Methods of diagnosing hepatocellular carcinoma and pancreatic cancer

Abstract

Disclosed herein are biomarkers for hepatocellular carcinoma and pancreatic cancer. The biomarkers may be laminin gamma 2 monomer, PIVKA-II, AFP, CEA, CA19-9, or combinations thereof. Also disclosed herein are methods of diagnosing, prognosing, classifying risk, and monitoring progression of hepatocellular carcinoma or pancreatic cancer by the detecting the level of laminin gamma 2 monomer, PIVKA-II, AFP, CEA, CA19-9, or combinations thereof.

What is claimed is: 
     
         1 . A method of diagnosing hepatocellular carcinoma (HCC) in a subject in need thereof, the method comprising:
 (a) obtaining a biological sample from the subject;   (b) determining a level of laminin gamma 2 monomer in the biological sample;   (c) comparing the level of laminin gamma 2 monomer to a reference level of laminin gamma 2 monomer; and   (d) identifying the subject as having HCC when the level of laminin gamma 2 monomer is greater than the reference level of laminin gamma 2 monomer.   
     
     
         2 . The method of  claim 1 , further comprising determining a level of at least one additional biomarker in the biological sample, wherein the at least one additional biomarker is selected from the group consisting of: protein induced vitamin K antagonist-II (PIVKA-II), alpha fetal protein (AFP), and the combination thereof. 
     
     
         3 . The method of  claim 2 , further comprising comparing the level of the at least one additional biomarker to a reference level of the at least one additional biomarker; and identifying the subject as having HCC when the level of the at least one additional biomarker is greater than the reference level of the at least on additional biomarker. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the levels of:
 (a) laminin gamma 2 monomer and PIVKA-II are determined in the biological sample;   (b) laminin gamma 2 monomer and AFP are determined in the biological sample; or   (c) laminin gamma 2 monomer, PIVKA-II, and AFP are determined in the biological sample.   
     
     
         5 . The method of  claim 4 , wherein the subject is identified as having HCC when:
 (a) the levels of laminin gamma 2 monomer and PIVKA-II in the biological sample are greater than the reference levels of laminin gamma 2 monomer and PIVKA-II;   (b) the levels of laminin gamma 2 monomer and AFP in the biological sample are greater than the reference levels of laminin gamma 2 monomer and AFP; or   (c) the levels of laminin gamma 2 monomer, PIVKA-II, and AFP in the biological sample are greater than the reference levels of laminin gamma 2 monomer, PIVKA-II, and AFP.   
     
     
         6 . The method of any one of  claims 1 - 5 , wherein the biological sample is selected from the group consisting of: a whole blood sample, a plasma sample, and a serum sample. 
     
     
         7 . The method of  claim 6 , wherein the biological sample is a serum sample. 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein determining the level of laminin gamma 2 monomer in the biological sample includes detecting laminin gamma 2 monomer with an immunoassay. 
     
     
         9 . The method of any one of  claims 2 - 8 , wherein determining the level of PIVKA-II and AFP in the biological sample includes detecting PIVKA-II and AFP with an immunoassay. 
     
     
         10 . The method of  claim 8  or  9 , wherein the immunoassay is a sandwich immunoassay. 
     
     
         11 . The method of any one of  claims 2 - 10 , wherein the reference level of PIVKA-II and AFP is a level of PIVKA-II and AFP in a control sample. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a control sample. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the reference level of laminin gamma 2 monomer is a cutoff level. 
     
     
         14 . The method of  claim 13 , wherein the cutoff level is determined by a mean plus 2 standard deviation analysis of multiple control samples. 
     
     
         15 . The method of any one of  claims 1 - 14 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a calibrator. 
     
     
         16 . A method of determining if a subject has or is at risk of developing hepatocellular carcinoma (HCC), the method comprising:
 (a) obtaining a biological sample from the subject;   (b) measuring a level of laminin gamma 2 monomer in the biological sample;   (c) comparing the level of laminin gamma 2 monomer to a reference level of laminin gamma 2 monomer; and   (d) determining the subject has or is at risk of developing HCC when the level of laminin gamma 2 monomer is greater than the reference level of laminin gamma 2 monomer.   
     
     
         17 . The method of  claim 16 , further comprising measuring a level of at least one additional biomarker in the biological sample, wherein the at least one additional biomarker is selected from the group consisting of: protein induced vitamin K antagonist-II (PIVKA-II), alpha fetal protein (AFP), and the combination thereof. 
     
     
         18 . The method of  claim 17 , further comprising comparing the level of the at least one additional biomarker to a reference level of the at least one additional biomarker; and determining the subject has or is at risk of developing HCC when the level of the at least one additional biomarker is greater than the reference level of the at least one additional biomarker. 
     
