KR-20260066163-A - TISSUE-SPECIFIC METHYLATION MARKER

Abstract

The present invention provides a composition comprising a tissue-specific marker that identifies the tissue of origin of cell-free nucleic acids, for example, cell-free DNA molecules. The present invention also provides a method, composition, and system for identifying the tissue of origin of cell-free nucleic acids by determining the absolute amount of cell-free nucleic acids containing said tissue-specific marker. The present invention also provides a method, composition, and system for detecting cancer in the tissues of an organism by analyzing the tissue-specific marker.

Inventors

• 로 육-밍 데니스
• 치우 로싸 와이 콴
• 챤 콴 치
• 가이 환크시아
• 지 루

Assignees

• 그레일, 인코포레이티드.

Dates

Publication Date

20260512

Application Date

20190315

Priority Date

20180315

Claims (20)

1. (a) A step of obtaining a cell-free DNA molecule from a first biological sample of an organism having cancer of the first tissue; (b) a step of performing an assay on a cell-free DNA molecule to determine a first methylation state of a target sequence within the cell-free DNA molecule, wherein the first methylation state of the target sequence indicates that the cell-free DNA molecule containing the target sequence is derived from a second tissue of an organism, and the first tissue and the second tissue are different; (c) determining the absolute amount of cell-free DNA molecules from a first biological sample containing a target sequence having a first methylation state; and (d) A step to determine whether the organism has cancer in the second tissue based on the absolute amount A method for determining whether an organism having cancer in a first tissue has cancer located in a second tissue, comprising
2. A method according to claim 1, wherein the methylation state includes a methylation level.
3. A method according to claim 1 or 2, wherein the assay comprises the step of isolating a cell-free DNA molecule containing a target sequence from a first biological sample.
4. A method according to paragraph 3, wherein the assay comprises the step of isolating a cell-free DNA molecule containing a target sequence from an oil emulsion.
5. A method according to any one of claims 1 to 4, wherein the assay comprises the step of hybridizing a cell-free DNA molecule containing a target sequence to a probe.
6. In paragraph 5, the method wherein the probe hybridizes to the target sequence.
7. A method according to claim 6, wherein the affinity of the hybridization of the probe to the target sequence is determined by the first methylation state of the target sequence in the first biological sample.
8. In claim 7, the probe is a method of hybridizing to the target sequence when the methylation site of the target sequence is methylated in the first biological sample.
9. In claim 7, the probe is a method of hybridizing to the target sequence when the methylated site of the target sequence is unmethylated in the first biological sample.
10. A method according to any one of claims 5 to 9, wherein the assay includes the step of detecting hybridization of a probe to a target sequence.
11. A method according to any one of claims 1 to 10, wherein the assay comprises the step of amplifying a cell-free DNA molecule.
12. A method according to claim 11, wherein the amplifying step involves the use of a pair of primers.
13. A method according to claim 12, wherein the affinity of hybridization of at least one primer of the primer pair to the target sequence is determined by the first methylation state of the target sequence.
14. A method according to claim 13, wherein at least one primer of the primer pair hybridizes to the target sequence when the methylation site of the target sequence is methylated in the first biological sample.
15. A method according to claim 13, wherein at least one primer of the primer pair hybridizes to the target sequence when the methylation site of the target sequence is unmethylated in the first biological sample.
16. A method according to any one of claims 1 to 15, wherein the assay comprises the step of converting unmethylated cytosine residues within a cell-free DNA molecule into uracil bisulfite.
17. A method according to any one of claims 1 to 16, wherein the assay comprises the step of performing a methylation recognition sequencing analysis of a cell-free DNA molecule from a first biological sample.
18. A method according to any one of claims 1 to 17, wherein the target sequence comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 methylation sites.
19. A method according to any one of claims 1 to 18, wherein the target sequence comprises at least 5 methylation sites.
20. A method according to any one of claims 1 to 19, wherein the first methylation state comprises a methylation density for an individual site within the target sequence, a distribution of methylated/non-methylated sites across adjacent regions within the target sequence, a pattern or level of methylation for each individual methylation site within the target sequence, or non-CpG methylation.

