Early Detection Of Hepatocellular Carcinoma

Abstract

Provided are methods, assays, and kits for detecting hepatocellular carcinoma, as well as methods for stratifying subjects among higher and lower risk categories for having hepatocellular carcinoma, and methods of treating and managing treatment of subjects that are suspected or at risk of having hepatocellular carcinoma. Although previous work has attempted to address the need for a highly sensitive, early predictor of hepatocellular carcinoma by assessment of one or more biological factors, none have approached the degree of sensitivity that is required for clinically relevant determination of whether a subject, especially a non-symptomatic subject, has that condition. The present inventors have discovered that certain combinations of factors fulfill this need by conferring a high level of accuracy that was not previously attainable.

1 .- 31 . (canceled) 
     
     
         32 . A method for detecting hepatocellular carcinoma in a subject comprising:
 measuring the amount of biomarkers comprising fucosylated kininogen, fucosylated alpha-1-antitrypsin, fucosylated fetuin A, fucosylated hemopexin, or any combination thereof in a biological fluid from the subject or in a separate sample derived from the biological fluid,   but prior to measuring the amount of a biomarker that is a fucose-containing glycoform, removing IgG and IgM proteins from a biological fluid obtained from the subject or the separate sample derived from the biological fluid;   and,   determining the absence or presence of hepatocellular carcinoma in the subject using an output of a function of the measured biomarkers in the biological fluid or the separate sample derived from the biological fluid.   
     
     
         33 . A kit for detecting hepatocellular carcinoma in a subject comprising:
 reagents for respectively measuring biomarkers comprising fucosylated kininogen, fucosylated alpha-1-antitrypsin, fucosylated fetuin A, fucosylated hemopexin, or any combination thereof in a biological fluid or a separate sample derived from the biological fluid   a component for removing IgG from the biological fluid from the subject or the separate sample derived from the biological fluid prior to measuring the amount of a biomarker that is a fucose-containing glycoform;   a component for removing IgM from the biological fluid from the subject or the separate sample derived from the biological fluid prior to measuring the amount of a biomarker that is a fucose-containing glycoform; and,   instructions for determining the absence or presence of hepatocellular carcinoma in the subject using an output of a function of the measured biomarkers in the biological fluid or the separate sample derived from the biological fluid.   
     
     
         34 . A method for managing treatment of a subject suspected of having hepatocellular carcinoma comprising:
 measuring the amount of biomarkers comprising fucosylated kininogen, fucosylated alpha-1-antitrypsin, fucosylated fetuin A, fucosylated hemopexin, or any combination thereof in a biological fluid from the subject or in a separate sample derived from the biological fluid;   but prior to measuring the amount of a biomarker that is a fucose-containing glycoform removing IgG and IgM proteins from a biological fluid obtained from the subject or the separate sample derived from the biological fluid;   and,   determining a treatment for the subject based on an output of a function of and the measured biomarkers in the biological fluid or the separate sample derived from the biological fluid, wherein the treatment is appropriate for hepatocellular carcinoma when the subject is determined to have that disease based on the output of the function.   
     
     
         35 . The method according to  claim 32 , wherein the function comprises respective weighting coefficients for the measured amounts of the one or more biomarkers. 
     
     
         36 . The method according to  claim 32 , wherein the biomarkers further comprise one or more of fucosylated fetuin-A, fucosylated alpha-1-antitrypsin, and alanine aminotransferase (ALT). 
     
     
         37 . The method according to  claim 32 , wherein the biomarkers are
 alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, and aspartate aminotransferase;   alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, and fucosylated alpha-1-antitrypsin;   alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, and fucosylated fetuin-A; or   alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, fucosylated fetuin-A, and fucosylated alpha-1-antitrypsin.   
     
     
         38 . The method according to  claim 32 , wherein the biomarkers are alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, and aspartate aminotransferase. 
     
     
         39 . The method according to  claim 32 , wherein the biomarkers are alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, and fucosylated alpha-1-antitrypsin. 
     
     
         40 . The method according to  claim 32 , wherein the biomarkers are alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, and fucosylated fetuin-A. 
     
     
         41 . The method according to  claim 32 , wherein the IgG is removed by incubating the biological fluid or the separate sample derived from the biological fluid with Protein A/G, and the IgM is removed by passing the biological fluid or the separate sample derived from the biological fluid through a molecular weight-based filter. 
     
