Method for Determining Hepatocellular Carcinoma Subtype and Detecting Hepatic Cancer Stem Cells

Abstract

Described herein are methods of determining an HCC subtype in a subject which includes a) obtaining a sample from the subject, b) assaying the sample to detect the expression of 1 or more biomarkers, and c) correlating the expression of the biomarkers with an HCC subtype in a subject. Also described are methods of detecting HCC stem cells in a sample, and methods and compositions for treating subjects with HCC that take advantage of the biomarkers associated with HCC stem cells.

What is claimed is: 
     
         1 . A method of determining a hepatocellular carcinoma (HCC) subtype in a subject comprising;
 a) analyzing a sample from the subject, by laboratory assay, for a change in the level of expression of one or more biomarkers relative to the level of expression of a corresponding biomarker in at least one control sample, wherein the biomarker consists of at least one of the biomarkers identified by SEQ ID NOs: 1-39;   b) correlating the change in the level of expression of the biomarkers, relative to the level of expression of the corresponding biomarker in the control sample, with the presence of the subtype of HCC in the subject; and   c) determining a hepatocellular carcinoma (HCC) subtype as the subtype if one or more of the biomarkers in the sample are high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample, and determining a hepatocellular subtype as not the HCC subtype if biomarkers in the sample are not high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample.   
     
     
         2 . A method of determining a hepatocellular carcinoma (HCC) subtype in a subject comprising
 a) analyzing a sample from the subject, by laboratory assay, for a change in the level of expression of one or more biomarkers relative to the level of expression of a corresponding biomarker in at least one control sample, wherein the biomarker consists of at least one of the biomarkers identified by SEQ ID NOs:1-39; and   b) correlating the change in the level of expression of the biomarkers, relative to the level of expression of the corresponding biomarker in the control sample, with the presence of the subtype of HCC in the subject,   c) determining a hepatocellular carcinoma (HCC) subtype as:
 i) hepatic stem cell-like hepatocellular carcinoma (HSC-HCC) subtype if one or more of the biomarkers identified by SEQ ID NOs: 1-19 in the sample are high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample, and 
 determining a hepatocellular carcinoma (HCC) subtype as not HSC-HCC subtype if one or more of the biomarkers identified by SEQ ID NOs:1-19 in the sample are not high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample; or, 
 ii) bile duct epithelium-like hepatocellular carcinoma (BDE-HCC) subtype if one or more of the biomarkers identified by SEQ ID NOs: 2, 9-12, 14-17 and 19-25 in the sample are high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample, and 
 determining a hepatocellular carcinoma (HCC) subtype as not BDE-HCC subtype if one or more of the biomarkers identified by SEQ ID NOs: 2, 9-12, 14-17 and 19-35 in the sample are not high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample; or, 
 iii) hepatocytic progenitor-like hepatocellular carcinoma (HP-HCC) subtype if one or more of the biomarkers identified by SEQ ID NOs: 1-8, 11-13, 17-18, 23, 28-29 and 33-39 in the sample are high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample, and 
 determining a hepatocellular carcinoma (HCC) subtype as not HP-HCC subtype if one or more of the biomarkers identified by SEQ ID NOs: 1-8, 11-13, 17-18, 23, 28-29 and 33-39 in the sample are not high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample; or 
   iv) mature hepatocyte-like hepatocellular carcinoma (MH-HCC) subtype if one or more of the biomarkers identified by SEQ ID NOs: 1, 8-12, 14-17, 19-24 and 26-39 in the sample are high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample, and
 determining a hepatocellular carcinoma (HCC) subtype as not MH-HCC subtype if one or more of the biomarkers identified by SEQ ID NOs: 1, 8-12, 14-17, 19-24 and 26-39 in the sample are not high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample 
   
     
     
         3 . The method of  claim 1 , wherein the sample is selected from the group consisting of liver tumor tissue, liver normal tissue, frozen biopsy tissue, paraffin-embedded biopsy tissue, serum, plasma, and combinations thereof. 
     
     
         4 . The method of  claim 1 , wherein the sample is analyzed by one or more methods selected from the group consisting of micro array techniques, PCR amplification, RNA hybridization, in situ hybridization, gel electrophoresis, and combinations thereof. 
     
     
         5 . The method of  claim 1 , further including a step (d) of further discriminating among subtypes: hepatic stem cell-like hepatocellular carcinoma (HSC-HCC) subtype, bile duct epithelial (BDE-HCC) subtype, hepatic stem cell (HSC-HCC) subtype, and hepatocytic progenitor subtype (HP-HCC) subtype,
 by correlating an alteration in the level of expression of one or more of the biomarkers, relative to the level of expression of the corresponding biomarker in the control sample.   
     
