invention, for determining the degree of systemic inflammation in a subject, which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has a severe systemic inflammation.

[0132] In a particular embodiment, the sixth method of the invention further comprises: a) determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28; and b) comparing the expression level of said genes with their reference values; wherein an altered expression level of said genes when compared to the reference value for said genes, wherein said altered expression level is: an increased expression level of the genes IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or a decreased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes, is indicative that said subject has a severe systemic inflammation.

[0133] Thus, in a particular embodiment, the sixth method of the invention comprises determining the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes and the expression level of at least 2 genes selected from the group consisting of the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28 genes.

[0134] Systemic inflammation is the result of release of pro-inflammatory cytokines from immune-related cells and the chronic activation of the innate immune system. It can contribute to the development or progression of certain conditions such as cardiovascular disease, cancer, diabetes mellitus, chronic kidney disease, non-alcoholic fatty liver disease, autoimmune and neurodegenerative disorders.

[0135] The terms “marker”, “subject” and “sample” have been defined or explained above, and these definitions are applicable to the fifth method of the invention.

[0136] In a particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood.

[0137] In a particular embodiment, the subject suffers from ACLF or an infection. The term ACLF has been defined in the first method of the invention, and this definition is applicable to the fifth method of the invention. In a more particular embodiment, the ACLF is ACLF1 , ACLF2, ACLF3 or early ACLF.

[0138] The term “infection” as used herein refers to the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmissible disease or communicable disease, is an illness resulting from an infection. Common types of infections in patients with liver diseases include spontaneous bacterial peritonitis (SBP), urinary tract infection (UTI), pneumonia, bacteremia and soft tissue infection.

[0139] In a particular embodiment the infection is a bacterial infection, a virus infection, a parasite infection, a fungi infection or a prion infection.

[0140] In a more particular embodiment, the infection is a bacterial infection. In a preferred embodiment, the bacterial infection is caused by E. coli, Klebsiella spp., Enterobacter spp, Pseudomonas aeruginosa, non-enterococal streptococci, Enterococci or Staphylococcus aureus.

[0141] In a first step, the sixth method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0142] In a particular embodiment, the sixth method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 .CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0143] The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the fifth method of the invention.

[0144] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0145] The second step of the sixth method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value.

[0146] In a particular embodiment, the sixth method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0147] The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the fifth method of the invention.

[0148] Once the comparison is made between the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject and the reference value for said markers, the fifth method of the invention allows determining if a subject has a severe systemic inflammation based on if the expression level of HMGB2, RETN, ZNF608 and PYCARD genes is increased with respect to the reference value.

[0149] In a particular embodiment, if the sixth method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28, then once the comparison is made between the expression level of these genes in the sample from the subject and the reference value for said markers, the fifth method of the invention allows determining if a subject has a severe systemic inflammation based on: if the expression level of IGFBP7,

[0150] ORM1 ,ACSL1,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11, OSCAR, IGKV2-28, IGKV2-33 genes is increased with respect to the reference value or;

[0151] - if the expression level of XCL1 , GZMH, GNLY, FASLG or SAMD3 genes is decreased with respect to the reference value.

[0152] Seventh method of the invention In another aspect the present invention relates to an in vitro method, hereinafter the sixth method of the invention, for determining the presence of a bacterial infection with sepsis in a subject which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has a bacterial infection with sepsis.

[0153] In a particular embodiment, the seventh method of the invention further comprises: c) determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11 , CRISPLD2, OSCAR, C3AR1 ,IGKV1 D-33.FPR1 ,SAMD3, MS4A4A, IGKV2-28; and d) comparing the expression level of said genes with their reference values; wherein an altered expression level of said genes when compared to the reference value for said genes, wherein said altered expression level is: an increased expression level of the genes IGFBP7, ORM1 ,ACSL1 ,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or a decreased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes, is indicative that said subject has said subject has a bacterial infection with sepsis.

[0154] Thus, in a particular embodiment, the seventh method of the invention comprises determining the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes and the expression level of at least 2 genes selected from the group consisting of the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28 genes.

[0155] The expression “bacterial infection” refers to that infections that are caused by bacteria. Common types of infections in patients with liver diseases include spontaneous bacterial peritonitis (SBP), urinary tract infection (UTI), pneumonia, bacteremia and soft tissue infection. In a particular embodiment the bacterial infection is caused by E. coli, Klebsiella spp., Enterobacter spp, Pseudomonas aeruginosa, non-enterococal streptococci, Enterococci or Staphylococcus aureus.

[0156] The terms “marker”, “subject” and “sample” have been defined or explained above, and these definitions are applicable to the sixth method of the invention.

[0157] In a particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood.

[0158] The term “sepsis” refers to a syndrome of life-threatening physiological, pathological and biochemical abnormalities associated with infection. These abnormalities are secondary to a disproportionate immune response to infection, which ultimately damages subject's own tissues and organs and leads to multi-organ dysfunction. Systemic inflammatory response syndrome is associated with sepsis, but is not an absolute cause of sepsis, as sepsis involves the activation of both proinflammatory and anti-inflammatory responses. Systemic inflammatory response syndrome is a serious condition related to systemic inflammation, organ dysfunction, and organ failure. It is a subset of cytokine storm, in which there is abnormal regulation of various cytokines.

[0159] In a particular embodiment, the subject has septic shock. The term “sepsis shock” refers to a type of sepsis that is accompanied by circulatory, cellular and metabolic abnormalities profound enough to substantially increase mortality.

[0160] In a first step, the seventh method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0161] In a particular embodiment, the seventh method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11.CRISPLD2, OSCAR, C3AR1 ,IGKV1 D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0162] The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the seventh method of the invention.

[0163] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0164] The second step of the seventh method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value.

[0165] In a particular embodiment, the seventh method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0166] The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the seventh method of the invention.

[0167] Once the comparison is made between the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject and the reference value for said markers, the seventh method of the invention allows determining if a subject has a bacterial infection with sepsis based on if the expression level of HMGB2, RETN, ZNF608 and PYCARD genes is increased with respect to the reference value.

[0168] In a particular embodiment, if the seventh method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28, then once the comparison is made between the expression level of these genes in the sample from the subject and the reference value for said markers, the sixth method of the invention allows determining if a subject has a bacterial infection with sepsis based on: if the expression level of IGFBP7,

[0169] ORM1 ,ACSL1,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11, OSCAR, IGKV2-28, IGKV2-33 genes is increased with respect to the reference value; or

[0170] - if the expression level of XCL1 , GZMH, GNLY, FASLG or SAMD3 genes is decreased with respect to the reference value.

[0171] Kits of the invention

[0172] In another aspect, the present invention relates to a kit or assay device, hereinafter first kit or assay device of the invention, comprising reagents adequate for the determination of the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes. It will be understood that, depending on the nature of the method, the reagents adequate for its implementation will vary.

[0173] In the context of the present invention, “kit” is understood as a product containing the different reagents required for carrying out the methods of the invention packaged such that it allows being transported and stored. The materials suitable for the packaging of the components of the kit include glass, plastic (polyethylene, polypropylene, polycarbonate, and the like), bottles, vials, paper, sachets, and the like. Where there are more than one component in a kit they may be packaged together if suitable or the kit will generally contain a second, third or other additional container into which the additional components may be separately placed. However, in some embodiments, certain combinations of components may be packaged together comprised in one container means. A kit can also include a means for containing any reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained. One or more compositions of a kit can be lyophilized. In some embodiments, all compositions of a kit of the disclosure will be lyophilized. In some embodiments, a kit of the disclosure with one or more lyophilized agents will be supplied with a re-constitution buffer. Reagents and components of kits may be comprised in one or more suitable container means. A container means may generally comprise at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted.

[0174] In a particular embodiment, said reagents are reagents for detecting and / or quantifying mRNA of the HMGB2, RETN, ZNF608 and PYCARD genes. In another particular embodiment, said reagents are reagents for detecting and / or quantifying the proteins encoded by the HMGB2, RETN, ZNF608 and PYCARD genes.

[0175] In a particular embodiment, said reagents for detecting and / or quantifying mRNA of the HMGB2, RETN, ZNF608 and PYCARD genes comprises probes which hybridizes with a cDNA to said mRNA of HMGB2, RETN, ZNF608 and PYCARD or a pair of oligonucleotide primers which hybridizes with said mRNA of HMGB2, RETN, ZNF608 and PYCARD or with said cDNA to said mRNA of HMGB2, RETN, ZNF608 and PYCARD.

