AU2018202716A1

AU2018202716A1 - Detection of liver disease

Info

Publication number: AU2018202716A1
Application number: AU2018202716A
Authority: AU
Inventors: Grant RAMM; Richard Skoien; Anna Weis
Original assignee: Queensland Institute of Medical Research QIMR
Current assignee: QIMR Berghofer Medical Research Institute
Priority date: 2018-04-18
Filing date: 2018-04-18
Publication date: 2019-11-07

Abstract

-50 Provided are methods for determining or predicting the diagnosis, prognosis, treatment and therapeutic efficacy of a hepatitis-associated disease, disorder or condition, such as hepatic fibrosis, cirrhosis and/or hepatocellular carcinoma, in a subject with hepatitis. The 5 present methods involve evaluating the expression level of one or more miRNA biomarkers including miRNA-122, miRNA-486, miRNA-151a, miRNA-142, miRNA 409 and miRNA-22 and fragments or variants thereof as an indication of whether the subject with hepatitis may have a hepatitis-associated disease, disorder or condition. 3615415vl

Description

DETECTION OF LIVER DISEASE

TECHNICAL FIELD

THIS INVENTION relates to the prognosis, diagnosis and/or treatment of liver disease. More particularly, this invention relates to determining expression levels of one or more micro-RNAs correlated with a hepatitis-associated liver disease in a biological sample from a subject having hepatitis.

BACKGROUND

Chronic Hepatitis C (CHC) is a major risk factor for the development of cirrhosis and subsequent hepatocellular carcinoma (HCC), a cancer whose incidence and mortality is increasing.¹ Chronic infection with the Hepatitis C virus (HCV) affects an estimated 80 million individuals worldwide.²

Next generation direct acting antivirals (DAA) have improved treatment of HCV with clearance rates of 85-100%, regardless of patient or viral factors.³ The impact of these new treatments on the global burden of liver disease due to CHC largely depends on when it is diagnosed and DAA availability.⁴ With an aging CHC population cohort, there is a higher probability of advanced fibrosis⁵ and cirrhosis conferring a risk of HCC.⁶Despite DAA treatment, the burden of HCV-related HCC in most countries is still increasing and expected to peak in the next decade.^{4, 5}’ ⁷ This highlights the clinical imperatives to increase treatment rates, accurately identify patients with cirrhosis and improve diagnostic tests for HCV-related HCC. Furthermore, whilst achieving a SVR has been shown to improve liver function in cirrhotic patients, the risk of complications like HCC remains.⁸

The gold standard for the identification of cirrhosis for the purposes of HCC surveillance is liver biopsy but this is invasive, prone to sampling error, does not necessary reflect the dynamic continuum of chronic liver disease (or cirrhosis itself)⁹ and is now rarely used. Non-invasive tools, such as liver stiffness assessment (e.g. transient elastography) have been employed to screen CHC patients for cirrhosis but there is still a clinically-significant indeterminate range affecting test accuracy.¹⁰ Non-invasive biomarker panels, such as the aminotransferase to platelet ratio (APRI)¹¹ and Fibrosis-4 (FIB-4)¹²’¹³ scores provide good sensitivity and specificity within defined cut-offs¹¹ but

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-2values falling short of these specific thresholds lack diagnostic accuracy. Access to these tools is also sometimes limited to specialist treatment, pathology or imaging facilities.

The only currently-accepted HCC tumour biomarker, alpha-fetoprotein (AFP), generally has suboptimal sensitivity and specificity for surveillance and diagnosis in most cases.¹⁴’ ¹³ Routine HCC surveillance comprises 6-monthly ultrasound with dynamic scanning (CT/MRI) used to further characterise suspicious lesions. For a proportion of tumours, diagnosis is necessarily delayed until interval growth can be demonstrated but this limits early access to definitive treatment. Indeed, although the sensitivity and specificity of ultrasound-based diagnostic strategies exceed 90% for detection of HCC across all stages, its sensitivity is only 63% for early HCC.¹⁴

Identification of cirrhotic patients at highest risk of cirrhosis-related complications and the early diagnosis of HCC remain unmet clinical needs. Further to this and despite the increasing number of studies investigating the use of miRNAs as biomarkers for chronic liver disease, there remains a need for biomarkers, such as miRNA biomarkers, that are useful in the diagnosis, prognosis, therapeutic response monitoring and/or disease monitoring with respect to liver diseases associated with and/or secondary to hepatitis in patients. Specifically, the use of such biomarkers may address the outstanding clinical needs for more sensitive tools to screen for hepatitis-associated liver diseases, inclusive of the progression of such liver diseases, in these patients having hepatitis, as well as prognostic tools to guide treatment decisions for patients infected with HCV, including liver fibrosis, cirrhosis and HCC.

SUMMARY

MicroRNAs represent an important class of biomarkers that provide opportunities for clinical translation. The invention is broadly directed to a method of diagnosis, prognosis, therapeutic response monitoring and/or disease response monitoring of a hepatitis-associated disease, disorder or condition in patients with hepatitis, and more particularly viral hepatitis.

More particularly, the inventors have discovered specific miRNA biomarkers that have proved to be useful in the diagnosis, prognosis and/or disease monitoring of liver disease in patients infected with a Hepatitis C virus (HCV). Subsequently, methods have been developed to diagnose liver disease and/or monitor liver disease progression in subjects having hepatitis as well as to provide an indication of liver disease prognosis.

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-3 Furthermore, it is envisaged that particular miRNA bio markers may serve as a prognostic marker of liver disease with respect to treatment response.

In a first aspect, the invention provides a method of determining whether or not a subject with hepatitis has a hepatitis-associated disease, disorder or condition, including:

determining an expression level of one or more miRNA biomarkers in a biological sample from the subject, wherein the one or more miRNA biomarkers are selected from the group consisting of miRNA-122, miRNA-486, miRNA-15la, miRNA-142, miRNA409 and miRNA-22, or a fragment or variant thereof, and wherein the hepatitis-associated disease, disorder or condition is detected if said expression level of one or more miRNA biomarkers, is altered or modulated in the biological sample.

In a second aspect, the invention provides a method of determining the prognosis of a subject with hepatitis, including the steps of:

determining an expression level of one or more miRNA biomarkers in a biological sample obtained from the subject, wherein the one or more miRNA biomarkers are selected from the group consisting of miRNA-122, miRNA-486, miRNA-151a, miRNA142, miRNA-409 and miRNA-22, or a fragment or variant thereof, to thereby evaluate the prognosis of the subject with hepatitis.

In one embodiment, the present method further includes the step of determining whether or not the subject with hepatitis has a hepatitis-associated disease, disorder or condition based on the expression level of the one or more miRNA biomarkers determined in the aforementioned step.

Suitably, if the expression level of said one or more miRNA biomarkers is altered or modulated in the biological sample, the prognosis may be negative or positive.

In one embodiment, the prognosis is used, at least in part, to determine whether the subject with hepatitis would benefit from treatment of the hepatitis-associated disease, disorder or condition.

In one embodiment, the prognosis is used, at least in part, to develop a treatment strategy for the subject with hepatitis.

In one embodiment, the prognosis is used, at least in part, to determine progression of the hepatitis-associated disease, disorder or condition in the subject with hepatitis.

Suitably, the present method further includes the step of determining suitability of the subject for treatment based, at least in part, on the prognosis.

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-4In one embodiment of the aforementioned aspects, the method further includes the step of determining a disease stage and/or grade for the hepatitis-associated disease, disorder or condition based on the expression level of the one or more miRNA biomarkers.

In one embodiment of the above aspects, the expression level of the one or more miRNA biomarkers is determined before, during and/or after treatment.

In a third aspect, the invention provides a method of treating a hepatitis-associated disease, disorder or condition in a subject with hepatitis including;

determining an expression level of one or more miRNA biomarkers in a biological sample from the subject, before, during and/or after treatment of the hepatitis-associated disease, disorder or condition, wherein the one or more miRNA biomarkers are selected from the group consisting of miRNA-122, miRNA-486, miRNA-151a, miRNA-142, miRNA-409 and miRNA-22, or a fragment or variant thereof, and based on the determination made, initiating, continuing, modifying or discontinuing a treatment of the hepatitis-associated disease, disorder or condition.

In a fourth aspect, the invention provides a method of evaluating treatment efficacy of a hepatitis-associated disease, disorder or condition in a subject with hepatitis including:

determining an expression level of one or more miRNA biomarkers in a biological sample from the subject before, during and/or after treatment, wherein the one or more miRNA biomarkers are selected from the group consisting of miRNA-122, miRNA-486, miRNA-151a, miRNA-142, miRNA-409 and miRNA-22, or a fragment or variant thereof; and determining whether or not the treatment is efficacious according to whether said expression level of one or more miRNA biomarkers is altered or modulated in the subject's biological sample.

In one embodiment of the third and fourth aspects, the method further comprises selecting a treatment for the hepatitis-associated disease, disorder or condition based on the expression level of the miRNA biomarkers.

In particular embodiments, the method of the aforementioned aspects further includes the step of measuring an expression level of one or more additional miRNA biomarkers.

Suitably, the method of the hereinbefore described aspects further includes the step of determining an expression level of a further biomarker. Preferably, the further

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- 5 biomarker is selected from the group consisting of an hepatic enzyme (e.g., aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), alkaline phosphatase (ALP)), alpha-fetoprotein, a platelet count, a Child-TurcottePugh (CTP) score, a Model for End-Stage Liver Disease (MELD) score, an International Normalized Ratio (INR) score, fibrosis-4 (FIB-4) index, aspartate aminotransferase (AST) to platelet ratio index (APRI), transient elastography, 2-D shear wave elastography and any combination thereof. Even more preferably, the further biomarker is or comprises FIB-4 index and/or APRI.

In particular embodiments of the methods of the above aspects, the one or more miRNA bio markers are miRNA-409 and/or miRNA-122, or a fragment or variant thereof. More preferably, the one or more miRNA biomarkers comprise miRNA-409 and miRNA122. With respect to these particular embodiments, the hepatitis-associated disease, disorder or condition preferably is or comprises cirrhosis.

In alternative embodiments of the methods of the above aspects, the one or more miRNA biomarkers are selected from the group consisting of miRNA-122, miRNA-486 and miRNA-142, or a fragment or variant thereof. More preferably, the one or more miRNA biomarkers are or comprise miRNA-142, miRNA-122 and miRNA-486. With respect to these alternative embodiments, the hepatitis-associated disease, disorder or condition preferably is or comprises a hepatic cancer, such as hepatocellular carcinoma.

With respect to the above aspects, the hepatitis-associated disease, disorder or condition suitably is selected from the group consisting of hepatic fibrosis, cirrhosis, a hepatic cancer and any combination thereof. Preferably, the hepatic cancer is or comprises hepatocellular carcinoma.

