Methods for diagnosing fibrotic nash

Abstract

The present invention provides methods for diagnosing, staging, or monitoring fibrotic non-alcoholic steatohepatitis (NASH) in a subject. The invention also provides monitoring a subject's compliance to a prescribed treatment for fibrotic NASH, as well as monitoring therapeutic effect of a prescribed treatment for fibrotic NASH. The invention also provides a kit suitable for use in the methods of the present invention.

Description

[0001] METHODS FOR DIAGNOSING FIBROTIC NASH

[0002] The present invention provides methods for diagnosing, staging, or monitoring fibrotic non-alcoholic steatohepatitis (NASH) in a subject. The invention also provides monitoring a subject’s compliance to a prescribed treatment for fibrotic NASH, as well as monitoring therapeutic effect of a prescribed treatment for fibrotic NASH. The invention also provides a kit suitable for use in the methods of the present invention.

[0003] Non-alcoholic fatty liver disease (NAFLD) is a disease of alarmingly increasing prevalence, linked to metabolic, cardiovascular and malignant morbidity1 2. It is a spectrum of liver disease, ranging from non-alcoholic isolated steatosis, in which the predominant histological characteristic is lipid accumulation in hepatocytes, to non-alcoholic steatohepatitis (NASH), with additional hepatic inflammation leading to fibrosis, and ultimately culminating in NASH-related cirrhosis and hepatocellular carcinoma (HCC)1. Obesity and insulin resistance are strongly associated with NAFLD, both via increased delivery of free fatty acids to the liver and through increases of hepatic lipogenesis associated with hyperglycemia and hyperinsulinemia2. With the global increase in obesity and insulin resistance / type 2 diabetes mellitus, NAFLD has become the most prevalent liver disease in the world with prevalence rising to 25-30% of adult populations34.

[0004] Increased liver fibrosis has been shown to be the most important feature associated with increased overall and liver-related mortality and increased likelihood of developing liver-related complications including HCC and the need for liver transplantation, while disease activity itself did not correlate with outcome56.

[0005] This renders NAFLD fibrosis staging essential in determining the severity of NAFLD during clinical work-up, in order to initiate the right and timely multidisciplinary treatment program for each individual patient, which can consist of lifestyle and dietary interventions, treatment in clinical trials and for some cases the consideration of bariatric surgery1 7.

[0006] Liver biopsy remains the clinical reference standard for fibrosis detection and staging, but has the potential for complications and suffers from sampling and reading errors89. Radiologic assessment of liver stiffness has gained credibility over the past decade as a diagnostic and staging tool for liver fibrosis with vibration-controlled transient elastography (i.e. Fibroscan) and magnetic resonance imaging10 11. Simple proxies of liver fibrosis including FIB-4 (age, platelet count, aminotransferases), NAFLD fibrosis score, and serologic panels of combined biomarkers like Enhanced Liver Fibrosis (ELF), have good accuracy in excluding advanced fibrosis, and could be used to identify individuals at low risk for advanced disease11 12. However, all these diagnostic tests do not discriminate those who are at risk for fibrosis development and / or progression from those who are not. Moreover, recently it was demonstrated that repeated measurements of these tests are not useful for monitoring the course of NAFLD13. These are great unmet clinical needs in daily practice for patients with NAFLD- NASH and their medical practitioners including hepatologist, internists, family physicians and diabetes nurses12. The present invention aims to, at least, partially address these long-felt, unmet clinical needs.

[0007] Summary of the invention

[0008] The invention is based on the inventors’ finding of novel biomarkers that are specific for fibrotic NASH. Surprisingly, these biomarkers are not only useful in the context of diagnosing fibrotic NASH, but also can be used for monitoring and staging fibrotic NASH.

[0009] As shown in the Examples section of the present disclosure, the inventors found that patients with fibrotic NASH have reduced a2,3-sialylation across most complex-type / V-glycans. Specifically, altered level of a2,3-sialylation was also observed in / V-glycans with two antennae (A2L), three antennae (A3L), and four antennae (A4L). Additionally, the combined level of a2,3-sialylation in complex-type / V-glycans and (mono-antennary) hybrid-type glycans was decreased, showing odds ratios (OR) of 0.36 and 0.33 for the two cohorts studied as compared to healthy controls. In contrast, a2,6-sialylation was found to be generally increased in patients with fibrotic NASH. This altered a2,6-sialylation was most pronounced for complex-type / V-glycans with three antennae (A3E), where the inventors noted ORs of 2.95 and 6.16 in the two cohorts studied as compared to healthy controls.

[0010] Furthermore, as shown for example in Figure 3, 11 and Tables 2, 4, 6, and 8 of the present disclosure, the inventors found that alteration in a2 ,3-sialylation and / or a2 ,6-sialylation levels may be proportional to the severity of fibrosis in patients with NASH. In other words, patients with more advanced liver fibrosis (for example patients with a Brunt fibrosis score of 4) may have lower a2,3-sialylation levels and / or higher a2,6-sialylation levels as compared to patients with less severe fibrosis (for example patients with a Brunt fibrosis score of 3, 2, 1), or no fibrosis. This proportional change in the levels of a2,3-sialylation and / or a2,6-sialylation renders these biomarkers useful for applications such as fibrosis staging and / or patient monitoring.

[0011] An expansion of the replication cohort, illustrated by Example 2, improved the statistical power of the analysis and confirmed the initial findings, illustrated by Example 1 . Notably, the combined level of a2,3-sialylation in complex-type / V-glycans and (mono-antennary) hybrid-type glycans and (mono- antennary) hybrid-type glycans and (mono-antennary) hybrid-type glycans was decreased, showing ORs of 0.36 and 0.11 for the two cohorts studied as compared to healthy controls. In contrast, a2,6- sialylation was found to be generally increased in patients with fibrotic NASH. This altered a2,6- sialylation was most pronounced for complex-type / V-glycans with three antennae (A3E), where the inventors noted ORs of 2.95 and 11 .6 in the two cohorts studied as compared to healthy controls.

[0012] Accordingly, in a first aspect provided herein is a method for diagnosing fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:

[0013] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a sample from the subject; and • comparing the determined levels of a2,3-sialylation and / or a2,6-sialylation to a reference value, wherein: i) an increase in the levels of a2,6-sialylation in the sample; and / or ii) a decrease in the levels of a2 ,3-sialylation in the sample is indicative of the subject having fibrotic NASH.

[0014] In a further aspect, provided herein is a method for staging fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:

[0015] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a sample from the subject; and

[0016] • comparing the determined levels of a2,3-sialylation and / or a2,6-sialylation to reference values indicative of a stage of fibrotic NASH, and thereby determining the subject’s stage of fibrotic NASH.

[0017] Suitably, the staging may be according to Brunt Fibrosis score.

[0018] In a further aspect, provided herein is a method for monitoring fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:

[0019] • determining the levels of a2,3-sialylation and / or a2 ,6-sialylation of / V-glycans in a first sample from the subject; and

[0020] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a second sample from the subject, wherein the second sample has been obtained from the subject at a later time point than the first sample, wherein: i) an increase in the levels of a2 ,6-sialylation in the second sample and / or a decrease in the levels of a2 ,3-sialylation in the second sample as compared to the first sample is indicative of fibrotic NASH progressing in the subject; or ii) a decrease in the levels of a2 ,6-sialylation in the second sample and / or an increase in the levels of a2-3-sialylation in the second sample as compared to the first sample is indicative of fibrotic NASH regressing in the subject.

[0021] In a further aspect, provided herein is a method for monitoring a subject’s compliance to a prescribed treatment for fibrotic non-alcoholic steatohepatitis (NASH), the method comprising the steps of:

[0022] • determining the levels of a2,3-sialylation and / or a2 ,6-sialylation of / V-glycans in a first sample from the subject; and

[0023] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a second sample from the subject, wherein the second sample has been obtained from the subject after the prescribed treatment, wherein a decrease or no change in the levels of a2,6-sialylation in the second sample, and / or an increase or no change in the levels of a2,3-sialylation in the second sample as compared to the first sample is indicative of a subject’s compliance to the prescribed treatment for fibrotic NASH.

[0024] In a further aspect, provided herein is a method of monitoring therapeutic effect of a prescribed treatment for fibrotic non-alcoholic steatohepatitis (NASH), the method comprising the steps of:

[0025] • determining the levels of a2 ,3-sialylation and / or a2 ,6-sialylation of / V-glycans in a first sample from the subject; and

[0026] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a second sample from the subject, wherein the second sample has been obtained from the subject after the prescribed treatment, wherein a decrease or no change in the levels of «2,6-sialylation in the second sample and / or an increase or no change in the levels of «2,3-sialylation in the second sample as compared to the first sample is indicative of a therapeutic effect of a prescribed treatment for fibrotic NASH.

[0027] Suitably, the / V-glycans may be complex / V-glycans.

[0028] Suitably, the / V-glycans may be selected from the group consisting of diantennary / V-glycans, triantennary / V-glycans, and tetraantennary / V-glycans, or a combination thereof.

[0029] Suitably, the / V-glycans may be selected from the group consisting of triantennary / V-glycans and tetraantennary / V-glycans.

[0030] Suitably, the sample may be a blood sample.

[0031] Suitably, the blood sample may be a dried blood spot sample.

[0032] Suitably, the blood sample may be selected from the group consisting of whole blood, blood plasma, and blood serum.

[0033] Suitably, the subject may have been diagnosed with or determined to be at risk of fibrotic NASH.

[0034] Suitably, the subject that has been determined to be at risk of fibrotic NASH: i) may have been diagnosed with NAFLD or non-fibrotic NASH; ii) may have been diagnosed with type 2 diabetes or insulin resistance; iii) may be obese; iv) may have been diagnosed with hypertension and / or dyslipidaemia; and / or v) may be suspected of having a genetic predisposing factor, optionally wherein the genetic predisposing factor is a mutation in a gene selected from the group consisting of PNPLA3, TM6SF2, MBOAT7, GCKR, and HSD18B13.

[0035] Suitably, the treatment may be selected from the group consisting of weight loss and lifestyle improvement.

[0036] Suitably, levels of a2,3-sialylation and / or a2,6-sialylation may be determined by a method selected from the group consisting of mass spectrometry, high-performance liquid chromatography, capillary (gel) electrophoresis with laser induced fluorescence detection, hydrophilic interaction liquid chromatography, lectin- or antibody-based binding assay, and an ELISA based assay.

[0037] Suitably, the method may further comprise determining the subject’s FIB-4 score, ELF score, and / or APRI score; and / or the subject’s levels of aspartate transaminase (AST), alanine transaminase (ALT); and / or performing liver imagining.

[0038] Suitably, the sample may have been subjected to / V-glycan release from blood proteins and linkagespecific chemical sialic acid derivatization prior to determining the levels of a2,3-sialylation and / or a2 ,6-sialylation of glycans, optionally wherein the proteins may be selected from the group consisting of plasma, serum and whole blood proteins.

[0039] Accordingly, in one aspect, the present invention provides a method for diagnosing fibrotic nonalcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:

[0040] • subjecting a sample from the subject to / V-glycan release from blood proteins and linkagespecific chemical sialic acid derivatization to provide a derivatized sample;

[0041] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in the derivatized sample; and

[0042] • comparing the determined levels of a2,3-sialylation and / or a2,6-sialylation to a reference value, wherein: i) an increase in the levels of a2,6-sialylation in the derivatized sample; and / or ii) a decrease in the levels of a2 ,3-sialylation in the derivatized sample is indicative of the subject having fibrotic NASH.

[0043] In a further aspect, provided herein is a method for staging fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:

[0044] • subjecting a sample from the subject to / V-glycan release from blood proteins and linkagespecific chemical sialic acid derivatization to provide a derivatized sample;

[0045] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in the derivatized sample; and • comparing the determined levels of a2 ,3-sialylation and / or a2,6-sialylation in the derivatized sample to reference values indicative of a stage of fibrotic NASH, and thereby determining the subject’s stage of fibrotic NASH.

[0046] Suitably, the staging may be according to Brunt Fibrosis score.

[0047] In a further aspect, provided herein is a method for monitoring fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:

[0048] • subjecting a first and second sample from the subject to / V-glycan release from blood proteins and linkage-specific chemical sialic acid derivatization to provide a first and second derivatized sample;

[0049] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in the first the derivatized sample; and

[0050] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in the second derivatized sample, wherein the second sample has been obtained from the subject at a later time point than the first sample, wherein: i) an increase in the levels of a2 ,6-sialylation in the second sample and / or a decrease in the levels of a2 ,3-sialylation in the second sample as compared to the first sample is indicative of fibrotic NASH progressing in the subject; ii) a decrease in the levels of a2 ,6-sialylation in the second sample and / or an increase in the levels of a2-3-sialylation in the second sample as compared to the first sample is indicative of fibrotic NASH regressing in the subject.

[0051] In a further aspect, provided herein is a method for monitoring a subject’s compliance to a prescribed treatment for fibrotic non-alcoholic steatohepatitis (NASH), the method comprising the steps of:

[0052] • subjecting a first and second sample from the subject to / V-glycan release from blood proteins and linkage-specific chemical sialic acid derivatization to provide a first and second derivatized sample;

[0053] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in the first derivatized sample; and

[0054] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in the second derivatized sample from the subject, wherein the second sample has been obtained from the subject after the prescribed treatment, wherein a decrease or no change in the levels of a2,6- sialylation in the second derivatized sample, and / or an increase or no change in the levels of a2 ,3-sialylation in the second derivatized sample as compared to the first derivatized sample is indicative of a subject’s compliance to the prescribed treatment for fibrotic NASH. In a further aspect, provided herein is a method of monitoring therapeutic effect of a prescribed treatment for fibrotic non-alcoholic steatohepatitis (NASH), the method comprising the steps of:

[0055] • subjecting a first and second sample from the subject to / V-glycan release from blood proteins and linkage-specific chemical sialic acid derivatization to provide a first and second derivatized sample;

[0056] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in the first derivatized sample; and

[0057] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in the second derivatized sample, wherein the second sample has been obtained from the subject after the prescribed treatment, wherein a decrease or no change in the levels of a2,6-sialylation in the second derivatized sample and / or an increase or no change in the levels of a2,3-sialylation in the second derivatized sample as compared to the first derivatized sample is indicative of a therapeutic effect of a prescribed treatment for fibrotic NASH.

[0058] Suitably, any one of the methods described herein may further comprise the steps of administering a fibrotic NASH treatment to a subject that has been diagnosed or determined to be at risk of fibrotic NASH.

[0059] In a further aspect, provided herein is a kit for use in a method of the invention, the kit comprising a detectably labelled agent that specifically binds to a2 ,3-sialylation and / or a2 ,6-sialylation of glycans.

[0060] Herein, the terms NAFLD and MASLD are used interchangeably. In view of a recent change in naming conventions, non-alcoholic fatty liver disease (NAFLD) is presently known as metabolic dysfunction- associated steatotic liver disease (MASLD).

[0061] Herein, the terms NASH and MASH are used interchangeably. In view of a recent change in naming conventions, non-alcoholic steatohepatitis (NASH) is presently known as metabolic dysfunction- associated steatohepatitis (MASH).

[0062] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0063] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0064] Various aspects of the invention are described in further detail below.