[0176] The term "primer" as used herein refers to oligonucleotides that can specifically hybridize to a target polynucleotide sequence, due to the sequence complementarity of at least part of the primer within a sequence of the target polynucleotide sequence. A primer can have a length of at least 8 nucleotides, typically 8 to 70 nucleotides, usually of 18 to 26 nucleotides. For proper hybridization to the target sequence, a primer can have at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence complementarity to the hybridized portion of the target polynucleotide sequence. Oligonucleotides useful as primers may be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. (1981) 22: 1859-1862, using an automated synthesizer, as described in Needham-Van Devanter et al, Nucleic Acids Res. (1984) 12: 6159-6168. Primers are useful in nucleic acid amplification reactions in which the primer is extended to produce a new strand of the polynucleotide. Primers can be readily designed by a skilled artisan using common knowledge known in the art, such that they can specifically anneal to the nucleotide sequence of the target nucleotide sequence of the at least one biomarker provided herein. Usually, the 3' nucleotide of the primer is designed to be complementary to the target sequence at the corresponding nucleotide position, to provide optimal primer extension by a polymerase. The term "probe" as used herein refers to oligonucleotides or analogs thereof that can specifically hybridize to a target polynucleotide sequence, due to the sequence complementarity of at least part of the probe within a sequence of the target polynucleotide sequence. Exemplary probes can be, for example DNA probes, RNA probes, or protein nucleic acid (PNA) probes. A probe can have a length of at least 8 nucleotides, typically 8 to 70 nucleotides, usually of 18 to 26 nucleotides. For proper hybridization to the target sequence, a probe can have at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent sequence complementarity to hybridized portion of the target polynucleotide sequence. Probes can also be chemically synthesized according to the solid phase phosphoramidite triester method as described above. Methods for preparation of DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition. Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11.

[0177] In a preferred embodiment, the reagents adequate for the determination of the expression levels of one or more genes comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents adequate for the determination of the expression levels of genes forming the kit.

[0178] In another particular embodiment, said reagents for detecting and / or quantifying the proteins encoded by the HMGB2, RETN, ZNF608 and PYCARD genes are antibodies that recognize HMGB2, RETN, ZNF608 and PYCARD.

[0179] Thus, in a particular embodiment, said reagents for detecting and / or quantifying the protein encoded by HMGB2, RETN, ZNF608 and PYCARD are antibodies that recognizes HMGB2 RETN, ZNF608 and PYCARD, respectively.

[0180] In another aspect, the present invention relates to a kit or assay device, hereinafter the second kit or assay device of the invention, comprising reagents adequate for the determination of the expression level of at least the HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28.

[0181] In a particular embodiment, said reagents are reagents for detecting and / or quantifying mRNA of at least HMGB2, RETN, ZNF608 and PYCARD genes and the mRNA of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2- 28.

[0182] In another particular embodiment, said reagents are reagents for detecting and / or quantifying the proteins encoded by the HMGB2, RETN, ZNF608 and PYCARD genes and one or more of the proteins encoded by the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1D- 33, FPR1 , SAMD3, MS4A4A and IGKV2-28.

[0183] In another particular embodiment, said reagents for detecting and / or quantifying mRNA of the HMGB2, RETN, ZNF608 and PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 comprises probes which hybridizes with a cDNA to said mRNA or a pair of oligonucleotide primers which hybridizes with said mRNA or with said cDNA to said mRNA.

[0184] In another particular embodiment, said reagents for detecting and / or quantifying the proteins encoded by the HMGB2, RETN, ZNF608 and PYCARD genes are antibodies that recognize the proteins encoded by HMGB2, RETN, ZNF608 and PYCARD genes, and the reagents for detecting and / or quantifying the proteins encoded by the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1, IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 genes are antibodies that recognize the proteins encoded by one or more of these genes.

[0185] In a particular embodiment, the second kit of the invention comprises one or more reagents for detecting and / or quantifying the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes and / or proteins encoded by said genes and one or more reagents for detecting and / or quantifying the expression level of at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes or at least 34 genes selected from the group consisting of the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 genes and / or proteins encoded by said genes.

[0186] In addition to these means, the first and the second kits of the invention can comprise other components useful in the implementation of the present invention, such as, buffers, material supports, positive and / or negative control components, etc. In addition to the aforementioned components, the kit can also include instructions for practicing the object of the invention. These instructions may be present in the aforementioned kit in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a sheet or sheets of paper on which the information is printed, on the kit packaging, on a package insert, etc. Another medium would be a computer readable medium, e.g., CD, USB, etc., on which the information has been recorded. Another medium that may be present is a website address that can be used via the Internet to access the information at a remote site. Any convenient medium may be present in the kits of the invention.

[0187] Uses of the invention In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes or the first kit of the invention for determining the presence of ACLF in a subject.

[0188] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes or the first kit of the invention for determining the risk of developing ACLF in a subject.

[0189] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes or the first kit of the invention for monitoring the progression of ACLF in a subject.

[0190] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes or the first kit of the invention for monitoring the effect of a therapy for ACLF in a subject.

[0191] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes or the first kit of the invention for selecting a patient suffering from ACLF for a therapy.

[0192] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes or the first kit of the invention for determining the degree of systemic inflammation in a subject.

[0193] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes or the first kit of the invention for determining the presence of a bacterial infection with sepsis in a subject.

[0194] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 or the second kit of the invention for determining the presence of ACLF in a subject.

[0195] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 or the second kit of the invention for determining the risk of developing ACLF in a subject.

[0196] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 or the second kit of the invention for monitoring the progression of ACLF in a subject.

[0197] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 or the second kit of the invention for monitoring the effect of a therapy for ACLF in a subject.

[0198] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 or the second kit of the invention for selecting a patient suffering from ACLF for a therapy.

[0199] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 or the second kit of the invention for determining the degree of systemic inflammation in a subject.

[0200] In another aspect, the present invention relates to the use of reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 or the second kit of the invention for determining the presence of a bacterial infection with sepsis in a subject.

[0201] Computer related aspects

[0202] In particular embodiments of any of the methods the invention, the method is computer- implemented.

[0203] In another aspect, the present invention relates to a computer system comprising one or more programs, wherein the one or more programs are include instructions for performing the methods of the invention.

[0204] In another aspect, the present invention relates to a computer-readable storage medium storing one or more programs, the one or more programs comprising instructions, which when executed by one or more processors of an electronic device, cause the electronic device to perform the methods of the invention. The term “computer implemented” as used herein refers to fact that the steps of the method are performed by a computer or a computer system. The term “computer implemented” is meant to also include the term “partially computer-implemented”, which refers to a method in which only particular steps, e.g., calculating steps, are computer- implemented, whereas other steps of the method are not.

[0205] A further aspect of the present invention relates to a computer system comprising one or more programs, wherein the one or more programs include instructions for performing the method I, II and / or III of the invention.

[0206] Another aspect of the present invention relates to a computer-readable storage medium storing one or more programs, the one or more programs comprising instructions, which when executed by one or more processors of an electronic device, cause the electronic device to perform the method I, II and / or III of the invention.

[0207] As will be appreciated by one of skill in the art, the computer readable instructions cause the computing system to carry out the method according to the invention. The device may take the form of an entire hardware embodiment or an embodiment combining software and hardware aspects. Furthermore, the present invention may include a computer program product on a computer-usable storage medium having computer- usable program code means embodied in the medium. Any suitable computer readable medium may be utilized including hard disks, CD-ROMs, optical storage devices, or magnetic storage devices. The computer-usable or computer-readable medium may be or include, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. More specific examples (a non-exhaustive list) of the computer readable medium would include the following: an electrical connection having one or more wires, a portable computer diskette, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, and a portable compact disc read-only memory (CD-ROM), a CD ROM, a DVD (digital video disk), or other electronic storage medium. Note that the computer- usable or computer-readable medium could even be paper or another. Suitable medium upon which the program is printed, as the program can be electronically captured, via, for instance, optical scanning of the paper or other medium, then compiled, interpreted or otherwise processed in a suitable manner if necessary, and then stored in a computer memory. Computer program code for carrying out operations of the present invention may be written in an object-oriented programming language such as R (this is the language that was used for study), Python, Matlab, Java, and Javascript, C #, Smalltalk or C++. However, the computer program code for carrying out operations of the present invention may also be written in conventional procedural programming languages, such as the “C” or FORTRAN programming language or even assembly language. The program code may execute entirely on the user's computer, partly on the user's computer, as a standalone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer. In the latter scenario, the remote computer may be connected to the user's computer through a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider).

[0208] Eighth method of the invention

[0209] In another aspect, the present invention relates to a method, hereinafter the eighth method of the invention, for determining the presence of acute-on chronic liver-failure (ACLF) in a subject suffering from cirrhosis or delayed pre-ACLF a patient suffering from ACLF and treating the patient comprising: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said patient; and b) comparing the expression level of said genes with reference values for each gene, wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the patient is greater than the respective reference values, said patient is treated with an adequate therapy including antibiotics, diuretics, albumin, lactulose, beta-adrenergic blocker agents, vasoconstrictors, sclerosing agents, intrahepatic portosystemic shunting, plasma exchange, artificial respiratory support, renal hemodialysis, liver hemodialysis, liver transplantation and / or combinations thereof.

