Tissue-specific methylation markers

Abstract

Provided herein are compositions containing tissue-specific markers for identifying the tissue of origin of cell-free nucleic acids, e.g., cell-free DNA molecules. Also provided herein are methods, compositions, and systems for identifying the tissue of origin of cell-free nucleic acids by measuring the absolute amount of cell-free nucleic acids containing tissue-specific markers. Also provided herein are methods, compositions, and systems for detecting cancer in the tissues of an organism by analyzing tissue-specific markers.

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 643,649, filed March 15, 2018, and U.S. Provisional Patent Application No. 62 / 769,928, filed November 20, 2018, each of which is incorporated by reference in its entirety into this specification. [Background technology]

[0002] background

[0002] Quantitative measurement of circulating DNA from different tissues can potentially provide important information regarding the presence of various pathologies. However, existing methods, including genome-wide bisulfite sequencing, are relatively expensive and can present analytical challenges. A more cost-effective approach to measuring DNA from different tissues would be useful. Summary of the Invention [Problem to be solved by the invention]

[0003]

[0003] Detection of circulating cell-free DNA from cancer cells, often known as liquid biopsy, is increasingly used in the management of cancer patients. For example, detection of epidermal growth factor receptor (EGFR) mutations in plasma can be well correlated with the mutation status in tumor tissue and can predict responsiveness to EGFR tyrosine kinase inhibitors. In addition to point mutations, even other cancer-related genetic and genomic abnormalities, including copy number changes and fragmentation pattern abnormalities, can be detected in cell-free plasma of cancer patients. Patients identified by screening of plasma DNA can potentially have a significantly earlier stage distribution and favorable progression-free survival compared to unscreened patients. [Means for solving the problem]

[0004] overview

[0004] 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, the method comprising: (a) obtaining a cell-free DNA molecule from a first biological sample of the organism having cancer in the 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, where 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, where the first tissue and the second tissue are different; (c) determining an absolute quantity 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 quantity.

[0005]

[0005] In some cases, the methylation state comprises a methylation level. In some cases, the assay comprises isolating a cell-free DNA molecule comprising the target sequence from a first biological sample. In some cases, the assay comprises isolating a cell-free DNA molecule comprising the target sequence in an oil emulsion. In some cases, the assay comprises hybridizing a cell-free DNA molecule comprising the target sequence to a probe. In some cases, the probe hybridizes to the target sequence. In some cases, the affinity of hybridization of the probe to the target sequence depends on a first methylation state of the target sequence in the first biological sample. In some cases, the probe hybridizes to the target sequence when a methylation site of the target sequence is methylated in the first biological sample. In some cases, the probe hybridizes to the target sequence when a methylation site of the target sequence is unmethylated in the first biological sample. In some cases, the assay comprises detecting hybridization of the probe to the target sequence.

[0006]

[0006] In some cases, the assay includes amplifying the cell-free DNA molecule. In some cases, the amplification includes the use of a primer pair. In some cases, the affinity of hybridization of at least one primer of the primer pair to the target sequence depends on a first methylation state of the target sequence. In some cases, at least one primer of the primer pair hybridizes to the target sequence when a methylation site of the target sequence is methylated in the first biological sample. In some cases, at least one primer of the primer pair hybridizes to the target sequence when a methylation site of the target sequence is unmethylated in the first biological sample. In some cases, the assay includes bisulfite conversion of unmethylated cytosine residues in the cell-free DNA molecule to uracil. In some cases, the assay includes performing methylation-aware sequencing of the cell-free DNA molecule from the first biological sample.

[0007]

[0007] 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 at individual sites in the target sequence, a distribution of methylated / non-methylated sites across adjacent regions in the target sequence, a pattern or level of methylation at each individual methylation site in the target sequence, or non-CpG methylation. In some cases, the target sequence comprises a higher methylation density in the first tissue than in the second tissue. In some cases, the first methylation state comprises a methylation density at individual sites with the target sequence, and the target sequence has a methylation density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% in the first tissue. In some cases, the target sequence comprises a methylation density of more than 50% in the first tissue. In some cases, the target sequence comprises a methylation density of up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or 0% in the second tissue. In some cases, the target sequence comprises a methylation density of less than 20% in the second tissue. In some cases, the target sequence comprises a lower methylation density in the first tissue than in the second tissue. In some cases, the target sequence comprises a methylation density of up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or 0% in the first tissue. In some cases, the target sequence comprises a methylation density of less than 50% in the first tissue. In some cases, the target sequence comprises a methylation density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% in the second tissue. In some cases, the target sequence comprises a methylation density of more than 80% in the second tissue.