[0065] Brief description of the Figures

[0066] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0067] Figure 1 shows monosaccharide constituents that build up human / V-glycans (bottom) and glycosylation traits calculated therefrom, as illustrated on a fully sialylated tetraantennary / V-glycan (top).

[0068] Figure 2 shows replicated associations between individuals with NAFLD and healthy controls, (a) Volcano plot based on the calculated 36 glycosylation traits in the discovery cohort, (b-e) Comparison of the relative abundance differences between healthy controls and NAFLD using the glycosylation traits with negative and (f, g) positive effect sizes. P-values, ORs and 95% Cis are shown in Table 2. *, **: p-value < 0.05, 0.01 , respectively.

[0069] Figure 3 shows replicated associations identified between patients with NAFLD and healthy controls as further stratified as per NASH-related fibrosis stage (Brunt). *, **, ***: p-value < 0.05, 0.01 , 0.001 , respectively. The shown data corresponds to the replication cohort.

[0070] Figure 4 shows principle component analysis (PCA) based on the calculated glycosylation traits in each cohort, illustrating the distribution of samples and standards or pools. Scores plot of the PCA analysis for the discovery (a) and replication (b) illustrating the separation between healthy controls and NAFLD patients along PC1 and PC2, as well as the clustering of standards (discovery cohort, (a)) or pools (replication cohort, (b)). Loadings plots visually represent the variables that contribute to the separation in the PCA model in each cohort. Plasma standards in the discovery cohort (a) were distributed across 5 plates, and have previously been shown to be randomly scattered, indicating the absence of systematic batch effects. The close clustering of plasma standards in the discovery cohort and pools in the replication cohort indicate the low technical variability of the method. In the scores plots, healthy controls are indicated by lines, NAFLD patients are indicated by filled circles, plasma standards are indicated by filled squares in (a), and serum pools are indicated by filled squares in (b).

[0071] Figure 5 shows age and sex distribution across disease groups (a) Overall and (b) sex stratified age distribution in the cohorts, (c) Overall and (d) sex stratified age distribution in the replication cohort. A significant age difference was observed for females in the discovery cohort based on simple statistical testing, although this effect has been corrected for by logistic regression analysis including age, sex and their interaction in the model as covariates.

[0072] Figure 6 shows the ratio of the glycosylation traits AL and AE, illustrating the observed effect of NAFLD (a-b), and more specifically of fibrosis (c-d) on the blood / V-glycome as a single glycosylation trait, (a) Differences in the relative levels of AL / AE in healthy controls versus NAFLD patients overall, and (b) sex stratified, (c) Overall and (d) sex stratified differences in AL / AE between healthy controls and patients with various degree of fibrosis (Brunt fibrosis scores) in the replication cohort. While this ratio represents the overall effect, due to its limitations described in the results and discussion of Example 1 , all significantly associated glycosylation traits are shown and described separately in Example 1 (Figure 2, 3; Table 2, 3). Note that females in Brunt fibrosis score groups (d) did not hold up to Kruskal-Wallis statistical testing, although visually the trend is clear. As age and sex is rather well- matched between the groups, the inventors believe the lack of statistical significance for the female group is due to low sample size.

[0073] Figure 7 shows comparison of overall sialylation (CS) between the disease groups per cohorts and sex. (a) Differences in the relative levels of CS in healthy controls versus NAFLD patients overall, and (b) sex stratified, (c) Overall and (d) sex stratified differences in CS between healthy controls and patients with various degree of fibrosis (Brunt fibrosis scores) in the replication cohort. The insignificant results of the statistical tests suggest that the observed fibrosis-specific effect is driven by a shift in the relative abundance of sialic acid linkages (Figure 6), rather than alterations in the relative levels of CS (Rs = -0.022; p-value = 0.89). (e) Forest plot for the comparison of logistic regression models for each predictor and their respective odds ratios. Gradient indicates -logw p-value. A4L and A3L display a -log p-value of 3.5 and 4, respectively. A2L displays a -logw p-value of about 2.8. AL displays a -logw p-value of about 2.5. (f) ROC curves for the composite and A4L model.

[0074] Figure 8 shows PCA based on the calculated glycosylation traits in each cohort, illustrating the distribution of samples and standards or pools. Scores plot of the PCA analysis for the discovery (a) and replication (b) illustrating the separation between healthy controls and MASLD (NAFLD) patients along PC1 and PC2, as well as the clustering of standards (discovery cohort, a) or pools (replication cohort, b). Loadings plots visually represent the variables that contribute to the separation in the PCA model in each cohort. Plasma standards in the discovery cohort (a) were distributed across 5 plates, and have previously been shown to be randomly scattered, indicating the absence of systematic batch effects30. The close clustering of plasma standards in the discovery cohort and pools in the replication cohort indicate the low technical variability of the method. In the scores plots, healthy controls are indicated by lines, MASLD patients are indicated by filled circles, plasma standards are indicated by filled squares in (a), and serum pools are indicated by filled squares in (b).

[0075] Figure 9 shows replicated associations between individuals with MASLD (NAFLD) and healthy controls, (a) Volcano plot based on the calculated 36 glycosylation traits in the discovery cohort, (b-g) Comparison of the relative abundance differences between healthy controls and MASLD (NAFLD) using the glycosylation traits with negative (b, e) and (f, g) positive effect sizes. P-values, ORs and 95% Cis are shown in Table 6. *, **: p-value < 0.05, 0.01 , respectively.

[0076] Figure 10 shows the ratio of the glycosylation traits AL and AE, illustrating the observed effect of MASLD (NAFLD) (a-b), and more specifically of fibrosis (c-d) on the blood / V-glycome as a single glycosylation trait, (a) Differences in the ratio of AL / AE in healthy controls versus MASLD (NAFLD) patients overall, and (b) sex stratified in the cohorts, (c) Overall and (d) sex stratified differences in AL / AE between healthy controls and patients with various degree of fibrosis (Brunt fibrosis scores) in the replication cohort. While this ratio represents the overall effect, due to its limitations described in the results and discussion of Example 2, all significantly associated glycosylation traits are shown and described separately in Example 2 (Figure 9, 11; Table 6, 7). Note that sex stratification results in lower power for statistical analysis, especially for females (d). As age and sex is rather well-matched between the groups, the inventors believe that the less differentiation between healthy & F0 vs. F1-F4 fibrosis scores for the female group is due to low sample size. Furthermore, neither sex nor age were founds as significant covariates in binary classification models.

[0077] Figure 11 shows replicated associations identified between patients with MASLD (NAFLD) and healthy controls as stratified as per MASH(NASH)-related fibrosis stage (Brunt). *, **, ***: p-value < 0.05, 0.01 , 0.001 , respectively. The shown data corresponds to the replication cohort.

[0078] Figure 12 shows age and sex distribution across disease groups (a) Overall and (b) sex stratified age distribution in the cohorts, (c) Overall and (d) sex stratified age distribution in the replication cohort. A significant age difference was observed for females in the discovery cohort and for males in the replication cohort based on simple statistical testing, although this effect has been accounted for by logistic regression analysis including age, sex and their interaction in the model as covariates (see “Statistics” section of the Examples). Furthermore, neither sex nor age were found as significant covariates in binary classification models.

[0079] Figure 13 shows influence of type 2 diabetes mellitus (T2DM). Association of glycosylation signatures and fibrosis in T2DM stratified groups (0 (left two plots) = no T2DM; 1 (right two plots) = T2DM). Healthy patients were considered F0 and non-diabetic. For the applicable groups, healthy and F0 patients were pooled for this analysis. Note that due to the low number of F0 patients in the T2DM group, and low number of patients upon further stratification per fibrosis stage, further univariate statistical analysis could not be performed. However, the direction of alteration upon fibrosis is similar in both the non-T2DM and T2DM groups, indicating a fibrosis- and not T2DM-specific effect.

[0080] Figure 14 shows comparison of overall sialylation (CS) between the disease groups per cohorts and sex. (a) Differences in the relative levels of CS in healthy controls versus MASLD (NAFLD) patients overall, and (b) sex stratified in the cohorts, (c) Overall and (d) sex stratified differences in CS between healthy controls and patients with various degree of fibrosis (Brunt fibrosis scores) in the replication cohort. While statistical testing suggested significant differences between the groups (c-d), this effect was explicit for patients with advanced fibrosis (F4; cirrhosis), and no differences were observed between healthy and / or F0 patients vs. those with F1-F3 fibrosis. These results suggest that the observed fibrosis-specific effect is driven by a shift in the relative abundance of sialic acid linkages (Figure 10), rather than alterations in the relative levels of CS (Rs = -0.15; p-value = 0.86).

[0081] Figure 15 shows ROC analysis and associated AUC value for the single predictor model including A4L, illustrating its power to predict fibrosis upon histological appearance.

[0082] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0083] Various aspects of the invention are described in further detail below.

[0084] Detailed Description

[0085] In one aspect, the present invention provides a method for diagnosing fibrotic non-alcoholic steatohepatitis (NASH) in a subject.

[0086] The methods described herein are based on the inventors’ finding of novel biomarkers that enable differential diagnosis, staging, and / or monitoring of fibrotic NASH. Currently, diagnosing and monitoring fibrotic NASH, without performing an invasive liver biopsy is difficult without further tests, such as liver imaging (for example Fibroscan). Even then, differentiating between certain types of liver diseases, for example autoimmune hepatitis and fibrotic NASH is sometimes difficult. Therefore a non- invasive test is a clinical unmet need.

[0087] Surprisingly, the inventors have found biomarkers which can be used to diagnose, stage and / or monitor fibrotic NASH. These biomarkers are unique N-glycosylation patterns. Specifically the inventors found that an increase in the levels of a2,6-sialylation in the sample; and / or a decrease in the levels of a2,3-sialylation in the sample is indicative of the subject having fibrotic NASH.

[0088] Non-alcoholic fatty liver disease (NAFLD) refers to a condition occurring when fat is deposited in the liver (steatosis) not due to excessive alcohol consumption. It may be caused by insulin resistance and the metabolic syndrome and may respond to treatments originally developed for other insulinresistant states (e.g. diabetes mellitus type 2) such as weight loss, metformin and thiazolidinediones. NAFLD ranges from simple steatosis to non-alcoholic steatohepatitis (NASH). “NASH” is the most severe form of NAFLD, and occurs when the build-up of fat in NAFLD causes inflammation and / or fibrosis, resulting in a hepatic disorder. The most severe form of NASH cirrhosis is a life threatening condition. Depending on the severity of NASH, it may be fibrotic (i.e. NASH with hepatic fibrosis) or non-fibrotic (NASH without hepatic fibrosis). Non-fibrotic NASH is the least advanced form of NASH. However, it may progress to fibrotic NASH. The extent of hepatic inflammation and / or hepatic fibrosis, and therefore the severity of NASH, can be assessed by methods well known in the art. An example method in the art is the histopathological evaluation of a liver biopsy from a subject using the Brunt fibrosis scoring system (which scores the amount of fibrosis in the liver) and / or Brunt activity scoring system (which scores the amount of inflammation in the liver).

[0089] A brunt fibrosis score of 0 indicates the subject has no hepatic fibrosis. A Brunt fibrosis score of 1 indicates the subject has portal fibrosis (minimal scarring). A Brunt fibrosis score of 2 indicates the subject has periportal fibrosis (significant scarring has occurred and extends outside the liver area). A Brunt fibrosis score of 3 indicates the subject has severe fibrosis (fibrosis spreading and forming bridges with other fibrotic liver areas). A Brunt fibrosis score of 4 indicates the subject has cirrhosis (advanced scarring).

[0090] A Brunt activity grade score of 0 indicates the subject has no hepatic inflammation. A Brunt activity grade score of 1 indicates the subject has mild hepatic inflammation. A Brunt activity grade score of 2 indicates the subject has moderate hepatic inflammation. A Brunt activity grade score of 3 indicates the subject has severe hepatic inflammation.

[0091] A liver biopsy is currently the clinical reference standard for fibrosis detection and staging, but is an invasive procedure and suffers from sampling errors as the sample is taken from one small area of the liver so is not a true representation of the overall condition of the liver. The invention described herein provides a non-invasive method of diagnosing and staging the severity of fibrotic non-alcoholic steatohepatitis (NASH) in a subject. Specifically, the method of diagnosing fibrotic NASH as described herein, may enable diagnosing a patient with fibrotic NASH and a Brunt fibrosis score of 1 or more. Following a subject’s Brunt fibrosis score over a period may allow the subject’s NASH to be monitored. A decrease in the Brunt score over time may be indicative of the disease regressing, whereas an increase in the Brunt score over time may be indicative of the disease progressing.

[0092] The term “diagnosing” as used herein means assessing whether a subject as referred to in accordance with the method of the present invention has fibrotic NASH.

[0093] In the context of the present disclosure, the “subject” may be any organism that has a liver. Suitably, the subject may be a mammal or a non-mammal. More suitably the subject may be a mammal. Most suitably, the subject may be a human. Suitably, the human subject is an adult human. The human subject may be male or female. Suitably, the adult subject is 20 or more years of age. For example, the subject may be 30, 40, 50, 60, 70, or more years of age.

[0094] Suitably, the subject may be symptomatic or asymptomatic. A subject that is symptomatic may exhibit one or more symptoms associated with NAFLD and / or NASH. Merely by way of example a symptom associated with NAFLD and / or NASH may be selected from the group consisting of intense itching, altered levels of a liver enzyme, abdominal swelling, easy bruising and bleeding, jaundice, increased presence of blood vessels beneath the skin's surface, ascites, and behaviour changes (such as confusion, fatigue, slurred speech, etc.).

[0095] Suitably, the subject may have been diagnosed with NAFLD and / or NASH (for example, the subject may have been diagnosed with non-fibrotic or fibrotic NASH). The subject may have been diagnosed with NAFLD and / or NASH by any method known in the art. A subject that is asymptomatic may be known to be predisposed to NAFLD and / or NASH, or may be suspected of being predisposed to NAFLD and / or NASH. A subject that is predisposed to NAFLD and / or NASH may be known to have a risk factor for NAFLD and / or NASH. Risk factors for NAFLD and / or NASH may include type 2 diabetes or insulin resistance, obesity, hypertension and / or dyslipidaemia, age, and / or a genetic predisposing factor. A genetic predisposing factor may include a mutation in a gene selected from the group consisting of PNPLA3, TM6SF2, MBOAT7, GCKR, and HSD18B13. The PNPLA3 gene encodes the enzyme adiponutrin, also known as patatin-like phospholipase domain-containing protein 3, acylglycerol O-acyltransferase, or calcium-independent phospholipase A2-epsilon. The TM6SF2 gene encodes the transmembrane 6 superfamily 2 protein which is associated with the hepatocytic secretion of tiglyceride-rich lipoproteins. The MBOAT7 gene encodes protein Membrane Bound O-Acyltransferase Domain Containing 7. The MBOAT7 gene encodes protein Membrane Bound O-Acyltransferase Domain Containing 7. The GCKR gene encodes the protein Glucokinase Regulator. The HSD18B13 gene encodes the protein hydroxysteroid 17-beta dehydrogenase 13 .