In particular embodiments of the method of the aforementioned aspects, the biological sample comprises tissue, blood, serum, plasma, urine or cerebrospinal fluid. Typically, the miRNAs described herein are obtainable from a non-cellular source. Accordingly, the biological sample suitably is, comprises, or is obtained from a noncellular source. To this end, the biological sample may be serum, plasma, or cerebrospinal fluid, although without limitation thereto.

Suitably, the subject’s hepatitis referred to herein is caused, at least in part, by a viral infection. Preferably, the subject’s hepatitis is caused, at least in part, by a hepatitis C virus.

Suitably, the subject referred to herein is a mammal. Preferably, the subject is a human.

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-6As used herein, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further elements, components, integers or steps but may include one or more unstated further elements, components, integers or steps.

It will be appreciated that the indefinite articles “a” and “an” are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, “a” cell includes one cell, one or more cells and a plurality of cells.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Association between circulating miRNA levels and severity of hepatic fibrosis in HCV. (A) miRNA-122-5p, (B) miRNA-486-5p, (C) miRNA-151a-5p, (D) miRNA-142-3p and (E) miRNA-409-3p. Lines represent mean expression levels (± SEM). * p < 0.05, ** p < 0.01, *** p < 0.001. FO-2 = mild disease; F4 = cirrhosis; HCC = hepatocellular carcinoma.

Figure 2. ROC curve for individual serum miRNAs to predict cirrhosis or HCC in chronic hepatitis C. (A). ROC curve performance of miRNA-122-5p with AUC 0.69 (CI 0.51 to 0.86, p = 0.023) and miRNA-409-3p with AUC 0.74 (CI 0.58 to 0.90, p = 0.004) to predict cirrhosis when compared to mild to moderate fibrosis. (B) ROC curve performance of selected miRNAs to predict HCC when compared to cirrhosis as follows: miRNA-486-5p AUC 0.78 (CI 0.63 to 0.93, p < 0.001); miRNA-I51a-5p AUC 0.70 (CI 0.54 to 0.87, p = 0.014); miRNA-122-5p AUC 0.85 (CI 0.72 to 0.97, p < 0.001); miRNA142-3p AUC 0.60 (CI 0.42 to 0.78, p = 0.14). HCC = hepatocellular carcinoma; CI = confidence interval.

Figure 3. ROC curve of serum miRNA panels to predict cirrhosis or HCC in chronic Hepatitis C. (A) miRNA panel (miRNA-122-5p and miRNA-409-3p) performance to predict cirrhosis, compared to mild disease, with AUC 0.80 (CI 0.66 to 0.95, p < 0.001). (B) miRNA panel to predict HCC, compared to cirrhosis, using miRNA-122-5p, miRNA486-5p and miRNA-142-3p with AUC 0.94 (CI 0.87 to 1.00, p < 0.001). FO-2 = mild disease; F4 = cirrhosis; HCC = hepatocellular carcinoma.

Figure 4. K-fold (5-fold) cross validation ROC curve of serum miRNA panels to predict cirrhosis or HCC in chronic Hepatitis C. (A) miRNA panel (miRNA-122-5p and miRNA-409-3p) cross validation performance to predict cirrhosis, compared to mild disease, with AUC 0.79 (Cl 0.65 to 0.93) (Fig. 2A). (B) miRNA panel to predict HCC,

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-7 compared to cirrhosis, using miRNA-122-5p, miRNA-486-5p and miRNA-142-3p with AUC 0.91 (CI 0.81 to 1.0) (Fig. 2B). FO-2 = mild disease; F4 = cirrhosis; HCC = hepatocellular carcinoma.

Figure 5. ROC curves to predict cirrhosis or HCC using FIB-4 or APRI alone or in combination with circulating miRNA panels in chronic Hepatitis C. (A) Performance of cirrhosis miRNA panel (miRNA-409-3p and miRNA-122-5p, as in Figure 3A), and FIB-4 (AUC 0.87, p < 0.001, using a cut-off of >1.45 to indicate advanced fibrosis) alone and in combination with an AUC 0.89 (p < 0.001). (B) Performance of cirrhosis miRNA panel (as in Figure 3A) and APRI (AUC of 0.84, p < 0.001, with a cut-off of >1.0 indicating advanced fibrosis) alone, and in combination with an AUC of 0.87 (p < 0.001).

(C) Performance of HCC miRNA panel (including miRNA-122-5p, miRNA-486-5p and miRNA-142-3p, see Figure 3B), and AFP (AUC 0.64, p = 0.065) using a cut-off of >20 to diagnose HCC) alone, and in combination (AUC 0.94, p < 0.001). HCC = hepatocellular carcinoma; Fib-4 = Fibrosis-4 biomarker panel; APRI = aspartate aminotransferase to platelet ratio; AFP = alpha-fetoprotein.

miRNA-, miRNA-,, miRNA-, miRNA-and miRNAFigure 6. The nucleic acid sequence and observed sequence variation of miRNA-122. Figure 7. The nucleic acid sequence and observed sequence variation of miRNA-486. Figure 8. The nucleic acid sequence and observed sequence variation of miRNA-151a. Figure 9. The nucleic acid sequence and observed sequence variation of miRNA-142. Figure 10. The nucleic acid sequence and observed sequence variation of miRNA-409. Figure 11. The nucleic acid sequence and observed sequence variation of miRNA-22.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 = nucleic acid sequence miRNA-122 of Figure 6 (mature sequence of miRNA-122)

SEQ ID NO: 2 = nucleic acid sequence miRNA-486 of Figure 7 (mature sequence of miRNA-486)

SEQ ID NO: 3 = nucleic acid sequence miRNA-151a of Figure 8 (mature sequence of miRNA-15 la)

SEQ ID NO: 4 = nucleic acid sequence miRNA-142 of Figure 9 (mature sequence of miRNA-142)

SEQ ID NO: 5 = nucleic acid sequence miRNA-409 of Figure 10 (mature sequence of miRNA-409)

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-8SEQ ID NO: 6 = nucleic acid sequence miRNA-22 of Figure 11 (mature sequence of miRNA-22)

DETAILED DESCRIPTION

The present invention is predicated, at least in part, on the surprising discovery that a number of micro RNAs (miRNAs or miRs), in isolation or together as a panel, may provide a serum-based prognostic and/or diagnostic biomarker for disease progression in patients with hepatitis. In this regard, the inventors demonstrate herein that particular miRNAs may be used to detect the onset and/or progression of liver disease in patients with hepatitis, and more particularly, patients infected with a hepatitis C virus, as well as monitor disease progression in patients with hepatitis, particularly in regards to the development of hepatic fibrosis, cirrhosis and/or hepatocellular carcinoma. Accordingly, the miRNAs disclosed herein may also have utility in methods of treating and/or evaluating treatment efficacy in patients with hepatitis.

In an aspect, the invention provides a method of determining whether or not a subject with hepatitis has a hepatitis-associated disease, disorder or condition, including:

As generally used herein, the term “hepatitis” refers to inflammation of the liver. A variety of different chemical, viral, and biological agents can induce hepatitis. In particular embodiments, the subject’s hepatitis is or comprises inflammation of the liver caused, at least in part, by a viral infection (z.e., a viral hepatitis), particularly a hepatotrophic viral infection. To this end, the terms “viral hepatitis” and “hepatitis virus infection” as used herein refer to infection of any hepatitis virus known in the art, including, but not limited to, those caused by a hepatitis A virus (HAV), a hepatitis B virus (HBV), a hepatitis C virus (HCV), a hepatitis D virus (HDV), a hepatitis E virus (HEV), a hepatitis F virus (HFV), a hepatitis G virus (HGV), or a cryptogenic hepatitis viruses.

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-9In one preferred embodiment, the subject’s hepatitis is a viral hepatitis caused, at least in part, by a hepatitis C virus (HCV). As used herein the term “hepatitis C virus” or “HCV” refers to any of the strains and isolates of hepatitis C that have been identified or are identifiable according to classifications and methods known in the art. It will further be appreciated that these terms are intended to encompass all currently known strains, types and subtypes of HCV as wells those discovered and classified as HCV in the future.

In particular preferred embodiments, the hepatitis-associated disease, disorder or condition is a HCV-associated disease, disorder or condition, such as HCV-associated hepatic fibrosis, HCV-associated cirrhosis and HCV-associated hepatocellular carcinoma. As used herein, the term “HCV-associated disease, disorder or condition” refers to those hepatitis-associated diseases, disorders or conditions, such as hepatic fibrosis, cirrhosis and hepatocellular carcinoma that are secondary to infection with a hepatitis C virus (HCV).

As used herein, the terms “hepatitis-associated disease, disorder or condition” or “hepatitis disease condition” refer to diseases, conditions, disorders and syndromes having a symptom or pathology involving or affecting the liver, which can be caused, at least in part, by hepatitis. Nonlimiting examples of such diseases, disorders or conditions include, but are not limited to, hepatitis, hepatic fibrosis, cirrhosis and hepatic cancer, such as hepatocellular carcinoma (HCC).

As used herein, “hepatic fibrosis” (inclusive of the term “liver fibrosis”) refers to morphological and/or histological indicators of fibrosis involving the liver that can occur in the context of hepatitis. Such indicators may include, but are not limited to, the deposition of fibrotic tissue or collagen in the liver, and activation of the fibrogenesis cascade as evidenced by increased MDA-adducts, stellate cell activation, and enhanced expression of c-myb and collagen al (I) mRNA in stellate cells.

The term “cirrhosis” refers to an advanced stage of hepatic fibrosis, defined by chronic progressive hepatic fibrosis characterized by replacement of liver tissue by fibrotic scar tissue and regenerative nodules (lumps that occur as a result of a process in which damaged tissue is regenerated), which leads to progressive loss of liver function. For the purposes of the present invention, “cirrhosis” is considered to be a type of liver fibrosis, and is included within the meaning of the term “hepatic fibrosis” used herein. Cirrhosis is most commonly caused by fatty liver disease, alcoholism and hepatitis B and C viral infections, but has many other possible causes. Cirrhosis has historically been thought to be generally irreversible once it occurs, and historical treatment focused on

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- 10preventing progression and complications. In the advanced stages of cirrhosis, the only option generally available to patients is a liver transplant. The present invention may be used to limit, inhibit or reduce the likelihood of cirrhosis occurring in a subject with hepatitis or treat cirrhosis of the liver.

The terms “hepatic cancer” and “liver cancer” as used herein refer to cancers or neoplasms found in liver, which may originate from liver (i.e., primary hepatic cancer), inclusive of hepatocellular carcinoma, cholangiocarcinoma, liver angiosarcoma and hepatoblastoma, or from other organs that have spread to the liver as a result of metastasis (i.e., secondary hepatic cancer). Preferably, the hepatic cancer is a primary hepatic cancer.

