Use of double-stranded DNA in exosomes: a novel biomarker in cancer detection
The present invention is directed to methods of prognosing, treating, or managing treatment of cancer in a subject. These methods involve selecting a subject having cancer, obtaining, from the selected subject, a sample containing exosomes, recovering the exosomes from the sample, and isolating the double-stranded DNA from within the exosomes. The isolated double-stranded DNA is then used to detect the presence or absence of one or more genetic mutations associated with cancer, quantify the amount of isolated double-stranded DNA from the recovered exosomes in the sample, detect the methylation status of the isolated double-stranded DNA, or quantify the amount isolated double-stranded DNA able to enter a recipient cell. The prognosing, treating, or managing treatment is carried out based on this information.
1. A method of prognosing cancer in a subject, said method comprising:
selecting a subject having cancer;
obtaining, from the selected subject, a sample containing exosomes;
recovering the exosomes from the sample;
removing DNA from outside of the recovered exosomes;
isolating double-stranded DNA, a majority of which has a size of less than 2500 bp, from within said exosomes after said removing;
contacting the isolated double-stranded DNA with one or more reagents suitable to: (1) detect the presence or absence of one or more genetic mutations in the isolated double-stranded DNA that are associated with cancer, (2) quantify the amount of isolated double-stranded DNA from the recovered exosomes in the sample, or (3) detect the methylation status of the isolated double-stranded DNA;
determining a difference between (1) the presence or absence of one or more genetic mutations in the isolated double-stranded DNA that are associated with cancer and a reference sample, (2) the quantified amount of isolated double-stranded DNA from the recovered exosomes in the sample and a reference sample, or (3) the methylation status of the isolated double-stranded DNA and a reference sample; and
prognosing the cancer based on said determining, wherein a difference in the presence or absence of one or more genetic mutations in the isolated double-stranded DNA, the quantified amount of isolated double-stranded DNA from the recovered exosomes, or the methylation status of the isolated double-stranded DNA relative to the reference sample prognoses the cancer.
2. The method of claim 1 , wherein said sample is blood.
3. The method of claim 1 , wherein said contacting is carried out by detecting the presence or absence of one or more genetic mutations in the isolated double- stranded DNA that are associated with cancer.
4. The method of claim 1 , wherein said contacting is suitable for quantifying the amount of isolated double-stranded DNA from the recovered exosomes in the sample.
5. The method of claim 4 , wherein said quantifying is carried out by comparing the amount of isolated double-stranded DNA to that in a prior sample obtained from the selected subject and subjected to said recovering, said isolating, and said contacting.
6. The method of claim 4 , wherein said quantifying is carried out by comparing the amount of isolated double-stranded DNA to a standard.
7. The method of claim 1 , wherein said contacting is suitable for detecting the methylation status of the isolated double-stranded DNA.
8. The method of claim 1 , wherein said prognosing is carried out to predict sites of metastasis, to determine the stage of the cancer, or to identify the location of a primary tumor in the subject.
9. The method of claim 1 further comprising:
selecting a suitable cancer therapeutic based on said prognosing and
administering the selected cancer therapeutic to said selected subject.
10. The method of claim 1 , wherein the cancer is selected from the group consisting of melanoma, breast cancer, lung cancer, and leukemia.
11. The method of claim 1 , wherein said prognosing is carried out to predict the metastatic potential of the cancer.
12. The method of claim 1 , wherein said one or more reagents suitable for detecting the presence or absence of one or more mutations in the sample are suitable for carrying out allele-specific polymerase chain reaction (PCR) or genomic sequencing.
13. The method of claim 3 , wherein the selected subject has melanoma and the presence or absence of a mutation in BRAF is detected.
14. The method of claim 3 , wherein the presence or absence of one or more mutations in EGFR is detected.
15. The method of claim 14 , wherein the one or more mutations in EGFR is selected from the group consisting of an exon 19 deletion, L858R, T790M, and any combination thereof.
16. The method of claim 3 , wherein said one or more genetic mutations are mutations in genes selected from the group consisting of BRA, EGFR, APC, NOTCH1, HRAS, KRAS, NRAS, MET, p53, PTEN, HER2, FLT3, BRCA1, BRCA2, PIK3CA, KIT, RET, AKT, ABL, CDK4, MYC, RAF, PDGFR, BCR-ABL, NPM1, CEBPalpha, and SRC.
17. The method of claim 8 , wherein said prognosing is carried out to predict sites of metastasis.
18. The method of claim 8 , wherein said prognosing is carried out to determine the stage of the cancer.
19. The method of claim 8 , wherein said prognosing is carried out to identify the location of a primary tumor in the subject.
20. The method of claim 1 , wherein the isolated double-stranded has a size range of 100 bp to 2500 bp.
21. A method comprising:
selecting a subject having cancer;
obtaining, from the selected subject, a sample containing exosomes;
recovering the exosomes from the sample;
removing DNA from outside of the recovered exosomes;
isolating double-stranded DNA, a majority of which has a size of less than 2500 bp, from within said exosomes after said removing; and
contacting the isolated double-stranded DNA with one or more reagents suitable to: (1) detect the presence or absence of one or more genetic mutations in the isolated double-stranded DNA that are associated with cancer, (2) quantify the amount of isolated double-stranded DNA from the recovered exosomes in the sample, or (3) detect the methylation status of the isolated double-stranded DNA.
22. The method of claim 21 further comprising:
determining a difference between (1) the presence or absence of one or more genetic mutations in the isolated double-stranded DNA that are associated with cancer and a reference sample, (2) the quantified amount of isolated double-stranded DNA from the recovered exosomes in the sample and a reference sample, or (3) the methylation status of the isolated double-stranded DNA and a reference sample after said contacting.
23. The method of claim 21 , wherein said sample is blood.
24. The method of claim 21 , wherein said one or more reagents suitable for detecting the presence or absence of one or more mutations in the sample are suitable for carrying out allele-specific polymerase chain reaction (PCR) or genomic sequencing.