     
         42 . The method according to  claim 32 , wherein the IgG and the IgM are removed by incubating the biological fluid or the separate sample derived from the biological fluid with polyethylene glycol. 
     
     
         43 . The method according to  claim 32 , wherein the hepatocellular carcinoma is early stage hepatocellular carcinoma, alpha fetoprotein negative hepatocellular carcinoma, or early stage hepatocellular carcinoma that is also alpha fetoprotein negative hepatocellular carcinoma. 
     
     
         44 . The method according to  claim 32 , wherein the biomarkers further comprise one or more of fucosylated alpha-1-antitrypsin, alanine aminotransferase, and fucosylated fetuin A. 
     
     
         45 . The method according to  claim 44 , wherein the biomarkers further comprise alanine aminotransferase. 
     
     
         46 . The method according to  claim 44 , wherein the biomarkers further comprise fucosylated fetuin A, fucosylated alpha-1-antitrypsin, or both. 
     
     
         47 . The method according to  claim 32 , wherein the biomarkers include:
 fucosylated kininogen in the absence of other fucosylated biomarkers;   fucosylated kininogen and fucosylated alpha-1-antitrypsin in the absence of other fucosylated biomarkers;   fucosylated fetuin A in the absence of other fucosylated biomarkers;   fucosylated hemopexin in the absence of other fucosylated biomarkers;   fucosylated fetuin A and fucosylated hemopexin in the absence of other fucosylated biomarkers;   fucosylated kininogen and fucosylated fetuin A in the absence of other fucosylated biomarkers; or,   fucosylated kininogen, fucosylated alpha-1-antitrypsin, and fucosylated fetuin A in the absence of other fucosylated biomarkers.   
     
     
         48 . The kit according to  claim 33 , wherein the function comprises respective weighting coefficients for the measured amounts of the one or more biomarkers. 
     
     
         49 . The kit according to  claim 33 , wherein the biomarkers further comprise one or more of fucosylated fetuin-A, fucosylated hemopexin, fucosylated alpha-1-antitrypsin, and alanine aminotransferase (ALT). 
     
     
         50 . The kit according to  claim 33 , wherein the biomarkers are
 alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, and aspartate aminotransferase;   alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, and fucosylated alpha-1-antitrypsin;   alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, and fucosylated fetuin-A; or   alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, fucosylated fetuin-A, and fucosylated alpha-1-antitrypsin.   
     
     
         51 . The kit according to  claim 33 , wherein the biomarkers are alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, and aspartate aminotransferase. 
     
     
         52 . The kit according to  claim 33 , wherein the biomarkers are alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, and fucosylated alpha-1-antitrypsin. 
     
     
         53 . The kit according to  claim 33 , wherein the biomarkers are alpha-fetoprotein, fucosylated kininogen, alkaline phosphatase, aspartate aminotransferase, and fucosylated fetuin-A. 
     
     
         54 . The kit according to  claim 33 , wherein the component for removing the IgG is Protein A/G, and the component for removing IgM is a molecular weight-based filter. 
     
     
         55 . The kit according to  claim 33 , wherein the biomarkers further comprise one or more of fucosylated alpha-1-antitrypsin, alanine aminotransferase, and fucosylated fetuin A. 
     
     
         56 . The kit according to  claim 55 , wherein the biomarkers further comprise alanine aminotransferase. 
     
     
         57 . The kit according to  claim 55 , wherein the biomarkers further comprise fucosylated fetuin A, fucosylated alpha-1-antitrypsin, or both. 
     
     
         58 . The kit according to  claim 33  wherein the biomarkers include:
 fucosylated kininogen in the absence of other fucosylated biomarkers; 
 fucosylated kininogen and fucosylated alpha-1-antitrypsin in the absence of other fucosylated biomarkers; 
 fucosylated fetuin A in the absence of other fucosylated biomarkers; 
 fucosylated hemopexin in the absence of other fucosylated biomarkers; 
 fucosylated fetuin A and fucosylated hemopexin in the absence of other fucosylated biomarkers; 
 fucosylated kininogen and fucosylated fetuin A in the absence of other fucosylated biomarkers; or, 
 fucosylated kininogen, fucosylated alpha-1-antitrypsin, and fucosylated fetuin A in the absence of other fucosylated biomarkers.