Granted Patent
B2
US 12,601,747 · App. 18/502,243 · Granted Apr 14, 2026
Biomarker combinations to simultaneously evaluate non-alcoholic steatohepatitis and hepatic fibrosis status
Inventors:
Isabelle Batxelli-Molina (Basel, CH); Florian Salipante (Basel, CH); Roger P. Walker (Benicia, CA); John Flanagan (Walnut Creek, CA); Jeannette Fareh-Michiel (Basel, CH)
Assignee:
BIO-RAD LABORATORIES, INC.
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Abstract
The invention provides a method of diagnosing Non-Alcoholic Steatohepatitis (NASH) and/or the hepatic fibrosis status of a subject, especially a subject afflicted with Non-alcoholic fatty liver disease (NAFLD) or NASH, based on the level of only three or more particular biomarkers. The invention further provides a kit suitable for performing said method and the use of said method and methods of treating patients diagnosed in accordance with the disclosed methods.
Claims (154)
1 . A kit for diagnosing NASH and/or the hepatic fibrosis status of a subject comprising:
(i) antibodies for determining, in a blood, plasma, serum, urine or cerebrospinal fluid sample obtained from a subject, levels of at least three biomarkers, said at least three biomarkers being a pro-inflammatory cytokine, a chemokine and a glycosaminoglycan contributing to cell-adhesion and tissue modeling, wherein:
(a) the pro-inflammatory cytokine is IL-8;
(b) the chemokine is CXCL10;
(c) the glycosaminoglycan contributing to cell-adhesion and tissue modeling is hyaluronic acid; and
(ii) instructions, said instructions disclosing:
contacting said sample with antibodies that bind to said at least three biomarkers and determining the levels of said IL-8, CXCL10 and HA in said sample;
calculating a value for f 1(NASH) and/or f 1(fibrosis) , wherein
f
1
(
NASH
)
=
exp
(
y
1
(
N
A
S
H
)
)
1
+
exp
(
y
1
(
N
A
S
H
)
)
wherein
f
1
(
NASH
)
:
y
1
(
NASH
)
=
(
-
2.3083
+
0.4079
×
IL
8
-
0.0129
×
HA
+
0.0019
×
CXCL
10
)
and
f
1
(
fibrosis
)
=
exp
(
y
1
(
fibrosis
)
)
1
+
exp
(
y
1
(
fibrosis
)
)
where
y
1
(
fibrosis
)
=
(
-
7.0457
+
1.0511
×
IL
8
+
0.067
×
HA
+
0.0004
×
CXCL
10
)
and
when f 1(NASH) >0.4997, the subject is diagnosed with NASH and when f 1(NASH) ≤0.4997, the subject is diagnosed with non-alcoholic fatty liver (NAFL) and when f 1(fibrosis) >0.1953, the subject is diagnosed with advanced liver fibrosis and when f 1(fibrosis) ≤0.1953, the subject is diagnosed with early-moderate liver fibrosis.
2 . The kit of claim 1 , further comprising antibodies for determining, in a sample obtained from the subject, the levels of one or more of ghrelin, haptoglobin and pre-HP2.
3 . The kit of claim 1 , wherein:
(i) the kit comprises labeled antibodies that bind the biomarkers and the measuring the levels of biomarkers is done by ELISA, Luminex multiplex technology, microarray or point of care testing; and/or
(ii) the sample is a plasma or serum blood.
4 . The kit according to claim 3 , wherein the antibodies are fluorescently labeled.
5 . The kit according to claim 2 , wherein the antibodies are fluorescently labeled.
Priority Claims (1)
EP 17290092
· Jul 19, 2017
· regional
Continuity (2)
Continuation
16632404
References Cited (137)
US 6631330B1
· Poynard
· 2003
[cited by applicant]
US 7138229B2
· Hu et al.
· 2006
[cited by applicant]
US 7824871B2
· Younossi et al.
· 2010
[cited by applicant]
US 7860656B2
· Poynard
· 2010
[cited by applicant]
US 8489335B2
· Cales
· 2013
[cited by applicant]
US 9624541B2
· Watelet et al.
· 2017
[cited by applicant]
US 20080161203A1
· Su et al.