[0008] In some cases, the first tissue comprises liver tissue and the target sequence comprises a polynucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 1. In some cases, the first tissue comprises liver tissue and the assay comprises amplification using a primer comprising SEQ ID NO: 2, a primer comprising SEQ ID NO: 3, or both, or using a detectably labeled probe comprising SEQ ID NO: 4 to detect the target sequence. In some cases, the amplification further comprises using a primer comprising SEQ ID NO: 5, a primer comprising SEQ ID NO: 6, or both, or using a detectably labeled probe comprising SEQ ID NO: 7 to detect the target sequence.

[0009] In some cases, the first tissue comprises colon tissue and the target sequence comprises a polynucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 8. In some cases, the first tissue comprises colon tissue and wherein the amplification comprises the use of a primer comprising SEQ ID NO: 9, a primer comprising SEQ ID NO: 10, or both, or the use of a detectably labeled probe comprising SEQ ID NO: 11 to detect the target sequence. In some cases, the methylation specific amplification further comprises the use of a primer comprising SEQ ID NO: 12, a primer comprising SEQ ID NO: 13, or both, or the use of a detectably labeled probe comprising SEQ ID NO: 14 to detect the target sequence.

[0010]

[0010] In some cases, the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, hematopoietic malignancies, 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, gastric cancer, melanoma, and thyroid cancer. In some cases, the cancer comprises hepatocellular carcinoma or colorectal cancer.

[0011]

[0011] Optionally, the method further comprises determining the classification of the cancer in the second tissue. Optionally, determining the classification of the cancer in the second tissue comprises evaluating cell-free nucleic acid molecules from a second biological sample derived from the organism. Optionally, evaluating comprises determining a methylation profile, copy number variation, single nucleotide polymorphism (SNP) profile, or fragmentation pattern of the cell-free nucleic acid molecules from the second biological sample. Optionally, evaluating comprises measuring the amount of cell-free nucleic acid molecules from the second biological sample derived from the pathogen. Optionally, the second biological sample is the same as the first biological sample. Optionally, the second biological sample is different from the first biological sample.

[0012]

[0012] One aspect of the present disclosure provides a system configured to perform the methods as provided herein.

[0013]

[0013] One aspect of the present disclosure provides a non-transitory computer-readable medium comprising a set of instructions for controlling a computer system to perform a method as disclosed herein.

[0014]

[0014] One aspect of the present disclosure provides a method for analyzing a biological sample of an organism. The method may include: amplifying a first tissue-specific marker in a cell-free DNA molecule from the biological sample based on a methylation state of the first tissue-specific marker; wherein the first tissue-specific marker comprises a predetermined sequence having one or more differential methylation sites with a first methylation state in a first tissue of the organism and a second methylation state in another tissue of the organism, and the first and second methylation states are different; identifying a tissue of origin in the cell-free DNA molecule by detecting the amplification of the first tissue-specific marker; and determining an absolute amount of the cell-free DNA molecule from the biological sample derived from the first tissue of the organism.

[0015]

[0015] In some cases, the method further comprises bisulfite conversion of unmethylated cytosine residues to uracil before amplification. In some cases, the method further comprises isolating the cell-free DNA molecule from other DNA molecules from the biological sample before amplification. In some cases, the amplification comprises using a methylation-specific primer complementary to the first tissue-specific marker and annealing to at least a portion of the one or more differential methylation sites. In some cases, identifying the tissue of origin comprises determining that the cell-free DNA molecule is derived from the first tissue if the first tissue-specific marker in the cell-free DNA molecule is amplified by a primer configured to amplify the first tissue-specific marker methylated at a first methylation state.

[0016]

[0016] In some cases, the one or more differentiated methylation sites have a higher methylation density in the first tissue than in the second tissue. In some cases, the one or more differentiated methylation sites have a methylation density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% in the first tissue. In some cases, the one or more differentiated methylation sites have a methylation density of more than 50% in the first tissue. In some cases, the one or more differentiated methylation sites have a methylation density of up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or 0% in the second tissue. In some cases, the one or more differentiated methylation sites have a methylation density of less than 20% in the second tissue.