[0096] A subject suspected of being predisposed to NAFLD and / or NASH may have a family history of NAFLD and / or NASH, and / or a family history of a risk factor for NAFLD and / or NASH. The method for diagnosing fibrotic non-alcoholic steatohepatitis (NASH) in a subject comprises the step of determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a sample from the subject.

[0097] As it would be clear to a person of skill in the art, the level of a2,3-sialylation and / or a2,6-sialylation is typically “determined” by measuring the level of a2,3-sialylation and / or a2,6-sialylation in the sample. The term “determining” can therefore be replaced with the term “measuring” or “determining by measuring” herein. The term “level” or levels” as used herein refers to the amount or accumulation of a biomarker. In the context of the present disclosure, the biomarker is a2 ,3-sialylation and / or a2,6- sialylation. The level may be an absolute amount of the biomarker in a sample or to a relative amount of the biomarker. The relative amount may be for example a percentage, fraction, or ratio. The ratio may be for example the ratio of a2,3-sialylation to a2,6-sialylation, or vice versa. As shown in the Examples section of the present disclosure, the level may be determined on a sample that has undergone / V-glycan release from plasma and linkage-specific chemical sialic acid derivatization prior to determining the levels of a2,3-sialylation and / or a2,6-sialylation of glycans. A sample that has undergone / V-glycan release and linkage-specific chemical sialic acid derivatization may be referred to herein as a “derivatized sample”.

[0098] Conventional "determining" methods may include sending a clinical sample to a commercial laboratory for measurement of the biomarker levels in the biological sample, or the use of commercially available assay kits for measuring the biomarker levels in the biological sample. Exemplary kits and suppliers will be apparent to a person of skill in the art. In various examples, biomarkers may be determined, detected and / or quantified using mass spectrometry, high-performance liquid chromatography, capillary (gel) electrophoresis with laser induced fluorescence detection, hydrophilic interaction liquid chromatography (for example with fluorescence or UV detection), lectin- or antibody-based binding assay, including but not limited to those relying on an ELISA based approach (for example an ELISA based assay using glycan binding proteins such as lectins, antibodies or aptamers). Exemplary methods for determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans are also provided in the Examples section of the present specification. Merely by way of example, a2,3- sialylation of N-glycans (mono-, di-, tri- and tetraantennary glycans combined) may be calculated as H7N6F1 L1 E3 + H7N6F2L3E1 + H7N6F2L2E2 )) [H = hexose; N = N-acetylhexosamine; F = deoxyhexose (fucose); L = lactonized N-acetylneuraminic acid (a2,3-linked); E = ethyl esterified N- acetylneuraminic acid (a2,6-linked). The structures may have varying isomers. Position and linkage of monosaccharides are well known in literature].

[0099] Merely by way of example, a2,6-sialylation of N-glycans (mono-, di-, tri- and tetraantennary glycans combined) may be calculated as follows: a 2 ,6-sialylation = ( ( 0 / 1 * ( H4N3F2L1 ) + 1 / 1 * ( H3N3E1 + + H5N4F1 + H4N5F1 + H5N5 + H4N4F1 E1 + H5N4E1 + H5N5F1 + H5N4F1 E1 + H4N5F1 E1 + H7N6L3E1 + H7N6F2L2E1 + H7N6L2E2 + H7N6L1 E3 + H7N6F1 L3E1 + H7N6F1 L2E2 + H7N6F1 L1 E3 + H7N6F2L3E1 + H7N6F2L2E2 )) [H = hexose; N = N-acetylhexosamine; F = deoxyhexose (fucose); L = lactonized N-acetylneuraminic acid (a2,3-linked); E = ethyl esterified N- acetylneuraminic acid (a2,6-linked). The structures may have varying isomers. Position and linkage of monosaccharides are well known in literature].

[0100] Suitably, a2,3-sialylation of / V-glycans may be determined on diantennary, triantennary and / or tetraantennary / V-glycans. In other words it can be said that the level of A2L, A3L and / or A4L is determined, respectively. Suitably, a2,3-sialylation of / V-glycans may be determined on triantennary and / or tetraantennary / V-glycans. Suitably, a2 ,6-sialylation of / V-glycans may be determined on triantennary and / or tetraantennary / V- glycans. In other words it can be said that the level of A3E and / or A4E, respectively is determined. Suitably, a2,6-sialylation of / V-glycans may be determined on triantennary / V-glycans.

[0101] In general, a glycan is a saccharide, which includes a monosaccharide, a disaccharide or a trisaccharide; it can include an oligosaccharide or a polysaccharide. An oligosaccharide is an oligomeric saccharide that contains two or more saccharides. The structure of an oligosaccharide is typically characterized by particular identity, order, linkage positions (including branch points), and linkage stereochemistry (a, P) of the monomers, and as a result has a defined molecular weight and composition. An oligosaccharide typically contains about 2 to about 20 or more saccharide monomers. In a polysaccharide, the identity, order, linkage positions (including branch points) and / or linkage stereochemistry can vary from molecule to molecule. A protein with a glycan may be referred to as a glycoprotein.

[0102] The glycan component of the glycoprotein can be / V-linked or O-linked. An / V-glycan is attached to a nitrogen atom, for example, at the side chain nitrogen atom of an asparagine amino acid within the peptide. An O-linked glycan is attached to an oxygen atom, for example at the side chain hydroxyl oxygen of a hydroxylysine, hydroxyproline, tyrosine, serine, or threonine amino acid within the peptide. “Glycosylation” refers to the covalent attachment of at least one saccharide moiety to a molecule. Glycosidic linkages include O-glycosidic linkages, / V-glycosidic linkages, S-glycosidic linkages and C- glycosidic linkages. An O-glycosidic linkage is formed between the anomeric carbon (C1) of a saccharide and an oxygen atom of another molecule (such as another saccharide or a polypeptide), while an / V-glycosidic linkage is formed between the anomeric carbon (C1) of a saccharide and a nitrogen atom of another molecule. Likewise, S-glycosidic linkages and C-glycosidic linkages involve a sulphur and carbon atom from another molecule, respectively. In addition, glycosidic linkages are classified according to the ring position of the carbon atoms participating in the bond. For example, a 1 ,4 glycosidic linkage is formed between the first carbon (C1) on a first saccharide and the fourth carbon (C4) on a second saccharide while a 1 ,6 glycosidic linkage is formed between the first carbon (C1) on a first saccharide and the sixth carbon (C6) on a second saccharide. As another example, a 2,3 glycosidic linkage is formed between the second carbon (C2) on a first saccharide and the third carbon (C3) on a second saccharide while a 2,6 glycosidic linkage is formed between the second carbon (C2) on a first saccharide and the sixth carbon (C6) on a second saccharide. Glycosidic linkages are further classified as a-glycosidic or p-glycosidic according to whether the substituent groups on the carbons flanking the oxygen in the saccharide are pointing in the same or opposite directions. The term “glycosylation” as used herein should be broadly construed so as to encompass the covalent linkage of any other carbohydrate moieties such as fucose and sialic acid, and as such includes fucosylation or sialylation. Most / V-linked glycans share a common structure, referred to as a core, which typically contains three mannose, and two / V-acetylglucosamine residues. The core may contain modifications such as sulfation or phosphorylation; the core may be intact or it may be truncated. Terminal modifications and core modifications of a glycan are feasible by various glycans. Core glycosylation refers to the addition of glycosyl moieties to a core / V-acetylglucosamine and / or mannose. Core fucosylation refers to the addition of a fucose residue to the core / V-acetylglucosamine. A glycan can be branched or unbranched. Non-core parts, and e.g. sialic acids, may also be prone to modification, such as acetylation.

[0103] Suitably, the / V-glycans may comprise or consist of complex-type / V-glycans. A “complex-type / V- glycan” is a glycan that contains at least one / V-acetylglucosamine on each of the two mannose branches of the core. Suitably, the complex-type / V-glycan may be selected from the group consisting of diantennary / V-glycan, triantennary / V-glycan, and tetraantennary / V-glycan. More suitably, the glycan may be selected from the group consisting of a diantennary, triantennary / V-glycan and tetraantennary / V-glycan. More suitably, the glycan may be selected from the group consisting of a triantennary / V-glycan and tetraantennary / V-glycan.

[0104] In a branched glycan, the monosaccharide at the branch point is covalently linked to two other saccharides at carbons other than C1 . For example, a branch point monosaccharide may be linked to other monosaccharides at C4 and C6, in addition to being linked to another monosaccharide or to an amino acid at C1.

[0105] A complex glycan may be, without limitation, biantennary (i.e. have two branches on the core structure), triantennary (i.e. have three branches on the core structure), or tetraantennary (i.e. have four branches on the core structure). One or more branch may be galactose-terminated. When the galactose moiety has a sialic acid moiety attached, it is said to be sialylated.

[0106] The present inventors have found that subjects with fibrotic NASH have altered levels of a2,3- sialylation and / or a2,6-sialylation of / V-glycans as compared to healthy controls or patients with NAFLD or non-fibrotic NASH. Specifically the inventors found that patients with fibrotic NASH have decreased levels of a2,3-sialylation and / or increased levels of a2,6-sialylation. The term “ct2,3-sialylation” refers to sialic acids linked to galactose by a2,3 linkages. In humans a2,3-sialyl linkages are determined - amongst other factors - mainly by the activity of sialyltransferases (genes: St3Gal1, St3Gal2. St3Gal3, St3Gal4, St3Gal5 and / or St3Gal6. By the same token, the term “a2 ,6-sialylation” refers to sialic acids linked to galactose by a2,6 linkages. In humans a2,6 linkages are determined mainly by the activity of the sialyltransferase genes St6Gal1 and St6Gal2. Other factors may include glycosidases, i.e. enzymes that specifically remove sialic acids in a certain linkage (sialidases; genes: NEUR1-4), or receptors that remove glycoproteins from the circulation (Asialoglycoprotein receptor; genes: ASGR1 , ASGR2).

[0107] Methods described herein comprise the step of comparing the level of a2,3-sialylation and / or a2,6- sialylation of / V-glycans to a reference value. It will be appreciated that the reference value may be derived from a suitable control sample.

[0108] Depending on the context, the reference value may be one which allows to determine whether a subject has or does not have fibrotic NASH. Such a reference value may be particularly relevant in the context of the method for diagnosing fibrotic NASH as disclosed herein. In this context, the reference value may be derived from a control sample that has normal levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans. Suitably, the control sample may be obtained from one or more subjects (referred to herein as control subject(s)) that do not have and / or are not at risk of having NAFLD and / or NASH (e.g. fibrotic or non-fibrotic NASH). Suitably, the reference value may be the level of a2,3- sialylation and / or a2 ,6-sialylation of / V-glycans in an individual or group of individuals that are believed to be of good overall health (i.e. with no diagnosed disorders and / or no symptoms indicative of a disorder).

[0109] In another embodiment, the reference value may be one which defines the stage of fibrotic NASH. In such an embodiment, the reference value may be obtained from a subject or group of subjects previously diagnosed with a specific stage of fibrosis, and optionally assigned a Brunt fibrosis score, and / or another score based on a known classification method for fibrotic NASH. Other classification methods (also referred to herein as scoring systems are described elsewhere herein). An exemplary method for determining a reference value is provided in the Examples section of the present disclosure.

[0110] “Derived from” as used herein means substantially corresponding to the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a suitable control subject(s).

[0111] Suitably, the control sample may be the same type of sample as the test sample (i.e. the sample from the subject who is being tested using the methods disclosed herein).

[0112] Suitably, the control sample may be from a subject that has been matched to the test subject (i.e. for example is the same sex or age). However, surprisingly the present inventors have found that a2,3- sialylation and / or a2,6-sialylation of / V-glycans is neither confounded by the age and sex of the subject. Therefore, advantageously, the sample does not have to be matched.

[0113] The control sample may be assayed at the same time, before or after, separately or simultaneously with the test sample to provide a reference value.

[0114] The reference value that is used in the comparison with the test sample may be a value that is calculated as an average or median of more than one (e.g. two or more, five or more, ten or more, a group etc.) control sample. Alternatively, the control sample may be a sample that originated from (i.e. is a mix of) more than one (e.g. two or more, five or more, ten or more, a group etc.) control samples. The reference value may be calculated as the average or median, taken from a group or population of control subjects. Suitably, the reference value may be single cut-off value, such as a median or mean. Alternatively, it can be a range of cut-off (or threshold) values. Ranges may be more suitable as reference values in methods for determining the stage of fibrosis.

[0115] The reference value may be a predetermined reference value of a2,3-sialylation and / or a2,6-sialylation of / V-glycans. Such a predetermined reference value may be obtained from a reference database. Therefore, it will be appreciated that in the context of the methods disclosed herein it is not necessary to determine the levels of a2,3-sialylation and / or a2 ,6-sialylation of / V-glycans to obtain a reference value in the same assay as determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V- glycans in the sample.

[0116] Suitably, the reference value may be derived from the average number of a2,3- and / or a2,6-linked sialic acids per antenna in a sample subjected to / V-glycan release from blood (e.g. plasma, serum, whole blood or dried blood) proteins and linkage-specific chemical sialic acid derivatization. Upon preparing the sample as described elsewhere herein it is possible to determine the total number of a2,3- and a2,6-sialyated antennae as well as the number of each of those separately, thereby making it possible to determine the relative abundance of a2,3- and / or a2,6-sialyation. Such relative abundance may be presented for example as a percentage or a fraction. a2,3-sialylation may be measured on monoantennary, diantennary, triantennary, and / or tetraantennary / V-glycans. A combination of mono-, di-, tri-, and tetraantennary glycans with a2,3- sialylation is referred to herein as “AL”. Bi-antennary glycans with a2 ,3-sialylation are referred to herein as “A2L”. Triantennary glycans with a2,3-sialylation are referred to herein as “A3L”. Tetraantennary glycans with a2 ,3-sialylation are referred to herein as “A4L”.

[0117] By the same token, a2 ,6-sialylation may be measured on monoantennary, diantennary, triantennary, and / or tetraantennary / V-glycans. A combination of mono-, di-, tri-, and tetraantennary glycans with a2,6-sialylation is referred to herein as “AE”. Tri-antennary glycans with a2,6-sialylation are referred to herein as “A3E”.

[0118] As shown in Table 4, the inventors have found that patients with fibrotic NASH have an average relative abundance of AL equal to 0.2316 compared to non-fibrotic patients where the relative abundance of AL is 0.2497. This means that in samples (that have undergone / V-glycan release from blood ( e.g. plasma, serum or whole blood) proteins and linkage-specific chemical sialic acid derivatization) from fibrotic patients, 23.16% of mono-, di-, tri-, and tetraantennary glycans are a2,3- sialylated as compared to 24.97% in non-fibrotic patients. In other words, it can be said that patients with fibrotic NASH have 1.81 % less AL glycans than non-fibrotic patients. From these results, the inventors have calculated an average cut-off value of 0.2406 as a suitable reference value for AL. Therefore, if a patient has a relative abundance of AL below this value it is indicative of the patient having fibrotic NASH. For clarity, in this context, the reference value of 0.2406 means that 24.06% of sialylated / V-glycan antennas in the sample which belong to the group of mono-, di-, tri-, and tetraantennary glycans are a2,3-sialylated.