· 2008
[cited by applicant]
US 20100136579A1
· Tseng et al.
· 2010
[cited by applicant]
US 20100203553A1
· Abdeen et al.
· 2010
[cited by applicant]
US 20140127819A1
· Takahashi et al.
· 2014
[cited by applicant]
US 20170032099A1
· Cales et al.
· 2017
[cited by applicant]
US 20180100867A1
· Okanoue et al.
· 2018
[cited by applicant]
US 20190265241A1
· Poynard
· 2019
[cited by applicant]
US 20200056235A1
· Watelet et al.
· 2020
[cited by applicant]
US 20210239713A1
· Lambert et al.
· 2021
[cited by applicant]
EP 2166358
· 2010
[cited by applicant]
JP 201340923
· 2013
[cited by applicant]
WO WO0216949
· 2002
[cited by applicant]
WO WO2006082522
· 2006
[cited by applicant]
WO WO2006103570
· 2006
[cited by applicant]
WO WO2007072290
· 2007
[cited by applicant]
WO WO2010000835
· 2010
[cited by applicant]
WO WO2012107528
· 2012
[cited by applicant]
WO WO2012107530
· 2012
[cited by applicant]
WO WO2015197934
· 2015
[cited by applicant]
Stratagene Catalog, 1988, p. 39.
[cited by examiner]
Anonymous, “Elevated Inflammatory Biomarkers Are Associated With Progression to AIDS and Death despite Viral Suppression” Jan. 1, 2010, 17
[cited by applicant]
Barsic, N. et al. “Overview and developments in noninvasive diagnosis of nonalcoholic fatty liver disease”
[cited by applicant]
Virakul, S. et al. “Histamine induces NF-κB controlled cytokine secretion by orbital fibroblasts via histamine receptor type-1”
[cited by applicant]
Written Opinion in International Application No. PCT/US2018/042836, Sep. 19, 2018, pp. 1-10.
[cited by applicant]
Shin, et al. “SPP1 polymorphisms associated with HBV clearance and HCC occurrence” International Journal of Epidemiology, Oct. 2007, pp. 1001-1008, vol. 36, No. 5.
[cited by applicant]
Schmilovitz-Weiss, H. et al. “Serum globulin levels in predicting the extent of hepatic fibrosis in patients with recurrent post-transplant hepatitis C infection” Clinical Transplantation, May 2007, pp. 391-397, vol. 21…
[cited by applicant]
Castera, et al. “Prospective Comparison of Transient Elastography, Fibrotest, APRI, and Liver Biopsy for the Assessment of Fibrosis in Chronic Hepatitis C”
[cited by applicant]
Shaheen, et al. “FibroTest and FibroScan for the Prediction of Hepatitis C-Related Fibrosis: A Systematic Review of Diagnostic Test Accuracy”
[cited by applicant]
Asselah, et al. “Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C”
[cited by applicant]
Asselah, et al. “Liver Gene Expression Signature of Mild Fibrosis in Patients With Chronic Hepatitis C” Gastroenterology, 2005, pp. 2064-2075, vol. 129.
[cited by applicant]
Bieche, et al. “Molecular profiling of early stage liver fibrosis in patients with chronic hepatitis C virus infection”
[cited by applicant]
Chen, et al. “Cell-Type Specific Gene Expression Signature in Liver Underlies Response to Interferon Therapy in Chronic Hepatitis C Infection”
[cited by applicant]
Huang, et al. “Plasma osteopontin concentration correlates with the severity of hepatic fibrosis and inflammation in HCV-infected subjects”
[cited by applicant]
Patouraux, et al. “The Osteopontin Level in Liver, Adipose Tissue and Serum Is Correlated with Fibrosis in Patients with Alcoholic Liver Disease”
[cited by applicant]
International Search Report for PCT/EP2012/052234 mailed Jun. 25, 2012.
[cited by applicant]
Written Opinion of the International Searching Authority for PCT/EP2012/052234 mailed Jun. 25, 2012.