[0210] The terms “ACLF”, “cirrhosis”, “ compensated cirrhosis”, “decompensated cirrhosis”, “marker”, “subject”, “sample” have been defined of explained in the first method of the invention, and these definitions are applicable to the seventh method of the invention.

[0211] In a particular embodiment, the subject suffers unstable or stable acute decompensated cirrhosis. In another particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood.

[0212] In a first step, the eighth method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0213] In a particular embodiment, the eighth method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0214] The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the eighth method of the invention.

[0215] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0216] The second step of the eighth method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value.

[0217] In a particular embodiment, the eighth method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 with a reference value. The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the seventh method of the invention.

[0218] Once the comparison is made between the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject and the reference value for said markers, if the expression level of these genes is greater than their respective reference values, then a therapy is administered to said subject who will benefit from the administration of said therapy.

[0219] In a particular embodiment, if the method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28, then:

[0220] - if the expression level of IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33 genes is increased with respect to the reference value, then a therapy is administered to said subject who will benefit from the administration of said therapy; or

[0221] - if the expression level of XCL1 , GZMH, GNLY, FASLG or SAMD3 genes is decreased with respect to the reference value, then a therapy is administered to said subject who will benefit from the administration of said therapy.

[0222] The terms “therapy” and “treatment” have been defined or explained above, and these definitions are applicable to the seventh method of the invention.

[0223] As it is well-known, the treatment of ACLF depends on the patient or the degree of the ACLF. Therapies which can be used to treat a patient suffering from ACLF are focused to treat the liver failure and the end-organ dysfunction associated to said condition. Examples of said therapies include but are not limited to therapies directed to attenuate the immune response, such as pentoxifylline, therapies to prevent translocation of aerobic bacteria from the gut, such as norfloxacin or rifaximin, therapies to treat portal hypertension and portal hypertensin related complications (e.g. variceal bleeding) such as somatostain or terlipressin, therapies to treat bacterial infection, including systemic administration of antibiotics, therapies to treat circulatory dysfunction, including the administration of vasopressor agents, therapies to treat hepatic encephalopathy including lactulose, lactitol, and cleansing enemas, therapies to treat respiratory failure, including oxygen administration or assisted ventilation, therapies to treat kidney failure, including hemodialysis or filtration therapies, therapies to treat liver failure including the so-called liver-assisted devices such as MARS or Prometheus or plasmapheresis or any other therapy aimed at improving the effects of liver failure . The effectiveness of the therapy after the treatment can be easily followed according to the teachings of this invention.

[0224] In a particular embodiment, the therapy includes antibiotics, diuretics, albumin, lactulose, beta-adrenergic blocker agents, vasoconstrictors, sclerosing agents, intrahepatic portosystemic shunting, plasma exchange, artificial respiratory support, renal hemodialysis, liver hemodialysis, liver transplantation and / or combinations thereof.

[0225] The term “antibiotic” refers to a type of antimicrobial substance active against bacteria including classical beta-lactams, carbapenems, new cephalosporins, tetracyclines, quinolones, glycopeptides and lipopeptides.

[0226] The term “diuretic” refers to any substance that promotes diuresis, the increased production of urine. This includes forced diuresis. In a particular embodiment, the diuretic is a high-ceiling / loop diuretic, a thiazide, a carbonic anhydrase inhibitor, a potassium- sparing diuretic, a calcium-sparing diuretic, an osmotic diuretic or a low-ceiling diuretic.

[0227] The term “albumin” refers to a family of globular proteins, the most common of which are the serum albumins. Albumins are commonly found in blood plasma and differ from other blood proteins in that they are not glycosylated. Albumin both supports the circulation and reduces systemic inflammation. In addition, to its oncotic function, it acts as an antioxidant, radical scavenger, and immune modulator. Long-term albumin therapy in patients with cirrhosis and ascites improves survival, prevents complications, simplifies ascites management, and lowers hospitalization rates.

[0228] The term “lactulose” refers to a non-absorbable sugar used in the treatment of constipation and hepatic encephalopathy. Specifically, it is effective as secondary prevention of hepatic encephalopathy in people with cirrhosis. The term “beta-adrenergic blocker agents” refers to medications that reduce blood pressure. Beta blockers work by blocking the effects of the hormone epinephrine, also known as adrenaline. Beta blockers are competitive antagonists that block the receptor sites for the endogenous catecholamines epinephrine (adrenaline) and norepinephrine (noradrenaline) on adrenergic beta receptors, of the sympathetic nervous system, which mediates the fight-or-flight response.

[0229] The term “vasoconstrictors” refers to medications causing vasoconstriction, which refers to the narrowing of the blood vessels resulting from contraction of the muscular wall of the vessels, in particular the large arteries and small arterioles. In a particular embodiment, the vasoconstrictor is selected from the group consisting of: norepinephrine, somatostatin, octreotide, terlipressin, and methylene blue, among others.

[0230] The term “sclerosing agents” refers to the agents that are used in sclerotherapy, typically, polidocanol and ethanolamine.

[0231] The term “intrahepatic portosystemic shunting” refers to an artificial channel within the liver that establishes communication between the inflow portal vein and the outflow hepatic vein. It is used to treat portal hypertension (which is often due to liver cirrhosis) which frequently leads to intestinal bleeding, life-threatening esophageal bleeding (esophageal varices) and the buildup of fluid within the abdomen (ascites). An interventional radiologist creates the shunt using an image-guided endovascular (via the blood vessels) approach, with the jugular vein as the usual entry site.

[0232] The term “plasma exchange” refers to a therapeutic procedure used to treat a wide variety of diseases through the bulk removal of plasma. Therapeutic plasma exchange (TPE) has been shown to reduce the levels of inflammatory cytokines, modulate adaptive immunity with the potential to lessen the susceptibility to infections, and reduce the levels of albumin-bound and water-bound toxins in liver failure.

[0233] The term “artificial respiratory support” refers to the medical term for using a machine called a ventilator to fully or partially provide artificial ventilation.

[0234] The term “hemodialysis” refers to a renal replacement therapy that aims to partially replace the function of the kidneys. In liver failure, hemodialysis (HD) is highly effective for the removal of small and medium-sized water-soluble, unbound molecules (e.g., ammonia, urea) from the circulation across a semipermeable membrane into hypotonic dialysate fluid along their concentration gradients.

[0235] Ninth method of the invention

[0236] In another aspect, the present invention relates to a method, hereinafter the ninth method of the invention, for determining the risk of developing ACLF in a subject suffering from cirrhosis or delayed pre-ACLF and treating the patient which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein if expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes, then the patient is treated with a therapy adequate for the prevention of ACLF including antibiotics, diuretics, albumin, lactulose, beta-adrenergic blocker agents, terlipressin, sclerosing agents, intrahepatic portosystemic shunting and / or combinations thereof.

[0237] The terms “ACLF”, “cirrhosis”, “compensated cirrhosis”, “decompensated cirrhosis”, “marker”, “subject”, “sample”, “determining the risk” have been defined of explained in the first method and in the second of the invention, and these definitions are applicable to the eight method of the invention.

[0238] In a particular embodiment, the subject suffers unstable or stable acute decompensated cirrhosis.

[0239] In another particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood.

[0240] In a first step, the ninth method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0241] In a particular embodiment, the ninth method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0242] The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the ninth method of the invention.

[0243] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11, CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0244] The second step of the eight method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value.

[0245] In a particular embodiment, the ninth method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0246] The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the eight method of the invention.

[0247] Once the comparison is made between the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject and the reference value for said markers, if the expression level of these genes is greater than their respective reference values, then a therapy is administered to said subject who will benefit from the administration of said therapy. In a particular embodiment, if the method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D-

[0248] 33.FPR1.SAMD3, MS4A4A, IGKV2-28, then:

[0249] - if the expression level of IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33 genes is increased with respect to the reference value, then a therapy is administered to said subject who will benefit from the administration of said therapy, or if the expression level of XCL1 , GZMH, GNLY, FASLG or SAMD3 genes is decreased with respect to the reference value, then a therapy is administered to said subject who will benefit from the administration of said therapy.

[0250] The terms “therapy” and “treatment” have been defined or explained above, and these definitions are applicable to the eight method of the invention.