[0119] By the same token, the present inventors have found that patients with fibrotic NASH have an average relative abundance of A2L equal to 0.0292 compared to non-fibrotic patients where the relative abundance of A2L is 0.0361. From these results, the inventors have calculated an average cut-off value of 0.0327 as a suitable reference value for A2L. Therefore, if a patient has a relative abundance of A2L below this value it is indicative of the patient having fibrotic NASH. Similarly, the present inventors have found that patients with fibrotic NASH have an average relative abundance of A3L equal to 0.2593 compared to non-fibrotic patients where the relative abundance of A3L is 0.2850. From these results, the inventors have calculated an average cut-off value of 0.2721 as a suitable reference value for A3L. Therefore, if a patient has a relative abundance of A3L below this value it is indicative of the patient having fibrotic NASH.

[0120] Similarly, the present inventors have found that patients with fibrotic NASH have an average relative abundance of A4L equal to 0.4438 compared to non-fibrotic patients where the relative abundance of A4L is 0.4936. From these results, the inventors have calculated an average cut-off value of 0.4687 as a suitable reference value for A4L. Therefore, if a patient has a relative abundance of A4L below this value it is indicative of the patient having fibrotic NASH.

[0121] As shown in Table 4, the present inventors have found that patients with fibrotic NASH have an average relative abundance of AE equal to 0.7684 compared to non-fibrotic patients where the relative abundance of AE is 0.7503. This means that in samples (that have undergone / V-glycan release from blood (e.g. plasma, serum, whole blood or dried blood) proteins and linkage-specific chemical sialic acid derivatization) from fibrotic patients, 76.84% of mono-, di-, tri-, and tetraantennary glycans are a2,6-sialylated as compared to 75.03% in non-fibrotic patients. In other words, it can be said that patients with fibrotic NASH have 1.81 % more AE glycans than non-fibrotic patients. From these results, the inventors have calculated an average cut-off value of 0.7594 as a suitable reference value for AE. Therefore, if a patient has a relative abundance of AE above this value it is indicative of the patient having fibrotic NASH. For clarity, in this context, the reference value of 0.7594 means that 75.94% of sialylated / V-glycan antennas in the sample which belong to the group of mono-, di-, tri-, and tetra-antennary glycans are a2,6-sialylated.

[0122] By the same token, the present inventors have found that patients with fibrotic NASH have an average relative abundance of A3E equal to 0.6678 compared to non-fibrotic patients where the relative abundance of A3E is 0.6463. From these results, the inventors have calculated an average cut-off value of 0.6571 as a suitable reference value for A3E. Therefore, if a patient has a relative abundance of A3E above this value it is indicative of the patient having fibrotic NASH.

[0123] As shown in Table 8, the inventors have found that patients with fibrotic NASH have an average relative abundance of AL equal to 0.215 compared to non-fibrotic patients where the relative abundance of AL is 0.227. This means that in samples (that have undergone / V-glycan release from blood (e.g. plasma, serum or whole blood) proteins and linkage-specific chemical sialic acid derivatization) from fibrotic patients, 21.5% of mono-, di-, tri-, and tetraantennary glycans are a2,3- sialylated as compared to 22.7% in non-fibrotic patients. In other words, it can be said that patients with fibrotic NASH have 1.2% less AL glycans than non-fibrotic patients. From these results, the inventors have calculated an average cut-off value of 0.221 as a suitable reference value for AL. Therefore, if a patient has a relative abundance of AL below this value it is indicative of the patient having fibrotic NASH. For clarity, in this context, the reference value of 0.221 means that 22.1 % of sialylated / V-glycan antennas in the sample which belong to the group of mono-, di-, tri-, and tetraantennary glycans are a2,3-sialylated.

[0124] By the same token, the present inventors have found that patients with fibrotic NASH have an average relative abundance of A2L equal to 0.031 compared to non-fibrotic patients where the relative abundance of A2L is 0.039. From these results, the inventors have calculated an average cut-off value of 0.035 as a suitable reference value for A2L. Therefore, if a patient has a relative abundance of A2L below this value it is indicative of the patient having fibrotic NASH.

[0125] Similarly, the present inventors have found that patients with fibrotic NASH have an average relative abundance of A3L equal to 0.272 compared to non-fibrotic patients where the relative abundance of A3L is 0.294. From these results, the inventors have calculated an average cut-off value of 0.283 as a suitable reference value for A3L. Therefore, if a patient has a relative abundance of A3L below this value it is indicative of the patient having fibrotic NASH.

[0126] Similarly, the present inventors have found that patients with fibrotic NASH have an average relative abundance of A4L equal to 0.508 compared to non-fibrotic patients where the relative abundance of A4L is 0.538. From these results, the inventors have calculated an average cut-off value of 0.523 as a suitable reference value for A4L. Therefore, if a patient has a relative abundance of A4L below this value it is indicative of the patient having fibrotic NASH.

[0127] As shown in Table 8, the present inventors have found that patients with fibrotic NASH have an average relative abundance of AE equal to 0.785 compared to non-fibrotic patients where the relative abundance of AE is 0.773. This means that in samples (that have undergone / V-glycan release from blood (e.g. plasma, serum, whole blood or dried blood) proteins and linkage-specific chemical sialic acid derivatization) from fibrotic patients, 78.5% of mono-, di-, tri-, and tetraantennary glycans are a2,6-sialylated as compared to 77.3% in non-fibrotic patients. In other words, it can be said that patients with fibrotic NASH have 1 .2% more AE glycans than non-fibrotic patients. From these results, the inventors have calculated an average cut-off value of 0.779 as a suitable reference value for AE. Therefore, if a patient has a relative abundance of AE above this value it is indicative of the patient having fibrotic NASH. For clarity, in this context, the reference value of 0.779 means that 77.9% of sialylated / V-glycan antennas in the sample which belong to the group of mono-, di-, tri-, and tetraantennary glycans are a2,6-sialylated.

[0128] By the same token, the present inventors have found that patients with fibrotic NASH have an average relative abundance of A3E equal to 0.672 compared to non-fibrotic patients where the relative abundance of A3E is 0.652. From these results, the inventors have calculated an average cut-off value of 0.662 as a suitable reference value for A3E. Therefore, if a patient has a relative abundance of A3E above this value it is indicative of the patient having fibrotic NASH.

[0129] In the context of the present disclosure, the term “change” refers to a statistically significant difference in the level of a2 ,3-sialylation and / or a2 ,6-sialylation of / V-glycans in the sample obtained from the test subject compared to the reference value. The difference (or change) may suitably be an increase or decrease in biomarker levels compared to the control sample or predetermined reference value. By the same token “no change” refers to a statistically insignificant difference in the level of a2,3- sialylation and / or a2,6-sialylation in the sample obtained from the test subject compared to the reference value. No change in the levels of a2 ,3-sialylation and / or a2,6-sialylation may indicate that the subject does not have fibrotic NASH. In other embodiment, no change may be indicative of fibrotic NASH not progressing. However, in view of the present disclosure, which teaches that an increase in the level of a2,6-sialylation and / or a decrease in the level of a2,3-sialylation is generally indicative of fibrotic NASH and / or progression of fibrotic NASH, it will be within the skilled person’s ability to interpret any changes (or lack thereof) observed in the levels of a2,3-sialylation and / or a2,6-sialylation.

[0130] The terms "increased", "increase" or “higher” are all used herein to generally mean an increase by a statistically significant amount; for the avoidance of any doubt, the terms "increased" or "increase" means an increase of at least about 0.5% as compared to a reference value, for example an increase of at least about 0.6%, or at least about 0.7%, or at least about 0.8%, or at least about 0.9%, or at least about 1 % as compared to a reference value. For example, the increase may be of at least about 1 .25%, or at least about 1 .5%, or at least about 1 .75%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, or more as compared to a reference value.

[0131] The terms "decreased", "decrease" or “lower” are all used herein to generally mean an increase by a statistically significant amount; for the avoidance of any doubt, these terms mean a decrease of at least about 0.5% as compared to a reference value, for example an decrease of at least about 0.6%, or at least about 0.7%, or at least about 0.8%, or at least about 0.9%, or at least about 1 % as compared to a reference value. For example, the decrease may be of at least about 1.25%, or at least about 1.5%, or at least about 1.75%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, or more as compared to a reference value.

[0132] The methods described herein may be carried out on a sample obtained from the subject. The term “sample” as used herein is intended to include biological matter from the subject. Suitably, the sample may be blood. Suitably, the blood may be arterial blood, capillary blood, venous blood or a mixture thereof.

[0133] In some examples, the blood sample may be selected from the group consisting of whole blood, blood plasma, and blood serum. The term “whole blood” as used herein refers to blood containing all its natural constituents, components, or elements or a substantial amount of the natural constituents, components, or elements. Accordingly, whole blood will comprise plasma, buffy coat (white cells and platelets), and red blood cells. The term “plasma” or “blood plasma” refers to the complete soluble fraction of the blood, whilst the term “serum” or “blood serum” refers to plasma devoid of coagulation factors, i.e. obtained after coagulation of blood.

[0134] In some examples, the blood sample may be fluid or non-fluid.

[0135] Suitably, the sample may be fluid blood. In some examples, the fluid blood sample is fresh. In this context, fresh means the fluid blood sample has been drawn from the subject less than 48 hours prior to being used in a method described herein. Suitably, the sample may have been refrigerated and / or frozen for some or all of the time between being drawn from the subject and being used in the method described herein. In some examples, the fluid blood sample has been thawed. In some examples, the fluid blood sample may be a rehydrated blood sample.

[0136] In some examples, the blood sample is non-fluid. The term “non-fluid blood sample” as used herein is intended to include a blood sample that was previously fluid, but has been dried through a process such as evaporation, freeze-drying, or dehydration. In some examples, the non-fluid blood sample is a dried blood spot sample. In some examples, the dried blood is rehydrated to form a fluid blood sample.

[0137] In some examples, the blood sample has been subjected to / V-glycan release from blood (e.g. plasma, serum, whole blood or dried blood) proteins and linkage-specific chemical sialic acid derivatization prior to determining the levels of a2,3-sialylation and / or a 2 ,6-sialylation of glycans. Accordingly, the methods described herein may include the step of subjecting the sample to / V-glycan release from blood proteins and linkage-specific chemical sialic acid derivatization prior to determining the levels of a 2 ,3-sialylation and / or a 2 ,6-sialylation of glycans. Suitably, the step of releasing / V-glycans may be performed as described in Vreeker, G. C. M. et al. 2018 (doi:10.1021 / acs.analchem.8b02391), which is incorporated herein by reference.

[0138] Merely by way of example, / V-glycan release may be performed by incubating the sample (such as a plasma sample) with SDS (for example 2% SDS). The incubation may be at a temperature of about 60°C and last from about 1 minute to about 1 hour (for example 10 minutes). Upon incubation, the sample may be incubated with a releasing mixture. The releasing mixture may comprise NP-40 (for example 4% NP-40), acidified PBS and PNGase F. Incubation with the releasing mixture may be at about 37 °C and last from about 1 hour to 24 hours, suitably for example around 8 to 10 hours.

[0139] It will be appreciated that when the sample is a dried blood spot, the step of / V-glycan release may be carried out prior to drying the blood sample. Alternatively, / V-glycan release may be carried out on a dry blood sample after it had been rehydrated.

[0140] The methods disclosed herein may be combined with other tests for diagnosing, staging and / or monitoring fibrotic NASH and disease monitoring. Whilst other tests useful in this context will be well known to those skilled in the art, merely by way of example they may include determining the subject’s FIB-4 score, ELF score, and / or APRI score. Additionally or alternatively, the methods described herein may further comprise liver imaging (such as Fibroscan), determining the levels of aspartate transaminase (AST), alanine transaminase (ALT) the subject (or a sample from the subject), and / or by histological analysis of a liver biopsy.

[0141] In a further aspect, the present invention provides a method for staging fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of: • determining the levels of a2,3-sialylation and / or a 2,6-sialylation of / V-glycans in a sample from the subject; and

[0142] • comparing the determined levels of a2 ,3-sialylation and / or a2 ,6-sialylation to reference values indicative of a stage of fibrotic NASH, and thereby determining the subject’s stage of fibrotic NASH.

[0143] The term "staging" as used herein refers to classifying the degree or severity of a disease in the subject. Suitably, the staging may comprise assigning the patient with a Brunt fibrosis score (for example score 0, 1 , 2, 3, or 4). The present inventors found that patients with different Brunt fibrosis scores showed different levels of a2 ,3-sialylation and / or a2,6-sialylation. Typically obtaining a Brunt fibrosis score involves obtaining a liver biopsy from the subject. However, due to inventors finding that the levels of a 2 , 3-sialy latio n and / or a 2,6-sialylation may differ in between patients with different Brunt scores, it may now be possible to measure the levels of a 2 ,3-sialylation and / or a 2,6-sialylation and assign the subject with a Brunt score without the need for a liver biopsy. Whilst the Examples of the present application show a correlation between different Brunt scores and levels of a 2 ,3-sialylation and / or a 2,6-sialylation, the skilled person will appreciate that correlations may also exist between the levels of a 2 ,3-sialylation and / or a 2,6-sialylation and other fibrosis staging methods, such as Brunt activity, Fibrosis-4 (FIB-4), Enhanced Liver Fibrosis (ELF), AST to Platelet Ratio Index (APRI) and / or Fibroscan scoring. Thus, in suitable embodiments the staging may be a according to a Brunt fibrosis score, Brunt activity score, FIB-4 score, ELF score, and / or APRI score.

[0144] Suitable reference values for the different fibrosis stages may be determined for example by receiver operating characteristic analysis (which assesses the sensitivity and specificity at different cut-off values) and determining an optimal cut-off based on the highest area under the curve (AUC). By way of another example, a linear regression equation could be obtained, indicative of the change in any of the glycosylation traits upon a unit change in Brunt score. Other methods for determining references values will be known to those skilled in the art.

[0145] In a further aspect, the present invention provides a method for monitoring fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:

[0146] • determining the levels of a2,3-sialylation and / or a2 ,6-sialylation of / V-glycans in a first sample from the subject; and

[0147] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a second sample from the subject, wherein the second sample has been obtained from the subject at a later time point than the first sample, wherein: i) an increase in the levels of a2 ,6-sialylation in the second sample and / or a decrease in the levels of a2 ,3-sialylation in the second sample as compared to the first sample is indicative of fibrotic NASH progressing in the subject; ii) a decrease in the levels of a2 ,6-sialylation in the second sample and / or an increase in the levels of a2 ,3-sialylation in the second sample as compared to the first sample is indicative of fibrotic NASH regressing in the subject. Suitably, no change in the levels of a2,6-sialylation and / or a2,3-sialylation in the second sample as compared to the first sample may be indicative of NASH neither progressing nor regressing in the subject.