     
         19 . The method of any one of  claims 16 - 18 , wherein the level of
 (a) laminin gamma 2 monomer and PIVKA-II are determined in the biological sample;   (b) laminin gamma 2 monomer and AFP are determined in the biological sample; or   (c) laminin gamma 2 monomer, PIVKA-II, and AFP are determined in the biological sample.   
     
     
         20 . The method of  claim 19 , wherein the subject is identified as having HCC when:
 (a) the levels of laminin gamma 2 monomer and PIVKA-II in the biological sample are greater than the reference levels of laminin gamma 2 monomer and PIVKA-II;   (b) the levels of laminin gamma 2 monomer and AFP in the biological sample are greater than the reference levels of laminin gamma 2 monomer and AFP; or   (c) the levels of laminin gamma 2 monomer, PIVKA-II, and AFP in the biological sample are greater than the reference levels of laminin gamma 2 monomer, PIVKA-II, and AFP.   
     
     
         21 . The method of any one of  claims 16 - 20 , wherein the biological sample is selected from the group consisting of: a whole blood sample, a plasma sample, and a serum sample. 
     
     
         22 . The method of  claim 21 , wherein the biological sample is a serum sample. 
     
     
         23 . The method of any one of  claims 16 - 22 , wherein determining the level of laminin gamma 2 monomer in the biological sample includes detecting laminin gamma 2 monomer with an immunoassay. 
     
     
         24 . The method of any one of  claims 17 - 23 , wherein determining the level of PIVKA-II and AFP in the biological sample includes detecting PIVKA-II and AFP with an immunoassay. 
     
     
         25 . The method of  claim 23  or  24 , wherein the immunoassay is a sandwich immunoassay. 
     
     
         26 . The method of any one of  claims 17 - 25 , wherein the reference level of PIVKA-II and AFP is a level of PIVKA-II and AFP in a control sample. 
     
     
         27 . The method of any one of  claims 16 - 26 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a control sample. 
     
     
         28 . The method of any one of  claims 16 - 27 , wherein the reference level of laminin gamma 2 monomer is a cutoff level. 
     
     
         29 . The method of  claim 28 , wherein the cutoff level is determined by a mean plus 2 standard deviation analysis of multiple control samples or ROC analysis compared HCC with multiple control samples. 
     
     
         30 . The method of any one of  claims 16 - 29 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a calibrator. 
     
     
         31 . A method of monitoring progression of hepatocellular carcinoma (HCC) in a subject in need thereof, the method comprising:
 (a) obtaining first and second biological samples from the subject;   (b) measuring a first level of laminin gamma 2 monomer in the first biological sample and a second level of laminin gamma 2 monomer in the second biological sample;   (c) comparing the first and second levels of laminin gamma 2 monomer; and   (d) determining
 (i) HCC has progressed in the subject when the second level of laminin gamma 2 monomer is greater than the first level of laminin gamma 2 monomer or 
 (ii) HCC has not progressed in the subject when the second level of laminin gamma 2 monomer is equivalent to or less than the first level of laminin gamma 2 monomer. 
   
     
     
         32 . The method of  claim 31 , further comprising measuring a first level of at least one additional biomarker in the first biological sample and a second level of the at least one additional biomarker in the second biological sample, wherein the at least one additional biomarker is selected from the group consisting of: protein induced vitamin K antagonist-II (PIVKA-II), alpha fetal protein (AFP), and the combination thereof. 
     
     
         33 . The method of  claim 32 , further comprising comparing the first and second levels of the at least one additional biomarker; and determining HCC has progressed in the subject when the second level of the at least one additional biomarker is greater than the first level of the at least one additional biomarker, or HCC has not progressed in the subject when the second level of the at least one additional biomarker is equivalent to or less than the first level of the at least one additional biomarker. 
     
     
         34 . The method of any one of  claims 31 - 33 , wherein the first and second levels of
 (a) laminin gamma 2 monomer and PIVKA-II are measured in the first and second biological samples;   (b) laminin gamma 2 monomer and AFP are measured in the first and second biological samples; or   (c) laminin gamma 2 monomer, PIVKA-II, and AFP are measured in the first and second biological samples.   
     
     
         35 . The method of  claim 34 , wherein the subject is identified as having HCC when:
 (a) the levels of laminin gamma 2 monomer and PIVKA-II in the biological sample are greater than the reference levels of laminin gamma 2 monomer and PIVKA-II;   (b) the levels of laminin gamma 2 monomer and AFP in the biological sample are greater than the reference levels of laminin gamma 2 monomer and AFP; or   (c) the levels of laminin gamma 2 monomer, PIVKA-II, and AFP in the biological sample are greater than the reference levels of laminin gamma 2 monomer, PIVKA-II, and AFP.   
     
     
         36 . The method of any one of  claims 31 - 35 , wherein the first and second biological samples are whole blood samples, plasma samples or serum samples. 
     
