# US 8,465,917 B2 · Methods for determining heptocellular carcinoma subtype and detecting hepatic cancer stem cells

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- Number: US 8,465,917 B2
- Document ID: US8465917B2
- Status: Granted
- Kind: B2
- Application number: 12/663,586
- Filed: 2008-06-09
- Published: 2010-08-05
- Granted: 2013-06-18
- Earliest claimed: 2007-06-08
- Assignees: The Ohio State University Research Foundation; The United States of America as represented by the Secretary, Department of Health and Human Services; National Institutes of Health, Office of Technology Transfer
- Applicants: Xin Wei Wang; Junfang Ji; Taro Yamashita; Carlo M. Croce
- Inventors: Xin Wei Wang; Junfang Ji; Taro Yamashita; Carlo M. Croce
- Web page: https://patentgrep.com/patent/US8465917B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- A61P 35/00: Antineoplastic agents
- G01N 33/6893: {related to diseases not provided for elsewhere}
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/158: Expression markers
- C12Q 2600/178: miRNA, siRNA or ncRNA
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention provides a method of determining an HCC subtype in a subject comprising 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. The invention further provides methods of detecting HCC stem cells in a sample. Additionally, the invention provides methods and compositions for treating subjects with HCC that take advantage of the biomarkers associated with HCC stem cells.

## Claims

### Claim 1

A method of determining a hepatocellular carcinoma (HCC) subtype in a subject comprising
a) obtaining a sample from the subject,
b) analyzing the sample, 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 miR-221 and miR-222, and
c) 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 bile duct epithelium-like hepatocellular carcinoma (BDE-HCC) subtype of HCC in the subject,
d) determining a hepatocellular carcinoma (HCC) subtype as bile duct epithelium-like hepatocellular carcinoma (BDE-HCC) subtype if biomarkers for at least one of miR-221 and miR-222 in the sample are high 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 biomarkers for at least one of miR-221 and miR-222 in the sample are not high relative to the level of expression of the corresponding biomarker in normal liver as the at least one control sample.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

The method of claim 1, further including a step (e) of: further discriminating bile duct epithelial (BDE-HCC) subtype from hepatic stem cell (HSC-HCC) subtype and hepatocytic progenitor subtype (HP-HCC) 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.

### Claim 5 (depends on 1)

The method of claim 1, wherein step (b) further comprises determining an increased level of expression of at least both of miR-221 and miR-222 biomarkers.

### Claim 6 (depends on 1)

The method of claim 1, wherein step (b) further includes analyzing the sample for a change in the level of expression of at least one or more additional biomarkers identified by miR-106a, miR-106b, and miR-17, and
wherein step (c) further includes correlating an increase in the level of expression of at least one or more additional biomarkers identified by miR-106a, miR-106b, and miR-17, 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.

### Claim 7 (depends on 1)

The method of claim 1, wherein step (b) further includes analyzing the sample for a change in the level of expression of at least one or more additional biomarkers identified by miR-25, miR-32, miR-92-1, miR-122a, miR-125b-1, and miR-125b-2, and
wherein step (c) further includes correlating a decrease in the level of expression of at least one or more additional biomarkers identified by miR-25, miR-32, miR-92-1, miR-122a, miR-125b-1, and miR-125b-2, 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.

### Claim 8 (depends on 1)

The method of claim 1, wherein step (b) further includes analyzing the sample for a change in the level of expression of at least one or more additional biomarkers identified by miR-106a, miR-17, miR-32, miR-92-1, miR-122a, miR-125b-1, and miR-125b-2, and
wherein step (c) further includes correlating a decrease in the level of expression of at least one or more additional biomarkers identified by miR-106a, miR-17, miR-32, miR-92-1, miR-122a, miR-125b-1, and miR-125b-2, relative to the level of expression of the corresponding additional biomarker in bile duct epithelium-like non-hepatocellular carcinoma (BDE-non-HCC) as the at least one control sample, with the presence of BDE-HCC subtype in the subject.