[0148] The term “monitoring” as used herein refers to determining whether fibrotic NASH in a subject is progressing or regressing between at least two different time points. It will be understood by a person skilled in the art that in order to monitor a subject, the samples used to determine the level of a2,3- sialylation and / or a2,6-sialylation of / V-glycans must be obtained at two different time points. The subject may or may not be receiving treatment for fibrotic NASH in the time between the first and second samples being obtained. In an embodiment, when the subject is receiving treatment between the first and second sample being obtained, monitoring can also be used to determine whether a subject is treated successfully or whether at least fibrotic NASH can be ameliorated over time by a certain therapy. Accordingly, the aforementioned method can be applied in order to determine whether a treatment against fibrotic NASH is therapeutically effective, and / or whether a subject is complying with a prescribed treatment for fibrotic NASH. These applications give rise to further aspects of the invention as described hereinbelow.

[0149] The term “progression” as used herein refers to the worsening of fibrotic NASH in a subject. It can be said that fibrotic NASH is progressing when an increase in the levels of a2 ,6-sialylation in the second sample and / or a decrease in the levels of a2,3-sialylation in the second sample as compared to the first sample is observed. By the same token, the term “regression” as used herein refers to an amelioration of fibrotic NASH in a subject. It can be said that fibrotic NASH is regressing when a decrease in the levels of a2,6-sialylation in the second sample and / or an increase in the levels of a2,3- sialylation in the second sample as compared to the first sample is observed.

[0150] In a further aspect, the present invention provides a method of monitoring therapeutic effect of a prescribed treatment for fibrotic non-alcoholic steatohepatitis (NASH), the method comprising the steps of:

[0151] • determining the levels of a2 ,3-sialylation and / or a2 ,6-sialylation of / V-glycans in a first sample from the subject; and

[0152] • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a second sample from the subject, wherein the second sample has been obtained from the subject after the prescribed treatment, wherein a decrease or no change in the levels of a2,6-sialylation in the second sample and / or an increase or no change in the levels of a2 ,3-sialylation in the second sample as compared to the first sample is indicative of a therapeutic effect of a prescribed treatment for fibrotic NASH. The term “prescribed treatment” (also referred to herein as “treatment”) means any intervention intended to ameliorate fibrotic NASH in a subject. Ameliorate in this context means prevent, stop, or slow down the progression of fibrotic NASH, or reverse fully or partially fibrotic NASH.

[0153] It will be appreciated that the treatment may directly or indirectly ameliorate fibrotic NASH. A treatment that indirectly ameliorates fibrotic NASH may be a treatment that is prescribed for a different, underlying disease (such as type 2 diabetes) and / or condition (such as obesity), and whilst treating said underlying disease and / or condition the treatment also may have a therapeutic effect on fibrotic NASH. A treatment that directly ameliorates fibrotic NASH may be a treatment that is prescribed specifically to ameliorate fibrotic NASH in a subject.

[0154] Suitably, the prescribed treatment may be a life style change (for example improved diet, increased physical activity, and / or weight loss) or a drug (for example a biotherapeutic or a small molecule).

[0155] Suitably, the lifestyle change or improvement may comprise a reduction in weight of the subject, an increase in the physical activity of the subject, for example via increased physical exercise, and / or an improvement of the diet of the subject, for example an increase in the (proportional) dietary intake of fruit and / or vegetables, and / or a reduction in the(proportional) dietary intake of ultra-processed food. The method may be useful as a screening tool for determining if a drug and / or drug regimens have a therapeutic effect on fibrotic NASH. The tested drugs and / or drug regiments may be new regimens or new drugs, modified regimens or modified drugs, or known regimens or known drugs, that need further testing. In this context, the term “drug regimen” refers to a dosage amount, dosage period, dosage interval, and / or combination of drugs.

[0156] A prescribed treatment may be identified as having a therapeutic effect if it results in a decrease in the levels of a2,6-sialylation in the second sample and / or an increase in the levels of a2,3-sialylation in the second sample as compared to the first sample.

[0157] In the context of the method of monitoring therapeutic effect of a prescribed treatment, it will be appreciated that the time interval between the first and second sample being obtained from the subject has to be sufficient to enable the detection of any potential therapeutic effect the prescribed treatment may have. Accordingly, the second sample may be obtained about 2 weeks, about 4 weeks, about 6 weeks, about 3 months, about 6 months, about 1 year, or more after the first sample was obtained. Additionally or alternatively, the second sample may be obtained from the subject about 2 weeks, about 4 weeks, about 6 weeks, about 3 months, about 6 months, about 1 year, or more after the commencement of the prescribed treatment. At the time of obtaining the first sample from the subject, the subject may or may not have already commenced the prescribed treatment.

[0158] Suitable time intervals for monitoring an improvement in disease status or symptoms (e.g. during treatment of the subject) can easily be identified by a person of skill in the art and will depend on the patient (e.g. cause of fibrotic NASH) being monitored.

[0159] In a related aspect, the invention provides a method for monitoring a subject’s compliance to a prescribed treatment for fibrotic NASH, the method comprising the steps of:

[0160] • determining the levels of a2 ,3-sialylation and / or a2 ,6-sialylation of / V-glycans in a first sample from the subject; and • determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a second sample from the subject, wherein the second sample has been obtained from the subject after the prescribed treatment, wherein a decrease or no change in the levels of a2,6-sialylation in the second sample, and / or an increase or no change in the levels of a2 ,3-sialylation in the second sample as compared to the first sample is indicative of a subject’s compliance to the prescribed treatment for fibrotic NASH.

[0161] The term “compliance” as used herein refers to the patient's adherence to the prescribed treatment, i.e. the degree to which the patient behaves according to the prescribed treatment.

[0162] In the context of the method for monitoring therapeutic effect or monitoring a subject’s compliance, a prescribed treatment may be one that is intended to reverse (regress) fibrotic NASH. In such an embodiment, it will be appreciated that a decrease in the levels of a2,6-sialylation and / or an increase in the levels of a2 , 3-sialy latio n in the second sample as compared to the first sample may be indicative of the treatment having a therapeutic effect or the subject’s compliance with the treatment.

[0163] In other examples, the prescribed treatment may be one that is intended to prevent development or stop the progression of fibrotic NASH. In such embodiments, no change in the levels of a2,3-sialylation and / or a2,6-sialylation may be indicative of the treatment having a therapeutic effect or the subject’s compliance with the treatment.

[0164] Furthermore, in other examples, the treatment may be intended to slow down the progression of NASH. In such embodiments, an increase in the levels of a2,6-sialylation and / or a decrease in the levels of a2 ,3-sialylation may in fact be indicative of the treatment having a therapeutic effect or the subject’s compliance with the treatment. In such embodiments, the increase in the levels of a2,6- sialylation and / or a decrease in the levels of a2,3-sialylation may be slower than otherwise expected (for example slower than the change in the levels of a2,6-sialylation and / or a2,3-sialylation observed prior to the subject beginning the prescribed treatment).

[0165] In some aspects, the present invention provides a method of diagnosing and treating a patient with fibrotic NASH, the method comprising:

[0166] • determining the levels of a2 ,3-sialylation and / or a2 ,6-sialylation of / V-glycans in a sample from the subject;

[0167] • comparing the determined levels of a2,3-sialylation and / or a2,6-sialylation to a reference value;

[0168] • identifying the subject as having fibrotic NASH if the levels of a2,6-sialylation are increased and / or levels of a2 ,3-sialylation were decreased; and

[0169] • administering to the subject that has been identified as having fibrotic NASH a treatment for fibrotic NASH. As used herein, the terms “treat”, “treating”, and “treatment” are taken to include an intervention performed with the intention of ameliorating fibrotic NASH. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom.

[0170] The methods disclosed herein may be combined with other tests for diagnosing or monitoring fibrotic NASH. Such other tests will be known to those skilled in the art. Merely by way of example they may include determining the levels of aspartate transaminase (AST), alanine transaminase (ALT) in the subject (or a sample from the subject), and / or by histological analysis of a liver biopsy, liver imaging (such as Fibroscan), liver disease scoring, etc.

[0171] In a further aspect, the present invention also provides a kit for use in the methods disclosed herein. The kits may include reagents suitable for determining levels of an analyte in a test sample (e.g., reagents suitable for determining levels of the biomarker disclosed herein).

[0172] Suitably, the kit comprises a detectably labelled agent that specifically binds to a2 ,3-sialylation and / or a2,6-sialylation of glycans.

[0173] Optionally, the kit may contain one or more control samples or references. Also, the kits, in some cases, may include written information (indicia) providing a reference (e.g., pre-determined values), wherein a comparison between the levels of the biomarkers in the subject and the reference (predetermined values) is indicative of a clinical status. In some cases, the kits comprise software useful for comparing biomarker levels or occurrences with a reference (e.g., a prediction model). Usually the software will be provided in a computer readable format such as a compact disc, but it also may be available for downloading via the internet. However, the kits are not so limited and other variations with will apparent to one of ordinary skill in the art.

[0174] The components of the kit may be housed in a container that is suitable for transportation.

[0175] The term “detectably labelled agent” refers to a binding partner that interacts (i.e. binds) specifically with the biomarker of interest (a2 ,3-sialylation and / or a2 ,6-sialylation) or complex-type glycans and is also capable of being detected e.g. directly (such as via a mass tag or a fluorescent or tag) or indirectly (such as via a labelled secondary antibody). The detectably labelled agent is therefore a selective binding partner for the biomarker of interest (and does not substantially bind to other glycans, proteins or the combination thereof (e.g. glycopeptides or glycoproteins)). Selective binding partners may include antibodies that selectively bind to one of the biomarker of interest.

[0176] As used herein, “specifically binds a2,3-sialylation and / or a2,6-sialylation” means that under certain conditions the binding partner that “specifically binds a2,3-sialylation and / or a2,6-sialylation” will selectively bind a2 ,3-sialylation and / or a2 ,6-sialylation and will not bind in a significant amount to other protein modifications, including other types of sialylation. Thus the binding partner may bind to a2,3- sialy lation and / or a2 ,6-sialylation with at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 fold more affinity than it binds other glycans, peptides, proteins, and / or protein modifications.

[0177] In some examples the kits include the detectably labelled agent(s) on a continuous (e.g. solid) surface, such as a lateral flow surface. Alternatively, in examples comprising more than one detectably labelled agent, the detectably labelled agent(s) may be located in distinct (i.e. spatially separate) zones on a (e.g. solid) surface, such as a multiwall micro-titre plate (e.g. for an ELISA assay). Other appropriate surfaces and containers that are well known in the art may also form part of the kits described herein. In one example, the kit further comprises one or more reagents for detecting the detectably labelled agent. Suitable reagents are well known in the art and include but are not limited to standard reagents and buffers required to perform any one of the appropriate detection methods that may be used (and are well known in the art). In one example, the kit comprises one or more of the following: a multi-well plate, ball bearing(s), extraction buffer, extraction bottle and a lateral flow device.

[0178] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0179] Aspects of the invention are demonstrated by the following non-limiting examples.

[0180] EXAMPLES

[0181] Here, the inventors employed a semi-automated high-throughput mass spectrometry-based glycomics approach to explore blood protein / V-glycosylation on the released glycan level, i.e. after enzymatic removal of / V-glycans from their carrier proteins. Their aim was to identify clinically translatable biomarkers allowing for early, non-invasive detection of NAFLD, and in particular to address the unmet clinical need to capture the progression of non-fibrotic NASH to NASH-associated fibrosis.

[0182] EXAMPLE 1

[0183] MATERIALS AND METHODS

[0184] Study design

[0185] In this study, samples were obtained from the biobanks of Leiden University Medical Center (discovery and replication cohort) and Amsterdam University Medical Center (replication cohort). The discovery and replication cohorts involved 30 NAFLD and 60 presumably healthy controls with plasma samples, and 45 NAFLD and 12 presumably healthy controls with serum samples, respectively (see also reference 30). The cohorts’ demographics can be found in Table 1 . The study protocol was approved a priori by the local ethical committees (B19.071 and B21.045) for the discovery and replication cohort, respectively). Healthy controls in the replication cohort were obtained via the Leiden University Medical Center voluntary donor service (“LUMC Vrijwillige Donoren Service”). Informed consent was obtained from all patients and healthy controls, and the studies complied with the latest version of the Declaration of Helsinki.

[0186] Table 1. Demographic and clinical characteristics of patients and healthy controls in the discovery and replication cohorts. Median and interquartile ranges are shown unless indicated otherwise.

[0187] Materials

[0188] Materials and reagents used in the study were of analytical grade and purchased from commercial suppliers. Type I Ultrapure Water (UP) was used to prepare solutions, which was produced by an ELGA Purelab Ultra system (Elga LabWater, High Wycombe, United Kingdom). Nonidet P-40 substitute (NP-40), super-DHB and 1 -hydroxybenzotriazole monohydrate (HOBt), ammonium bicarbonate (NH4HCO3), potassium chloride (KCI), disodium hydrogen phosphate hydrate (Na2HPO4'7H2O) and 85% phosphoric acid (H3PO4) were obtained from Sigma-Aldrich (Steinheim, Germany). Ethanol, sodium hydroxide (NaOH), sodium dodecyl sulphate (SDS), and trifluoroacetic acid, disodium hydrogen phosphate dihydrate (Na2HPO4'2H2O), potassium dihydrogen phosphate (KH2PO4), and sodium chloride (NaCI) were purchased from Merck (Darmstadt, Germany). 1 -ethyl-3- (3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) was obtained from Fluorochem (Hadfield, United Kingdom), while peptideW-glycosidase F (PNGase F) was purchased from Roche Diagnostics (Mannheim, Germany). HPLC-supra-gradient acetonitrile (ACN) and ethanol (EtOH) were obtained from Biosolve (Valkenswaard, The Netherlands) and Merck (Darmstadt, Germany), respectively. The Visucon-F healthy human plasma standard originated from Affinity Biologicals (Ancaster, Canada). Peptide Calibration Mix II was obtained from Bruker Daltonics (Billerica, MA).

[0189] Liver biopsy

[0190] In the replication cohort, fibrosis scores were defined according to the Brunt scoring system14, based on histological examination of liver biopsies by two independent pathologists. FO: no fibrosis (no scarring); F1 : portal fibrosis (minimal scarring); F2: periportal fibrosis (significant scarring has occurred and extends outside the liver area); F3: severe fibrosis (fibrosis spreading and forming bridges with other fibrotic liver areas); F4: cirrhosis (advanced scarring). No such fibrosis-specific readout was available for patients enrolled in the discovery cohort, although the presence of cirrhosis was defined on the basis of histology or - if unavailable - using liver elastography or liver ultrasound. Decompensated liver cirrhosis was defined as presence of ascites, varices bleeding, hepatocellular carcinoma, hepatorenal or hepatopulmonary syndrome.

[0191] Mass spectrometry glycomics and data processing

[0192] The total blood / V-glycome was analyzed by matrix-assisted laser desorption / ionization - Fourier- transform ion cyclotron resonance - mass spectrometry (MALDI-FTICR-MS) after linkage-specific sialic acid derivatization, as described previously15and as outlined below. After initial data preprocessing including data quality control, similarly to preceding reports16and as outlined below, the relative abundances of individual glycans were calculated in the total plasma and total serum. Glycosylation traits summarizing specific glycosylation features that reflect biosynthetic pathways were calculated using relative abundance of individual glycans (Fig. 1).