[cited by applicant]
Gene List, “Human Genome CGH Microarray 44B G4410B”
[cited by applicant]
Ducés, A. et al. “Liver Gene Expression Signature of Mild Fibrosis in Chronic Hepatitis C”
[cited by applicant]
Ducés, A. et al. “Liver Gene Expression Signature to Predict Response to Pegylated Interferon Plus Ribavirin in Chronic Hepatitis C”
[cited by applicant]
Ducés, A. et al. “Liver Gene Expression Signature to Predict Response to Pegylated Interferon Plus Ribavirin in Chronic Hepatitis C”
[cited by applicant]
Poster shown at the Meeting of the American of the American Association of the Study of Liver Diseases (AASLD) on Sep. 2, 2010.
[cited by applicant]
French Search Report dated Jan. 31, 2012, issued in connection with FR 1151022.
[cited by applicant]
Claims pending in U.S. Appl. No. 16/553,213, filed Nov. 11, 2019, pp. 1-2.
[cited by applicant]
Seidler, S. et al. “Elevated circulating soluble interleukin-2 receptor in patients with chronic liver diseases is associated with non-classical monocytes”
[cited by applicant]
Abe, T. et al. “CD44 Participates in IP-10 Induction in Cells in Which Hepatitis C Virus RNA Is Replicating, through an Interaction with Toll-Like Receptor 2 and Hyaluronan”
[cited by applicant]
Mak, T.-M. et al. “Liver Fibrosis Assessment Using Transient Elastography Guided With Real-Time B-Mode Ultrasound Imaging: A Feasibility Study”
[cited by applicant]
Micheloud, D. et al. “Serum Levels of Fibrosis Progression Biomarkers Measured Early After LT are Associated to Severe HCV Recurrence”
[cited by applicant]
Patel, K. et al. “Multiplex Protein Analysis to Determine Fibrosis Stage and Progression in Patients With Chronic Hepatitis C”
[cited by applicant]
El Raziky, M. et al. “A Novel Prediction Model for Liver Fibrosis in Patients With Chronic Hepatitis C Virus Using Fibroscan and Routine Laboratory Data”
[cited by applicant]
Wu, Y.-M. et al. “A simple noninvasive index to predict significant liver fibrosis in patients with advanced schistosomiasis japonica”
[cited by applicant]
Written Opinion in International Application No. PCT/FR2015/051518, Aug. 14, 2015, pp. 1-8.
[cited by applicant]
Abd El-Kader, S.M. et al. “Non-alcoholic fatty liver disease: The diagnosis and management”
[cited by applicant]
Vernon, G. et al. “Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults”
[cited by applicant]
Hashimoto, E. et al. “Characteristics and diagnosis of NAFLD/NASH”
[cited by applicant]
Angulo, P. et al. “Non-alcoholic fatty liver disease”
[cited by applicant]
Beymer, C. et al. “Prevalence and Predictors of Asymptomatic Liver Disease in Patients Undergoing Gastric Bypass Surgery”
[cited by applicant]
Crespo, J. et al. “Are there Predictive Factors of Severe Liver Fibrosis in Morbidly Obese Patients with Non-alcoholic Steatohepatitis?”