[0251] An adequate treatment for the prevention of ACLF include, but is not limited to, antibiotics, diuretics, albumin, lactulose, beta-adrenergic blocker agents, terlipressin, sclerosing agents, intrahepatic portosystemic shunting and / or combinations thereof.

[0252] Tenth method of the invention

[0253] In another aspect, the present invention relates to a method, hereinafter the tenth method of the invention, for determining the degree of systemic inflammation in a subject and treating the subject which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject is greater than the reference value for said genes, then the patient is treated with a therapy adequate for severe systemic inflammation including antibiotics, corticosteroids, albumin, plasma exchange and / or combinations thereof. The terms “systemic inflammation”, “marker”, “subject”, “sample” have been defined of explained above and these definitions are applicable to the ninth method of the invention.

[0254] In a particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood.

[0255] In a first step, the tenth method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0256] In a particular embodiment, the tenth method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study.

[0257] The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the tenth method of the invention.

[0258] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0259] The second step of the tenth method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value.

[0260] In a particular embodiment, the tenth method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0261] The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the ninth method of the invention.

[0262] Once the comparison is made between the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject and the reference value for said markers, if the expression level of these genes is greater than their respective reference values, then a therapy is administered to said subject who will benefit from the administration of said therapy.

[0263] In a particular embodiment, if the method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11, CRISPLD2, OSCAR, C3AR1 , IGKV1 D- 33.FPR1.SAMD3, MS4A4A, IGKV2-28, then:

[0264] - if the expression level of IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11, OSCAR, IGKV2-28, IGKV2-33 genes is increased with respect to the reference value, then a therapy is administered to said subject who will benefit from the administration of said therapy, or if the expression level of XCL1 , GZMH, GNLY, FASLG or SAMD3 genes is decreased with respect to the reference value, then a therapy is administered to said subject who will benefit from the administration of said therapy.

[0265] The terms “therapy” and “treatment” have been defined or explained above, and these definitions are applicable to the tenth method of the invention.

[0266] An adequate therapy for the treatment of severe systemic inflammation include, but is not limited to, antibiotics, corticosteroids, albumin, plasma exchange and / or combinations thereof.

[0267] Eleventh method of the invention In another aspect, the present invention relates to a method, hereinafter the eleventh method of the invention, for determining the presence of a bacterial infection with sepsis in a subject and treating the subject which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject is greater than the reference value for said genes, then the patient is treated with a therapy adequate for bacterial infection and / or sepsis including antibiotics, vasoconstrictors, organ support including renal hemodialysis and / or artificial respiratory support.

[0268] The terms “bacterial infection”, “sepsis”, “marker”, “subject”, “sample”, have been defined of explained in the first method of the invention, and these definition are applicable to the tenth method of the invention.

[0269] In a particular embodiment, the sample is a biofluid. In a more particular embodiment, the biofluid is blood.

[0270] In a particular embodiment, the bacterial infection is caused by E. coli, Klebsiella spp., Enterobacter spp, Pseudomonas aeruginosa, non-enterococal streptococci, Enterococci and Staphylococcus aureus.

[0271] In a first step, the eleventh method of the invention comprises determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from the subject under study.

[0272] In a particular embodiment, the eleventh method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11.CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 in a sample from the subject under study. The term “expression level” has been defined or explained in the first method of the invention and this definition is applicable to the eleventh method of the invention.

[0273] In a particular embodiment, the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

[0274] The second step of the eleventh method of the invention comprises comparing the expression level of HMGB2, RETN, ZNF608 and PYCARD genes obtained in the first step of said method with a reference value.

[0275] In a particular embodiment, the eleventh method of the invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 .CRISPLD2, OSCAR, C3AR1JGKV1D- 33,FPR1 ,SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0276] The terms “reference value”, “increased”, “decreased” and “equal” have been defined or explained in the first method of the invention and these definitions are applicable to the tenth method of the invention.

[0277] Once the comparison is made between the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject and the reference value for said markers, and if the expression level of these genes is greater than their respective reference values, then a therapy is administered to said subject who will benefit from the administration of said therapy.

[0278] In a particular embodiment, if the method of the invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28, then: - if the expression level of IGFBP7, ORM1, ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33 genes is increased with respect to the reference value, then a therapy is administered to said subject who will benefit from the administration of said therapy, or if the expression level of XCL1 , GZMH, GNLY, FASLG or SAMD3 genes is decreased with respect to the reference value, then a therapy is administered to said subject who will benefit from the administration of said therapy.

[0279] As it is well-known, the treatment of a bacterial infection and / or sepsis depends on the patient or the degree of the infection. An adequate therapy for the treatment of a bacterial infection and / or sepsis include, but is not limited to, antibiotics, vasoconstrictors, organ support including renal hemodialysis and / or artificial respiratory support.

[0280] The invention is described below by way of the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention.

[0281] EXAMPLES

[0282] Material and methods

[0283] Patients.

[0284] The study was performed using data and biobanking material collected during the Predict and Aclara studies, two multicenter prospective observational investigations in 1273 and 1274 patients, respectively, hospitalized with ADC. These studies aimed to characterize the ACLF syndrome and other phenotypes of ADC, both clinically and through genomic, transcriptomic and metabolomic assessment. Informed consents were obtained from the patients or their representatives, and both studies were approved by the corresponding research and ethics committees at each center. Authorization was also given to use data and samples for ancillary studies.

[0285] The study was performed in 700 patients (Cohort 1), 383 from the Predict and 317 from the Aclara study, hospitalized with ADC with and without ACLF and explored by wholeblood RNA sequencing (RNA-seq) within 2 days after hospital admission (time 1 , T1). In 375 patients (Sub-cohort 1a), a second RNA-seq was performed at T2 (median duration of the T1-T2 period 7 days; 75% Cl: 5- 8 days). Finally, 59 patients from Sub-cohort 1a, who were explored by one or two additional RNA-seq after T2, formed the Sub-cohort 1 b. The CLF-SIG score was designed in Cohort 1. The early course of CLIF-SIG score was estimated in Sub-cohort 1a and more in detail in Sub-cohort 1 b. Cohort 1 and Subcohort 1a included patients with and without ACLF at T1 (Tables 1 and 2). Sub-cohort 1 b included patients without ACLF at T1.

[0286] According to disease severity at T1 , patients were distributed into a “low-severity group”, which included 403 patients with “delayed” pre-ACLF and unstable or stable decompensated cirrhosis, and a “high-severity group”, which included 297 patients with “early” pre-ACLF (development of ACLF within 21 days after T1), or with ACLF.

[0287] Clinical features, biomarkers of systemic inflammation [CRP, WBC, neutrophil-to- lymphocyte ratio, and cytokines), and precipitating events (acute alcoholic hepatitis or bacterial infection) were recorded at T1 in patients from Cohort 1 , at T1 and T2 in Subcohort 1a, and at T1 , T2, T3 and in some cases at T4, in Sub-cohort 1b. Acute alcoholic hepatitis was diagnosed according to the NIAAA criteria.

[0288] New Scores.

[0289] The CLIF-SIG score. It was designed in the 700 patients from Cohort 1 , based on top genes discriminating patients from the high- and low-severity groups, and aimed to obtain a single continuous variable estimating the individual magnitude of systemic inflammation at a given time point. Changes in CLIF-SIG score between T2 and T1 (CLIF-SIG delta score) estimate the course of systemic inflammation during the study period.

[0290] The strategy to design the CLIF-SIG score relied on two evidence-based concepts and one assumption. The first concept was based on studies supporting that chronic systemic inflammation, a characteristic feature of patients with decompensated cirrhosis [patients with prior episodes of acute decompensation (AD)], is the result of continuous translocation of bacteria and / or pathogen-associated molecular patterns (PAMPs) through a leaky intestinal mucosa. ADC would occur within the context of transient bursts of systemic inflammation, over a background of moderate chronic systemic inflammation, likely related to transiently enhanced bacterial translocation, or to exogenous proinflammatory precipitants, mainly acute alcoholic hepatitis, or bacterial infections. Therefore, systemic inflammation in cirrhosis is the result of an innate immune response involving neutrophils, basophils, monocytes, macrophages, NK cells and dendritic cells, and B lymphocytes, which respond to PAMPs by rapid release of low specific polyclonal antibodies. The second concept relied on unpublished data from the Predict study, specifically obtained for the design of the current study, indicating that whereas systemic inflammation in patients with “delayed” pre-ACLF, or unstable and stable decompensated cirrhosis is moderate, it is severe in patients with “early” pre-ACLF or with ACLF.

[0291] As a corollary of these two concepts, inventors assumed that top genes related to innate immune cells and B cells discriminating patients from the high- and low-severity groups at T1 , would include sensitive genes estimating the magnitude of systemic inflammation in patients with ADC.

