5-hydroxymethylcytosine in human cancer

• US 9,297,806 B2
• Filed: 08/01/2013
• Issued: 03/29/2016
• Est. Priority Date: 08/01/2012
• Status: Active Grant

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Yegnasubramanian, Srinivasan, Haffner, Michael Christoph, Chaux, Alcides, Nelson, William G., DeMarzo, Angelo M.
• Primary Examiner(s): Cheu, Jacob
• Application Number:
  US13/956,879
• Publication Number:
  US 20140038183A1
• Time in Patent Office: 971 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: G01N 2800/50 Determining the risk of developing a disease
• CPC Class Codes: G01N 33/57496 involving intracellular compounds

Abstract

The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions useful for diagnosing or predicting cancer in a patient. In one embodiment, a method for identifying a patient as having cancer comprises the steps of (a) providing a formalin-fixed, paraffin-embedded or fresh frozen sample of patient tissue; (b) steaming the sample in antigen retrieval buffer; (c) incubating the sample in hydrochloric acid (HCl); (d) incubating the sample with an affinity reagent specific for 5hmC under conditions to form a complex between the affinity reagent and 5-hydroxymethylcytosine (5hmC) present in the sample; (e) detecting the complexes formed between 5hmC and the affinity reagent with secondary detection reagents; (f) quantifying 5hmC levels; and (g) identifying the patient as having cancer if the 5hmC levels in the sample are reduced as compared to a control.

Claims

1. A method for identifying a patient as having cancer comprising the steps of:

a. providing a formalin-fixed, paraffin-embedded or fresh frozen sample from the patient tissue;

b. steaming the sample in antigen retrieval buffer;

c. incubating the sample in hydrochloric acid (HCl);

d. incubating the sample with an affinity reagent specific for 5hmC under conditions to form a complex between the affinity reagent and 5-hydroxymethylcytosine (5hmC) present in the sample;

e. detecting the complexes formed between 5hmC and the affinity reagent with secondary detection reagents;

f. quantifying 5hmC levels; and

g. identifying the patient as having cancer if the 5hmC levels in the sample are reduced as compared to a control.

2. The method of claim 1, wherein the patient sample is from any tissue with a suspected neoplastic growth.

3. The method of claim 2, wherein the neoplastic growth is a cancer from prostate, breast, colon or leukemia.

4. The method of claim 3, wherein the quantifying step is accomplished by semi-quantitative scoring or by using an image analysis software program.

5. A method for identifying a patient as having cancer comprising the steps of:

a. providing a formalin-fixed, paraffin-embedded sample from the patient tissue;

b. steaming the sample in citrate buffer;

c. incubating the sample in hydrochloric acid (HCl);

d. immunolabeling 5-hydroxymethylcytosine (5-hmC) with an antibody;

e. detecting the immunocomplexes formed between 5-hmC and the antibody;

f. quantifying 5-hmC levels; and

g. identifying the patient as having cancer if the 5-hmC levels in the sample are reduced as compared to a control.

6. The method of claim 5, wherein the patient tissue is from the prostate, breast or colon.

7. The method of claim 5, wherein the detection step comprises labeling the immunocomplexes with secondary antibodies conjugated with a fluorescent dye and visualizing using a fluorescence microscope.

8. The method of claim 7, wherein the quantifying step is accomplished using an image analysis software program.