As used herein, the terms “hepatocellular carcinoma” and “hepatoma” denote a cancer derived from hepatic cells or hepatocytes, and encompasses primary hepatocellular carcinoma that begins within the liver and metastatic hepatocellular carcinoma that has spread to the liver from other sites. The exact cause of hepatocellular carcinoma can be unclear, but most hepatocellular carcinoma patients have liver cirrhosis, with hepatocellular carcinoma frequently occurring in patients with liver cirrhosis and/or chronic active hepatitis B and/or C viral infections, or are hepatitis B and/or C viral carriers.

As generally used herein, the term “microRNA” (inclusive of “miRNA”, “miR” and “pRNA”) typically refers to single-stranded RNA molecules of around 17-23 nucleotides in length, which have roles in regulating gene expression. miRNAs are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein (i.e. they are non-coding RNAs). The genes encoding miRNAs are much longer than the processed mature miRNA molecule; miRNAs are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, 60- to 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is typically integrated into the RISC. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC, on the basis of the stability of the 5'

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- 11 end. The remaining strand, known as the anti-guide or passenger strand, can be subsequently degraded as a RISC substrate.

When two mature microRNAs originate from opposite arms of the same premiRNA they are denoted with a -3p or -5p suffix and this distinction may also be made with “s” (sense) and “as” (antisense). It will be appreciated that the miRNAs described herein (e.g., miRNA-122, miRNA-486, miRNA-151a, miRNA-142, miRNA-409 and miRNA-22) may broadly be both or either of the -3p or -5p designat ions unless expressly stated otherwise. The mature miRNA, however, found from one arm of the hairpin is usually much more abundant than that miRNA found from the complementary arm, as alluded to above. Accordingly, in particular embodiments, the miRNAs described herein may be preferably afforded the -3p or the -5p designation (e.g., miRNA-122-3p, miRNA122-5p, miRNA-486-3p, miRNA-486-5p, miRNA-151a-3p, miRNA-15la-5p, miRNA142-3p, miRNA-142-5p, miRNA-409-3p, miRNA-409-5p, miRNA-22-3p and miRNA22-5p).

The term “determining” includes any form of measurement, and includes determining if an element is present or not. As used herein, the terms “determining”, “measuring”, “evaluating”, “assessing” and “assaying” are used interchangeably and include quantitative and qualitative determinations. Determining may be relative or absolute. “Determining the presence of includes determining the amount of something present (e.g., an miRNA and/or protein/enzyme biomarker), and/or determining whether it is present or absent.

As would be understood by the skilled person, the expression level of the one or more miRNA biomarkers is deemed to be “altered” or “modulated” when the amount or expression level of the respective miRNA biomarker is increased or up regulated or decreased or down regulated, as defined herein.

In one embodiment, the hepatitis-associated disease, disorder or condition is detected in the subject with hepatitis if the one or more miRNA biomarkers (e.g., miRNA122, miRNA-151a, miRNA-142) are at a reduced level, down regulated or absent in the biological sample. With respect to such embodiments, the hepatitis-associated disease, disorder or condition preferably is or comprises hepatocellular carcinoma.

In an alternative embodiment, the hepatitis-associated disease, disorder or condition is detected in the subject with hepatitis if the one or more miRNA biomarkers (e.g., miRNA-486, miRNA-409, miRNA-22) are at an increased level, up regulated or present

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- 12in the biological sample. Referring to these embodiments, the hepatitis-associated disease, disorder or condition preferably is or comprises cirrhosis and/or hepatocellular carcinoma.

It will be appreciated that in a further embodiment, the hepatitis-associated disease, disorder or condition is detected in the subject with hepatitis if a first subset of the one or more miRNA biomarkers are at a reduced level, down regulated or absent in the biological sample and a second subset of the one or more miRNA biomarkers are at an increased level, up regulated or present in the biological sample.

By “enhanced”, “increased” or “up regulated” as used herein to describe the expression level of miRNA biomarkers, additional miRNA biomarkers, and further biomarkers hereinafter described refers to the increase in and/or amount or level of the one or more miRNA biomarkers, additional miRNA biomarkers, and further biomarkers, including variants, in a biological sample when compared to a control or reference sample or a further biological sample from a subject. The expression level of a biomarker may be relative or absolute. In some embodiments, the expression level of the one or more miRNA biomarkers, additional miRNA biomarkers, and further biomarkers is increased if its level of expression is more than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400% or at least about 500% higher than the level of expression of the corresponding miRNA biomarkers, additional miRNA biomarkers, and further biomarkers in a control sample or further biological sample from a subject.

The terms, “reduced” and “down regulated”, as used herein to describe the expression level of miRNA biomarkers, additional miRNA biomarkers, and further biomarkers, refer to a reduction in and/or amount or level of one or more miRNA biomarkers, additional miRNA biomarkers, and further biomarkers, including variants, in a biological sample when compared to a control or reference sample or further biological sample from a subject. The expression level of a biomarker may be relative or absolute. In some embodiments, the expression level of one or more miRNA biomarkers, additional miRNA bio markers, and further bio markers is reduced or down regulated if its level of expression is more than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or 0.0001% of the level of expression of the corresponding miRNA biomarkers, additional miRNA biomarkers, and further biomarkers in a control sample or further biological sample from a subject.

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- 13 The term control sample' typically refers to a biological sample from a healthy or non-diseased individual not having hepatitis. In one embodiment, the control sample may be from a subject known to be free of a hepatic disease, disorder or condition. Alternatively, the control sample may be from a subject with hepatitis known to be free of a hepatitis-associated disease, disorder or condition. The control sample may be a pooled, average or an individual sample. An internal control is a marker from the same biological sample being tested.

Suitably, the expression level of any combination of the miRNA biomarkers, miRNA-122, miRNA-486, miRNA-151a, miRNA-142, miRNA-409 and miRNA-22, in a biological sample may be determined, including for example: miRNA-122 and miRNA409; miRNA-122 and miRNA-486; miRNA-486 and miRNA-142; miRNA-409, miRNA22 and miRNA-142; miRNA-122, miRNA-486 and miRNA-142; miRNA-151a, miRNA22, miRNA-142 and miRNA-409; miRNA-122 and miRNA-142; miRNA-409 and miRNA-151a; or miRNA-151a, miRNA-122 and miRNA-409.

In one embodiment the method of determining whether or not a subject with hepatitis has a hepatitis-associated disease, disorder or condition includes the step of determining the expression level of one or more further or additional miRNA biomarkers in addition to miRNA-122, miRNA-486, miRNA-I51a, miRNA-142, miRNA-409 and miRNA-22, and as are known in the art, in a biological sample from the subject and wherein a hepatitis-associated disease, disorder or condition may be detected if at least one of the miRNA biomarkers, inclusive of additional miRNA biomarkers, is at an altered or modulated level or expression in the biological sample when compared to a control or reference sample. By way of example, an increase in the level or up regulation in the biological sample of a further miRNA biomarker, may indicate the presence of a hepatitisassociated disease, disorder or condition in the subject with hepatitis. Further, a decrease in the level, down regulation or absence in the biological sample of a further miRNA biomarker, may indicate the presence of hepatitis-associated disease, disorder or condition in the subject with hepatitis.

It will be appreciated that the methods of the invention include methods of determining the expression level of the one or more miRNA biomarkers alone or in combination with one or more further biomarkers, such as protein and/or nucleic acid biomarkers, which have been identified as being diagnostic for hepatitis-associated diseases, disorders or conditions in subjects with hepatitis. In certain embodiments, the expression level of one or more protein and/or nucleic acid biomarkers may also be

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- 14determined. In this regard, the one or more further biomarkers may include, for example, alpha-fetoprotein, a platelet count, a Child-Turcotte-Pugh (CTP) score, a Model for EndStage Liver Disease (MELD) score, an International Normalized Ratio (INR) score, aspartate aminotransferase/platelet ratio index (APRI), Fibrosis-4 index, an hepatic enzyme (e.g., aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), alkaline phosphatase (ALP)), ultrasound and modified ultrasound modalities, such as transient elastography and 2-D shear wave elastography (SWE), albeit without limitation thereto.

The expression level of the miRNA biomarkers, further miRNA biomarkers and further biomarkers, such as protein and/or nucleic acid biomarkers, may be determined by any method known in the art. By way of example, the expression level of miRNA biomarkers may be determined by hybridization-based techniques (e.g., Northern blots, in situ hybridization, RT-PCR, and microarrays), amplification-based techniques (e.g., realtime quantitative PCR; gold nanoparticle-initiated silver enhancement) and cloning-based techniques (e.g., miRAGE).

Suitably, when the expression level of the one or more miRNA biomarkers, one or more further miRNA biomarkers and/or one or more further biomarkers are determined, they can be derived from the same or different samples. For example, the expression level of the one or more miRNA biomarkers can be determined in a blood-derived sample (e.g., serum) and the expression level of a protein biomarker can be determined in a tissue sample.

In particular embodiments, the biological sample comprises tissue, blood, serum, plasma, urine or cerebrospinal fluid. Typically, the miRNAs described herein are obtainable from a non-cellular source. Accordingly, the biological sample suitably is, comprises, or is obtained from a non-cellular source. To this end, the biological sample may be serum, plasma, or cerebrospinal fluid, although without limitation thereto.

In some embodiments, the method of determining whether or not a subject with hepatitis has a hepatitis-associated disease, disorder or condition may be performed in “high throughput” diagnostic tests or procedures such as performed by commercial pathology laboratories or in hospitals. Furthermore or alternatively, the method of the present aspect may be used to confirm a diagnosis of a hepatitis-associated disease, disorder or condition, including hepatic fibrosis, cirrhosis and hepatocellular carcinoma, such as that initially detected by a different or alternative diagnostic test or procedure.

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- 15 It would be further appreciated, that such methods of determining miRNA expression in the biological sample from a hepatitis patient may have potential utility in characterising disease progression and/or severity of a given patient. Additionally, such methods may be used for selecting patients for particular treatments.

In one embodiment, the method determines whether the subject with hepatitis has hepatic fibrosis, cirrhosis and/or hepatocellular carcinoma.

In another aspect, the invention provides a method of determining the prognosis of a subject with hepatitis, including the steps of:

Suitably, if the expression level of said one or more miRNA biomarkers, is altered or modulated in the biological sample, the prognosis may be negative or positive.