[0292] The Chronic Liver Failure (CLIF)-Systemic Inflammation Composite (SIC) score (CLIF- SIC score). It was designed in 654 patients who had a complete set of well-recognized markers of systemic inflammation at T1 , including proteins such as cytokines known to be highly increased in patients with ACLF as compared to patients without ACLF (IL-6, IL-8, IL-1 RA, IP-10, MCP1 , MIP1 / ?, and TNFa) and CRP, and cells such as WBC, and the neutrophil-to-lymphocyte ratio.

[0293] Computation of the scores. Inventors randomly divided Cohort 1 in a training set (n=463) and a test set (n=237). Then, using the training set, inventors compared whole blood transcriptome from the high-severity group with that from the low-severity group with the use of GSEA analysis of 1 MSigDB collections (Gene Ontology) and the Blood Transcription Module (BTM) space. Doing that, inventors identified four gene sets of interest that were significantly enriched in the high-severity group. These gene sets included the BTM related to immune activation-generic cluster (M37.0), the BTM related to monocyte surface signature (S4), the BTM enriched in NK cells (I) (M7.2) and the immunoglobulin complex (GOCC 0019814). There were 220 leading-edge genes across the four gene sets which were used to compute the CLIF-SIG score (supplementary Table 1). This was performed with the use of the glmnet R package to compute a Lasso solution and find an optimal A by 10-fold CV to select the genes that optimize the misclassification error when comparing patients from the above-mentioned groups. The resulting score was a risk probability.

[0294] Patients with complete set of inflammatory markers were randomly selected for the training (n=430) and testing (n=224) sets. The CLIF-SIG score was computed by a stepwise selection method comparing high- and low-severity group patients in a logistic regression. During computation, however, neither cytokines nor the neutrophil-to- lymphocyte ratio remained in the model. Therefore, the CLIF-SIC score was based only on CRP, WBC.

[0295] Characterization of the CLIF-SIG score and comparison to other markers of systemic inflammation. The characterization of the new scores, CRP, WBC count, and neutrophil to lymphocyte ratio differentiating the high- and low-severity groups were performed at T1 in patients from the Predict and Aclara studies. The characterization of CLF-SIG score in each of the 7 phenotypes of ADC, however, was based only in the Predict study patients, since follow-up clinical and laboratory data during the period between day-28 to day-90 after T 1 were not available in the Aclara study. Indeed, the phenotypes of stable and unstable decompensated cirrhosis, and “delayed” pre-ACLF require the availability of these data for identification. The CLIF-SIG score was also characterized in patients with different types and number of major complications and precipitating events.

[0296] Testing the CLIF-SIG score as a marker of systemic inflammation.

[0297] To test if a score, like the CLIF-SIG score, based on gene transcription, estimates the magnitude of systemic inflammation, we correlated this score with the CLIF-SIC score at T1.

[0298] In addition, since ACLF is known to have an extremely dynamic clinical course, resolving, improving, following a steady course, or worsening within few days after hospital admission, in close association with changes in systemic inflammation, inventors explored in the high-severity group if CLIF-SIG score detects these follow-up changes in systemic inflammation.

[0299] Limitations of Sub-cohort 1a to estimate the course of the CLIF-SIG score.

[0300] The course of the CLIF-SIG score in Sub-cohort 1a was based in only two time-points and a median study period of seven days. To limit potential misinterpretations in the characteristics of the acute inflammatory reaction inventors extended the investigation to Sub-cohort 1 b, whose patients had 1 or 2 additional RNA-seq and a longer period of observation. Sub-cohort 1 b included 15 patients with “early” pre-ACLF, 4 with “delayed” pre-ACLF (ACLF development after 21 days from T1), and 38 with unstable or stable decompensated cirrhosis.

[0301] Characterization of CLIF-SIG score genes in a public RNA-seq dataset from unselected patients with bacterial infections.

[0302] To assess if genes selected for CLIF-SIG score also estimate the magnitude of systemic inflammation in other diseases associated with systemic inflammation, inventors explored a recently public RNA-seq dataset reported by Herwanto et al, BMC Res Notes (2021) 14:76 including 40 healthy subjects, 32 patients with infections (20 with sepsis) and 19 patients with septic shock.

[0303] Methods for RNA-seq and other statistical and bioinformatic assessments. Results were expressed as median and IQR for not normally distributed data, as mean and SD for normally distributed data, and as count and relative percentage for categorical data. Statistical testing was performed accordingly. Monaco database signatures were used to identify the cell populations associated with the CLIF-SIG score genes.

[0304] RNA Preparation from Whole Blood

[0305] RNA was isolated from blood stored in Tempus tubes using the Tempus™ Spin RNA Isolation Kit (reference 4380204, Applied Biosystems). RNA quality was assessed using Agilent RNA 6000 Nano and Pico Chips (Agilent Technologies, Cat.# 5067-1511 and Cat.# 5067-151 , respectively) and concentration via Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Cat.# Q32855). Sequencing libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Globin kit (Illumina Inc., Cat.# 20020612) and TruSeq RNA CD Index Plate (Illumina Inc., Cat.# 20019792) following TruSeq Stranded Total RNA Sample Prep-guide (Part # 15031048 Rev. E). Briefly, starting from 500 ng of total RNA, rRNA and globin mRNA were depleted and remaining RNA was purified, fragmented and primed for cDNA synthesis. cDNA first strand was synthesized with SuperScript-ll Reverse Transcriptase (Thermo Fisher Scientific, Cat.#18064-014) for 10 min at 25°C, 15 min at 42°C, 15 min at 70°C and pause at 4°C. cDNA second strand was synthesized with Illumina reagents at 16°C for 1 hour. Then, A-tailing and adaptor ligation were performed. Finally, enrichment of libraries was achieved by PCR (30 sec at 98°C; 15 cycles of 10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C; 5min at 72°C and pause at 4°C). Afterwards, libraries were visualized on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (Agilent Technologies, Cat. # 5067-4626), quantified using Qubit dsDNA HS DNA Kit (Thermo Fisher Scientific, Cat. # Q32854) and sequenced in a NovaSeq-6000 (Illumina Inc.) by at least 100 million paired-end 100nt reads. The reads were aligned to the hg38 genome assembly and the transcriptome using STAR v2.5.3a and GENCODE 26 annotation.

[0306] Gene expression was quantified using STAR (-quantMode GeneCounts) and VSN- transformed (variance stabilizing normalization). Then a quantile normalization method was applied to normalize the nonbiological variability. Using the HUGO Genome Nomenclature Committee (HGNC), which is a resource for approved human gene nomenclature, inventors included protein-coding genes, and among other gene locus types, immunoglobulin genes, and T cell receptor genes. Genes brought by sexual chromosomes and mitochondrial DNA were not included in the analysis.

[0307] Determining the correlation between the gene expression of the 28 genes composing the CLIF-SIG score and their gene expression The expression of the 28 genes composing the CLIF-SIG score was determined by realtime PGR using low-density microfluidic arrays (Fluidigm, Standard BioTools Inc., South San Francisco, CA) in whole blood collected in Tempus tubes from 40 patients with acutely decompensated cirrhosis, in whom the expression of these genes was previously measured by RNA-seq. The expression of the 28 genes composing the CLIF-SIG score in the same group of patients was also determined by digital quantification of nucleic acids using the nCounter technology (Nanostring, Seattle, WA).

[0308] Results

[0309] Patients’ characteristics.

[0310] Table 1 shows the unpaired comparison of clinical features, laboratory data and scores at T1 , and the 28-day and 90-day mortality rates, between patients from the low- and high-severity groups in Cohort 1 . There were no differences with respect to demographic data and etiology of cirrhosis. Among the major complications at hospital admission, ascites and hepatic encephalopathy were more frequent, and gastrointestinal hemorrhage less frequent in patients from the high-severity group. The prevalence of acute alcoholic hepatitis and bacterial infections were higher, liver and renal function worse, and biomarkers and scores estimating systemic inflammation increased in the high-severity group. Mortality rates at 28 days and 90 days after admission were low in the low severity group and very high in the high severity group.

[0311] Comparing changes in both severity groups, significant differences were only found in some parameters of systemic inflammation, including a marked decrease of CRP and moderate reduction of IL-6 in the low-severity group. The CLIF-SIC score decreased markedly in these patients from the low-severity group but not in patients from the high- severity group in concordance with the reduction in CRP observed in the former group of patients. The high-but not the low-severity group, showed significant reduction in CLI F- SIG score. Improvement in CLIF-SIG score in the high-severity group coincided with a significant reduction in the prevalence of ACLF at T2 mainly due to resolution of ACLF- 1 in many patients and of ACLF 2 in some. Improvement in the CLIF-SIG score in the high-severity group occurred despite that some the 52 patients with “early” pre-ACLF developed ACLF during the T1-T2 period. There was no change in MELD-sodium scores in any of the two groups. The rate of patients infected at T1 or who acquired infections during the study period were 22% and 7% respectively in the low-severity group, and 38% and 16% in high-severity group.