     
         37 . The method of  claim 36 , wherein the first and second biological samples are serum samples. 
     
     
         38 . The method of any one of  claims 31 - 37 , wherein determining the level of laminin gamma 2 monomer in the biological sample includes detecting laminin gamma 2 monomer with an immunoassay. 
     
     
         39 . The method of any one of  claims 32 - 38 , wherein determining the level of PIVKA-II and AFP in the biological sample includes detecting PIVKA-II and AFP with an immunoassay. 
     
     
         40 . The method of  claim 38  or  39 , wherein the immunoassay is a sandwich immunoassay. 
     
     
         41 . The method of any one of  claims 32 - 40 , wherein the reference level of PIVKA-II and AFP is a level of PIVKA-II and AFP in a control sample. 
     
     
         42 . The method of any one of  claims 31 - 41 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a control sample. 
     
     
         43 . The method of any one of  claims 31 - 42 , wherein the reference level of laminin gamma 2 monomer is a cutoff level. 
     
     
         44 . The method of  claim 43 , wherein the cutoff level is determined by a mean plus 2 standard deviation analysis of multiple control samples. 
     
     
         45 . The method of any one of  claims 31 - 44 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a calibrator. 
     
     
         46 . A kit for detecting HCC in a subject in need thereof, the kit comprising one or more reagents for detecting laminin gamma 2 monomer. 
     
     
         47 . The kit of  claim 46 , further comprising
 (a) one or more reagents for detecting protein induced vitamin K antagonist-II (PIVKA-II); and   (b) one or more reagents for detecting alpha fetal protein (AFP).   
     
     
         48 . A method for diagnosing pancreatic cancer in a subject in need thereof, the method comprising:
 (a) obtaining a biological sample from the subject;   (b) determining a level of laminin gamma 2 monomer in the biological sample;   (c) determining a level of at least one additional biomarker in the biological sample, wherein the at least one additional biomarker is selected from the group consisting of: carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9);   (d) comparing the level of laminin gamma 2 monomer to a reference level of laminin gamma 2 monomer and the level of the at least one additional biomarker to a reference level of the at least one additional biomarker; and   (e) identifying the subject has having pancreatic cancer when the levels of laminin gamma 2 monomer and the at least one additional biomarker are greater than the reference levels of laminin gamma 2 monomer and the at least one additional biomarker.   
     
     
         49 . The method of  claim 48 , wherein the levels of:
 (a) laminin gamma 2 monomer and CEA are determined in the biological sample;   (b) laminin gamma 2 monomer and CA19-9 are determined in the biological sample; or   (c) laminin gamma 2 monomer, CEA, and CA19-9 are determined in the biological sample.   
     
     
         50 . The method of  claim 48  or  49 , wherein the biological sample is selected from the group consisting of: a whole blood sample, a plasma sample, and a serum sample. 
     
     
         51 . The method of  claim 50 , wherein the biological sample is a serum sample. 
     
     
         52 . The method of any one of  claims 48 - 51 , wherein determining the level of laminin gamma 2 monomer in the biological sample includes detecting laminin gamma 2 monomer with an immunoassay. 
     
     
         53 . The method of any one of  claims 49 - 52 , wherein determining the level of CEA and CA19-9 in the biological sample includes detecting CEA and CA19-9 with an immunoassay. 
     
     
         54 . The method of  claim 52  or  53 , wherein the immunoassay is a sandwich immunoassay. 
     
     
         55 . The method of any one of  claims 49 - 54 , wherein the reference level of CEA and CA19-9 is a level of CEA and CA19-9 in a control sample. 
     
     
         56 . The method of any one of  claims 48 - 55 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a control sample. 
     
     
         57 . The method of any one of  claims 48 - 56 , wherein the reference level of laminin gamma 2 monomer is a cutoff level. 
     
     
         58 . The method of  claim 57 , wherein the cutoff value is determined by a mean plus 2 standard deviation analysis of multiple control samples. 
     
     
         59 . The method of any one of  claims 48 - 58 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a calibrator. 
     
     
         60 . A method of determining if a subject has or is at risk of developing pancreatic cancer, the method comprising:
 (a) obtaining a biological sample from the subject;   (b) measuring a level of laminin gamma 2 monomer in the biological sample;   (c) measuring a level of at least one additional biomarker in the biological sample, wherein the at least one additional biomarker is selected from the group consisting of: carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9);   (d) comparing the level of laminin gamma 2 monomer to a reference level of laminin gamma 2 monomer and the level of the at least one additional biomarker to a reference level of the at least one additional biomarker; and   (e) determining the subject has or is at risk of developing pancreatic cancer when the levels of laminin gamma 2 monomer and the at least one additional biomarker are greater than the reference levels of laminin gamma 2 monomer and the at least one additional biomarker.   
     