The terms “prognosis” and “prognostic” are used herein to include making a prognosis, which can provide for predicting a clinical outcome (with or without medical treatment), selecting an appropriate course of treatment (or whether treatment would be effective) and/or monitoring a current treatment and potentially changing the treatment. This may be at least partly based on determining expression levels of one or more miRNA biomarkers by the methods of the invention, which may be in combination with determining the expression levels of further biomarkers hereinbefore described, such as protein and/or other nucleic acid bio markers. A prognosis may also include a prediction, forecast or anticipation of any lasting or permanent physical or psychological effects of the hepatitis-associated disease, disorder or condition suffered by the subject with hepatitis after the hepatitis-associated disease, disorder or condition has been successfully treated or otherwise resolved. Furthermore, prognosis may include one or more of determining a degree of hepatic fibrosis or occurrence, the presence of cirrhosis and/or hepatocellular carcinoma, therapeutic responsiveness, implementing appropriate treatment regimes and determining the probability, likelihood or potential for treatment of the

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- 16hepatitis-associated disease, disorder or condition with therapy. It would be appreciated that a positive prognosis typically refers to a beneficial clinical outcome or outlook, such as the hepatitis subject being considered not to have a hepatitis-associated disease, disorder or condition, whereas a negative prognosis typically refers to a negative clinical outcome or outlook, such as the presence (or recurrence) of a hepatitis-associated disease, disorder or condition in the subject with hepatitis and/or progression thereof.

As described herein, the expression levels of miRNA-486, miRNA-409 and miRNA-22 are typically increased or upregulated, whilst the expression levels of miRNA122, miRNA-151a and miRNA-142 are typically decreased or down regulated, upon the initial development of a hepatitis-associated disease, disorder or condition in a subject with hepatitis. The expression levels of these miRNAs, however, may be shown to be altered or modulated (z.e., they may increase and/or decrease) with disease progression, such as with the development of cirrhosis and/or hepatocellular carcinoma.

In one embodiment, the method further includes the step of measuring an expression level of one or more additional miRNA biomarkers. In this regard, an increase or up regulation of the level in the biological sample of an additional miRNA biomarker, may indicate a negative prognosis for the subject with hepatitis. Further, a decrease or down regulation of the level in the biological sample of an additional miRNA biomarker, may indicate a negative prognosis for the subject.

In one particular embodiment, the one or more miRNA biomarkers are or comprise miRNA-409 and/or miRNA-122, or a fragment or variant thereof. More preferably, the one or more miRNA biomarkers are or comprise miRNA-409 and miRNA-122, or a fragment or variant thereof. With respect to the present embodiments, the hepatitis-associated disease, disorder or condition preferably is or comprises cirrhosis.

In an alternative embodiment, the one or more miRNA biomarkers are or comprise miRNA-142, miRNA-122 and/or miRNA-486, or a fragment or variant thereof. More preferably, the one or more miRNA biomarkers are or comprise miRNA-142, miRNA122 and miRNA-486, or a fragment or variant thereof. Regarding the present embodiments, the hepatitis-associated disease, disorder or condition suitably is or comprises hepatocellular carcinoma.

In certain embodiments, the expression level of one or more further biomarkers, such as protein and/or nucleic acid biomarkers, as hereinbefore described, may also be determined.

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- 17In particular embodiments, the biological sample comprises tissue, blood, serum, plasma, urine or cerebrospinal fluid. Preferably, the biological sample comprises serum, plasma, or cerebrospinal fluid, although without limitation thereto.

In one embodiment, the method further comprises determining a stage and/or grade for the subject’s hepatitis-associated disease, disorder or condition, such as hepatic fibrosis, based on the expression level of the one or more miRNA biomarkers.

In one embodiment, the expression level of the one or more miRNA biomarkers is determined before, during and/or after treatment.

In one embodiment, the prognosis is used, at least in part, to develop a treatment strategy for the subject.

In one embodiment, the prognosis is used, at least in part, to determine disease progression or recurrence in the subject.

In one embodiment, the method of this aspect further includes the step of determining suitability of the subject for treatment based, at least in part, on the prognosis.

Suitably, the hepatitis-associated disease, disorder or condition suitably is or comprises hepatic fibrosis, cirrhosis and/or hepatocellular carcinoma.

In a further aspect, the invention provides a method of treating a hepatitisassociated disease, disorder or condition in a subject with hepatitis including;

As used herein, “treating”, “treat” or “treatment” refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of the hepatitis-associated disease, disorder or condition after such a disease, disorder or condition and/or its symptoms have at least started to develop. As used herein, “preventing”, “prevent” or “prevention” refers to therapeutic intervention, course of action or protocol initiated prior to the onset of the hepatitis-associated disease, disorder

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- 18or condition and/or a thereof so as to prevent, inhibit or delay development or progression of the hepatitis-associated disease, disorder or condition or the symptom.

In one embodiment, the method further comprises measuring an expression level of one or more additional miRNA biomarkers.

In an alternative embodiment, the one or more miRNA bio markers are or comprise miRNA-142, miRNA-122 and/or miRNA-486, or a fragment or variant thereof. More preferably, the one or more miRNA bio markers are or comprise miRNA-142, miRNA122 and miRNA-486, or a fragment or variant thereof. Regarding the present embodiments, the hepatitis-associated disease, disorder or condition suitably is or comprises hepatocellular carcinoma.

In certain embodiments, the expression level of one or more further biomarkers, such as protein and/or nucleic acid biomarkers, as herein before described, may also be determined.

In particular embodiments, the biological sample comprises tissue, blood, serum, plasma, urine or cerebrospinal fluid. Preferably, the biological sample comprises serum, plasma, or cerebrospinal fluid, although without limitation thereto.

In one embodiment, the method further comprises selecting a treatment for the hepatitis-associated disease, disorder or condition based on the expression level of the miRNA biomarkers.

It will be appreciated that the method of treating the hepatitis-associated disease, disorder or condition may include administration of one or more therapeutic agents that facilitate treatment or prevention thereof. By way of example only, these include antifibrotic agents such as hepatocyte growth factor (HGF), pirfenidone, inhibitors of the Renin-Angiotensin System (RAS) (e.g., angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors), endothelin receptor antagonists, anti-inflammatory agents, such as NSAIDs and corticosteroids, ursodeoxycholic acid (UDCA), liver transplantation, surgical resection, radiofrequency ablation (RFA), anti-cancer agents, although without limitation thereto.

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- 19With respect to hepatocellular carcinoma, suitable cancer treatments may include drug therapy, chemotherapy, antibody, nucleic acid and other biomolecular therapies, radiation therapy, surgery, nutritional therapy, relaxation or meditational therapy and other natural or holistic therapies, although without limitation thereto.

Generally, drugs, biomolecules (e.g antibodies, inhibitory nucleic acids such as siRNA) or chemotherapeutic agents are referred to herein as “anti-cancer agents”. In certain embodiments, the anticancer agent is or comprises an ALK inhibitor (e.g., TAE684), an Aurora kinase inhibitor (e.g., Alisertib, AMG-900, BI-847325, GSK1070916A, ilorasertib, MK-8745, danusertib), a BCR-ABL inhibitor (e.g., Nilotinib, Dasatinib, Ponatinib), a HSP90 inhibitor (e.g., Tanespimycin (17-AAG), PF0429113, AUY922, Luminespib, ganetespib, Debio-0932), an EGFR inhibitor (e.g., Afatinib, Erlotinib, Lapatinib, cetuximab), a PARP inhibitor (e.g., ABT-888, AZD-2281), retinoic acid (e.g., all-trans retinoic acid or ATRA), a Bcl2 inhibitor (e.g., ABT-263), a gluconeogenesis inhibitor (e.g., metformin), a p38 MAPK inhibitor (e.g., B1RB0796, LY2228820), a MEK1/2 inhibitor (e.g., trametinib, cobimetinib, binimetinib, selumetinib, pimasertib, refametinib, TAK-733), a mTOR inhibitor (e.g., BEZ235, JW-7-25-1), a PI3K inhibitor (e.g., Idelalisib, buparlisib/apelisib, copanlisib, GSK-2636771, pictilisib, AMG319, AZD-8186), an IGF1R inhibitor (e.g., BMS-754807, dalotuzumab, ganitumab, linsitinib), a PLCy inhibitor (e.g., U73122), a JNK inhibitor (e.g., SP600125), a ΡΑΚΙ inhibitor (e.g., IPA3), a SYK inhibitor (e.g., BAY613606), a HDAC inhibitor (e.g., Vorinostat), an FGFR inhibitor (e.g., Dovitinib), a X1AP inhibitor (e.g., Embelin), a PLK1 inhibitor (e.g., Volasertib, P-937), an ERK5 inhibitor (e.g., XMD8-92), a MPS1/TTK inhibitor (e.g., BAY-1161909), a multi-kinase inhibitor (e.g., sorafenib, trametinib, dabrafenib, vemurafenib, crizotinib, sunitinib, axitinib, ponatinib, ruxolitinib, vandetanib, cabozantinib, afatinib, ibrutinib and regorafenib) and any combination thereof.

In yet another aspect, the invention provides a method of evaluating treatment efficacy of a hepatitis-associated disease, disorder or condition in a subject with hepatitis including:

determining an expression level of one or more miRNA biomarkers in a biological sample from the subject before, during and/or after treatment, wherein the one or more miRNA biomarkers are selected from the group consisting of miRNA-122, miRNA-486,

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-20miRNA-151a, miRNA-142, miRNA-409 and miRNA-22, or a fragment or variant thereof; and determining whether or not the treatment is efficacious according to whether said expression level of one or more miRNA biomarkers is altered or modulated in the subject's biological sample.

In this regard, an increase or up regulation and/or a decrease or down regulation of the level in the biological sample of the one of more miRNA biomarkers in the subject with hepatitis, may indicate disease treatment or regression in the subject, and as such the treatment is efficacious, or alternatively it may indicate disease progression, such as hepatic fibrosis, cirrhosis and/or hepatocellular carcinoma, and that the treatment is inefficacious.

In one embodiment, the method further comprises measuring the expression level of one or more additional miRNA biomarkers. In this regard, a decrease or down regulation of the level in the biological sample of an additional biomarker, may indicate the treatment is efficacious or conversely it may indicate that the treatment is inefficacious.

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-21 It would be appreciated by the skilled artisan that the methods of present invention also include within their scope fragments and variants of the miRNA biomarkers described herein.

In this regard, a miRNA ‘fragment” includes a nucleic acid sequence that constitutes less than 100%, but at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of said miRNA sequence. In particular embodiments, a miRNA fragment may comprise, for example, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 contiguous nucleotides of said miRNA biomarkers.

As used herein, “variant” refers to those miRNAs described herein that have one or more nucleic acids deleted, added or substituted by different nucleotides. In particular embodiments, a miRNA variant may comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotide deletions, additions and/or substitutions. In this regard, it is well known in the art that miRNAs can demonstrate some degree of variation from their respective reference miRNA sequence. By way of example and referring to FIGS. 6-11, it will be appreciated that the nucleotide sequences of SEQ ID NOS: 1-6 are “canonical” or preferred sequences and that there may be significant variability with respect to the length of the nucleotide sequence of each miRNA bio marker, in particular.

In some embodiments, a diagnostic, prognostic and/or treatment expression level of the one or more miRNA biomarkers described herein is correlated to the hepatitisassociated disease, disorder or condition by merely its presence or absence. In other embodiments, a threshold level of a diagnostic, prognostic and or treatment expression level of the one or more miRNA biomarkers can be established, and the level of the one or more miRNA biomarkers in a subject’s biological sample can simply be compared to the threshold level.