[0017]

[0017] In some cases, the one or more differentiated methylation sites have a lower methylation density in the first tissue than in the second tissue. In some cases, the one or more differentiated methylation sites have a methylation density of up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or 0% in the first tissue. In some cases, the one or more differentiated methylation sites have a methylation density of less than 50% in the first tissue. In some cases, the one or more differentiated methylation sites have a methylation density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% in the second tissue. In some cases, the one or more differentiated methylation sites have a methylation density of more than 80% in the second tissue.

[0018]

[0018] In some cases, the first tissue comprises a liver tissue, and the first tissue-specific marker comprises a polynucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:1. In some cases, the first tissue comprises a liver tissue, and the amplification comprises the use of a primer comprising SEQ ID NO:2, a primer comprising SEQ ID NO:3, or both, or the use of a detectably labeled probe comprising SEQ ID NO:4 for the detection of the first tissue-specific marker. In some cases, the amplification further comprises the use of a primer comprising SEQ ID NO:5, a primer comprising SEQ ID NO:6, or both, or the use of a detectably labeled probe comprising SEQ ID NO:7 for the detection of the first tissue-specific marker. In some cases, the first tissue comprises a colon tissue, and the first tissue-specific marker comprises a polynucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:8. In some cases, the first tissue comprises colon tissue, and wherein the amplification comprises the use of a primer comprising SEQ ID NO:9, a primer comprising SEQ ID NO:10, or both, or the use of a detectably labeled probe comprising SEQ ID NO:11 for detection of the first tissue-specific marker. In some cases, the methylation specific amplification further comprises the use of a primer comprising SEQ ID NO:12, a primer comprising SEQ ID NO:13, or both, or the use of a detectably labeled probe comprising SEQ ID NO:14 for detection of the first tissue-specific marker.

[0019]

[0019] Optionally, the method further comprises determining the amount of cell-free DNA molecules from the biological sample derived from a second tissue based on the methylation pattern of the second tissue-specific marker, where the first and second tissues are different. Optionally, the second tissue belongs to an organism.

[0020]

[0020] In some cases, the method further comprises diagnosing, monitoring, or prognosing the cancer in the first tissue based on the absolute amount of cell-free DNA molecules from a biological sample derived from the first tissue of the organism. In some cases, the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, hematopoietic malignancies, 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, gastric cancer, melanoma, and thyroid cancer. In some cases, the cancer comprises hepatocellular carcinoma or colorectal cancer. In some cases, the diagnosis or monitoring comprises determining the size of the tumor in the first tissue based on the absolute amount of cell-free DNA molecules from a biological sample derived from the first tissue of the organism. In some cases, the diagnosis or monitoring includes determining whether the cancer has metastasized to a first tissue based on the absolute amount of cell-free DNA molecules from a biological sample derived from the first tissue of the organism.

[0021]

[0021] In some cases, the first tissue comprises a transplanted organ. In some cases, the method provided herein further comprises assessing organ transplantation based on the absolute amount of cell-free DNA molecules from a biological sample derived from the first tissue.

[0022]

[0022] Another aspect of the present disclosure provides a composition for determining the amount of cell-free DNA molecules from a biological sample derived from the liver of an organism. The composition may include a primer pair for amplification of a liver-specific marker based on the methylation status of the liver-specific marker, where the liver-specific marker comprises a polynucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:1. In some cases, the primer pair includes a primer including SEQ ID NO:2 and a primer including SEQ ID NO:3. In some cases, the composition further includes a detectable-labeled probe including SEQ ID NO:4 for detection of the liver-specific marker. In some cases, the composition further includes a primer including SEQ ID NO:5 and a primer including SEQ ID NO:6. In some cases, the composition further includes a detectable-labeled probe including SEQ ID NO:7 for detection of the liver-specific marker.

[0023]

[0023] Another aspect of the present disclosure provides a composition for determining the amount of cell-free DNA molecules from a biological sample derived from the colon of an organism, the composition comprising a primer pair for amplification of a colon-specific marker based on the methylation status of the colon-specific marker, wherein the colon-specific marker comprises a polynucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:8. In some cases, the primer pair comprises a primer comprising SEQ ID NO:9 and a primer comprising SEQ ID NO:10. In some cases, the composition further comprises a detectably labeled probe comprising SEQ ID NO:11 for detection of the colon-specific marker. In some cases, the composition further comprises a primer comprising SEQ ID NO:12 and a primer comprising SEQ ID NO:13. In some cases, the composition further comprises a detectably labeled probe comprising SEQ ID NO:14 for detection of the colon-specific marker.