[0312] The 44 patients from the low-severity group and the 15 patients with “early” pre-ACLF differed only in serum bilirubin and MELD-score, but not in any clinical feature or biomarkers of systemic inflammation, including cytokines and CLIF-SIC score. However, the CLIF-SIG score was significantly increased in patients with “early” pre-ACLF.

[0313] Table 1. Demographic, clinical and laboratory data and 28-day and 90-day mortality rates in patients from Cohort 1.

[0314] Low-severity group High-severity group .

[0315] (n=403) (n=297)P value

[0316] Demographic data

[0317] Age (Years) 58.6+ / - 11.59 57.9+ / - 12.46 0.47

[0318] Sex (Female, %) 148(37%) 92(31 %) 0.13

[0319] Ethiology of cirrhosis data

[0320] Alcohol 177(44%) 137(46%) 0.61

[0321] Hepatitis C 36(9%) 19(6%) 0.28

[0322] NASH 44(11 %) 28(9%) 0.61

[0323] Mixed 60(15%) 51(17%) 0.48

[0324] Other 86(21 %) 62(21 %) 0.96

[0325] Complications at admission

[0326] Ascites 267(66%) 222(75%) 0.02

[0327] Hepatic encephalopathy 123(31%) 149(50%) p < 0.01

[0328] Gastrointestinal bleeding 95(24%) 47(16%) 0.02

[0329] Bacterial infections 129(32%) 145(49%) p < 0.01

[0330] Acute alcoholic hepatitis 72(21 %) 83(31 %) 0.01

[0331] Laboratory data

[0332] Serum bilirubin (mg / dL) 2.03(1.23-4.16) 5.6(1.92-13.16) p < 0.01

[0333] INR 1.44(1.27-1.66) 1.71(1.4-2.2) p < 0.01

[0334] Serum creatinine (mg / dL) 0.89(0.7-1.2) 1.36(0.86-2.1) p < 0.01

[0335] Serum albumin (g / dL) 2.8(2.47-3.3) 27(2.2-3.2) p < 0.01

[0336] Biomarkers of systemic inflammation and scores

[0337] White blood cells (x109 / L) 5.9(4.16-8.46) 7.54(5.3-11.0) p < 0.01

[0338] CRP (mg / L) 16.9(7.3-38.25) 29.4(15-64.8) p < 0.01

[0339] Neutrophile Lymphocyte

[0340] Ratio 3.86(2.43-6.23) 6.23(3.86-9.52) p < 0.01

[0341] IL_1 RA (pg / mL) 3.31 (1.73-7.44) 4.99(2.3-10.88) p < 0.01

[0342] IL_6 (pg / mL) 11.52(5.63-23.15) 21.06(9.22-49.62) p < 0.01

[0343] IL_8 (pg / mL) 3.4(1.4-7.71) 4.72(2.06-11.97) p < 0.01

[0344] 203.16(118.49- 253.54(123.45-

[0345] IP_10 (pg / mL) 362.09) 434.58) 0.02

[0346] 167.11 (123.89-

[0347] MCP_1 (pg / mL) 231.75) 227.5(137.02-357.39) p < 0.01

[0348] MIP_1_Beta (pg / mL) 15.34(10.89-20.65) 17.62(12.59-26.06) p < 0.01

[0349] TNF_Alpha (pg / mL) 24(14.83-47.66) 36.38(23-63.81) p < 0.01

[0350] Treatments

[0351] Antibiotics* 271 (68%) 201 (69%) 0.91

[0352] Vasopressors 41(10%) 36(12%) 0.48

[0353] Diuretics 250(63%) 131 (45%) p < 0.01

[0354] Betablockers 179(45%) 102(35%) 0.01

[0355] Albumin 104(26%) 89(30%) 0.24

[0356] Scores and ACLF prevalence and grades

[0357] MELD-Na 18(15-22) 26(22-30) p < 0.01 CLIF-SIC score 0.59(0.38-0.87) 0.8(0.56-1.24) p < 0.01

[0358] CLIF-SIG score 0.3+ / - 0.18 0.6+ / - 0.22 p < 0.01

[0359] ACLF prevalence - 166(56%) p < 0.01

[0360] ACLF - 1 - 97(33%)

[0361] ACLF - 2 - 53(18%)

[0362] ACLF - 3 - 16(5%)

[0363] Mortality 28-day mortality 4(1 %) 101 (34%) p < 0.01

[0364] 90-day mortality 50(12%) 151 (52%) p < 0.01

[0365] *Non-absorbable or poorly absorbable oral antibiotics included

[0366] The CLIF-SIG score as a marker of systemic inflammation

[0367] Authors identified 28 genes, 4 related to B cell and 24 to innate immune cells, as independent variables discriminating high- from low-severity group patients and were used to compute the CLIF-SIG score. The relative influence of each of these genes on the CLF-SIG score is given in Figure 1A; and although for simplicity reasons they are homogeneously represented, 24 genes were upregulated and 4, those related to NK cells (XCL1 , GZMH, GNLY and FASLG), were downregulated. The CLIF-SIG score at T 1 was markedly increased in the high-severity group (“early” pre-ACLF and ACLF are represented separately) versus the low-severity group and healthy subjects (Figure 1 B), and in patients with ACLF versus patients with “early” pre-ACLF. The CLIF-SIC score also differentiated patients from the high- versus the low-severity groups and “early” pre- ACLF from ACLF. However, there was marked overlap between both groups (Figure 1C). To allow comparative estimation, median values of CLIF-SIG and CLIF-SIC scores are also given as fold increases for each comparative in parenthesis at the top of the figure. Figure 1 D shows that the area under the ROC curves differentiating high-severity from low-severity groups in the train and test cohorts at T1 were significantly higher with the CLIF-SIG score than with CLIF-SIC score, indicating higher accuracy for the CLIF- SIG score. Figure 1 E shows that CLIF-SIG score and CLIF-SIC score were directly and significantly correlated.

[0368] Figures 2A-to-2D illustrate the characterization of CLIF-SIG and CLF-SIC scores, CRP, and WBC among the different phenotypes of ADC in the Predict study. None of these variables differentiated stable and unstable cirrhosis and “delayed” pre-ACLF, likely indicating that these different phenotypes develop in the setting of a similar magnitude of moderate systemic inflammation. The CLIF-SIG score was the only variable discriminating “early” pre-ACLF from ACLF, and WBC was the only variable differentiating ACLF-1 from ACLF-2 and ACLF. Finally, all variables except WBC differentiated high- from low-severity groups, although overlapping was intense in all except the CLIF-SIG score.

[0369] The course of the CLIF-SIG score correlates with patients’ clinical course.

[0370] Definitions

[0371] Authors first identified the magnitude of the delta CLIF-SIG score that better stratifies the three inflammatory courses detected in Sub-cohort 1a: upregulation, minimal or no change, or downregulation (Figure 3A).

[0372] The course of patients from the low-severity group during the study period was uneventful, except for a low percentage of patients developing infections. In contrast, the course of patients from the high severity group was extremely variable, being stratified as “good” if patients resolved ACLF (no ACLF at T2, 60 patients), presented steady ACLF-1 (ACLF-1 at T1 and T2, 20 patients), or improved ACLF-3 or ACLF-2 to ACLF-1 (5 patients) within the first 21 -days after enrolment; and “poor” if they had “early” pre- ACLF (42 patients), because they all progressed from no ACLF to ACLF of any grade within 21 days after T1 , if they presented ACLF-1 at T1 which progressed to ACLF-2 or ACLF-3, or ACLF-2 which progressed to ACLF-3 (9 patients), if they presented ACLF-3 at T1 which improved only to ACLF-2 or steady ACLF-2 or ACLF-3 (27 patients).

[0373] Finally, outcome was defined according to whether patients died within the first 28 days after hospital admission or survived.

[0374] The course of systemic inflammation in Sub-cohorts 1a and 1b indicates a significant mismatching between the systemic inflammatory reaction and hospital admission.