In some embodiments, multiple time points prior to, during and/or after treatment of a subject with the hepatitis-associated disease, disorder or condition may be selected to determine the expression level of the one or more miRNA biomarkers, with or without other biomarkers, to determine a diagnosis, prognosis or treatment efficacy. For example, the expression level of the one or more miRNA biomarkers with or without additional specific miRNA biomarkers, protein biomarkers and/or nucleic acid biomarkers can be determined at an initial time point and then again at one, two, three or more time points.

Suitably, the time points may be selected throughout a treatment cycle or over a desired time period. Over a desired time period, for example, the time points may be prior to treatment, mid way through treatment and/or after treatment has been completed.

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-22Suitably, an altered or modulated expression level, such as a decrease or reduction (e.g., miRNA-486, miRNA-409 and miRNA-22) and/or an increase or upregulation (e.g., miRNA-122, miRNA-151a and miRNA-142) in the expression level, of the one or more miRNA biomarkers and/or the one or more additional miRNA biomarkers, such as, may be utilised by the methods of the invention from the first to second and/or third time points may provide a good or positive prognosis for a subject with the hepatitis-associated disease, disorder or condition. Alternatively, an altered or modulated expression level, such as a decrease or reduction (e.g., miRNA-122, miRNA-151a and miRNA-142) and/or an increase or upregulation (e.g., miRNA-486, miRNA-409 and miRNA-22) in the expression level, of the one or more miRNA biomarkers and/or the one or more additional miRNA biomarkers, utilised by the methods of the invention from the first to second and/or third time points may provide a poor prognosis for a subject with the hepatitisassociated disease, disorder or condition.

As would be appreciated by the skilled artisan, the alteration or modulation in expression level of the one or more miRNA biomarkers and/or the one or more additional miRNA biomarkers may also be related to the severity, stage, recurrence or progression of the hepatitis-associated disease, disorder or condition and/or the efficacy of the treatment. By way of example, differences in the expression levels of the one or more miRNA biomarkers and/or the one or more additional miRNA biomarkers may be used to delineate different stages of progression of hepatic fibrosis, cirrhosis, and/or hepatocellular carcinoma.

In one embodiment, biological samples may be sourced and/or collected from a subject at diagnosis and then prior to each cycle of treatment. Suitably, there may be any number of treatment cycles, depending on the subject and the nature and/or stage of the hepatitis-associated disease, disorder or condition, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 and/or 20 cycles. The treatment cycles may be close together, spread out over a period of time and/or intense cycles at defined time points over a period of time, or any combination of the above.

In further embodiments, samples may be taken both during treatment and/or after treatment has been completed. Suitably, samples may be sourced from a subject at any time point after treatment has been completed, examples of which include 1, 2, 3, 4, 5, 10, 15, 20, 25 and/or 30 days post treatment, 1, 2 and/or 3 weeks post treatment and/or 1, 3, 6 and/or 9 months post treatment and/or 1, 2, 3, 4, 5, 10, 15, 20 and/or 30 years post treatment. The treatment may be completed once the subject is considered treated or after

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-23 at least one or more treatment cycles, depending on the subject and the hepatitisassociated disease, disorder or condition.

In one embodiment, an increase, a decrease and/or no change in the expression level of the one or more miRNA biomarkers and/or the one or more additional miRNA biomarkers in a second, third, fourth and/or fifth etc., biological sample from a subject collected either after treatment or during treatment as compared to the expression level in a first earlier sample, may indicate progression of the hepatitis-associated disease, disorder or condition or failure of the treatment.

In one embodiment, an increase and/or decrease in the expression level of the one or more miRNA biomarkers and/or the one or more additional miRNA biomarkers in a second, third, fourth and/or fifth etc., biological sample from a subject collected either after treatment or during treatment as compared to the expression level in a first earlier sample, may indicate that the treatment is efficacious.

The methods described herein may be suitable for any biological sample from a subject. In particular embodiments, the biological sample is or comprises tissue, blood, serum, plasma, urine or cerebrospinal fluid. Typically, the miRNAs are obtainable from a non-cellular source. Accordingly, the biological sample is, comprises, or is obtained from a non-cellular source. The biological sample may be serum, plasma, or cerebrospinal fluid, although without limitation thereto.

Additionally, the one or more biomarkers described herein may be used in in vitro assays of response by subject-derived test cells or tissue to predict an in vivo response to the treatment. As such, the one or more biomarkers described herein, may be used, for example, to predict and monitor how particular subjects with the hepatitis-associated disease, disorder or condition respond to therapeutic intervention with the treatment.

It would be understood that the methods described herein may be applicable to any mammal. In particular embodiments, the term “mammal” includes but is not limited to humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). Preferably, the subject is a human.

So that the present invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting examples.

EXAMPLE I

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-24New biomarkers, such as microRNAs (miRNA), are being actively investigated for their diagnostic potential. The roles of miRNAs in viral hepatitis, hepatic fibrosis and HCC have been discussed extensively elsewhere.¹⁶'¹⁸ MiRNAs are small non-coding RNAs involved in regulation of gene expression at a post-transcriptional level. The stability of miRNAs in the circulation, as well as both healthy and diseased tissue, highlights their potential to function as biomarkers.¹⁹ Preliminary studies of miRNAs in CHC-related HCC have identified some promising candidates but these results require further validation.^{20, 21} To improve diagnostic accuracy, the development of panels of miRNAs associated with different disease severities and HCC are now being explored.

In the present Example, using a liver-specific miRNA microarray and qRT-PCR validation, we sought to identify differentially-expressed serum miRNAs in patients with differing severities of HCV-related chronic liver disease, including those with HCC. We also sought to evaluate the diagnostic accuracy of the most promising candidates as miRNA panels that could serve as non-invasive diagnostic tests.

MATERIALS AND METHODS

Patient recruitment and characteristics

This study was approved by the Human Research and Ethics Committees of the Royal Brisbane and Women’s Hospital and QIMR Berghofer Medical Research Institute. Informed consent was obtained from all study participants. Sixty HCV-posifive patients were retrospectively subdivided into 3 cohorts based on expert clinical assessment, transient elastography (FibroScan™; Echosens, Paris, France) and medical imaging as follows: mild disease without advanced fibrosis (FO-2; n=20); cirrhosis (F4; n=20); and cirrhosis with HCC (HCC; n=20). The diagnosis of HCC was established according to currently-accepted professional guidelines.² Patient demographics, biochemistry and relevant medical information were obtained from patients’ medical records. All hepatocellular carcinomas were stratified using the Barcelona clinic liver cancer (BCLC) criteria, as summarised by Forner et al.^2j Serum samples were obtained through Pathology Queensland collection centres. All samples were processed within 8 hours of blood draw and stored at -80°C. For each patient APRI¹¹, FIB-4¹²’¹³, Child-Turcotte-Pugh (CTP)²’’²⁴and model for end-stage liver disease (MELD)²⁵'²⁷ scores were calculated at the time of serum sample collection.

RNA extractions and reverse transcription.

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-25 For the screening phase, RNA was extracted from 200 μΐ of serum using the miRNeasy Serum/Plasma Kit (Qiagen; Hilden, Germany). Isolations were performed according to manufacturer’s instructions with minor modifications to optimise results. Following the phenol-chloro form phase separation, the interphase and organic layers were rehydrated with RNAse free water equal to the volume removed during the aqueous phase collection.¹ Thus, a second aqueous phase was obtained maximizing RNA recovery. Both aqueous phases were combined into one RNeasy MiniElute spin column and RNA isolation was continued.¹ Following RNA elution, a second elution with identical settings was performed using 14μ 1 RNAse free water in a new collection tube. All samples were assessed for ethanol contamination and RNA yield by Nanodrop^1M (Thermo Fisher Scientific; Waltham, MA, USA). Serum extracted RNA was reverse transcribed using the miScript II RT Kit (Qiagen) following the manufacturer’s instructions and RNA input recommendation for miScript miRNA PCR Arrays with 250 ng RNA. cDNA products were diluted 10-fold prior to microRNA PCR array run. During the validation phase, serum RNA was extracted using the Plasma/Serum RNA Purification Mini Kit (Norgen Biotek Corp; Thorold, Ontario, Canada) following the manufacturer’s instructions with minor modifications. Prior to the transferring the sample onto the Micro Spin Column the mixture was passed through a syringe with a 26G needle to break down lysis debris and prevent column clogging. RNA was eluted in 15 μΐ of RNAse free water and assessed on a Nanodrop^IM. Extracted RNA was reversed transcribed using the miRCURY LNA^rMuniversal RT microRNA PCR Kit (Exiqon, Vedbaek, Denmark) following manufacturer’s instructions. A fixed RNA volume input of 4 pL was used (at the manufacturer’s suggestion) due to limitations in quantifying circulatory microRNAs. cDNA products were diluted 1:40 prior to qRT-PCR use.

MiRNA PCR Array and qRT-PCR.

During the screening phase, a miRNA PCR Array (Human Liver miFinder miScript miRNA PCR Array MIHS-3116ZG; Qiagen) was used that simultaneously measures the expression of 372 liver-related miRNAs in all 60 samples. Eight leading miRNA candidates, based on differential expression, were selected for independent validation in the entire cohort by qRT-PCR (using the miRCURY UNA™ miRNA kit and commercially available primers (Exiqon; see Table 3). The miRNA PCR Array and miRCURY miRNA qRT-PCR were performed according to the manufacturers’

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-26instructions using the Lightcylcer480 (Roche; Basel, Switzerland) and HEPATITISX384 (Bio-Rad; Hercules, California, United States) thermal cycler, respectively.

Data normalisation and statistical analysis.

MiRNA PCR Array and miRCURY qRT-PCR data were reported as crossing points (CP) and quantification cycles (Cq), respectively. Following the screening phase, the array data for 372 miRNAs for the cohort (n=60) were normalised using quantile normalisation and then analysed for differentially-expressed miRNAs using pairwise comparison of the groups of interest with the modified t-test (limma package, R). MiRNA candidates were selected based on statistical significance with P-value < 0.05. Housekeepers for further validation studies were selected based on distance metrics ranking (limma package, R). Validation qRT-PCR data were analysed using the 2^Acrmethod and expression values were normalized to the expression levels of the let-7i-5p and miRNA-23a-3p. Significant differential expression was tested using one-way ANOVA with Tukey’s post hoc pairwise comparison. Results were presented as mean with standard error of the mean and p < 0.05 was used to define statistical significance. Data normalisation and statistical analysis were performed using GraphPad Prism 7 (GraphPad Software Inc; California, USA) and the limma package in R (version 3.3.3).²⁹

Panel design and k-fold cross validation.