Description

Tissue-Specific Methylation Marker Cross-reference The present application claims the benefit of U.S. provisional application No. 62/643,649, filed March 15, 2018, and U.S. provisional application No. 62/769,928, filed November 20, 2018, respectively incorporated herein by reference. Quantitative measurement of DNA, ranging from different tissues to circulating DNA, can potentially provide important information regarding the presence of many different pathological conditions. However, existing methods involving genome-wide bisulfite sequencing are relatively expensive and can present difficulties in analysis. A more cost-effective method for measuring DNA derived from different tissues would be useful. The detection of circulating cell-free DNA derived from cancer cells, often known as liquid biopsy, is increasingly being used for the management of cancer patients. For example, the detection of epidermal growth factor receptor (EGFR) mutations in plasma can correlate highly with the mutation status in tumor tissue and can predict responsiveness to EGFR tyrosine kinase inhibitors. In addition to point mutations, other cancer-related genetic and genomic alterations, including copy number changes and altered fragmentation patterns, have also been detected in the cell-free plasma of cancer patients. Patients identified by plasma DNA screening may potentially have a significantly earlier stage distribution and superior progression-free survival compared to unscreened patients. One aspect of the present disclosure provides a method for determining whether an organism having cancer in a first tissue has cancer located in a second tissue, comprising: (a) obtaining a cell-free DNA molecule from a first biological sample of an organism having cancer in a first tissue; (b) performing an assay on the cell-free DNA molecule to determine a first methylation state of a target sequence in the cell-free DNA molecule, wherein the first methylation state of the target sequence indicates that the cell-free DNA molecule containing the target sequence is derived from a second tissue of the organism, and the first tissue and the second tissue are different; (c) determining an absolute amount of the cell-free DNA molecule from the first biological sample containing the target sequence having the first methylation state; and (d) determining whether the organism has cancer in the second tissue based on the absolute amount. In some cases, the methylation state includes the methylation level. In some cases, the assay includes the step of isolating a cell-free DNA molecule containing the target sequence from a first biological sample. In some cases, the assay includes the step of isolating a cell-free DNA molecule containing the target sequence from an oil emulsion. In some cases, the assay includes the step of hybridizing the cell-free DNA molecule containing the target sequence to a probe. In some cases, the probe hybridizes to the target sequence. In some cases, the affinity of the probe's hybridization to the target sequence depends on the first methylation state of the target sequence in the first biological sample. In some cases, the probe hybridizes to the target sequence when the methylation site of the target sequence is methylated in the first biological sample. In some cases, the probe hybridizes to the target sequence when the methylation site of the target sequence is unmethylated in the first biological sample. In some cases, the assay includes the step of detecting the hybridization of the probe and the target sequence. In some cases, the assay includes a step of amplifying a cell-free DNA molecule. In some cases, the amplification step includes the use of a pair of primers. In some cases, the affinity for hybridization of at least one of the pair of primers to the target sequence is determined by the first methylation state of the target sequence. In some cases, at least one of the pair of primers hybridizes to the target sequence when the methylation site of the target sequence is methylated in the first biological sample. In some cases, at least one of the pair of primers hybridizes to the target sequence when the methylation site of the target sequence is unmethylated in the first biological sample. In some cases, the assay includes a step of converting unmethylated cytosine residues of the cell-free DNA molecule into uracil bisulfites. In some cases, the assay includes a step of performing a methylation recognition sequencing of the cell-free DNA molecule from the first biological sample. In some cases, the target sequence comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 methylation sites. In some cases, the target sequence comprises at least 5 methylation sites. In some cases, the first methylation state comprises a methylation density for an individual site within the target sequence, a distribution of methylated/non-methylated sites across adjacent regio