DNA integrity assay (DIA) for cancer diagnostics, using confocal fluorescence spectroscopy

• US 8,835,110 B2
• Filed: 11/04/2009
• Issued: 09/16/2014
• Est. Priority Date: 11/04/2008
• Status: Active Grant

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Wang, Tza-Huei, Liu, Kelvin J., Shih, Ie-Ming
• Primary Examiner(s): MARSCHEL, ARDIN H
• Application Number:
  US13/120,060
• Publication Number:
  US 20110171741A1
• Time in Patent Office: 1,777 Days
• Field of Search: 435/6.1, 435/6.14
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates, e.g., to a method for determining the size distribution of DNA molecules in a sample comprising cell-free nucleic acid, comprising labeling the DNA with a fluorescent dye in a stoichiometric manner, subjecting the DNA to molecular spectroscopy (e.g., cylindrical illumination confocal spectroscopy), analyzing suitable fluorescent burst parameters of the labeled DNA, and conducting single molecule DNA integrity analysis of the labeled DNA molecules in the sample. In one embodiment of the invention, the method is used as a diagnostic method for detecting cancer.

Claims

1. A method for determining the size distribution of cell-free DNA molecules in a sample, comprising

labeling the cell-free DNA molecules in the sample with a fluorescent dye in a stoichiometric manner,

subjecting the labeled DNA to single molecule spectroscopy, and

measuring fluorescent burst parameters and fluorescent burst rate of the labeled DNA,

wherein the fluorescent burst parameters, which comprise burst size, burst height and/or transit time, indicate the length of each of the DNA molecules;

and the fluorescent burst rate indicates the abundance of each size of DNA molecule, and

conducting single molecule DNA integrity analysis (smDIA) of the labeled DNA molecules in the sample, thereby determining the size distribution of the cell-free DNA molecules.

2. The method of claim 1, wherein the single molecule spectroscopy in carried out using flow cytometry.

3. The method of claim 1, wherein the single molecule spectroscopy is carried out using nanochannels.

4. The method of claim 1, wherein the single molecule spectroscopy is cylindrical illumination confocal spectroscopy (CICS).

5. The method of claim 1, wherein the single molecule spectroscopy is microfluidic cylindrical illumination confocal spectroscopy (μ

CICS).

6. The method of claim 1, wherein the single molecule spectroscopy is conducted by

causing the sample comprising the labeled DNA molecules to flow through a channel of a fluidic device,

illuminating a portion of the fluid flowing through the channel substantially uniformly with a sheet-like beam of light that activates the fluorescent dye,

directing fluorescing light from the labeled DNA molecules to be detected through a substantially rectangular aperture of an aperture stop to be detected,

wherein the substantially rectangular aperture is constructed and arranged to substantially match a width of said channel, and

detecting the labeled DNA molecules based on light directed through the substantially rectangular aperture.

7. The method of claim 1, wherein the smDIA comprises

correlating substantially quantized light pulses with the lengths of the DNA molecules detected, and

calculating the single molecule DNA integrity (smDI, the ratio of long bursts to short bursts) of DNA molecules in the sample.

8. The method of claim 7, wherein the distinction between long and short DNA bursts is defined by a predetermined cut-off value or range of values.

9. The method of claim 7, further comprising comparing the smDI to a positive and/or a negative reference standard.

10. The method of claim 1, wherein the sample is a body fluid.

11. The method of claim 1, which is a method for determining if a subject is likely to have a cancer, wherein

the sample is a body fluid from the subject,

the single molecule spectroscopy is CICS or μ

CICS, and

the smDIA comprises correlating substantially quantized light pulses with the lengths of the DNA molecules detected, and calculating the single molecule DNA integrity (smDI) of DNA molecules in the sample,

wherein the smDI is compared to a predetermined cut-off value, and

wherein an smDI of at least about 0.04, 0.06, 0.08, 0.10, 0.125, 0.15, 0.175, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 indicates that the subject is likely to have the cancer.

12. The method of claim 1, which is a method for determining the tumor load in a subject compared to one or more reference standards, wherein

the sample is a body fluid from the subject,

the single molecule spectroscopy is CICS or μ

CICS, and

the single molecule DNA integrity analysis (smDIA) comprises correlating substantially quantized light pulses with the lengths of the DNA molecules detected, and calculating the single molecule DNA integrity (smDI) of DNA molecules in the sample,

further comprising

comparing the smDI of the DNA molecules in the sample to a positive and/or a negative reference standard,

wherein the negative and positive reference standards are representative of defined amounts of tumor load.