Stepwise logistic regression using forward selection and backward elimination was used to derive microRNA panels for i) mild disease (FO-2) vs cirrhosis (F4), and ii) cirrhosis (F4) vs HCC. Model selection was based on the Akaike Information Criteria, the likelihood ratio test based on the change of the residual deviance, and by assessing the stability of the coefficient estimates.

The resulting model equation is as follows:

Function A: F4 vs FO-2

InQ^) = 4.03 - (0.89 x ACT miR-122-5p) - (0.83 x ACT miR-409-3p)

Function B: HCC vs F4 lⁿ(^·) = 3.59 + (1.78 X ACT miR-122-5p) - (1.99 x ACT miR-486-5p) + (2.32 x ACT miR-142-3p)

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-27To obtain the p_t use the following formula:

p. zz --------------------------¹ 1 + exp (—function)

Pi is the probability of the i^th subject having the outcome of cirrhosis (function A) or HCC (function B). The probability, ρ_έ, will range from 0 to 1 and using the Youden’s index a panel specific cut-point was determined. Using function A, a value above the cutpoint of 0.44 will indicate cirrhosis, while in function B a value above the cut-point of 0.65 indicates HCC.

K-fold cross-validation (5-fold) was used to assess the performance of the selected microRNA panels. Univariate and multivariate analyses were used to perform receiver operating characteristic (ROC) curve analysis resulting in area under the curve (AUC) with 95% confidence interval (CI), positive predictive value (PPV), negative predictive value (NPV), sensitivity, specificity and accuracy. All calculations were performed using the cvAUC and the ROCRpackage on R version (version 3.3.3).²

Methodology References

1. Burgos KL, Javaherian A, Bomprezzi R, et al. Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing. RNA 2013;!9:71222.

2. Team RC. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2013: ISBN 3-900051-07-0, 2014.

RESULTS

Patient characteristics.

The patient demographics and clinical characteristics of the CHC patient cohort (n=60) are summarised in Table 1. All patients with mild disease (FO-2) and cirrhosis (F4) were followed in the hepatology clinic for a mean of 16.7 (SD 7.2) and 21.2 (SD 6.7) months from sample collection, respectively, and showed no evidence of clinical decompensation. Cirrhotic patients with HCC were followed until a treatment endpoint (being surgical resection, locoregional therapy, transplant listing, or referral for best supportive care). The mean follow-up for cirrhotic patients with HCC was 3.3 (SD 3.0)

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-28months from sample collection to treatment endpoint. There were no significant differences amongst the groups according to HCV genotypes or treatment histories (p > 0.05).

Paired serum samples for miRNA testing and routine laboratory assessment of disease status were obtained with 95% of patients’ samples taken within 4 months of each other. As expected, traditional markers of disease severity (e.g. ALT, AST, sodium and platelet levels) were significantly different amongst the three study groups (p = 0.0002, p = 0.0005, p = 0.002 and p < 0.0001, respectively), whereas creatinine levels were similar. Post hoc analysis revealed significant differences in platelet counts when comparing FO-2 vs. F4 (p = 0.0014) and FO-2 vs. F4 (p = 0.0014) and FO-2 vs. HCC (p < 0.001). Markers of hepatic function (i.e. bilirubin, albumin and INR) were significantly worse in patients with cirrhosis and HCC compared with those with cirrhosis alone (all p < 0.001). Interestingly, the tumour marker AFP was not significantly different between the F4 and HCC groups.

MiRNA expression screening phase.

The microarray data provided evidence of significant differential expression of circulating miRNAs comparing patients with (i) mild disease and cirrhosis (FO-2 vs. F4), and (ii) cirrhosis and cirrhosis with HCC (F4 vs. HCC). The most differentially-expressed miRNAs, based on a greater than 2-fold change in expression were selected, leaving 27 miRNA candidates in total. Of these miRNA candidates all miRNAs with statistical significance (p < 0.05), were selected for further validation. This analysis identified 8 candidate miRNAs: miRNA-19b-3p, miRNA-151a-5p, miRNA-122-5p, miRNA-409-3p, miRNA-22-3p, miRNA-142-3p, miRNA-1281 and miRNA-486-5p.

MiRNA expression validation phase.

Differential expression levels of the selected miRNA candidates in all 60 serum samples are presented in Figure 1. Although the PCR array study showed significant differences for miRNA-22-3p and miRNA-1281, qRT-PCR validation did not show any significant differences with miRNA-22-3p only showed a trend towards increased expression in cirrhotic patients compared with mild disease (p = 0.0554; see Table 2). Comparing serum miRNA levels in patients with mild disease (FO-2) versus cirrhotic patients (F4), miRNA-409-3p was found to be significantly upregulated in cirrhosis (p = 0.023). Serum levels of miRNA-122-5p and miRNA-15la-5p were significantly

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-29decreased in HCC (p = 0.0002 and p = 0.039, respectively), whereas expression of miRNA-486-5p was significantly increased in HCC patients (p = 0.001), compared with F4 patients. Expression of miRNA-142-3p was also significantly decreased in HCC compared to FO-2 alone (p = 0.039).

Discriminating cirrhosis and HCC using miRNAs.

Receiver operating characteristics (ROC) curve analysis was performed to assess the potential of individual candidate miRNAs to discriminate cirrhosis (compared with mild disease) and HCC (compared with cirrhosis alone). Using miRNA-409-3p and miRNA-122-5p to distinguish cirrhosis from mild disease resulted in an AUC of 0.74 (p = 0.004) and AUC of 0.69 (p = 0.023), respectively. Several miRNA candidates distinguished HCC patients from those with cirrhosis alone with AUCs of 0.85 (p < 0.001) for miRNA-122-5p, 0.78 (p < 0.001) for miRNA-486-5p, 0.70 (p = 0.014) for miRNA-151a-5p and 0.60 (p = 0.14) for miRNA-142-3p (Figure 2).

Stepwise logistic regression, using forward selection and backward elimination, was used to select a miRNA panel that could distinguish cirrhosis from mild disease (F4 vs. FO-2). A panel comprising miRNA-122-5p and miRNA-409-3p provided an AUC of 0.80 (p <0.001) (Figure 3A). Applying logistic regression to distinguish patients with HCC compared with cirrhosis alone resulted in a miRNA panel of miRNA-122-5p, miRNA-486-5p and miRNA-142-3p with an AUC of 0.94 (p <0.001) (Figure 3B).

Cross validation of miRNA panels.

To further assess the miRNA panel performance, k-fold cross validation (5-fold) was performed. Using 5-fold cross-validation, the cirrhosis panel of miRNA-409-3p and miRNA-122-5p resulted in an AUC of 0.79 (95% CI 0.65 - 0.93), with a sensitivity of 85.3%, specificity of 65.3%, NPV of 76%, PPV of 75%, and overall accuracy of 77.5% (Figure 4A). These calculations used a panel specific cut-point of 0.44. Using 5-fold cross-validation, the performance of the HCC miRNA panel, combining miRNA-122-5p, miRNA-486-5p and miRNA-142-3p, resulted in an AUC of 0.91 (95% Cl of 0.81 - 1.0), sensitivity of 81.3%, specificity of 86.7%, NPV of 80%, PPV of 91%, and overall accuracy of 85%. (Figure 4B).The panel specific cut-point was 0.65.

Comparative performance of APRI and FIB-4 to discriminate cirrhosis.

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-30The abilities of the biochemical indices APRI and FIB-4 to discriminate cirrhosis (F4) from mild disease (FO-2) in our cohort were also assessed. Applying an APR! cut-off of 1.0 to indicate severe fibrosis resulted in an AUC = 0.84 (p < 0.001) with a sensitivity of 80%, specificity of 75%, NPV of 78.9%, PPV of 76.2%, and overall accuracy of 77.5%. Combining the proposed miRNA panel (of miRNA-409-3p and miRNA-122-5p) with APRI improved the diagnostic performance with an AUC of 0.87 (p < 0.001) (Figure 5B).

Using the recommended FIB-4 cut-off of 1.45 to exclude cirrhosis resulted in an AUC = 0.87 (p < 0.001, sensitivity of 95%, specificity of 50%, NPV of 92.3%, PPV of 70.4%, and overall accuracy of 77.5%) in this cohort. Combining the proposed cirrhosis miRNA panel (of miRNA-409-3p and miRNA-122-5p) with FIB-4 produced an AUC of 0.89 (p < 0.001), which was better than either FIB-4 or the cirrhosis miRNA panel alone (Figure 5A).

Performance of AFP in identifying HCC.

In this cohort, the traditional tumour marker AFP was a poor discriminator of HCC with an AUC of 0.64 (p = 0.065). Using an AFP cut-off of > 20 pg/L to identify HCC produced a sensitivity of 25% and specificity of 90% (NPV of 54.5%, PPV of 71.4%, and overall accuracy of 57.5%). In comparison, the proposed HCC miRNA panel (of miRNA-122-5p, miRNA-486-5p and miRNA-142-3p) produced an AUC of 0.94 (p < 0.001). Combining AFP with the proposed miRNA panel did not improve its performance (AUC of 0.94; see Figure 5C).

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-31 2018202716 18 Apr 2018

Table 1. Demographic and clinical characteristics of patients across all study groups

	Mild fibrosis (n=20)	Cirrhosis (n=20)	HCC (n=20)	Pvalue
Gender¹				0.0453
Male (n/%)	10 (50%)	17 (85%)	15 (75%)
Female (n/%)	10 (50%)	3(15%)	5 (25%)
Age (years)²	49.6 (SD 10.1)	53.8 (SD 7.4)	58.3 (SD 5.3)	0.0041
BMI²	26.3 (SD 6.0)	27.2 (SD 5.1)	27.1 (SD5.2)	ns
HCV genotype (n)¹				ns
1	1 (5%)	-	2(10%)
1a	8 (40%)	8 (40%)	6 (30%)
1b	3(15%)	4 (20%)	2 (10%)
Mixed 1/3	1 (5%)	-	-
2	1 (5%)	-	-
3	6 (30%)	6 (30%)	9 (45%)
4	NA	1 (5%)	NA
Prior SVR	NA	1 (5%)	1 (5%)
HCV treatment history¹				ns
Treatment experienced	6 (30%)	6 (30%)	6 (30%)
Treatment naive	14 (70%)	14 (70%)	10 (50%)
On DAA treatment	-	-	4 (20%)
Baseline routine laboratory tests
Sodium (mmol/L)²	138.5 (SD2.7)	137.6 (SD 1.4)	135.9 (SD2.4)	0.0021
creatinine (umol/L)²	68.2 (SD 14.6)	71.3 (SD 13.4)	68.2 (SD 16.9)	ns
ALT (U/L)³	83.6 (SD 61.0)	209.6 (SD 178.7)	64.6 (SD 39.6)	0.0002
AST (U/L)³	57.5 (SD 44.2)	156.0 (SD 115.7)	85.7 (SD 57.9)	0.0005
PLT (x10^A9/L)²	226.3 (SD 57.6)	164.4 (SD32.8)	124.7 (SD63.2)	<0.0001
bilirubin (umol/L)⁴	NA	14.5 (SD 4.5)	25.3 (SD 10.0)	<0.0001
albumin (g/L)⁴	NA	40.4 (SD 2.8)	33.7 (SD 6.1)	<0.0001
Proximity of sample collection to
baseline routine laboratory tests
(months	1.47 (SD6.40)	0.22 (SD 1.45)	0.01 (SD 0.76)	NA
Other laboratory tests
I NR⁴	NA	1.1 (SD0.1)	1.2 (SD0.1)	<0.0001
AFP (ug/L)^b	NA	11.8 (SD 16.1)	375.2 (SD 1578.6)	ns
Sample collection to AFP (months)	NA	0.03 (SD 2.48)	0.48 (SD 1.55)	NA
Clinical follow up (months)	16.7 (SD 7.2)	21.2 (SD 6.7)	3.3 (SD3.0)	NA
Disease severity assessment
Transient elastography LSM (kPa)⁴	6.1 (SD 1.2)	19.2 (SD 8.5)	NA	<0.0001
Fib-4⁴	1.53 (SD 1.10)	3.53 (SD 1.63)	NA	<0.0001
APRI⁴	0.90 (SD0.91)	2.90 (SD2.15)	NA	0.0005
CTP				NA
A	NA	20 (100%)	8 (40%)
B	NA	-	11 (55%)
C	NA	-	1 (5%)
MELD	NA	7.31 (SD 0.67)	10.26 (SD2.38)	NA
BCLC (n/%)				NA
0	NA	NA	2(10%)
A	NA	NA	8 (40%)
B	NA	NA	8 (40%)
C	NA	NA	1 (5%)
D	NA	NA	1 (5%)