     
         61 . The method of  claim 60 , wherein the levels of:
 (a) laminin gamma 2 monomer and CEA in the biological sample are measured;   (b) laminin gamma 2 monomer and CA19-9 in the biological sample are measured; or   (c) laminin gamma 2 monomer, CEA, and CA19-9 in the biological sample are measured.   
     
     
         62 . The method of  claim 60  or  61 , wherein the biological sample is selected from the group consisting of: a whole blood sample, a plasma sample, and a serum sample. 
     
     
         63 . The method of  claim 62 , wherein the biological sample is a serum sample. 
     
     
         64 . The method of any one of  claims 60 - 63 , wherein determining the level of laminin gamma 2 monomer in the biological sample includes detecting laminin gamma 2 monomer with an immunoassay. 
     
     
         65 . The method of any one of  claims 61 - 64 , wherein determining the level of CEA and CA19-9 in the biological sample includes detecting CEA and CA19-9 with an immunoassay. 
     
     
         66 . The method of  claim 64  or  65 , wherein the immunoassay is a sandwich immunoassay. 
     
     
         67 . The method of any one of  claims 61 - 66 , wherein the reference level of CEA and CA19-9 is a level of CEA and CA19-9 in a control sample. 
     
     
         68 . The method of any one of  claims 60 - 67 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a control sample. 
     
     
         69 . The method of any one of  claims 60 - 68 , wherein the reference level of laminin gamma 2 monomer is a cutoff level. 
     
     
         70 . The method of  claim 69 , wherein the cutoff value is determined by a mean plus 2 standard deviation analysis of multiple control samples. 
     
     
         71 . The method of any one of  claims 60 - 70 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a calibrator. 
     
     
         72 . A method of monitoring progression of pancreatic cancer in a subject in need thereof, the method comprising:
 (a) obtaining first and second biological samples from the subject;   (b) measuring a first level of laminin gamma 2 monomer in the first biological sample and a second level of laminin gamma 2 monomer in the second biological sample;   (c) measuring a first level of at least one additional biomarker in the first biological sample and a second level of the at least one additional biomarker in the second biological sample, wherein the at least one additional biomarker is selected from the group consisting of: carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9);   (d) comparing the first and second levels of laminin gamma 2 monomer;   (e) comparing the first and second levels of the at least one additional biomarker; and   (f) determining
 (i) the pancreatic cancer has progressed in the subject when the second levels of laminin gamma 2 monomer and the at least one additional biomarker are greater than the first levels of laminin gamma 2 monomer and the at least one additional biomarker or 
 (ii) the pancreatic cancer has not progressed in the subject when the second levels of laminin gamma 2 monomer and the at least one additional biomarker are equivalent to or less than the first levels of laminin gamma 2 monomer and the at least one additional biomarker. 
   
     
     
         73 . The method of  claim 72 , wherein:
 (i) the first and second levels of laminin gamma 2 monomer and CEA in the first and second biological samples are measured;   (ii) the first and second levels of laminin gamma 2 monomer and CA19-9 in the first and second biological samples are measured; or   (iii) the first and second levels of laminin gamma 2 monomer, CEA, and CA19-9 in the first and second biological samples are measured.   
     
     
         74 . The method of  claim 72  or  73 , wherein the first and second biological samples are whole blood samples, plasma samples, and serum samples. 
     
     
         75 . The method of  claim 74  wherein the first and second biological samples are serum samples. 
     
     
         76 . The method of any one of  claims 72 - 75 , wherein determining the level of laminin gamma 2 monomer in the biological sample includes detecting laminin gamma 2 monomer with an immunoassay. 
     
     
         77 . The method of any one of  claims 73 - 76 , wherein determining the level of CEA and CA19-9 in the biological sample includes detecting CEA and CA19-9 with an immunoassay. 
     
     
         78 . The method of  claim 76  or  77 , wherein the immunoassay is a sandwich immunoassay. 
     
     
         79 . The method of any one of  claims 73 - 78 , wherein the reference level of CEA and CA19-9 is a level of CEA and CA19-9 in a control sample. 
     
     
         80 . The method of any one of  claims 72 - 79 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a control sample. 
     
     
         81 . The method of any one of  claims 72 - 80 , wherein the reference level of laminin gamma 2 monomer is a cutoff level. 
     
     
         82 . The method of  claim 81 , wherein the cutoff value is determined by a mean plus 2 standard deviation analysis of multiple control samples. 
     
     
         83 . The method of any one of  claims 72 - 82 , wherein the reference level of laminin gamma 2 monomer is a level of laminin gamma 2 monomer in a calibrator. 
     
     
         84 . A kit for detecting pancreatic cancer in a subject in need thereof, the kit comprising:
 (a) one or more reagents for detecting laminin gamma 2 monomer; and   (b) one or more reagents for detecting at least one additional biomarker, wherein the at least one additional biomarker is selected from the group consisting of: carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9).