[cited by applicant]
Dixon, J.B. et al. “Nonalcoholic Fatty Liver Disease: Predictors of Nonalcoholic Steatohepatitis and Liver Fibrosis in the Severely Obese”
[cited by applicant]
Gholam, P.M. et al. “Liver Pathology in Morbidly Obese Patients Undergoing Roux-en-Y Gastric Bypass Surgery”
[cited by applicant]
Moretto, M. et al. “Hepatic Steatosis In Patients Undergoing Bariatric Surgery and its Relationship to Body Mass Index and Co-Morbidities”
[cited by applicant]
Hsiao, P.-J. et al. “Significant correlations between severe fatty liver and risk factors for metabolic syndrome”
[cited by applicant]
Fracanzani, A.L. et al. “Risk of Severe Liver Disease in Nonalcoholic Fatty Liver Disease with Normal Aminotransferase Levels: A Role for Insulin Resistance and Diabetes”
[cited by applicant]
Angulo, P. et al., “Independent Predictors of Liver Fibrosis in Patients With Nonalcoholic Steatohepatitis”
[cited by applicant]
Chalasani, N. et al. “The Diagnosis and Management of Non-alcoholic Fatty Liver Disease: Practice Guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and Amer…
[cited by applicant]
European Association for the Study of the Liver, et al. “EASL-EASD-EASO Clinical Practice Guidelines for the Management of Non-Alcoholic Fatty Liver Disease”
[cited by applicant]
Chalasani, N. et al. “The Diagnosis and Management of Non-alcoholic Fatty Liver Disease: Practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the Amer…
[cited by applicant]
Farrell, G.C., et al. “Guidelines for the assessment and management of non-alcoholic fatty liver disease in the Asia-Pacific region: Executive summary”
[cited by applicant]
Ratziu, V. et al. “A position statement on NAFLD/NASH based on the EASL 2009 special conference”
[cited by applicant]
Sanyal, A.J. et al. “AGA Technical Review on Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Bedossa, P. et al. “Utility and Appropriateness of the Fatty Liver Inhibition of Progression (FLIP) Algorithm and Steatosis, Activity, and Fibrosis (SAF) Score in the Evaluation of Biopsies of Nonalcoholic Fatty Liver D…
[cited by applicant]
Kleiner, D.E. et al. “Nonalcoholic Fatty Liver Disease: Pathologic Patterns and Biopsy Evaluation in Clinical Research”
[cited by applicant]
Kleiner, D.E. et al. “Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Ratziu, V. et al. “Sampling Variability of Liver Biopsy in Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Vuppalanchi, R. et al. “Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis: Selected Practical Issues in Their Evaluation and Management”
[cited by applicant]
Vuppalanchi, R. et al. “Non-alcoholic fatty liver disease and nonalcoholic steatohepatitis: Selected practical issues in their evaluation and management”
[cited by applicant]
Musso, G. et al. “Meta-analysis: Natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity”
[cited by applicant]
Ryan, M.C. et al. “Associations Between Liver Histology and Severity of the Metabolic Syndrome in Subjects With Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Marchesini, G. et al. “Nonalcoholic Fatty Liver, Steatohepatitis, and the Metabolic Syndrome”
[cited by applicant]
Bedogni, G. et al. “The Fatty Liver Index: a simple and accurate predictor of hepatic steatosis in the general population”
[cited by applicant]
Lee, J.-H. et al. “Hepatic steatosis index: A simple screening tool reflecting nonalcoholic fatty liver disease”
[cited by applicant]
Poynard, T. et al., “The diagnostic value of biomarkers (SteatoTest) for the prediction of liver steatosis”
[cited by applicant]
Fedchuk, L. et al. “Performance and limitations of steatosis biomarkers in patients with nonalcoholic fatty liver disease”
[cited by applicant]
Fishbein, M. et al. “Hepatic MRI for Fat Quantitation: Its Relationship to Fat Morphology, Diagnosis, and Ultrasound”
[cited by applicant]
Saadeh, S. et al. “The Utility of Radiological Imaging in Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Angulo, P. et al. “The NAFLD Fibrosis Score: A Noninvasive System That Identifies Liver Fibrosis in Patients with NAFLD”
[cited by applicant]
Boursier, J. et al. “Diagnostic accuracy and prognostic significance of blood fibrosis tests and liver stiffness measurement by FibroScan in non-alcoholic fatty liver disease”
[cited by applicant]
Koplay, M. et al. “Importance of imaging and recent developments in diagnosis of nonalcoholic fatty liver disease”
[cited by applicant]
Castera, L. et al. “Pitfalls of Liver Stiffness Measurement: A 5-Year Prospective Study of 13,369 Examinations”
[cited by applicant]