Following normality test, P-values were calculated using the 'Chi-Square test, ²ANOVA, ³Kruskal-Wallis test, unpaired t-test and ⁵Mann-Whitney test with significance defined as a p-value < 0.05. (Abbreviations: HCC, cirrhosis with hepatocellular carcinoma; BMI, 5 body mass index; HCV, Hepatitis C virus; SVR, sustained virological response; DAA,

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2018202716 18 Apr 2018 direct acting antiviral drug; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PLT, platelet; INR, international normalized ratio; AFP, alphafetoprotein; LSM, liver stiffness measurement; Fib-4; Fibrosis-4 biomarker panel; APRI, aspartate aminotransferase to platelet ratio; CTP, Child-Turcotte Pugh Score; MELD, 5 model for end-stage liver disease score; BCLC, Barcelona clinic liver cancer; SD, standard deviation; ns, not significant; NA, not applicable).

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-33 2018202716 18 Apr 2018

Table 2. Associations of circulating miRNA expression with severity of disease in chronic Hepatitis C: Validation of selected miRNA expression levels using real-time PCR.

miRNA	Overall P-value (ANOVA)	P-value
FO-2 vs.F4	FO-2 vs HCC	F4 vs. HCC
miRNA-122-5p	0.0003	ns	ns	0.0002
miRNA-486-5p	0.0003	ns	0.001	0.001
miRNA-151a-5p	0.025	ns	0.057	0.039
miRNA-409-3p	0.03	0.023	ns	ns
miRNA-19b-3p	0.015	ns	0.0145	ns
miRNA- 142-3p	0.047	ns	0.039	ns

P-values were calculated using 2^AC1 normalisation method and ANOVA with Tukey’s post hoc test with significance designated by a P-value < 0.05. (Abbreviations: FO-2, mild disease; F4, cirrhosis; HCC, cirrhosis with hepatocellular carcinoma; ns, not significant).

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-342018202716 18 Apr 2018

Table 3: microRNA primer target sequences

N 0.	microRNA name	Description	Target Sequence	Product No. Exiqon
1	miR-19b-3p	hsa-miR-19b-3p LNA™ PCR primer set, UniRT	UGUGCAAAUCCAUGCA AAACUGA	204450
2	miR- 151a- 5p	hsa-miR-151a-5p LNA™ PCR primer set, UniRT	UCGAGGAGCUCACAGU CUAGU	204007
3	miR-122-5p	hsa-miR-122-5p LNA™ PCR primer set, UniRT	UGGAGUGUGACAAUGG UGUUUG	205664
4	miR-409-3p	hsa-miR-409-3p LNA™ PCR primer set, UniRT	GAAUGUUGCUCGGUGA ACCCCU	204358
5	miR-22-3p	hsa-miR-22-3p LNA™ PCR primer set, UniRT	AAGCUGCCAGUUGAAG AACUGU	204606
6	miR-142-3p	hsa-miR-142-3p LNA™ PCR primer set, UniRT	UGUAGUGUUUCCUACU UUAUGGA	204291
7	miR-1281	hsa-miR-1281 LNA™ PCR primer set, UniRT	UCGCCUCCUCCUCUCCC	2118520
8	miR-486-5p	hsa-miR-486-5p LNA™ PCR primer set, UniRT	UCCUGUACUGAGCUGCC CCGAG	204001
9	let-7i-5p	hsa-let-7i-5p LNA™ PCR primer set, UniRT	UGAGGUAGUAGUUUGU GCUGUU	204394
10	miR-23a-3p	hsa-miR-23a-3p LNA™ PCR primer set, UniRT	AUCACAUUGCCAGGGA UUUCC	204772

microRNA target sequences for commercially available miRCURY primers (Exiqon,

Vedbaek, Denmark) used for qRT-PCR validation study

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-35DISCUSSION

Despite the availability of new highly efficacious treatments, HCV remains a huge public health burden with millions of CHC patients worldwide affected by cirrhosis.²Distinguishing cirrhosis from mild disease is clinically important as these patients warrant ongoing surveillance for complications, such as HCC, even if the virus is successfully cleared. The invasive nature of the gold standard liver biopsy and the absence of highly accurate non-invasive biomarkers remain important challenges in routine patient care.²²MiRNAs and their potential utility as biomarkers have been studied in many diseases, including HCC.¹⁶ And several candidate miRNAs have been identified but an optimal diagnostic panel has not been validated.

In the current study, we used serum samples from 60 CHC patients across the spectrum of disease, including cirrhosis and HCC, to identify potential miRNA biomarkers. During the discovery phase we screened for differentially expressed miRNAs using a miRNA PCR-based microarray. Validation, using independent qRT-PCR, was then undertaken to identify the most promising candidate miRNAs that could be combined into biomarker panels with improved diagnostic accuracy over currently accepted clinical tools.

The circulating level of miRNA-409-3p was significantly increased in cirrhotic patients (F4) compared with those with only mild disease (FO-2). To-date there is little in the literature describing the role of miRNA-409-3p in fibrosis progression in liver disease, although increased plasma expression levels correlated with markers of liver injury (gamma-glutamyl-transferase, alkaline phosphatase) in a murine neoplastic model of hereditary tyrosinemia type l.^J° Interestingly, studies using in vitro models of prostate cancer have reported increased miRNA-409-3p levels in association with epithelial to mesenchymal transition, facilitating tumour growth and metastasis by targeting Ras suppressor protein l.^j1’ ^j2 In contrast, reduced expression of miRNA-409-3p has been described in other cancers, such as breast cancer^3j and colorectal cancer,³⁴’³⁵ suggesting a tissue-specific role for miRNA-409-3p. These reported associations with tumour development and evidence from the current study of increased circulating levels in patients with cirrhosis suggest a potential role for miRNA-409-3p as a mediator of progressive liver injury in CHC.

MiRNA-122-5p is the most abundant miRNA expressed in liver.³⁶ Increased levels of circulating miRNA-122-5p have been shown in liver diseases of different aetiologies such as Hepatitis B Virus (HBV) infection³⁷, alcoholic liver disease³⁷,

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-36paediatric hepatitis liver disease³⁸ and HCV³⁹. Furthermore, in CHC, circulating miRNA122-5p levels have been shown to correlate with serum ALT and AST levels and tissue necrosis and inflammation. ^21-39

In the current study, we demonstrated that circulating serum levels of miRNA122-5p were significantly decreased in CHC patients with HCC compared with cirrhosis alone. A previous study reported no significant differences in serum miR122-5p levels in HCC patients⁴⁰ but included patients with multiple disease aetiologies, suggesting miR122-5p may have be specific for CHC-related HCC. Increased levels of miRNA-1225p have been described in CHC-related HCC tissue samples,⁴¹’⁴² while HBV-related HCC is associated with decreased levels of tissue expression.^41,4j Poorer overall survival has been described in HCC patients with lower levels of circulating miRNA-122-5p⁴⁰ and lower expression of miRNA-122-5p in HCC tissue has been shown to correlate with poor prognosis and metastasis.⁴¹ These data clearly suggest the clinical importance of miRNA122-5p in HCC development and patient outcomes. While the precise mechanisms involved require further investigation, the current study provides consistent evidence that serum levels of miRNA-122-5p may be useful as a non-invasive diagnostic biomarker of HCC.

In the current study, circulating miRNA-486-5p was the only miRNA with an increased peripheral expression in CHC patients with HCC compared with those with cirrhosis alone. Higher circulating miRNA-486-5p has been previously described in patients with HCC compared with healthy controls, although the aetiology of HCC and cirrhosis status was not reported.⁴⁴ As a potential diagnostic test, the current study’s finding of differential expression in cirrhotic patients with and without HCC is clinically significant. Interestingly, higher serum expression of miRNA-486-5p has been shown to be associated with longer recurrence-free survival in HCC patients.⁴⁵ Decreased tissue expression of miRNA-486-5p in HCC, compared with adjacent non-tumorous tissue, has been reported in multiple aetiologies, including CHC-related HCC.⁴⁶'⁴⁸ Expression levels also appear to correlate with earlier HCC recurrence following resection.⁴⁶ Downstream targets of miRNA-486-5p include Phosphoinositide-3-Kinase Regulatory Subunit 1 (PIK3R1) and N1MA Related Kinase 2 (ΝΈΚ2), which have been shown to play important roles in HCC proliferation, migration and invasion.^{46, 47} Furthermore, in vitro studies have reported that miRNA-486 downregulates Insulin Like Growth Factor 1 Receptor (IGF-IR), Mechanistic Target Of Rapamycin Kinase (mTOR) and Signal Transducer And Activator Of Transcription 3 (STAT3),⁴⁸ which are part of the IGF