[0193] Sample preparation for matrix-assisted laser desorption / ionization - Fourier transform ion cyclotron resonance - mass spectrometry (MALDI-FTICR-MS)-based high-throughput glycosylation analysis

[0194] Plasma samples of the discovery cohort were part of a larger preceding study on autoimmune hepatitis30, and were randomized on overall 5 96-well plates, together with 4 Visucon F standards and 2 or 3 blanks per plate. Serum samples of the replication study were randomized on a single 96-well plate, togetherwith 4 pools (i.e. a pool generated by pooling equal amounts of serum from each patient in the cohort). Age and sex were taken into account for an optimal distribution of cases and controls per plate.

[0195] Glycan release from sera / plasma, linkage-specific sialic acid stabilization and MALDI-FTICR- MS analysis

[0196] Release of / V-glycans from plasma proteins and linkage-specific chemical sialic acid derivatization was performed as previously described in similar high-throughput, robotized workflow, using 2 uL of plasma / serum for the release1 2. For MALDI-FTICR-MS measurement, 1 uL sDHB matrix was topped by 1 uL HILIC-purified sample and left to dry by air1 2. The measurement was performed on a 15T Bruker SolariX XR FTICR mass spectrometer equipped with a ParaCell, a Smartbeam-ll laser and a Combisource (Bruker Daltonics, Bremen, Germany) in positive ionization mode2. Prior to measurement, calibration was performed with Peptide Calibration Mix II (Bruker Daltonics). For each spot, an average spectrum was obtained from the acquisition of 10 spectra in the m / z range of 1000- 5000 using 1 M data points. As it would be understood by a skilled person, stabilization of sialic acids may be obtained as a desired “by-product” of linkage-specific derivatization. Generally, stabilization may be desired for the MALDI measurement (otherwise the sialic acids would be lost), and linkagespecificity is required for the differentiation of a2,3 and a2,6-linkages (otherwise they would be indistinguishable due to identical mass).

[0197] Data processing

[0198] MALDI-FTICR-MS raw spectra were converted into xy files. Extraction of these raw data was performed using in-house developed software MassyTools3. For the targeted extraction of glycan peaks, analyte lists were created based on manual annotation of summed mass spectra. The assignment of glycoforms was based on exact mass and previous reports24'6. The 1+charge state was used for extraction. Signals were integrated by covering minimum 95% of the area of the isotopic envelope of glycan peaks. An analyte was included in the final data analysis if its signal-to-noise was above 27, its isotopic pattern did not deviate more than 25% from the theoretical one, and if its mass error was within a ±20 parts per million range. Additionally, the same analyte had to be present in at least 1 out of 4 spectra (25%) in spectra per disease group for inclusion to the final data analysis. The relative intensity values of glycan compositions that passed quality criteria were calculated by normalizing to the sum of their total areas.

[0199] Glycosylation trait calculation

[0200] Based on the measured blood-derived glycan traits (n = 81 and 72 for the discovery and replication cohort, respectively), glycosylation traits were calculated based on common structural characteristics, including the number of antennae (A), and the levels of fucosylation (F), antennary fucosylation (Fa) bisection (B), galactosylation (G), or sialylation (S), (Figure 1).

[0201] Method repeatability and robustness

[0202] To assess the MALDI-FTICR-MS method repeatability, the inter-plate coefficient of variation of the most abundant glycan peak H5N4E2 was calculated for the plasma standards in the discovery cohort and the intra-plate variation of the pools for the same glycan in the replication cohort and were 4.9% and 1.2%, respectively.

[0203] Statistical analysis

[0204] For both cohorts, a logistic regression model on standardized data (subtraction of the mean and division by the SD) including age, sex and their interaction as co-variates was used to study the associations between glycosylation of healthy controls and NAFLD patients (Healthy=0; NAFLD=1) (Table 2, method outlined below). To compare how the aforementioned significantly different glycosylation traits differ between fibrosis scores, a Kruskal-Wallis test was performed, which in case of a significant result, was followed by the post-hoc Dunn’s test (Figure 3). Spearman’s ranked correlation was performed to assess the correlation of glycosylation traits with Brunt fibrosis score as well as with age (Table 3). To account for multiple testing, during the evaluation of statistical significance per statistical question, the Benjamini-Hochberg procedure with a false discovery rate (FDR) of 5% was used (Table 2 (discovery cohort)). Statistical testing in the replication cohort was performed without multiple testing correction using a cut-off of p < 0.05 (Table 2).

[0205] Statistics

[0206] Age, sex and their interaction were included as co-variates in a logistic regression model to find disease specific associations of HC (0) versus NAFLD (1). The odds ratios (OR) were calculated with their 95% confidence intervals (Cl) and represent single standard deviation increases in the tested derived traits. Multiple testing correction was performed using the Benjamini-Hochberg procedure and was based on a false discovery rate (FDR) of 5% in the discovery cohort. On the analogy of genome wide association studies, the inventors used the discovery cohort to identify potential glycomic associations between healthy individuals and those with NAFLD. In the discovery study, multiple testing correction was applied to avoid / limit the finding of false positive associations. In contrast, the replication cohort was used to confirm associations previously found in the discovery cohort. To avoid overlooking valid associations, the inventors found it less necessary to perform multiple testing correction in the replication cohort. The statistical analyses were performed in R, version 4.2.2 (R Foundation for Statistical Computing, Vienna, Austria) and RStudio, version 2022.12.0, Build 353 (RStudio, Boston, MA).

[0207] RESULTS

[0208] The blood protein / V-glycome was analyzed by mass spectrometry, resulting in the identification of 81 and 72 / V-glycans (80% overlap) for the discovery and replication cohort, respectively. The annotated glycoforms were relatively quantified and summarized in 36 glycosylation traits based on their structural features including fucosylation, antennary fucosylation, bisection, galactosylation, sialylation, antennarity and / V-glycan type for the discovery and replication cohort, respectively (Figure 1). The identified glycoforms were consistent with those commonly found on blood proteins, although the structures are putative and could include collections of isomers15 17.

[0209] Blood protein N-glycosylation associates with NAFLD

[0210] In a first step, the inventors aimed to explore the differences between NAFLD and healthy controls by principle component analysis, which suggested that the key distinction between the two groups is driven by the modification of / V-glycans with sialic acids in a linkage-specific fashion (Figure 4). Further statistical analysis revealed 9 glycosylation traits that were statistically different between NAFLD and healthy controls in the discovery cohort, out of which 6 were replicated (Figure 2, Table 2). The replicated glycosylation traits could be classified into two main categories depending on sialic acid linkage variants (i.e. a2,3- or a2,6-linkages) (Figure 2 b-g, Table 2).

[0211] Table 2. Associations between plasma / V-glycan traits and NAFLD as compared to healthy controls. Logistic regression was performed between NAFLD (1) and HC (0), including age, sex and their interaction as co-variates. Only significant associations that passed the set Iog2 odds ratio threshold (0.5) are shown. To account for multiple testing, p-values in the discovery cohort were corrected by the Benjamini-Hochberg procedure using a 5% FDR. *P-values of the discovery cohort were corrected for multiple testing using the Benjamini-Hochberg procedure (5% FDR). Cl: confidence interval.

[0212] The inventors observed lower a2,3-linked sialylation with NAFLD across most complex-type / V-glycans Specifically, the combined level of a2,3-sialylation over all complex-type glycans (AL) showed odds ratios (OR) of 0.36 and 0.33 for discovery and replication cohort, respectively (Figure 2 b-e, Table 2). In contrast, a2,6-sialylation (AE) was found to be generally increased in NAFLD. This was most pronounced for / V-glycans with three antennae (A3E; ORs of 2.95 and 6.46 in the discovery and replication cohort, respectively) (Figure 2 f, g). The apparent shift from a2,6-sialylation to a2,3- sialylation was also conveyed by the AL to AE ratio (Figure 6).

[0213] Associations ofa2,3- and a2,6-sialylation with NASH-related fibrosis

[0214] As changes in a2,3- and a2,6-sialylation appeared to be a feature of NAFLD the inventors further investigated these glycosylation traits in relation to the degree of fibrosis in patients enrolled in the replication cohort (Figure 3). Patients without fibrotic scarring (non-fibrotic NASH; Brunt fibrosis score 0) did not differ in the degree of their a2,3- (Figure 3 a-d) and a2,6-linked sialylation (Figure 3 e, f) from healthy individuals. On the other hand, sialylation effects were observed upon the histological manifestation of fibrosis (NASH with various degrees of fibrosis; Brunt fibrosis score 1-4). Importantly, these associations were not affected by the age and sex of the individuals (Figure 5).

[0215] Correlation of glycosylation and fibrosis stage

[0216] To further investigate the found associations, a Spearman correlation analysis was performed between the replicated glycosylation traits and Brunt fibrosis score of patients with NASH-related fibrosis. Using this approach, the inventors identified that a2,3-sialylation of tri- (A3L) and tetra- antennary (A4L) glycans and a2,6-sialylation of tri-antennary glycans (A3E) significantly correlated with the progression of fibrosis, suggesting that these glycosylation traits follow a unidirectional trend and might be phenotype-indicative (Table 3). Interestingly, no correlation was found between levels of overall sialyation (i.e. without the distinction of linkage isomers) and Brunt fibrosis score (Figure 7 a-d).

[0217] Table 3. Correlation of glycosylation traits with Brunt fibrosis score. Shown are the Spearman’s correlation coefficients (Rs). Significant correlations are highlighted in bold (p < 0.05).

[0218] Determination of cut-off values forglycan biomarkers of fibrosis

[0219] As the histological manifestation of NASH-related fibrosis (F1-F4, Brunt fibrosis score) coincided with lower relative levels of a2,3-siaylation and higher relative levels of a2 ,6-sialylation in comparison to healthy controls or F0 (non-fibrotic) NASH patients, the inventors intended to establish a method to define cut-off values for the fibrosis-indicative glycosylation traits, which could then be used to detect the occurrence and / or stage of fibrosis in a clinical setting later on. To establish such threshold values, the inventors first examined median relative levels of the glycosylation traits across the severity groups. Based on the distribution, trends and statistics observed, the inventors next pooled the healthy controls and the F0 group (i.e. groups without fibrosis). Next, the inventors decided pooled the F1-F4 groups (i.e. with fibrosis). Based on these results, the inventors suggest the median value of the non- fibrotic group as a reference point for cut-off determination (Table 4). Specifically, the inventors suggest that if levels of a2,3-sialylation (A2L, A3L, A4L, and / or AL) fall lower than the cut-off defined in the non-fibrotic groups, this suggests that the patient may have fibrotic NASH. In otherwords, if the determined levels of A2L, A3L, A4L, and / or AL are below the average of the group cut-offs per glycosylation trait, this may be indicative of the patient having fibrotic NASH. In line with the afore analogy, the inventors suggest that if levels of a2,6-sialylation (A3E and / or AE) rise higher than the cut-off defined in the non-fibrotic groups, the patient may have fibrotic NASH. In other words, if the determined levels of A3E, and / or AE are above the average of the group cut-offs per glycosylation trait, this may be indicative of the patient having fibrotic NASH.

[0220] Table 4. Exemplary cut-off values as defined by the proposed methodology described above. Cut-off values must be defined for every measurement batch against respective linkage-specific sialylation levels of a non-fibrotic (healthy) control.

[0221] Binary classification model generation for the prediction of fibrosis

[0222] To evaluate the discriminative power of the glycosylation traits alone as well as in combination to predict fibrosis, the inventors decided to conduct multiple Receiver Operating Characteristic (ROC) analyses and compared their predictive accuracy. First, the inventors established a composite model with six predictors (A2L, A3L, A4L, AL, A3E, AE). The results of this model suggested that except for A3L and A4L, the predictors behave in a non-linear space. AE was excluded from the composite model. The resulting composite model (including A2L, A3L, A4L, AL, A3E) had the following characteristics: AUC = 0.92, Cl = 0.84-0.97; Cross-validated accuracy = 0.77, accuracy SD = 0.14 (Figure 7e). Despite the obtained high AUC value, the inventors considered the composite model to be insufficient due to non-linear behaviour of many of the variables as well as multicollinearity between the variables that may inflate the model, hence decided to move toward single predictor models. Using a “step” function for the model the inventors obtained an optimal model with A4L as a single predictor, with the following results: Odds Ratio: 0.07, Cl = 0.01-0.24, p < 0.001 (Figure 7f). Additional model parameters were: AUC = 0.90, Cl = 0.82-0.97; Cross-validated accuracy = 0.79, accuracy SD = 0.13. Similar results were obtained for A3L as a single predictor. Inclusion of potential confounding factors such as age and sex improve did not improve this model. Overall, these results suggest that the best performing predictors of fibrotic NASH are A4L and A3L.

[0223] DISCUSSION

[0224] Utilizing a mass spectrometry-based approach capable of discriminating sialic acid linkage variants15, the inventors studied the total blood protein / V-glycome of overall 72 presumably healthy controls and 75 individuals who developed NAFLD and various stages of NASH-related fibrosis. The study sheds light on a novel, replicated fibrosis-specific blood / V-glycomics signature in NASH patients. The inventors believe the results have potential for the development of a non-invasive diagnostic approach for the detection of non-fibrotic to fibrotic NASH transition, as well as fibrotic NASH

[0225] As reliable diagnosis and follow-up of fibrosis is biopsy-based, there is a significant need for non- invasive markers that could guide the selection of NAFLD patients who most benefit from a biopsy18. Alterations in blood protein / V-glycosylation patterns can serve as biomarkers of liver diseases as well as to potentially contribute to their development and progression22. In the present study, the inventors identified a characteristic, and to our knowledge unreported hallmark of NASH-related fibrosis, as the inventors show in two independent cohorts that NAFLD patients display (1) globally lower a2,3-linked sialylation as compared to healthy individuals, (2) globally elevated a2,6-linked sialylation of their triantennary / V-glycans, and (3) that a defined subset of glycosylation traits reflect the stage of NASH- related fibrosis.

[0226] Importantly, the aforementioned glycosylation signatures discerned fibrotic from non-fibrotic NASH within the NAFLD patient population, which is difficult with existing non-invasive diagnostics. Therefore, this new glycosylation pattern potentially targets the unmet clinical need to non-invasively identify patients with NAFLD at risk for fibrosis progression. The inventors believe that some of the results, such as those shown in Figure 3, are not statistically significant between the different fibrosis score groups, due to the size of the sample. Therefore, increased sample sizes would likely show statistically significant results.

