Methylation markers for diagnosing hepatocellular carcinoma and lung cancer

• US 10,513,739 B2
• Filed: 03/02/2018
• Issued: 12/24/2019
• Est. Priority Date: 03/02/2017
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California), Youhealth Oncotech Limited
• Original Assignee: Youhealth Oncotech Limited
• Inventors: Zhang, Kang, Hou, Rui, Zheng, Lianghong
• Primary Examiner(s): Myers, Carla J
• Application Number:
  US15/910,857
• Publication Number:
  US 20180274039A1
• Time in Patent Office: 662 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/154 Methylation markers

Abstract

Disclosed herein, in certain embodiments, are methods and kits for diagnosing a subject as having hepatocellular carcinoma (HCC) or lung cancer. In some instances, also described herein are methods of determining the prognosis of the subject having HCC or lung cancer. In additional instances, described herein are methods of determining the specific staging of HCC or lung cancer in a subject.

Claims

1. A method of detecting the methylation pattern of a plurality of markers in a human subject suspected of having hepatocellular carcinoma (HCC), comprising:

a) processing a biological sample obtained from the human subject with a deaminating agent to generate treated DNA comprising deaminated nucleotides; and

b) detecting the methylation pattern of cg26668608, cg11606215, cg18196829, cg25459300, cg25574765, cg11397370, and cg11825899, and one or more markers selected from the group consisting of cg10428836, cg25754195, cg05205842, cg24067911, cg24067911, cg23211949, cg17213048, cg23461741, cg06482904, cg07459019, cg20490031, and cg01643250, by

i) contacting the treated DNA with a first set and a second set of nucleic acid probes, wherein the first set of nucleic acid probes hybridizes to seven markers and no more than seven markers, wherein the markers are cg26668608, cg11606215, cg18196829, cg25459300, cg25574765, cg11397370, and cg11825899, and wherein the second set of nucleic acid probes hybridizes to at least one but no more than 11 markers selected from the group consisting of cg10428836, cg25754195, cg05205842, cg24067911, cg24067911, cg23211949, cg17213048, cg23461741, cg06482904, cg07459019, cg20490031, and cg01643250, and combinations thereof; and

ii) performing a DNA sequencing analysis to determine the methylation pattern of the plurality of markers.

2. The method of claim 1, wherein the method comprises detecting the methylation pattern of cg10428836, cg26668608, cg25754195, cg05205842, cg11606215, cg24067911, cg18196829, cg23211949, cg17213048, cg25459300, cg23461741, cg06482904, cg25574765, cg07459019, cg20490031, cg01643250, cg11397370, and cg11825899.

3. The method of claim 1, wherein the human subject has a stage I, stage II, stage III, or stage IV hepatocellular carcinoma (HCC).

4. The method of claim 1, wherein the biological sample comprises a blood sample, a tissue biopsy sample, or circulating tumor cells.

5. The method of claim 1, wherein the probe is a padlock probe.

6. The method of claim 1, wherein the DNA sequencing analysis comprises a digital droplet PCR analysis.

7. A method of detecting the methylation pattern of a plurality of markers in a human subject suspected of having hepatocellular carcinoma (HCC), the method comprising:

(a) processing a biological sample obtained from the human subject with a deaminating agent to generate treated DNA comprising deaminated nucleotides; and

(b) detecting the methylation pattern of cg26668608, cg11606215, cg18196829, cg25459300, cg25574765, cg11397370, and cg11825899, by

(i) contacting the treated DNA with a set of nucleic acid probes that hybridizes to seven markers and no more than seven markers, and wherein the markers are cg26668608, cg11606215, cg18196829, cg25459300, cg25574765, cg11397370, and cg11825899; and

(ii) performing a DNA sequencing analysis to determine the methylation pattern of the plurality of markers.

8. The method of claim 7, wherein the probe is a padlock probe.

9. The method of claim 7, wherein the DNA sequencing analysis comprises a digital droplet PCR analysis.

10. The method of claim 7, wherein the human subject has a stage I, stage II, stage III, or stage IV HCC.

11. The method of claim 7, wherein the biological sample comprises a blood sample, a tissue biopsy sample, or circulating tumor cells.