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-37signalling pathway , which in turn has been linked with HCC pathogenesis.⁴⁹ In other cancers, such as non-small-cell-lung cancer, increased circulating expression levels and reciprocal reduced tissue expression levels of miRNA-486-5p have been demonstrated.⁵⁰’ ⁵¹ Lower tissue expression of miRNA-486-5p has also been demonstrated in advanced lung cancer and metastatic disease.⁵¹ In this study, the pro-tumorigenic Rho GTPase Activating Protein 5 (ARHGAP5) was identified as a potential target of miRNA-486-5p, supporting its potential role as a tumour suppressor. In contrast, the oncogenic potential of miRNA-486-5p has been reported with increased tissue expression in cervical and prostate cancer through the targeting of Phosphatase and Tensin Homolog (PTEN).⁵²’^5jInterestingly, miRNA-486-5p is located on chromosome 8ql 1.21, an area commonly deleted in HCC, which possibly accounts for reduced tissue expression in HCC and supports its potential role as a tumour suppressor.⁵⁴

The current study demonstrated significantly decreased expression of circulating miRNA-151a-5p in cirrhotic patients with HCC compared with cirrhosis alone. While another study has described increased plasma expression of miRNA-151a-5p in viral hepatitis HCC, this was compared to healthy controls and non-viral hepatitis-related HCC patients.⁵⁵ Interestingly, increased tissue expression levels of miRNA-151a-5p have been described in HCC tissue, compared with adjacent non-tumorous tissue,⁵⁴’⁵⁶ and miRNA151a-5p is located on chromosome 8q24.3, which is an area that has been described as frequently amplified in HCC.⁵⁴’⁵⁶ High expression levels of miRNA-151a-5p have also been correlated with intrahepatic metastasis, cell migration and invasion through the targeting of Rho GDP Dissociation Inhibitor Alpha (RhoGDIA),⁵⁴ while FAK (the host gene of miRNA-15 la-5p) is usually co-expressed with miRNA-15la-5p and it has been suggested that the two function synergistically to enhance HCC cell motility.⁵⁴ Other cancer tissues, such as gastric cancer⁵⁷ and papillary thyroid carcinoma,⁵⁸ also show increased expression of miR-151a-5p, while studies of urothelial carcinoma did not find differential expression,³⁹ suggesting that the role of miRNA-151a-5p in cancer could be tissue specific. Overall, however, there is growing evidence that miRNA-151a-5p is associated with cancer progression and that it may have a pro-oncogenic function, although further studies of the downstream targets of miRNA-151a-5p are needed.

When comparing miRNA expression levels in patients with mild disease and cirrhotic patients with HCC, we found significantly decreased expression of circulating miRNA-142-3p in the HCC group. Several previous studies have reported miRNA-1423p to be downregulated in HCC tissue⁶⁰ ⁶² and functional studies have demonstrated a

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-38potential role for miR-142-3p in the inhibition of migration, proliferation and invasion in vitro by targeting Rac Family Small GTPase 1 (RAC1) and TGF-β.⁶¹’⁶^ One study has reported increased levels of circulating and tissue miRNA-142-3p⁶⁴ in HCC, although discrepancies may be accounted for by methodological differences.

We performed ROC curve analysis using stepwise logistic regression to identify a miRNA panel for the diagnosis of cirrhosis compared with mild disease in CHC. The resulting cirrhosis miRNA panel (comprising niiRNA-122-5p and miRNA-409-3p) resulted in an AUC of 0.80 (Figure 5). When combined with APRI, its performance improved with an AUC of 0.87 and the combination of our miRNA panel with FIB-4 resulted in an AUC of 0.89. Although these results need to be validated in a larger study cohort, the combination of this miRNA panel and other tools highlights the clinical potential of miRNAs to non-invasively identify patients with cirrhosis with very good diagnostic accuracy.

We also performed ROC curve analysis using our proposed HCC miRNA panel and demonstrated the ability of miRNA-122-5p, miRNA-486-5p and miRNA-142-3p to discriminate HCC from cirrhosis with an AUC of 0.94. This result was substantially better than the diagnostic performance of AFP to identify HCC (AUC of 0.64) highlighting the potential advantages of our miRNA panel over the only HCC tumour biomarker used in clinical practice. Combining AFP with our miRNA panel did not improve the diagnostic performance (Figure 5C). Further prospective validation of our miRNA panel (comprising miRNA-122-5p, miRNA-486-5p and miRNA-142-3p) in a larger study cohort is obviously required but the diagnostic potential of this panel is certainly promising.

This study contributes to the considerable interest over recent years in miRNAs as biomarkers of chronic liver disease. Two recent meta-analyses⁶^⁶⁶ have demonstrated that miRNAs have great potential as non-invasive biomarkers, especially in the diagnosis of cirrhosis and HCC, and highlighted a number of candidate miRNAs. These studies found, however, that the quality of studies in this area is variable, significant heterogeneity exists, and results are often contradictory. Meta-analysis identified that high frequency miRNAs (e.g. miRNA- 122-5p in HCC) may be most specific and combinations of noninvasive biomarkers and/or panels of miRNAs appear to be more accurate in distinguishing disease. These findings are consistent w ith those of the current study, as is the conclusion that further high-quality work is needed to validate promising preliminary results. In comparison to recent studies, strengths of the current study include: the use of controls with clinically-relevant disease (i.e. mild disease vs. cirrhosis, cirrhosis vs. HCC)

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-392018202716 18 Apr 2018 instead of healthy controls; the measurement of circulating rather than tissue-based miRNA levels (which are much less accessible to clinicians); and the use of serum expression levels (rather than plasma, which generally delivers poorer results).

CONCLUSION

This study has identified the differential expression of several miRNAs in the circulation across the spectrum of disease severity due to CHC. We have identified circulating miRNAs, such as miRNA-409-3p and miRNA-486-5p that are appealing as possible biomarkers of the development of cirrhosis and HCC, respectively. Combining 10 the expression levels of different miRNAs, we have developed diagnostic panels that could potentially be used to stratify CHC and diagnose early HCC. Further validation of our miRNA panels is required before translation into clinical practice but excellent preliminary results support the potential use of miRNAs as non-invasive biomarkers to improve the clinical assessment of patients with cirrhosis and HCC. The current study 15 also identifies a number of candidate miRNAs that could be investigated in functional studies to identify downstream targets and elucidate their roles in disease development and new therapeutic strategies.

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Claims

1. A method of determining whether or not a subject with hepatitis has a hepatitisassociated disease, disorder or condition, including the step of:

determining an expression level of one or more miRNA biomarkers in a biological sample from the subject, wherein the one or more miRNA biomarkers are selected from the group consisting of miRNA-122, miRNA-486, miRNA-151a, miRNA-142, miRNA409 and miRNA-22, or a fragment or variant thereof, and wherein the hepatitis-associated disease, disorder or condition is detected if said expression level of one or more miRNA biomarkers, is altered or modulated in the biological sample.

2. A method of determining the prognosis of a subject with hepatitis, including the step of:

3. The method of Claim 2, further including the step of determining whether or not the subject with hepatitis has a hepatitis-associated disease, disorder or condition based on the expression level of the one or more miRNA biomarkers.

4. The method of Claim 2 or Claim 3, wherein if the expression level of said one or more miRNA biomarkers is altered or modulated in the biological sample, the prognosis may be negative or positive.

5. The method of Claim 4, wherein the prognosis is used, at least in part, to determine whether the subject with hepatitis would benefit from treatment of the hepatitisassociated disease, disorder or condition.

6. The method of Claims 4 or 5, wherein the prognosis is used, at least in part, to develop a treatment strategy for the subject.

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7. The method of any one of Claims 2 to 6, wherein the prognosis is used, at least in part, to determine progression of the hepatitis-associated disease, disorder or condition in the subject with hepatitis.

8. The method of any one of Claims 2 to 7, further including the step of determining suitability of the subject for treatment based, at least in part, on the prognosis.

9. The method of any one Claims 1 to 8 further including the step of determining a disease stage and/or grade for the hepatitis-associated disease, disorder or condition based on the expression level of the one or more miRNA biomarkers.

10. The method of any one of Claims 1 to 9, wherein the expression level of the one or more miRNA biomarkers is determined before, during and/or after treatment.

11. A method of treating a hepatitis-associated disease, disorder or condition in a subject with hepatitis including;

12. A method of evaluating treatment efficacy of a hepatitis-associated disease, disorder or condition in a subject with hepatitis including:

determining an expression level of one or more miRNA biomarkers in a biological sample from the subject before, during and/or after treatment, wherein the one or more miRNA bio markers are selected from the group consisting of miRNA-122, miRNA-486, miRNA-151a, miRNA-142, miRNA-409 and miRNA-22, or a fragment or variant thereof; and

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-482018202716 18 Apr 2018 determining whether or not the treatment is efficacious according to whether said expression level of one or more miRNA biomarkers is altered or modulated in the subject's biological sample.

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13. The method of Claims 11 or 12, further including the step of selecting a treatment for the hepatitis-associated disease, disorder or condition based on the expression level of the miRNA biomarkers.

14. The method of any one of the preceding claims, further including the step of 10 measuring an expression level of one or more additional miRNA biomarkers, or a fragment or variant thereof.

15. The method of any one of the preceding claims, further includes the step of determining an expression level of a further biomarker.

16. The method of Claim 15, wherein the further biomarker is selected from the group consisting of an hepatic enzyme, alpha-fetoprotein, a platelet count, a Child-TurcottePugh (CTP) score, a Model for End-Stage Liver Disease (MELD) score, an International Normalized Ratio (INR) score, fibrosis-4 (FIB-4) index, aspartate aminotransferase

20 (AST) to platelet ratio index (APRI), transient elastography, 2-D shear wave elastography and any combination thereof.

17. The method of Claim 16, wherein the further biomarker is or comprises FIB-4 index and/or APRI.

18. The method of any one of the preceding claims, wherein the hepatitis-associated disease, disorder or condition is selected from the group consisting of hepatic fibrosis, cirrhosis, a hepatic cancer and any combination thereof.

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19. The method of Claim 18, wherein the hepatic cancer is or comprises hepatocellular carcinoma.

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20. The method of any one of the preceding claims, wherein the one or more miRNA biomarkers are or comprise miRNA-122 and/or miRNA-409, or a fragment or variant thereof.

21. The method of Claim 20, wherein the hepatitis-associated disease, disorder or condition is or comprises cirrhosis.

22. The method of any one of Claims 1 to 19, wherein the one or more miRNA biomarkers are or comprise miRNA-122, miRNA-486, and/or miRNA-142, or a fragment or variant thereof.

23. The method of Claim 22, wherein the hepatitis-associated disease, disorder or condition is or comprises hepatocellular carcinoma.

24. The method of any one of the preceding claims, wherein the subject’s hepatitis is caused, at least in part, by a viral infection.

25. The method of Claim 24, wherein the subject’s hepatitis is caused, at least in part, by a hepatitis C virus.

26. The method of any one of the preceding claims, wherein the biological sample is or comprises tissue, blood, serum, plasma, urine or cerebrospinal fluid.

27. The method of Claim 26, wherein the biological sample is or comprises serum, plasma, or cerebrospinal fluid.

28. The method of any one of the preceding claims, wherein the subject is a mammal.

29. The method of Claim 28, wherein the subject is a human.