     
         6 . The method of  claim 1 , wherein step (a) further comprises determining an increased level of expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 biomarkers. 
     
     
         7 . The method of  claim 1 , wherein step (a) further includes analyzing the sample for a change in the level of expression of at least one or more biomarkers identified by SEQ ID NOs:1- 15; and,    wherein step (b) further includes correlating an increase in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 1-15, relative to the level of expression of the corresponding additional biomarker in normal liver as the at least one control sample, with the presence of HSC-HCC subtype in the subject.   
     
     
         8 . The method of  claim 1 , wherein step (a) further includes analyzing the sample for a change in the level of expression of at least one or biomarkers identified by SEQ ID NOs: 16-19; and
 wherein step (b) further includes correlating a decrease in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 16-19, relative to the level of expression of the corresponding additional biomarker in normal live as the at least one control sample, with the presence of HSC-HCC subtype in the subject.   
     
     
         9 . The method of  claim 1 , wherein step (a) further includes analyzing the sample for a change in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 9, 11-12, 14-17 and 19-30; and,
 wherein step (b) further includes correlating an increase in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 9, 11-12, 14-17and 19-30, relative to the level of expression of the corresponding additional biomarker in normal liver as the at least one control sample, with the presence of BDE-HCC subtype in the subject.   
     
     
         10 . The method of  claim 1 , wherein step (a) further includes analyzing the sample for a change in the level of expression of at least one or biomarkers identified by SEQ ID NOs: 2, 10 and 31-35; and
 wherein step (b) further includes correlating a decrease in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 2, 10 and 31-35, relative to the level of expression of the corresponding additional biomarker in normal live as the at least one control sample, with the presence of BDE-HCC subtype in the subject.   
     
     
         11 . The method of  claim 1 , wherein step (a) further includes analyzing the sample for a change in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 18, 28-29 and 36-37; and,
 wherein step (b) further includes correlating an increase in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 18, 28-29 and 36-37, relative to the level of expression of the corresponding additional biomarker in normal liver as the at least one control sample, with the presence of HP-HCC subtype in the subject.   
     
     
         12 . The method of  claim 1 , wherein step (a) further includes analyzing the sample for a change in the level of expression of at least one or biomarkers identified by SEQ ID NOs: 1-8, 11-13, 17, 23 and 33-35; and
 wherein step (b) further includes correlating a decrease in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 1-8, 11-13, 17, 23 and 33-35, relative to the level of expression of the corresponding additional biomarker in normal live as the at least one control sample, with the presence of HP-HCC subtype in the subject.   
     
     
         13 . The method of  claim 1 , wherein step (a) further includes analyzing the sample for a change in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 1, 10, 31-35 and 38-39; and,
 wherein step (b) further includes correlating an increase in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 1, 10, 31-35 and 38-39, relative to the level of expression of the corresponding additional biomarker in normal liver as the at least one control sample, with the presence of HP-HCC subtype in the subject.   
     
     
         14 . The method of  claim 1 , wherein step (a) further includes analyzing the sample for a change in the level of expression of at least one or biomarkers identified by SEQ ID NOs :8-9, 11-12, 14-17, 19-24, 26-30 and 36-37; and
 wherein step (b) further includes correlating a decrease in the level of expression of at least one or more biomarkers identified by SEQ ID NOs: 8-9, 11-12, 14-17, 19-24, 26-30 and 36-37, relative to the level of expression of the corresponding additional biomarker in normal live as the at least one control sample, with the presence of HP-HCC subtype in the subject.   
     
     
         15 . The method of  claim 1 , wherein the sample is selected from the group consisting of liver tumor tissue, liver normal tissue, frozen biopsy tissue, paraffin-embedded biopsy tissue, serum, plasma, and combinations thereof. 
     
     
         16 . The method of  claim 1 , wherein the sample is analyzed by one or more methods selected from the group consisting of micro array techniques, PCR amplification, RNA hybridization, in situ hybridization, gel electrophoresis, and combinations thereof. 
     
     
         17 . The method of  claim 1 , wherein the sample is analyzed for 5 or more of the biomarkers; 10 or more of the biomarkers; 15 or more of the biomarkers; 20 or more of the biomarkers; 25 or more of the biomarkers; or for 30 or more of the biomarkers. 
     
     
         18 . A method of detecting a hepatocellular carcinoma (HCC) stem cell in a biological sample comprising:
 a) analyzing a sample from the subject, by laboratory assay, for a change in the level of expression of one or more biomarkers relative to the level of expression of a corresponding biomarker in at least one control sample, wherein the biomarker consists of at least one miR-181 biomarker;   b) correlating the change in the level of expression of the miR-181 biomarkers, relative to the level of expression of the corresponding biomarker in the control sample, with the presence of the HCC stem cell; and   c) determining the presence of the HCC stem cell if one or more of the miR-181 biomarkers in the sample are high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample, and   determining the absence of the HCC stem cell if biomarkers in the sample are not high or low relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample.   
     