[0375] Acute systemic inflammatory reaction in sepsis follows an explosive course with an initial ascending-developing (A-D) phase, a short intermediate top-steady inflammation (ITS) phase, and a final descending-recovery phase (D-R), overall lasting approximately one week in patients responding to treatment (Mantovani A, Garlanda C. Humoral innate immunity and acute phase protein. N Engl J Med 2023;388:439-452). In contrast, authors have reported previously that systemic inflammation in decompensated cirrhosis also develops in the form of bursts but with a longer duration (Fernandez J, Claria J, Amoros A, et al. Effects of albumin treatment on systemic and portal hemodynamics and systemic inflammation in patients with decompensated cirrhosis. Gastroenterology 2019;157:149- 162). This is likely the reason why, with two study points and a 7-day median duration of the study period, authors could detect only single phases of the inflammatory process (Figure 3B). Among the 375 patients from Sub-cohort 1a, 76 patients (20.3%) showed upregulation, 167 (44.5%) minor or no-changes, and 132 (35.2%) downregulation of the CLIF-SIG score. Mismatching between the onset of systemic inflammation and hospital admission was an expected finding, since ascites or encephalopathy may take few days before they are clinically evident. However, authors were surprised by the high frequency and particularly the large duration of this period in most patients. Indeed, they detected the initial A-D phase in only 76 patients, and the D-R phase in 132 patients, indicating that the initial A- D phase or both the A-D phase plus the TSI phase occurred prior hospitalization in 167 and 132 patients, respectively. Therefore, phases A-D or A-D plus TSI took place between 1 to 2 weeks prior to hospital admission in most of our patients. Although the duration of the systemic inflammatory reaction is difficult to estimate with our data, the one-week duration of the observation period for each phase suggests that it may average 3 weeks or more.

[0376] Authors identified a group of only 10 patients (2.6%) with undetectable systemic inflammation (CLIF-SIG score at T1 and T2 below the median CLIF-SIG score in healthy subjects) (Figure 3B).

[0377] Figure 30 shows the ROC curve estimating the accuracy of the CLIF-SIG score discriminating all patients from the low- and high-severity groups in Cohort 1 , and the score value of 0.386 (detected by the best Youden’s index) as the better cut-off level discriminating these groups at T1. This critical CLIF-SIG score level (CSL) was subsequently applied at T2 to stratify the magnitude of systemic inflammation into moderate (< 0.386) or severe (> 0.386).

[0378] Figure 3D shows the complex net of lines joining the 3 to 4 consecutive CLIF-SIG scores measured in patients from Sub-cohort 1b. At first glance, however, it is remarkable the high frequency of patients with intense downregulation of the score from day 0 or between day 7 and 14, reflecting patients in whom the A-D plus TSI or the A-D phases of the inflammatory reaction occurred before hospital admission, confirming the observations in Sub-cohort 1a.

[0379] Figures 3E-to-3H illustrate the estimated graphical expression of the most relevant clinical courses identified in Figure 3D. There were only 4 patients in whom the three phases of the inflammatory reaction were totally or partially detected within the first three weeks after hospital admission (Figure 3E). Figures 3F and 3G show representative examples of systemic inflammation curves lacking the TSI plus the A-D phases, or the A-D phase alone. Finally, Figure 3H shows existence of few patients in whom ADC develope in the absence of detectable systemic inflammatory reaction in Sub-cohort 1b and illustrates patients developing recurrent systemic inflammatory reactions as manifested by a descending-recovery phase from a prior burst of systemic inflammation, followed by the ascending phase (3 cases) or a complete course of a second inflammatory reaction.

[0380] Magnitude of systemic inflammation and patients’ clinical course

[0381] To improve the graphical expression on the relationship between the magnitude of systemic inflammation and clinical course, patients from the Sub-cohort 1a were assigned two colors, red or green, according to the course of the CLIF-SIG score (Figure 4A). Patients in whom the score upregulated during the study period to a level > 0.386 at T2, or followed a steady but highly upregulated course, as defined also by a score at T2 > 0.386 or downregulated but without reaching a score at T2 < 0.386 received the red color, indicating severe systemic inflammation. The remaining patients received the green color, indicating moderate systemic inflammation.

[0382] The course of the CLIF-SIG score in the 195 patients from the low-severity group and in the 180 patients from the high-severity group in Sub-cohort 1a was upregulated in 41 and 35 patients respectively, (21% and 19.4%), showed minor changes in 96 and 71 (49.2% and 39.4%) and downregulated in 58 and 74 (29.7% and 41.1 %) (Figure 4A). Severe systemic inflammation was extremely frequent (77,4%) in patients from the high- severity group, and infrequent (22.6%) in the low-severity groups. The time of hospitalization were 14 (9-22) and 7(5-10) days, respectively.

[0383] Two lines of evidence from Sub-Cohort 1a supports that severity of systemic inflammation correlated with patients’ clinical course. First, the prevalence of poor clinical course and the 28-day mortality rate were 53% and 32%, respectively, in patients with severe systemic inflammation, and 32% and 23% in patients with moderate systemic inflammation (p <0.001 and p<0.05, respectively; data below Figure 5B). Second, whereas the frequencies of patients developing poor clinical course were 60.6% (20 out of 33 patients) and 63.2% (43 out of 68 patients) during the A-D and the TSI phases of the inflammatory reaction, respectively, it was only of 24.3% (17 out of 70 patients) during the D-R phase, indicating that the risk of developing poor clinical course decreases markedly during the D-R phase.

[0384] However, the clearest evidence that systemic inflammation correlates with clinical course was observed in Sub-cohort 1b (Figure 4B). Whereas most patients whose clinical course took place within the virtual high inflammation zone (CSL > 0.386) developed ACLF, this was extremely infrequent in patients whose clinical course took place in the low inflammation zone.

[0385] CLIF-SIG score genes in unselected patients with bacterial infections. Since the RNA prepared by Herwanto V, Tang B, Wan Ya, et al. Blood trancriptome analysis of patients with uncomplicated bacterial infection and sepsis. BMC Res Notes (2021) 14:76 in patients with bacterial infections was depleted from beta-globin RNA, authors could validate 26 genes of their score, but not IGKV2D-30 and IGKV2D-28. Figure 5A show expression of CLIF-SIG score genes across the 3 groups (healthy subjects and non-cirrhotic patients with bacterial infections (with sepsis or septic shock)), represented in heatmap. Figures 5B and 50 show that the expression of these genes, represented in heatmap and PCA plots, were markedly increased in patients with bacterial infections as compared to healthy subjects and discriminated patients with and without septic shock, as surrogate of severity of systemic inflammation. Figure 5B illustrates that down-regulated genes (XCL1 , GZNH, GNLY and FASLG) were as accurate as upregulated genes discriminating these subsets of subjects.

[0386] CLIF-SIG score composed of 4 genes

[0387] The results in Figure 6 show that a model composed of 4 genes (HMGB2, RETN, ZNF608, PYCARD) was able to find significant differences in the distributions of healthy subjects and Low-severity group and High-severity group patients (Figure 6A). Even with only 4 genes the model was better than the CLIF-SIC score composed of 28 genes when discriminating patients from the low-severity group and the high severity group in both, the train and test sets, as shown in the Figure 6B. This score also showed a positive and significant association with the CLIF-SIC score, indicating an association with the score and systemic inflammation (Figure 6C).

[0388] CLIF-SIG score composed of 28 genes

[0389] The expression of the 28 genes composing the CLIF-SIG score were measured by two additional platforms that provide similar precise and accurate guantification of individual RNA molecules to that of the RNA seguencing platform. These two platforms were Fluidigm, a low-density microfluidic array system, and Nanostring, a digital molecular barcoding technology, both commonly available in tertiary hospitals. The results of these additional measurements showed high significant correlation between the expression data from Fluidigm and Nanostring and the expression results of the 28 genes obtained by RNA seg. These findings support the feasibility of measuring the CLIF-SIG score in the clinical setting closer to the patient bedside (Figure 7).

Claims

CLAIMS1. An in vitro method for determining the presence of acute-on chronic liver-failure (ACLF) in a subject suffering from cirrhosis or delayed pre-ACLF which comprises: a) determining the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has ACLF.

2. The in vitro method according to claim 1 , wherein the method further comprises: a) determining the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11 , CRISPLD2, OSCAR, C3AR1 ,IGKV1 D-33.FPR1 ,SAMD3, MS4A4A, IGKV2-28; and b) comparing the expression level of said genes with their reference values; wherein an altered expression level of said genes when compared to the reference value for said genes, wherein said altered expression level is: an increased expression level of the genes IGFBP7, ORM1 ,ACSL1 ,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or a decreased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes, is indicative that said subject has ACLF.

3. An in vitro method for determining the risk of developing ACLF in a subject suffering from cirrhosis or delayed pre-ACLF which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values;wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has high risk of developing ACLF.