[0024] Incorporation by Reference

[0024] All publications, patents, and patent applications mentioned in this specification are incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The novel features of the present disclosure are set forth with particularity in the appended claims. A more complete understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and the accompanying drawings that set forth illustrative embodiments in which the principles of the disclosure are utilized. [Brief description of the drawings]

[0026] [Figure 1]

[0026] Schematic diagram of the origin of plasma cell-free DNA in patients with colorectal cancer with liver metastasis. [Diagram 2]

[0027] Correlation between fractional concentrations of liver-derived DNA in plasma of liver transplant recipients based on liver-specific methylation marker analysis (droplet digital PCR) and donor-specific allele analysis by sequencing. [Figure 3A]

[0028] Absolute concentrations of liver-derived DNA using droplet digital PCR in plasma of healthy subjects, chronic HBV carriers, cirrhosis patients and HCC patients. [Figure 3B]

[0028] Fractional concentrations of liver-derived DNA using droplet digital PCR in plasma of healthy subjects, chronic HBV carriers, cirrhosis patients and HCC patients. [Figure 4A]

[0029] 1 shows the correlation between maximum tumor size and absolute concentration of liver-derived DNA using droplet digital PCR in plasma of HCC patients. [Figure 4B]

[0029] Correlation between maximum tumor size and fractional concentration of liver-derived DNA using droplet digital PCR in plasma of HCC patients. [Figure 5A]

[0030] Plasma concentrations of colon- and liver-derived DNA by droplet digital PCR in healthy subjects and CRC patients with and without liver metastases. Absolute concentration of colon-derived DNA. [Figure 5B]

[0030] Plasma concentrations of colon- and liver-derived DNA by droplet digital PCR in healthy subjects and CRC patients with and without liver metastases. Fractional concentration of colon-derived DNA. [Figure 5C]

[0030] Plasma concentrations of colon- and liver-derived DNA by droplet digital PCR in healthy subjects and CRC patients with and without liver metastases. Absolute concentration of liver-derived DNA. [Figure 5D]

[0030] Plasma concentrations of colon- and liver-derived DNA by droplet digital PCR in healthy subjects and CRC patients with and without liver metastases. Fractional concentration of liver-derived DNA. [Figure 6]

[0031] 1 shows the ROC curve for using absolute and fractional concentrations of DNA from liver and colon to distinguish CRC patients with and without liver metastases. "AUC" means area under the curve. [Figure 7]

[0032] Methylation density of CpG sites within the protein tyrosine kinase 2β (PTK2B) gene region is shown. [Figure 8]

[0033] Methylation density of CpG sites within the Sestrin 3 (SESN3) gene region is shown. [Figure 9]

[0034] 4 shows ROC curves for using absolute and fractional concentrations of liver-derived DNA to distinguish HCC patients from non-HCC subjects. [Figure 10]

[0035] 1 illustrates a computer control system that can be programmed or otherwise configured to carry out the methods provided herein. [Figure 11]

[0036] 1 illustrates the method and system as disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description

[0037] Provided herein are methods, compositions, and systems for quantifying cell-free nucleic acid molecules, such as cell-free DNA molecules, such as plasma DNA, derived from specific tissues using tissue-specific markers. Provided herein are clinical applications of these markers, including, but not limited to, cancer diagnosis, monitoring, and prognosis, detection of metastatic cancer, and in some cases, evaluation of organ transplantation.

[0028]

[0038] In addition to cancer-specific changes, there may be a general increase in DNA released from cancer-affected organs into circulation. For example, the level of liver-derived DNA may increase in patients with liver cancer. Without wishing to be bound by a particular theory, the increased level of DNA released by cancer-affected organs may be due to direct release of DNA from tumor cells or increased turnover of non-tumor cells infiltrated by cancer. Such an increase in tissue-specific DNA release may also be observed in organ transplant recipients who have experienced acute rejection as a result of increased cell turnover. In some cases, the proportional contribution of cells in transplanted organs to plasma DNA, as determined by deconvolution of methylation, correlates well with that determined based on the analysis of donor-specific alleles.

