Methods and compositions for the diagnosis for early hepatocellular carcinoma

• US 8,030,013 B2
• Filed: 04/13/2007
• Issued: 10/04/2011
• Est. Priority Date: 04/14/2006
• Status: Active Grant

• Current Assignee: Mount Sinai School of Medicine (Sinai Health System)
• Original Assignee: Mount Sinai School of Medicine (Sinai Health System)
• Inventors: Llovet, Josep M., Friedman, Scott L., Schwartz, Myron
• Primary Examiner(s): BRISTOL, LYNN ANNE
• Application Number:
  US11/735,402
• Publication Number:
  US 20080038736A1
• Time in Patent Office: 1,635 Days
• Field of Search: 435/7.23
• US Class Current: 435/7.23
• CPC Class Codes: G01N 33/57438 of liver, pancreas or kidney
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods and compositions are provide to allow discrimination of dysplastic nodules from early HCC nodules. More specifically, it has been determined that TERT, GPC3, gankyrin, survivin, TOP2A, LYVE1, Ecadherin, IGFBP3, PDGFRA, TGFA, cyclin D1 and HGF are differentially expressed in HCC as compared to normal liver cells and liver cells that have dysplastic, non-cancerous nodules.

Claims

1. A method of determining whether a patient is afflicted with hepatocellular carcinoma (HCC), the method comprising:

a) determining the level of expression of at least two markers in a patient sample comprising a liver nodule two centimeters or less in diameter, wherein the markers are selected from the group consisting of GPC3, survivin and LYVE1;

b) determining the level of expression of the markers in a control sample;

c) comparing the level of expression of each of the markers in the patient sample and in the control sample; and

d) identifying the patient as being afflicted with HCC when there is a difference between the level of expression of the markers in the patient sample and the control sample, thereby determining whether the patient is afflicted with HCC.

2. The method of claim 1, wherein the level of expression from a control sample is determined by a method selected from:

a) a level determined from liver cells from the patient which are non-cancerous;

b) a level determined from liver cells from a subject having benign dysplasia or normal tissue; and

c) a predetermined level using an average of the levels of expression from a population of subjects having benign dysplastic nodules or normal liver cells.

3. The method of claim 1, wherein the markers correspond to a secreted protein.

4. The method of claim 1, wherein the markers comprise transcribed polynucleotides or portions thereof.

5. The method of claim 3, wherein the presence of the markers are detected using reagents which specifically bind with the markers.

6. The method of claim 5, wherein the reagents are each selected from the group consisting of an antibody, and an antibody fragment.

7. The method of claim 4, wherein detecting the transcribed polynucleotides comprises amplifying the transcribed polynucleotides.

8. The method of claim 4, wherein the levels of expression of the markers in the patient sample are determined by detecting the presence in the patient sample of a transcribed polynucleotide which anneals with at least one of the markers or a portion thereof under stringent hybridization conditions comprising 42°

C. in a hybridization solution comprising 50% formamide, 1% SDS, 1M NaCl, and 10% dextran sulfate followed by washing twice for 30 minutes at 60°

C. in a wash solution comprising 0.1×

SSC and 1% SDS.

9. The method of claim 1, wherein the level of expression of at least one of the markers in the patient sample differs from the level of expression of that marker in the control sample by a factor of at least 2.

10. The method of claim 1, wherein said patient is identified as having HCC when said patient sample comprises up-regulation of GPC3 and survivin.

11. The method of claim 1, wherein said patient is identified as having HCC when said patient sample comprises a down-regulation of LYVE1.

12. The method of claim 1, wherein said method further comprises identification of the patient as having HCC when said patient sample comprises a down-regulation of LYVE1.

13. The method of claim 1, wherein said at least two markers are GPC3 and LYVE1.

14. The method of claim 1, wherein the expression of the markers is determined by using real-time RT-PCR.

15. The method according to claim 1 wherein the patient sample liver nodule is 1.5 centimeters or less in diameter.

16. The method according to claim 15 wherein the patient sample liver nodule is 1.2 centimeters or less in diameter.

17. A method for monitoring the progression of HCC in a patient, the method comprising:

a) determining the level or expression of at least two markers in a patient sample comprising a liver nodule two centimeters or less in diameter from a first point in time, wherein the markers are selected from the group consisting of GPC3, survivin and LYVE1;

b) determining the level of expression of the markers in a sample comprising a liver nodule of any size from the patient at a subsequent point in time; and

c) comparing the levels of expression detected in steps a) and b), thereby monitoring the progression of HCC in the patient, wherein a change in expression of the markers is indicative of either progression or regression of HCC.