[0227] Sialic acid linkage isomers exhibit functionally distinct roles, and changes in the abundance of one linkage variant over the other have been reported in cancer and inflammatory bowel disease19-21. On the landscape of glycomics of NAFLD, larger-scale studies relying on sialic acid linkage isomer differentiation have been lacking. A study of 15 patients across 3 fibrosis categories has found a decrease in the ratio of nonfucosylated to fucosylated glycans that are fully sialylated and carry a single a2,3-linked sialic acid, concluding that the observed signatures are antennary fucosylation- dependent and likely originate from the acute phase protein alpha-1 -antitrypsin (AAT)23. Another research group found a signature linked to the aforementioned composition (i.e. triantennary fully sialylated / V-glycans) as being linked to both AAT and alpha-1 -acid-glycoprotein (AGP), even though without details on the type of fucosylation and sialic acid linkages24. Furthermore, composite markers such as the GlycoFibroTest25and the more recent GlycoFibroTyper26rely on total serum or affinity- enriched immunoglobulin G / V-glycosylation analysis, and both suggest the presence of a bisecting / V- acetlyglucosamine (i.e. bisection) as an important glycosylation feature differentiating clinical phenotypes of NAFLD. Glycomic studies have reported / V-glycan signatures that associate with fibrosis, and directly24or indirectly2325related these glycans to candidate proteins22. Of note, glycoprotein abundance changes (i.e. decreased or elevated hepatic production) of such candidate proteins may to some extent explain the observed relative glycoform frequency changes. Noticeably, our data points towards globally lower a2,3-sialylation, indicating that the noted shifts are likely sialic acid linkage- and not protein abundance-dependent. This observation in the blood / V-glycome of patients with fibrosis suggests altered / V-glycan biosynthetic pathways in the liver or the modified clearance of circulatory glycoproteins. Future studies studying glycosyltransferase expression in hepatocytes or liver tissue sections could provide valuable insights into the pathophysiology of NASH-related fibrosis. The plasma half-life of glycoproteins is regulated by glycan-recognizing receptors, such as the asialoglycoprotein receptor (ASGPR) clearing non-sialylated glycoproteins from the circulation. As this receptor also has affinity for a2,6-linked sialic acids27, a decrease in the expression of ASGPR can also result in an increase in a2,6-linked sialylation on blood proteins.

[0228] Interestingly, a study using a hepatocyte-specific conditional knockout of glycosyltransferase p- galactoside a2,6-sialyltransferase 1 (St6gal1 encoding for the enzyme responsible for the addition of sialic acids in an a2,6-linkage) in mice connected the loss of hepatocyte and circulatory glycoprotein a2,6-sialylation to the spontaneous development of fatty liver disease and inflammation28. In contrast, in humans the inventors found low a2,3-sialylation and elevated a2,6-sialylation on triantennary N- glycans in patients with NASH-related fibrosis. As this latter feature was restricted to triantennary glycans, it may be linked to a change in the abundance of proteins carrying such glycoforms, including but not limited to previously reported acute phase proteins such as AAT24. To unravel this, it would be necessary to subject AAT-associated glycans or glycopeptides for a sialic acid linkage-specific analysis.

[0229] The current study has potential implication for the diagnosis of fibrosis. Unfortunately, neither the clinically available GlycoFibroTest, nor the GlycoFibroTyper include (linkage-specific) sialylation analysis2325.

[0230] CONCLUSIONS

[0231] In conclusion, a replicated fibrosis-specific blood / V-glycosylation signature was found in patients with NAFLD, which allows the detection of fibrotic NASH early-on. The global decrease of a2,3-sialylation, a unique feature on circulatory proteins produced by the fibrotic liver, offers the possibility for developing novel non-invasive diagnostic tests that facilitate early diagnosis of NASH-related fibrosis. Furthermore, the found signature provides new insights in the molecular mechanisms that may play a role in the development of fibrosis in NASH. Clinical translation of the glycomic signature could make diagnosis and follow-up more comfortable for both NAFLD patients and physicians in the future.

[0232] EXAMPLE 2

[0233] MATERIALS AND METHODS

[0234] Materials and methods for Example 2 were generally the same as for Example 1 as outlined above, with the following modifications. Study design

[0235] In this study, samples were obtained from the biobanks of Leiden University Medical Center (discovery and replication cohort) and Amsterdam University Medical Center (replication cohort). The discovery and replication cohorts involved 30 NAFLD and 60 presumably healthy controls with plasma samples, and 102 NAFLD and 29 presumably healthy controls with serum samples, respectively (Pongracz et al. 2024, Blood N-glycomic signature of fibrosis in metabolic-dysfunction associated steatotic liver disease shows low level of global a2,3-sialylation, unpublished manuscript). The cohorts’ demographics can be found in Table 5. The study protocol was approved a priori by the local ethical committees (B19.071 and B21.045) for the discovery and replication cohort, respectively). Healthy controls in the replication cohort were obtained via the Leiden University Medical Center voluntary donor service (“LUMC Vrijwillige Donoren Service”). Informed consent was obtained from all patients and healthy controls, and the studies complied with the latest version of the Declaration of Helsinki.

[0236]

[0237]

[0238] ‘Incomplete observations

[0239] Table 5. Demographic and clinical characteristics of patients and healthy controls in the discovery and replication cohorts.

[0240] Materials

[0241] As outlined in Example 1.

[0242] Liver biopsy

[0243] Performed as outlined in Example 1 .

[0244] Mass spectrometry glycomics and data processing

[0245] Performed as outlined in Example 1 .

[0246] Sample preparation for matrix-assisted laser desorption / ionization - Fourier transform ion cyclotron resonance - mass spectrometry (MALDI-FTICR-MS)-based high-throughput glycosylation analysis

[0247] Performed as outlined in Example 1 .

[0248] Glycan release from sera / plasma, linkage-specific sialic acid stabilization and MALDI-FTICR- MS analysis

[0249] Performed as outlined in Example 1 .

[0250] Data processing

[0251] Performed as outlined in Example 1 .

[0252] Glycosylation trait calculation

[0253] Performed as outlined in Example 1 .

[0254] Method repeatability and robustness

[0255] Performed as outlined in Example 1 .

[0256] Statistical analysis

[0257] For both cohorts, a logistic regression model on standardized data (subtraction of the mean and division by the SD) including age, sex and their interaction as co-variates was used to study the associations between glycosylation of healthy controls and NAFLD patients (Healthy=0; NAFLD=1) (Table 6, method outlined below). To compare how the aforementioned significantly different glycosylation traits differ between fibrosis scores, a Kruskal-Wallis test was performed, which in case of a significant result, was followed by the post-hoc Dunn’s test (Figure 11). Spearman’s ranked correlation was performed to assess the correlation of glycosylation traits with Brunt fibrosis score as well as with age and type 2 diabetes mellitus status (Table 7, Figure 13). To account for multiple testing, during the evaluation of statistical significance per statistical question, the Benjamini-Hochberg procedure with a false discovery rate (FDR) of 5% was used (Figure 9 and Table 6 (discovery cohort), Figure 15). Statistical testing in the replication cohort was performed without multiple testing correction using a cut-off of p < 0.05 (Table 6).

[0258] Statistics

[0259] Performed as outlined in Example 1 .

[0260] RESULTS

[0261] The blood protein / V-glycome was analyzed by mass spectrometry, resulting in the identification of 81 and 72 / V-glycans (80% overlap) for the discovery and replication cohort, respectively. The annotated glycoforms were relatively quantified and summarized in 36 glycosylation traits based on their structural features including fucosylation, antennary fucosylation, bisection, galactosylation, sialylation , antennarity and / V-glycan type for the discovery and replication cohort, respectively (Figure 1). The identified glycoforms were consistent with those commonly found on blood proteins, although the structures are putative and could include collections of isomers15 17.

[0262] Blood protein N-glycosylation associates with NAFLD In a first step, the inventors aimed to explore the differences between NAFLD and healthy controls by principle component analysis, which suggested that the key distinction between the two groups is driven by the modification of / V-glycans with sialic acids in a linkage-specific fashion (Figure 8). Further statistical analysis revealed 9 glycosylation traits that were statistically different between NAFLD and healthy controls in the discovery cohort, out of which 6 were replicated (Figure 9, Table 6). The replicated glycosylation traits could be classified into two main categories depending on sialic acid linkage variants (i.e. a2,3- or a2,6-linkages) (Figure 9 b-g, Table 6). A4L Of A4 0.035 8.12 x 1007

[0263] Table 6. Associations between plasma / V-glycan traits and NAFLD as compared to healthy controls. Logistic regression was performed between NAFLD (1) and HC (0), including age, sex and their interaction as co-variates. Only significant associations that passed the set In odds ratio threshold (0.345) are shown. To account for multiple testing, p-values in the discovery cohort were corrected by the Benjamini-Hochberg procedure using a 5% FDR. Cl: confidence interval.

[0264] The inventors observed lower a2,3-linked sialylation with NAFLD across most complex-type / V-glycans Specifically, the combined level of a2,3-sialylation over all complex-type glycans (AL) showed odds ratios (OR) of 0.36 and 0.11 for discovery and replication cohort, respectively (Figure 9 b-e, Table 6). In contrast, a2,6-sialylation (AE) was found to be generally increased in NAFLD. This was most pronounced for / V-glycans with three antennae (A3E; ORs of 2.95 and 11.6 in the discovery and replication cohort, respectively) (Figure 9 f, g, Table 6). The apparent shift from a2,6-sialylation to a2,3-sialylation was also conveyed by the AL to AE ratio (Figure 10).

[0265] Associations ofa2,3- and a2,6-sialylation with NASH-related fibrosis

[0266] As changes in a2,3- and a2,6-sialylation appeared to be a feature of NAFLD the inventors further investigated these glycosylation traits in relation to the degree of fibrosis in patients enrolled in the replication cohort (Figure 11). Patients without fibrotic scarring (non-fibrotic NASH; Brunt fibrosis score 0) did not differ in the degree of their a2,3- (Figure 11 a-d) and a2,6-linked sialylation (Figure 11 e, f) from healthy individuals. On the other hand, linkage-specific sialylation effects were observed upon the histological manifestation of fibrosis (NASH with various degrees of fibrosis; Brunt fibrosis score 1-4). Importantly, these associations were not affected by the age and sex (Figure 12) nor by comorbidities (type 2 diabetes mellitus status (Figure 13)) of the individuals, and no association was found between levels of overall sialylation (i.e. without the distinction of linkage isomers) and fibrosis (Figure 14).

[0267] Correlation of glycosylation and fibrosis stage

[0268] To further investigate the found associations, a Spearman correlation analysis was performed between the replicated glycosylation traits and Brunt fibrosis score of patients with NASH-related fibrosis. Using this approach, the inventors identified that a2,3-sialylation over all complex-type glycans (AL) and a2 ,6-sialylation over all complex-type glycans (AE), a2 ,3-sialylation of bi- (A2L), tri- (A3L) and tetra-antennary (A4L) glycans and a2,6-sialylation of tri-antennary glycans (A3E) significantly correlated with the progression of fibrosis, suggesting that these glycosylation traits follow a unidirectional trend and might be phenotype-indicative (Table 7).

[0269] Table 7. Correlation of glycosylation traits with Brunt fibrosis score. Shown are the Spearman’s correlation coefficients (Rs). All correlations were significant (p < 0.05). Healthy controls were considered as F0 and pooled with patients with normal histology (F0) for this analysis.

[0270] Determination of cut-off values forglycan biomarkers of fibrosis

[0271] As the histological manifestation of NASH-related fibrosis (F1-F4, Brunt fibrosis score) coincided with lower relative levels of a2,3-siaylation and higher relative levels of a2 ,6-sialylation in comparison to healthy controls or F0 (non-fibrotic) NASH patients, the inventors intended to establish a method to define cut-off values for the fibrosis-indicative glycosylation traits, which could then be used to detect the occurrence and / or stage of fibrosis in a clinical setting later on. To establish such threshold values, the inventors first examined median relative levels of the glycosylation traits across the severity groups. Based on the distribution, trends and statistics observed, the inventors next pooled the healthy controls and the F0 group (i.e. groups without fibrosis). Next, the inventors pooled the F1-F4 groups (i.e. with fibrosis). Based on these results, the inventors suggest the median value of the non-fibrotic group as a reference point for cut-off determination (Table 8). Specifically, the inventors suggest that if levels of a2,3-sialylation (A2L, A3L, A4L, and / or AL) fall lower than the cut-off defined in the non- fibrotic groups, this suggests that the patient may have fibrotic NASH. In otherwords, if the determined levels of A2L, A3L, A4L, and / or AL are below the average of the group cut-offs per glycosylation trait, this may be indicative of the patient having fibrotic NASH. In line with the afore analogy, the inventors suggest that if levels of a2,6-sialylation (A3E and / or AE) rise higher than the cut-off defined in the non- fibrotic groups, the patient may have fibrotic NASH. In other words, if the determined levels of A3E, and / or AE are above the average of the group cut-offs per glycosylation trait, this may be indicative of the patient having fibrotic NASH.

[0272] Table 8. Exemplary cut-off values as defined by the proposed methodology described above. Cut-off values must be defined for every measurement batch against respective linkage-specific sialylation levels of a non-fibrotic (healthy) control.

[0273] Binary classification model generation for the prediction of fibrosis

[0274] Motivated by the found associations, the inventors conducted a receiver operating characteristic (ROC) analysis to evaluate the discriminative power of the glycosylation traits to predict the histological manifestation of fibrosis. Using a “step” function, the inventors obtained an optimal model with A4L as a single predictor (Figure 15). Additional model parameters were: AUC = 0.87, Cl = 0.80-0.94. Similar results were obtained for A3L as a single predictor, suggesting that the best performing predictors of fibrotic MASH are A4L and A3L. Inclusion of potential confounding factors such as age, sex, BMI and type 2 diabetes mellitus status did not improve these models. Due to limited sample numbers in the F0 group (n=12), the same analysis was not performed for the conventional diagnostic markers FIB-4 and NFS.

[0275] Overall, these results suggest that the best performing predictors of fibrotic NASH are A4L and A3L. DISCUSSION

[0276] Utilizing a mass spectrometry-based approach capable of discriminating sialic acid linkage variants15, the inventors studied the total blood protein N-glycome of 75 individuals who developed NAFLD and various stages of NASH-related fibrosis compared to overall 72 controls. The study sheds light on a novel, replicated, liver fibrosis-specific blood N-glycomic signature in patients with NAFLD-NASH. The results may have potential for the development of a non-invasive diagnostic approach for the detection of non-fibrotic to fibrotic transition in NAFLD.

[0277] Alterations in blood protein N-glycosylation patterns can serve as biomarkers of liver diseases as well as to potentially contribute to their development and progression22. In the present study, the inventors identified a characteristic unreported hallmark of fibrotic NAFLD-NASH, as the inventors show in two independent cohorts that patients display globally lower a2,3-linked sialylation as compared to healthy individuals. Importantly, the aforementioned glycosylation signatures discerned fibrotic NASH from non-fibrotic counterparts within the NAFLD patient population from the point of histological signs of fibrosis, where existing non-invasive diagnostics falls short.

[0278] Sialic acid linkage isomers exhibit functionally distinct roles, and changes in the abundance of one linkage variant over the other have been reported in cancer, inflammatory bowel disease and type 2 diabetes mellitus.

[0279] Noticeably, the above data points towards globally lower a2,3-sialylation, indicating that the noted shifts are rather sialic acid linkage- and not protein abundance-dependent, as no significant decrease in major plasma glycoprotein concentrations have been described by proteomics studies that would support such a hypothesis. Thus, the above observation in the blood N-glycome of patients with fibrosis suggests altered N-glycan biosynthetic pathways in the liver or the modified clearance of secreted glycoproteins from the circulation.