4. The in vitro method according to claim 3, wherein the method further comprises: a) determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2.STX11 , CRISPLD2, OSCAR, C3AR1 ,IGKV1 D-33.FPR1 ,SAMD3, MS4A4A, IGKV2-28 ; and b) comparing the expression level of said genes with their reference values; wherein an altered expression level of said genes when compared to the reference value for said genes, wherein said altered expression level is: an increased expression level of the genes IGFBP7, ORM1,ACSL1,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1 , STX11, OSCAR, IGKV2-28, IGKV2-33, or a decreased expression level of the XCL1, GZMH, GNLY, FASLG or SAMD3 genes, is indicative that said subject has high risk of developing ACLF.

5. The in vitro method according to any one of claims 1 to 4 wherein the subject suffers unstable or stable acute decompensated cirrhosis.

6. An in vitro method for monitoring the progression of an ACLF in a subject which comprises: a) determining the expression levels of the HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject, b) comparing the expression levels of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point after the onset of ACLF, wherein if the expression level of HMGB2, RETN, ZNF608 and PYCARD genes are decreased with respect to said reference value, then it is indicative of ACLF resolution.

7. The in vitro method according to claim 6, wherein the method further comprises: a) determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2.STX11 , CRISPLD2, OSCAR, C3AR1 ,IGKV1 D-33.FPR1 ,SAMD3, MS4A4A, IGKV2-28 ; and b) comparing the expression level of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point after the onset of ACLF, wherein an altered expression level of said genes when compared to reference value of said genes in a sample is indicative of ACLF resolution, wherein said altered expression level is: a decreased expression level of the genes IGFBP7, ORM1 ,ACSL1,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1 , STX11, OSCAR, IGKV2-28, IGKV2-33, or an increased expression level of the XCL1, GZMH, GNLY, FASLG or SAMD3 genes.

8. An in vitro method for monitoring the effect of a therapy in a patient suffering from ACLF which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point, wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject are decreased with respect to said reference value, there is indicative of the therapy is being effective.

9. The in vitro method according to claim 8, wherein the method further comprises: a) determining the expression level of one or more of the following genes:IGFBP7, ORM1, ACSL1 , BCL6, OLFM4, SERPINB10, STK32B,CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11 , CRISPLD2, OSCAR, C3AR1 ,IGKV1 D-33.FPR1 ,SAMD3, MS4A4A, IGKV2-28 ; and b) comparing the expression level of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point, wherein an altered expression level of said genes when compared to reference value of said genes in a sample is indicative that the therapy is being effective, wherein said altered expression level is: a decreased expression level of the genes IGFBP7, ORM1 ,ACSL1 ,BCL6,OLFM4,SERPINB10,STK32B,CYP4F2,CDK14,P CBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or an increased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes.

10. An in vitro method for selecting a therapy for a patient suffering from ACLF which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes with reference values, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point, wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject are increased with respect to said reference value, then it is indicative that patient is to be treated with a therapy aimed at treating the effects of liver failure and / or for liver transplantation.11 . The in vitro method according to claim 10, wherein the method further comprises: a) determining the expression level of one or more of the following genes:IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2.STX11 , CRISPLD2, OSCAR, C3AR1 ,IGKV1 D-33.FPR1 ,SAMD3, MS4A4A, IGKV2-28; andb) comparing the expression level of said genes with a reference sample, wherein the reference values are expression levels of the same genes which have been obtained from said patient at an earlier time point; wherein an altered expression level of said genes when compared to the reference sample, wherein said altered expression level is: an increased expression level of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1 , STX11, OSCAR, IGKV2-28, IGKV2-33, or a decreased expression level of the XCL1, GZMH, GNLY, FASLG or SAMD3 genes, is indicative that said patient is a candidate for receiving a therapy aimed at treating the effects of liver failure and / or for liver transplantation.

12. An in vitro method for determining the degree of systemic inflammation in a subject, which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has a severe systemic inflammation.

13. The in vitro method according to claim 12, wherein the method further comprises: a) determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2,CDK14,PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28; and b) comparing the expression level of said genes with their reference values; wherein an altered expression level of said genes when compared to the reference value for said genes, wherein said altered expression level is: an increased expression level of the genes IGFBP7, ORM1, ACSL1,BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1 , CRISPLD2, C3AR1 , STX11 , OSCAR, IGKV2-28, IGKV2-33, or a decreased expression level of the XCL1 , GZMH, GNLY, FASLG or SAMD3 genes, is indicative that said subject has a severe systemic inflammation.

14. The in vitro method according to any one of claims 12 or 13, wherein the subject suffers from ACLF or an infection.

15. The method according to claim 14, wherein the infection is a bacterial infection.

16. The method according to any one of claims 1 to 14, wherein the ACLF is ACLF1 , ACLF2, ACLF3 or early ACLF.

17. An in vitro method for determining the presence of a bacterial infection with sepsis in a subject which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein an expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes is indicative that said subject has a bacterial infection with sepsis.

18. The method according to claim 17 wherein the subject has septic shock.

19. The method according to any one of claims 2, 4, 7, 9, 11 and 13 which comprises determining the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes and the expression level of at least 2 genes selected from the group consisting of the IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A, IGKV2-28 genes.

20. The method according to any one of claims 1 to 19, wherein the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D- 30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 comprises the determination of the mRNA level of said genes or a fragment thereof.

21. The method according to claim 20, wherein the determination of the expression level of at least HMGB2, RETN, ZNF608, PYCARD genes and one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28 is carried out by RNA-seq.

22. The method according to any one of claims 1 to 21 , wherein the sample is a biofluid.

23. The method according to claim 22, wherein the biofluid is blood.

24. A kit or assay device comprising reagents adequate for the determination of the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes.

25. A kit or assay device comprising reagents adequate for the determination of the expression level of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2- 28.

26. Use of: reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes; or a kit or assay device according to claim 24;for determining the presence of ACLF, for determining the risk of developing ACLF, for monitoring the progression of ACLF, for monitoring the effect of a therapy, for selecting a patient suffering from ACLF for a therapy, for determining the degree of systemic inflammation, or for determining the presence of a bacterial infection with sepsis in a subject.

27. Use of: reagents specific for the determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes and for the determination of the expression level of one or more of the following genes: IGFBP7, ORM1 , ACSL1 , BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1 , IGKV2D-28, IGKV2D-30, CD163, ABCA1 , LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1 , RNASE2, STX11 , CRISPLD2, OSCAR, C3AR1 , IGKV1 D-33, FPR1 , SAMD3, MS4A4A and IGKV2-28, or a kit or assay device according to claim 25; for determining the presence of ACLF, for determining the risk of developing ACLF, for monitoring the progression of ACLF, for monitoring the effect of a therapy, for selecting a patient suffering from ACLF for a therapy, for determining the degree of systemic inflammation, or for determining the presence of a bacterial infection with sepsis in a subject.

28. A computer system comprising one or more programs, wherein the one or more programs are include instructions for performing the method of any one of claims 1 to 23.

29. A computer-readable storage medium storing one or more programs, the one or more programs comprising instructions, which when executed by one or more processors of an electronic device, cause the electronic device to perform the method of any one of claims 1 to 23.

30. A method for determining the presence of acute-on chronic liver-failure (ACLF) in a subject suffering from cirrhosis or delayed pre-ACLF and treating the patient comprising: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said patient; and b) comparing the expression level of said genes with reference values for eachgene, wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the patient is greater than the respective reference values, said patient is treated with an adequate therapy including antibiotics, diuretics, albumin, lactulose, beta-adrenergic blocker agents, vasoconstrictors, sclerosing agents, intrahepatic portosystemic shunting, plasma exchange, artificial respiratory support, renal hemodialysis, albumin dialysis, liver transplantation and / or combinations thereof.31 . A method for determining the risk of developing ACLF in a subject suffering from cirrhosis or delayed pre-ACLF and treating the patient which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein if expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject greater than the reference value for said genes, then the patient is treated with a therapy adequate for the prevention of ACLF including antibiotics, diuretics, albumin, lactulose, beta-adrenergic blocker agents, terlipressin, sclerosing agents, intrahepatic portosystemic shunting and / or combinations thereof.

32. A method for determining the degree of systemic inflammation in a subject and treating the subject which comprises: a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject is greater than the reference value for said genes, then the patient is treated with a therapy adequate for severe systemic inflammation including antibiotics, corticosteroids, albumin, plasma exchange and / or combinations thereof.

33. A method for determining the presence of a bacterial infection with sepsis in a subject and treating the subject which comprises:a) determining the expression level of HMGB2, RETN, ZNF608 and PYCARD genes in a sample from said subject; and b) comparing the expression level of said genes in said sample with their reference values; wherein if the expression level of the HMGB2, RETN, ZNF608 and PYCARD genes in the sample from the subject is greater than the reference value for said genes, then the patient is treated with a therapy adequate for bacterial infection and / or sepsis including antibiotics, vasoconstrictors, organ support including renal hemodialysis and / or artificial respiratory support.

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