Tissue-specific methylation marker
Provided herein are compositions comprising tissue-specific markers for identifying a tissue of origin of a cell-free nucleic acid, e.g., a cell-free DNA molecule. Also provided herein are methods, compositions, and systems for identifying a tissue of origin of a cell-free nucleic acid by determining an absolute amount of cell-free nucleic acids comprising the tissue-specific marker. Also provided herein are methods, compositions, and systems for detecting a cancer in a tissue of an organism by analyzing tissue-specific markers.
1. A method of detecting and treating a metastasis in a subject, the method comprising selecting a subject having a metastasis of a cancer with a tissue of origin, and administering a treatment to the subject effective to treat the metastasis, wherein:
(a) the selecting comprises (i) receiving results of an assay of a first biological sample of the subject measuring an absolute amount of cell-free DNA comprising a target sequence having a first methylation status (ii) identifying the measured absolute amount as being above a reference amount, and (iii) identifying a first non-cancer tissue as a location of the metastasis;
(b) the first biological sample comprises cell-free DNA molecules from the cancer, the first non-cancer tissue, and one or more second non-cancer tissues;
(c) the tissue of origin, the first non-cancer tissue, and the one or more second non-cancer tissues are different;
(d) cell-free DNA molecules from the first non-cancer tissue comprise the first methylation status of the target sequence;
(e) the first methylation status is distinct from one or more second methylation statuses of the target sequence in cell-free DNA molecules from the cancer and from the one or more second non-cancer tissues;
(f) the reference amount is an absolute amount of cell-free DNA comprising the target sequence having the first methylation status for one or more subjects without metastasis in the first non-cancer tissue; and
(g) the treatment is selected from the group consisting of chemotherapy, radiation therapy, surgical removal of tumor tissues, immunotherapy, hormone therapy, and stem cell therapy.
2. The method of claim 1 , wherein the first biological sample comprises isolated cell-free DNA molecules comprising the target sequence.
3. The method of claim 2 , wherein the assay comprises isolating the cell-free DNA molecules comprising the target sequence in an oil emulsion.
4. The method of claim 1 , wherein the assay comprises hybridizing the cell-free DNA molecules comprising the target sequence to a probe, and wherein the probe hybridizes to the target sequence when a methylation site of the target sequence is methylated in the first biological sample, or the probe hybridizes to the target sequence when a methylation site of the target sequence is unmethylated in the first biological sample.
5. The method of claim 1 , wherein the assay comprises amplifying the cell-free DNA molecules using a pair of primers, wherein an affinity of hybridization of at least one primer of the pair of primers to the target sequence depends on the first methylation status of the target sequence, and wherein (i) the at least one primer of the pair of primers hybridizes to the target sequence when a methylation site of the target sequence is methylated in the first biological sample, or (ii) the at least one primer of the pair of primers hybridizes to the target sequence when a methylation site of the target sequence is unmethylated in the first biological sample.
6. The method of claim 1 , wherein the assay comprises bisulfite conversion of unmethylated cytosine residues in the cell-free DNA molecules to uracil.
7. The method of claim 1 , wherein the assay comprises performing methylation-aware sequencing of cell-free DNA molecules from the first biological sample.
8. The method of claim 1 , wherein the target sequence comprises at least 5 methylation sites.
9. The method of claim 1 , wherein the first methylation status comprises methylation density for individual sites within the target sequence, a distribution of methylated/unmethylated sites over a contiguous region within the target sequence, a pattern or level of methylation for each individual methylation site within the target sequence, or non-CpG methylation.
10. The method of claim 1 , wherein the target sequence comprises a higher methylation density in the first non-cancer tissue than in the cancer and in the one or more second non-cancer tissues.
11. The method of claim 10 , wherein the target sequence comprises a methylation density in the first non-cancer tissue that is more than 50%, and wherein the target sequence comprises a methylation density in the cancer and in the one or more second non-cancer tissues that is less than 20%.
12. The method of claim 1 , wherein the first non-cancer tissue comprises liver tissue, and the target sequence comprises a polynucleotide sequence having at least 60% identity to SEQ ID NO: 1.
13. The method of claim 12 , wherein the first non-cancer tissue comprises liver tissue, and wherein the assay comprises amplification using a primer comprising SEQ ID NO: 2, a primer comprising SEQ ID NO: 3, or both, or use of a detectably-labeled probe comprising SEQ ID NO: 4 for detection of the target sequence.
14. The method of claim 13 , wherein the amplification further comprises use of a primer comprising SEQ ID NO: 5, a primer comprising SEQ ID NO: 6, or both, or use of a detectably-labeled probe comprising SEQ ID NO: 7 for detection of the target sequence.
15. The method of claim 1 , wherein the first non-cancer tissue comprises colon tissue, and the target sequence comprises a polynucleotide sequence having at least 60% identity to SEQ ID NO: 8.
16. The method of claim 15 , wherein the first non-cancer tissue comprises colon tissue, and wherein the assay comprises amplification using a primer comprising SEQ ID NO: 9, a primer comprising SEQ ID NO: 10, or both, or use of a detectably-labeled probe comprising SEQ ID NO: 11 for detection of the target sequence.
17. The method of claim 16 , wherein the amplification further comprises use of a primer comprising SEQ ID NO: 12, a primer comprising SEQ ID NO: 13, or both, or use of a detectably-labeled probe comprising SEQ ID NO: 14 for detection of the target sequence.
18. The method of claim 1 , wherein the cancer is selected from the group consisting of: bladder cancer, bone cancer, brain tumor, breast cancer, carcinoma of cervix, colorectal cancer, esophageal cancer, gastrointestinal cancer, hematopoietic malignancy, head and neck squamous cell carcinoma, leukemia, liver cancer, lung cancer, lymphoma, myeloma, nasal cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, ovarian cancer, prostate cancer, sarcoma, stomach cancer, melanoma, and thyroid cancer.
19. The method of claim 1 , further comprising determining a classification of the cancer in the first non-cancer tissue, which comprises receiving results of an assay assessing cell-free nucleic acid molecules from a second biological sample from the subject, and wherein the assessing comprises determining methylation profile, copy number variations, single polymorphism (SNP) profile, or fragmentation pattern of the cell-free nucleic acid molecules from the second biological sample.
20. The method of claim 1 , wherein the treatment is selected from the group consisting of: chemotherapy, radiation therapy, and surgical removal of tumor tissues.