[0029]

[0039] In some cases, deconvolution of methylation using genome-wide bisulfite sequencing can be difficult due to the relatively high cost and long turnaround time. Furthermore, in some cases, deconvolution of methylation can determine the relative or proportional contribution of different organs, rather than the absolute concentration of DNA from each organ. In scenarios where DNA from more than one organ is released into the circulation, measuring the absolute concentration of DNA from a particular organ may be more informative. For example, in a patient with colorectal cancer (CRC) that has metastasized to the liver, DNA will be released from the liver into the circulation in increasing amounts. However, the fraction of hepatic DNA in plasma may show a paradoxical decrease due to the even greater increase in DNA released by tumor cells originating from the colon. Therefore, it may be useful to develop a method that can accurately determine the absolute amount of DNA using tissue-specific methylation patterns.

[0030]

[0040] I. Tissue-specific markers

[0041] Provided herein is a tissue-specific marker that can identify the tissue of origin in a cell-free DNA molecule. In some cases, the tissue-specific marker is a polynucleotide sequence of the genome of an organism. In some cases, the tissue-specific marker comprises a differential methylation region (DMR) that is identified based on the methylation status of one or more differential methylation sites contained within the marker polynucleotide sequence. In some cases, the one or more differential methylation sites comprise one or more CpG sites. In some cases, the one or more differential methylation sites comprise one or more non-CpG sites. In some cases, the tissue-specific marker as discussed herein can be referred to as a target sequence.

[0031]

[0042] In some cases, a differential methylation site of a tissue-specific marker has a first methylation state in a first tissue of an organism and a second methylation state in a different second tissue of the organism, where the first and second methylation states can be different such that the first and second tissues are distinguishable based on the methylation state of the tissue-specific marker.

[0032]

[0043] In some cases, a differential methylation site of a tissue-specific marker has a first methylation state in a first tissue of an organism, and a second methylation state in all other tissues of the organism, and the first and second methylation states can be different such that the first tissue is distinguishable from all other tissues of the organism based on the methylation state of the tissue-specific marker.

[0033]

[0044] In some cases, the tissue-specific marker comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 50 differential methylation sites. In some cases, the tissue-specific marker comprises at least 5 differential methylation sites. A methylated nucleotide or nucleotide base can refer to the presence of a methyl moiety on a nucleotide base, where the methyl moiety is not present in a recognized typical nucleotide base. For example, cytosine does not have a methyl moiety on its pyrimidine ring, but 5-methylcytosine has a methyl moiety at the 5-position of its pyrimidine ring. In this case, cytosine is not a methylated nucleotide and 5-methylcytosine is a methylated nucleotide. In another example, thymine has a methyl moiety at the 5-position of its pyrimidine ring, but for purposes herein, thymine is not considered a methylated nucleotide when present in DNA because it is a typical nucleotide base of DNA. Typical nucleoside bases in DNA are thymine, adenine, cytosine, and guanine. Typical bases in RNA are uracil, adenine, cytosine, and guanine. In contrast, a "methylation site" can be a position in a target gene nucleic acid region where methylation has occurred or has the potential to occur. For example, a position with a CpG is a methylation site, where the cytosine may or may not be methylated. A "site" can correspond to a single site, which can be a single base position or a group of base positions correlated thereto, such as CpG sites. A methylation site can refer to a CpG site or a non-CpG site of a DNA molecule that has the potential to be methylated. A CpG site can be a region of a DNA molecule where a cytosine nucleotide is followed by a guanine nucleotide in a linear sequence along the 5' to 3' direction of the bases, and is susceptible to methylation, either by events that occur naturally in vivo or events that initiate chemical methylation of the nucleotide in vitro.A non-CpG site may be a region that does not have a CpG dinucleotide sequence, but is still susceptible to methylation, either by naturally occurring events in vivo or by events that initiate chemical methylation of nucleotides in vitro. A locus or region may correspond to a region that includes multiple sites.

[0034]

[0045] The methylation state of a tissue-specific marker may include the methylation density at individual sites within the marker region, the distribution of methylated / unmethylated sites across adjacent regions within the marker, the pattern or level of methylation at each individual methylation site within a marker having two or more sites, and non-CpG methylation. In some cases, the methylation state of a tissue-specific marker includes the methylation level (or methylation density) at individual differentiated methylation sites. Methylation density may refer to the proportion of nucleic acid molecules methylated at a given methylation site to the total number of nucleic acid molecules of interest having such methylation site at a given methylation site. For example, the methylation density of a first methylation site in liver tissue may refer to the proportion of liver DNA molecules methylated at the first site to the total liver DNA molecules. In some cases, the methylation state includes the coherence of methylation / unmethylation status among individual differentiated methylation sites.