     
         19 . The method of  claim 18 , wherein the miR-181 biomarker is selected from the group consisting of miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, and miR-181c. 
     
     
         20 . The method of  claim 19 , wherein the presence of: 2, 3, 4 and/or 5 miR-181 biomarkers are detected. 
     
     
         21 . The method of  claim 18 , wherein the sample is selected from the group consisting of liver tumor tissue, liver normal tissue, frozen biopsy tissue, paraffin-embedded biopsy tissue, serum, plasma, and combinations thereof. 
     
     
         22 . The method of  claim 18 , further comprising: d) correlating the presence of the HCC stem cell with the presence of a hepatocellular carcinoma cell in the sample. 
     
     
         23 . The method of  claim 22 , wherein the presence or absence of the miR-181 biomarker in the sample is analyzed by one or more of the techniques selected from the group consisting of micro array techniques, PCR amplification, RNA hybridization, in situ hybridization, gel electrophoresis, and combinations thereof. 
     
     
         24 . The method of  claim 18 , further comprising determining the prognosis of the subject. 
     
     
         25 . The method of  claim 24 , which further comprises treating the subject for the HCC subtype. 
     
     
         26 . The method of  claim 25 , wherein the treatment comprises at least one procedure selected from the group consisting of: hepatic resection, transplantation, percutaneous ethanol injection, radiofrequency ablation, chemoembolisation, chemotherapy, gene therapy, beta-catenin inhibition, and combinations thereof. 
     
     
         27 . The method of  claim 25 , wherein the treatment comprises administering to the subject a beta-catenin inhibitor. 
     
     
         28 . The method of  claim 25 , wherein the treatment comprises administering an effective amount of a nucleic acid complementary to a miR-181 selected from the group consisting of miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, miR-181c, and combinations thereof. 
     
     
         29 . A method of detecting the HSC-HCC subtype in a biological sample from a subject, comprising:
 a) analyzing a sample from the subject, by laboratory analysis, for the presence of an EpCAM+AFP+stem cells, and   b) correlating the presence of EpCAM+ AFP+ stem cells with the presence of the HSC-HCC subtype in the sample.   
     
     
         30 . The method of  claim 29 , wherein the stem cells are detected by assaying the sample for a miR-181 biomarker. 
     
     
         31 . The method of  claim 30 , wherein the miR-181 biomarkers are selected from the group consisting of miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, miR-181c, and combinations thereof. 
     
     
         32 . The method of  claim 29 , wherein the stem cells are detected by methods selected from the group consisting of immunofluorescence, in situ hybridization, immunohistochemical analysis, frozen activator cell sorting, side population analysis, cell surface marker detection methods, and combinations thereof. 
     
     
         33 . The method of  claim 1 , further including a step (d) of: treating a subject with HSC HCC subtype by administering a therapeutically effective amount of an agent selected from the group consisting of a beta-catenin inhibitor, at least one miR-181 biomarker inhibitor, and combinations thereof. 
     
     
         34 . The method of  claim 33 , wherein the miR-181 biomarker inhibitor comprises a reagent comprising a nucleic acid complementary to at least one biomarker selected from the group consisting of miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, miR-181c, and combinations thereof. 
     
     
         35 . The method of  claim 1 , further including a step (d) of:
 i) treating a subject with HSC-HCC subtype by administering an effective amount of a reagent comprising nucleic acids complementary to at least 5 biomarkers selected from the group consisting of biomarkers identified by SEQ ID NOs: 1-19; or   ii) treating a subject with BDE-HCC subtype by administering an effective amount of a reagent comprising nucleic acids complementary to at least 5 biomarkers selected from the group consisting of biomarkers identified by SEQ ID NOs: 2, 9-12, 14-17, 19-24, and 26-35; or,   iii) treating a subject with HP-HCC subtype by administering an effective amount of a reagent comprising nucleic acids complementary to at least 5 biomarkers selected from the group consisting of biomarkers identified by SEQ ID NOs: 1-8, 11-13, 17-18, 23, 28-29 and 33-39; or,   iv) treating a subject with MH-HCC subtype comprising administering an effective amount of a reagent comprising nucleic acids complementary to at least 5 biomarkers selected from the group consisting of biomarkers identified by SEQ ID NOs: 1, 8-12, 14-17, 19-24 and 26-39.   
     
     
         36 . The method of  claim 35 , wherein the treatment further comprises at least one procedure selected from the group consisting of hepatic resection, hepatic transplantation, percutaneous ethanol injection, radiofrequency ablation, chemoembolisation, chemotherapy, gene therapy, beta-catenin inhibition, and combinations thereof.