[0280] Interestingly, a study using a hepatocyte-specific conditional knockout of glycosyltransferase p- galactoside a2,6-sialyltransferase 1 (St6gal1 ; encoding the enzyme responsible for the addition of sialic acids in an a2,6-linkage) in mice connected the loss of hepatocyte and circulatory glycoprotein a2,6-sialylation to the spontaneous development of fatty liver disease and a shift towards a proinflammatory immune-phenotype20. In contrast, the inventors found low a2,3-sialylation and elevated a2 ,6-sialylation on triantennary N-glycans in patients with NASH-related fibrosis. As this latter feature was restricted to triantennary glycans, it may be linked to an increase in the abundance of proteins carrying such glycoforms, including but not limited to previously reported acute phase proteins such as AAT24. To further unravel this, it would be necessary to subject AAT-associated glycans or glycopeptides for a sialic acid linkage-specific analysis.

[0281] The current study has potential implication for the diagnosis of fibrosis. Unfortunately, neither the clinically available GlycoFibroTest, nor the GlycoFibroTyper include (linkage-specific) sialylation analysis2325.

[0282] CONCLUSIONS

[0283] In conclusion, a replicated fibrosis-specific blood / V-glycosylation signature was found in patients with NAFLD, which allows the detection of fibrotic NASH early-on. The global decrease of a2,3-sialylation, a unique feature on circulatory proteins produced by the fibrotic liver, offers the possibility for developing novel non-invasive diagnostic tests that facilitate early diagnosis of NASH-related fibrosis. Furthermore, the found signature provides new insights in the molecular mechanisms that may play a role in the development of fibrosis in NASH. Clinical translation of the glycomic signature could make diagnosis and follow-up more comfortable for both NAFLD patients and physicians in the future, and may allow for timely intervention and improved disease management.

[0284] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0285] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0286] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0287] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0288] References

[0289] 1 Ruissen, M. M., Mak, A. L., Beuers, U., Tushuizen, M. E. & Holleboom, A. G. Non-alcoholic fatty liver disease: a multidisciplinary approach towards a cardiometabolic liver disease. EurJ Endocrinol 3, R57-R73 (2020). https: / / doi.org: 10.1530 / EJE-20-0065

[0290] 2 Loomba, R., Friedman, S. L. & Shulman, G. I. Mechanisms and disease consequences of nonalcoholic fatty liver disease. Cell 184, 2537-2564 (2021). https: / / doi.org:10.1016 / j.cell.2021.04.015

[0291] 3 Younossi, Z. et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol 15, 11-20 (2018). https: / / doi.org: 10.1038 / nrgastro.2017.109

[0292] 4 Estes, C. et al. Modeling NAFLD disease burden in China, France, Germany, Italy, Japan, Spain, United Kingdom, and United States for the period 2016-2030. J Hepatol 69, 896-904 (2018). https: / / doi.org: 10.1016 / j.jhep.2018.05.036 5 Taylor, R. S. et al. Association Between Fibrosis Stage and Outcomes of Patients With Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis. Gastroenterology 158, 1611-1625 e1612 (2020). https: / / doi.org:10.1053 / j.gastro.2020.01.043

[0293] 6 Dulai, P. S. et al. Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Systematic review and meta-analysis. Hepatology 65, 1557-1565 (2017). https: / / doi.org: 10.1002 / hep.29085

[0294] 7 Byrne, C. D. & Targher, G. EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease: is universal screening appropriate? Diabetologia 59, 1141-1144 (2016). https: / / doi.org: 10.1007 / s00125-016-3910-y

[0295] 8 Gilmore, I. T. et al. Indications, methods, and outcomes of percutaneous liver biopsy in England and Wales: an audit by the British Society of Gastroenterology and the Royal College of Physicians of London. Gut 36, 437-441 (1995). https: / / doi.org: 10.1136 / gut.36.3.437

[0296] 9 Seeff, L. B. et al. Complication rate of percutaneous liver biopsies among persons with advanced chronic liver disease in the HALT-C trial. Clin Gastroenterol Hepatol 8, 877-883 (2010). https: / / doi.org:10.1016 / j.cgh.2010.03.025

[0297] 10 Eddowes, P. J. et al. Accuracy of FibroScan Controlled Attenuation Parameter and Liver Stiffness Measurement in Assessing Steatosis and Fibrosis in Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology 156, 1717-1730 (2019). https: / / doi.org:10.1053 / j.gastro.2019.01.042

[0298] 11 Troelstra, M. A. et al. Assessment of Imaging Modalities Against Liver Biopsy in Nonalcoholic Fatty Liver Disease: The Amsterdam NAFLD-NASH Cohort. J Magn Reson Imaging 54, 1937- 1949 (2021). https: / / doi.org: 10.1002 / jmri.27703

[0299] 12 van Dijk, A. M., Schattenberg, J. M., Holleboom, A. G. & Tushuizen, M. E. Referral care paths for non-alcoholic fatty liver disease-Gearing up for an ever more prevalent and severe liver disease. United European Gastroenterol J 9, 903-909 (2021). https: / / doi.org: 10.1002 / ueg2.12150

[0300] 13 Balkhed, W., Aberg, F. O., Nasr, P., Ekstedt, M. & Kechagias, S. Repeated measurements of non-invasive fibrosis tests to monitor the progression of non-alcoholic fatty liver disease: A long-term follow-up study. Liver Int 42, 1545-1556 (2022). https: / / doi.org: 10.1111 / liv.15255

[0301] 14 Bedossa, P. Intraobserver and Interobserver Variations in Liver Biopsy Interpretation in Patients with Chronic Hepatitis C. Hepatology 20, 15-20 (1994). https: / / doi.org: 10.1002 / hep.1840200104

[0302] 15 Vreeker, G. C. M. et al. Automated Plasma Glycomics with Linkage-Specific Sialic Acid Esterification and Ultrahigh Resolution MS. Anal Chem 90, 11955-11961 (2018). https: / / doi.org: 10.1021 / acs.analchem.8b02391

[0303] 16 Dotz, V. et al. Plasma protein N-glycan signatures of type 2 diabetes. Biochimica et Biophysica Acta (BBA) - General Subjects 1862, 2613-2622 (2018). https: / / doi.org:https: / / doi.org / 10.1016 / j.bbagen.2018.08.005 17 Clerc, F. et al. Human plasma protein N-glycosylation. Glycoconj J 33, 309-343 (2016). https: / / doi.org: 10.1007 / s10719-015-9626-2

[0304] 18 Wong, G. L. Non-invasive assessments for liver fibrosis: The crystal ball we long for. J Gastroenterol Hepatol 33, 1009-1015 (2018). https: / / doi.org: 10.1111 / jgh.14103

[0305] 19 Park, E. I., Yilling, M., Unverzagt, C., Gabius, H.-J. & Baenziger, J. U. The asialoglycoprotein receptor clears glycoconjugates terminating with sialic acida2,6GalNAc. PNAS 102, 17125- 17129 (2005). https: / / doi.org:https: / / doi.org / 10.1073 / pnas.050853710

[0306] 20 Oswald, D. M., Jones, M. B. & Cobb, B. A. Modulation of hepatocyte sialylation drives spontaneous fatty liver disease and inflammation. Glycobiology (2019). https: / / doi.org: 10.1093 / glycob / cwz096

[0307] 21 Cummings, R. D., Darvill, A. G., Etzler, M. E. & Hahn, M. G. in Essentials of Glycobiology (eds rd etal.) 611-625 (2015).

[0308] 22 Verheist, X. et al. Protein Glycosylation as a Diagnostic and Prognostic Marker of Chronic Inflammatory Gastrointestinal and Liver Diseases. Gastroenterology 158, 95-110 (2020).

[0309] 23 Hanamatsu, H. et al. Comparative Glycomic Analysis of Sialyl Linkage Isomers by Sialic Acid Linkage-Specific Alkylamidation in Combination with Stable Isotope Labeling of alpha2,3- Linked Sialic Acid Residues. Anal Chem 91 , 13343-13348 (2019). https: / / doi.org: 10.1021 / acs.analchem.9b03617

[0310] 24 Ramachandran, P. et al. Serum Glycoprotein Markers in Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma. J Proteome Res 21 , 1083-1094 (2022). https: / / doi.org: 10.1021 / acs.jproteome.1 c00965

[0311] 25 Vanderschaeghe, D. et al. GlycoFibroTest is a highly performant liver fibrosis biomarker derived from DNA sequencer-based serum protein glycomics. Mol Cell Proteomics 8, 986-994 (2009). https: / / doi.org:10.1074 / mcp.M800470-MCP200

[0312] 26 Scott, D. A. et al. GlycoFibroTyper: A Novel Method for the Glycan Analysis of IgG and the Development of a Biomarker Signature of Liver Fibrosis. Front Immunol 13, 797460 (2022). https: / / doi.org: 10.3389 / fimmu.2022.797460

[0313] 27 Fernandez-Poza, S. et al. Tailor-made recombinant prokaryotic lectins for characterisation of glycoproteins. Anal Chim Acta 1155, 338352 (2021). https: / / doi.org: 10.1016 / j.aca.2021 .338352

[0314] 28 Ruhaak, L. R. et al. Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF. J Proteome Res 9, 6655-6664 (2010). https: / / doi.org: 10.1021 / pr100802f

[0315] 29 Mitra, I. et al. Structural Characterization of Serum N-Glycans by Methylamidation, Fluorescent Labeling, and Analysis by Microchip Electrophoresis. Anal Chem 88, 8965-8971 (2016). https: / / doi.org: 10.1021 / acs.analchem.6b00882 Pongracz, T. et al. Autoimmune hepatitis displays distinctively high multi-antennary sialylation on plasma N-glycans compared to other liver diseases; J Transl Med. 2024 May 14;22(1):456. doi: 10.1186 / sl 2967-024-05173-z

Claims

CLAIMS1 . A method for diagnosing fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:• determining the levels of a2 , 3-sialy lation and / or a2 ,6-sialy lation of / V-glycans in a sample from the subject; and• comparing the determined levels of a2,3-sialylation and / or a2,6-sialylation to a reference value, wherein: i) an increase in the levels of a2,6-sialylation in the sample; and / or ii) a decrease in the levels of a2 ,3-sialylation in the sample is indicative of the subject having fibrotic NASH.

2. A method for staging fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:• determining the levels of a2 , 3-sialy lation and / or a2 ,6-sialy lation of / V-glycans in a sample from the subject; and• comparing the determined levels of a2,3-sialylation and / or a2,6-sialylation to reference values indicative of a stage of fibrotic NASH, and thereby determining the subject’s stage of fibrotic NASH.

3. The method of claim 2, wherein the staging is according to Brunt Fibrosis score.

4. A method for monitoring fibrotic non-alcoholic steatohepatitis (NASH) in a subject, the method comprising the steps of:• determining the levels of a2 ,3-sialylation and / or a2 ,6-sialylation of / V-glycans in a first sample from the subject; and• determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a second sample from the subject, wherein the second sample has been obtained from the subject at a later time point than the first sample, wherein: i) an increase in the levels of a2 ,6-sialylation in the second sample and / or a decrease in the levels of a2 ,3-sialylation in the second sample as compared to the first sample is indicative of fibrotic NASH progressing in the subject; or ii) a decrease in the levels of a2 ,6-sialylation in the second sample and / or an increase in the levels of a2-3-sialylation in the second sample as compared to the first sample is indicative of fibrotic NASH regressing in the subject.

5. A method for monitoring a subject’s compliance to a prescribed treatment for fibrotic nonalcoholic steatohepatitis (NASH), the method comprising the steps of:• determining the levels of a2 ,3-sialylation and / or a2 ,6-sialylation of / V-glycans in a first sample from the subject; and• determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a second sample from the subject, wherein the second sample has been obtained from the subject after the prescribed treatment, wherein a decrease or no change in the levels of a2,6-sialylation in the second sample, and / or an increase or no change in the levels of a2 ,3-sialylation in the second sample as compared to the first sample is indicative of a subject’s compliance to the prescribed treatment for fibrotic NASH6. A method of monitoring therapeutic effect of a prescribed treatment for fibrotic non-alcoholic steatohepatitis (NASH), the method comprising the steps of:• determining the levels of cz2 ,3-sialylation and / or cz2 ,6-sialylation of / V-glycans in a first sample from the subject; and• determining the levels of a2,3-sialylation and / or a2,6-sialylation of / V-glycans in a second sample from the subject, wherein the second sample has been obtained from the subject after the prescribed treatment, wherein a decrease or no change in the levels of «2,6-sialylation in the second sample and / or an increase or no change in the levels of cz2 ,3-sialylation in the second sample as compared to the first sample is indicative of a therapeutic effect of a prescribed treatment for fibrotic NASH.

7. The method according to any preceding claim, wherein the / V-glycans are complex / V-glycans and / or hybrid / V-glycans.

8. The method according to any preceding claim, wherein the / V-glycans are selected from the group consisting of monoantennary / V-glycans, diantennary / V-glycans, triantennary / V-glycans, tetraantennary / V-glycans, and a combination thereof.

9. The method according to claim 7, wherein the / V-glycans are complex / V-glycans, optionally selected from the group consisting of triantennary / V-glycans and tetraantennary / V-glycans.

10. The method according to any preceding claim, wherein the sample is a blood sample.11 . The method according to claim 10, wherein the blood sample is a dried blood spot sample.

12. The method according to claim 10 or 11 , wherein the blood sample is selected from the group consisting of whole blood, blood plasma, and blood serum.

13. The method according to any preceding claim, wherein the subject has been diagnosed with or determined to be at risk of fibrotic NASH.

14. The method according to claim 13, wherein the subject determined to be at risk of fibrotic NASH: i) has been diagnosed with NAFLD or non-fibrotic NASH; ii) has been diagnosed with type 2 diabetes or insulin resistance; iii) is obese; iv) has been diagnosed with hypertension and / or dyslipidaemia; and / or v) has or is suspected of having a genetic predisposing factor, optionally wherein the genetic predisposing factor is a mutation in a gene selected from the group consisting of PNPLA3, TM6SF2, MBOAT7, GCKR, and HSD18B13.

15. The method according to any one of claims 5 to 14, wherein the treatment is selected from the group consisting of weight loss and lifestyle improvement.

16. The method according to any preceding claim, wherein the levels of a2,3-sialylation and / or a2,6- sialy lation are determined by a method selected from the group consisting of mass spectrometry, high- performance liquid chromatography, capillary (gel) electrophoresis with laser induced fluorescence detection, hydrophilic interaction liquid chromatography, lectin- or antibody-based binding assay, and an ELISA based assay.

17. The method according to any preceding claim, wherein the method further comprises determining the subject’s FIB-4 score, ELF score, and / or APRI score; and / or the subject’s levels of aspartate transaminase (AST), alanine transaminase (ALT); and / or performing liver imaging.

18. The method according to any one of claims 1-17, wherein the sample has been subjected to N- glycan release from blood proteins and linkage-specific chemical sialic acid derivatization prior to determining the levels of a2,3-sialylation and / or a2,6-sialylation of glycans, optionally wherein the proteins are selected from the group consisting of plasma, serum and whole blood proteins.

19. A method of treating fibrotic NASH in a subject, the method comprising the steps of administering a fibrotic NASH treatment to a subject that has been diagnosed or determined to be at risk of fibrotic NASH, by a method of any one of claims 1 to 18.

20. A kit for use in a method of any one of claims 1 to 19, the kit comprising a detectably labelled agent that specifically binds to a2,3-sialylation and / or a2,6-sialylation of glycans.

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