Wong, V. W-S. et al. “Diagnosis of Fibrosis and Cirrhosis Using Liver Stiffness Measurement in Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Wong, V. W-S. et al. “Liver Stiffness Measurement Using XL Probe in Patients With Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Tilg, H. “Adipocytokines in Nonalcoholic Fatty Liver Disease: Key Players Regulating Steatosis, Inflammation and Fibrosis”
[cited by applicant]
Jamali, R. et al. “Serum adipokines might predict liver histology findings in non-alcoholic fatty liver disease”
[cited by applicant]
Cusi, K. et al. “Limited value of plasma cytokeratin-18 as a biomarker for NASH and fibrosis in patients with non-alcoholic fatty liver disease”
[cited by applicant]
Grigorescu, M. et al. “A novel pathophysiological-based panel of biomarkers for the diagnosis of nonalcoholic steatohepatitis”
[cited by applicant]
Pirvulescu, I. et al. “Noninvasive Clinical Model for the Diagnosis of Nonalcoholic Steatohepatitis in Overweight and Morbidly Obese Patients undergoing Bariatric Surgery”
[cited by applicant]
Polyzos, S.A. et al. “Adipokines in nonalcoholic fatty liver disease”
[cited by applicant]
Larter, C.Z. et al. “A fresh look at NASH pathogenesis. Part 1: The metabolic movers”
[cited by applicant]
Park, S.H. et al. “Body Fat Distribution and Insulin Resistance: Beyond Obesity in Nonalcoholic Fatty Liver Disease among Overweight Men”
[cited by applicant]
Park, B.J. et al. “Visceral adipose tissue area is an independent risk factor for hepatic steatosis”
[cited by applicant]
Harris, R.B.S. et al., “Location, Location, Location . . . ”
[cited by applicant]
Zhang, X. et al., “CXCL10 plays a key role as an inflammatory mediator and a non-invasive biomarker of non-alcoholic steatohepatitis”
[cited by applicant]
Du Plessis, J. et al. “Association of Adipose Tissue Inflammation With Histologic Severity of Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Shah, A.G. et al. “Comparison of Noninvasive Markers of Fibrosis in Patients With Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Angulo, P. et al. “Simple Noninvasive Systems Predict Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease”
[cited by applicant]
Yoneda, M. et al. “Noninvasive scoring systems in patients with nonalcoholic fatty liver disease with normal alanine aminotransferase levels”
[cited by applicant]
Brunt, E.M. et al. “Nonalcoholic Steatohepatitis: A Proposal for Grading and Staging the Histological Lesions”
[cited by applicant]
Castera, L. et al. “Non-invasive evaluation of liver fibrosis using transient elastography”
[cited by applicant]
Imbert-Bismut, F. et al. “Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study”
[cited by applicant]
Adams, L.A. et al. “Hepascore: An Accurate Validated Predictor of Liver Fibrosis in Chronic Hepatitis C Infection”
[cited by applicant]
Forns, X. et al. “Identification of Chronic Hepatitis C Patients Without Hepatic Fibrosis by a Simple Predictive Model”
[cited by applicant]
Wai, C-T. et al. “A Simple Noninvasive Index Can Predict Both Significant Fibrosis and Cirrhosis in Patients With Chronic Hepatitis C”
[cited by applicant]
Sterling, R.K. et al. “Development of a Simple Noninvasive Index to Predict Significant Fibrosis in Patients With HIV/HCV Coinfection”
[cited by applicant]
Flanagan, J.J. et al. “Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA)”
[cited by applicant]
Team, R.C.,
[cited by applicant]
Benjamini, Y. et al. “Controlling the False Discovery Rate: a Practical and Powerful Approach to Multiple Testing”
[cited by applicant]
Youden, W.J. “Index for Rating Diagnostic Tests” Cancer, Jan. 1950, pp. 32-35.
[cited by applicant]
Hastie, T. et al. “The Elements of Statistical Learning: Data Mining, Inference, and Prediction” Springer Science & Business Media, 2001, pp. 1-764.
[cited by applicant]
Irvine, K. M. et al. “Multiplex Serum Protein Analysis Identifies Novel Biomarkers of Advanced Fibrosis in Patients with Chronic Liver Disease with the Potential to Improve Diagnostic Accuracy of Established Biomarkers”
[cited by applicant]
Zhou, J-H. et al. “Autoantibodies against MMP-7 as a novel diagnostic biomarker in esophageal squamous cell carcinoma”
[cited by applicant]
Xie, W. et al. “Anti-POSTN and Anti-TIMP1 Autoantibodies as Diagnostic Markers in Esophageal Squamous Cell Carcinoma”
[cited by applicant]
Liao, C-C. et al. “Isotypes of autoantibodies against differentially expressed novel malondialdehyde-modified peptide adducts in serum of Taiwanese women with rheumatoid arthritis”
[cited by applicant]