13. The method of claim 12, which is a method to determine if a subject is likely to have a cancer,

wherein the negative reference standard is representative of the tumor load in a subject that does not have the cancer;

and the positive reference standard is representative of the tumor load in a subject that has the cancer,

wherein an smDI of the DNA molecules in the sample that is statistically significantly greater than the negative reference standard, and/or is approximately the same as the positive reference standard, indicates that the subject is likely to have the cancer.

14. The method of claim 13, which is a method for detecting a cancer at stage 1 or stage 2.

15. The method of claim 12, which is a method to stage a cancer in the subject,

wherein the negative reference standard is representative of the tumor load in a subject that does not have the cancer, or has an early stage cancer, and the positive reference standard is representative of the tumor load in a subject that has a late stage cancer,

wherein an smDI that is approximately the same as the negative standard indicates that the subject is likely to have an early stage cancer, and an smDI that is statistically significantly greater than the negative reference standard, or is approximately the same as the positive standard, indicates that the subject is likely to have a more advanced stage of the cancer.

16. The method of claim 12, which is a method to determine if a tumor is benign or malignant,

wherein the negative reference standard is representative of the tumor load in a subject that has a benign tumor, and the positive reference standard is representative of tumor load in a subject that has a malignant cancer,

wherein an smDI that is approximately the same as the negative standard indicates that the subject is likely to have a benign tumor, and an smDI that is statistically significantly greater than the negative reference standard, or is approximately the same as the positive standard, indicates that the subject is likely to have a malignant tumor.

17. The method of claim 12, which is a method for monitoring the progress or prognosis of a cancer in a subject, comprising determining the smDI at various times during the course of the cancer,

wherein a decrease in the smDI over the course of the analysis indicates that cancer is going into remission and that the prognosis is likely to be good, and an increase in the smDI over the course of the analysis indicates that cancer is progressing and that the prognosis is not likely to be good.

18. The method of claim 12, which is a method for evaluating the efficacy of a cancer treatment, comprising measuring the smDI at different times during the treatment,

wherein a change in the smDI over the course of the analysis indicates whether the cancer treatment is efficacious.

19. The method of claim 1, wherein the fluorescent dye is an intercalating dye, is covalently bound to the DNA through a coupling reaction, is introduced into the DNA though an enzymatic reaction, or is incorporated into the DNA by the binding of a labeled fluorescent probe.

20. The method of claim 1, wherein the fluorescent dye is TOTO-3.

21. The method of claim 1, wherein the method is high throughput.

22. The method of claim 1, wherein the subject is human.

23. The method of claim 1, wherein DNA in the sample is not separated from other components in the sample.

24. The method of claim 1, further comprising introducing a fluorescent tracer particle during single molecule spectroscopy to control for flow velocity, focus position and/or fluorescent intensity.

25. The method of claim 11, wherein the cancer is ovarian, breast, lung, prostate, colorectal, esophageal, pancreatic, prostate, head and neck, gastrointestinal, bladder, kidney, liver, lung, or brain cancer, gynecological, urological or brain cancer, or a leukemia, lymphoma, myeloma or melanoma.

26. The method of claim 11, wherein the sample is generated from a pleural effusion, ascites sample, plasma, serum, whole blood, urine, ductal lavage or sputum.

27. A method for determining the size distribution of DNA molecules in a sample, comprising

labeling the DNA molecules in the sample with a fluorescent dye in a stoichiometric manner,

subjecting the labeled DNA to single molecule cylindrical illumination confocal spectroscopy (CICS), such that a uniform detection profile is produced, and

measuring fluorescent burst parameters and fluorescent burst rate of the labeled DNA,

wherein the fluorescent burst parameters, which comprise burst size, burst height and/or transit time, indicate the length of each of the DNA molecules;

and the fluorescent burst rate indicates the abundance of each size of DNA molecule, and

conducting single molecule DNA integrity analysis (smDIA) of the labeled DNA molecules in the sample, thereby determining the size distribution of the DNA molecules.

28. The method of claim 27, wherein the CICS is conducted in a cylindrical illumination confocal spectroscopy system as described in Example III.