[0035]

[0046] In some cases, a tissue-specific marker comprises a methylation site that is hypermethylated in a first tissue but hypomethylated in a second tissue. For example, a tissue-specific marker may comprise one or more methylation sites that are hypermethylated in liver tissue, thereby meaning that one or more methylation sites have a methylation density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% in liver tissue; in contrast, it may mean that one or more methylation sites have a methylation density of up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or 0% in other tissues, such as, but not limited to, blood cells, lung, esophagus, stomach, small intestine, colon, pancreas, bladder, heart, and brain. A tissue-specific marker may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 50 methylation sites that are hypermethylated in a first tissue but hypomethylated in a second tissue. In some cases, a tissue-specific marker comprises at least 5 methylation sites that are hypermethylated in a first tissue but hypomethylated in a second tissue.

[0036]

[0047] The tissue specific marker may comprise up to 300 base pairs (bp), up to 250 bp, up to 225 bp, up to 200 bp, up to 190 bp, up to 185 bp, up to 180 bp, up to 175 bp, up to 170 bp, up to 169 bp, up to 168 bp, up to 167 bp, up to 166 bp, up to 165 bp, up to 164 bp, up to 163 bp, up to 162 bp, up to 161 bp, up to 160 bp, up to 150 bp, up to 140 bp, up to 120 bp, or up to 100 bp. In some cases, the tissue specific marker comprises up to 166 bp.

[0037]

[0048] In some cases, a liver-specific marker as provided herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 50 methylation sites that are hypermethylated in liver tissue but hypomethylated in other tissues. In some cases, a liver-specific marker as provided herein comprises at least 5 methylation sites that are hypermethylated in liver tissue but hypomethylated in other tissues. Each of the methylation sites that are hypermethylated in liver tissue may have a methylation density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% in liver tissue and up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or 0% in other tissues, such as, but not limited to, blood, brain, thymus, pancreas, kidney. In some cases, each of the methylation sites that are hypermethylated in liver tissue may have a methylation density of greater than 50% in liver tissue and less than 20% in other tissues, such as, but not limited to, blood cells, lung, esophagus, stomach, small intestine, colon, pancreas, bladder, heart, and brain.

[0038]

[0049] In some cases, the tissue-specific marker comprises a methylation site that is hypomethylated in a first tissue but hypermethylated in a second tissue. In some cases, the tissue-specific marker comprises a methylation site that is hypomethylated in a first tissue but hypermethylated in other tissues. For example, the tissue-specific marker may comprise one or more methylation sites that are hypomethylated in liver tissue, thereby meaning that one or more methylation sites have a methylation density of at most 50%, at most 40%, at most 30%, at most 20%, at most 10%, at most 5%, or 0% in liver tissue; in contrast, it may mean that one or more methylation sites have a methylation density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% in other tissues, such as, but not limited to, blood cells, lung, esophagus, stomach, small intestine, colon, pancreas, bladder, heart, and brain. A tissue-specific marker may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 50 methylation sites that are hypomethylated in a first tissue but hypermethylated in a second tissue. In some cases, a tissue-specific marker comprises at least 5 methylation sites that are hypomethylated in a first tissue but hypermethylated in a second tissue.

[0039]

[0050] In some cases, a liver-specific marker as provided herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 50 methylation sites that are hypomethylated in liver tissue but hypermethylated in other tissues. In some cases, a liver-specific marker as provided herein comprises at least 5 methylation sites that are hypomethylated in liver tissue but hypermethylated in other tissues. Each of the methylation sites that are hypomethylated in liver tissue may have a methylation density of at most 50%, at most 40%, at most 30%, at most 20%, at most 10%, or at most 5% in liver tissue, and at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% in other tissues, such as, but not limited to, blood, brain, thymus, pancreas, kidney. In some cases, each of the methylation sites that are hypomethylated in liver tissue may have a methylation density of less than 50% in liver tissue and greater than 80% in other tissues, such as, but not limited to, blood cells, lung, esophagus, stomach, small intestine, colon, pancreas, bladder, heart, and brain.

[0040]

[0051] In some cases, colon-specific markers as provided herein comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 50 methylation sites that are hypermethylated in colon tissue but hypomethylated in other tissues. In some cases, colon-specific markers as provided herein comprise at least 5 methylation sites that are hypermethylated in colon tissue but hypomethylated in other tissues. Each of the methylation sites that are hypermethylated in colon tissue may have a methylation density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% in colon tissue, and up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or 0% in other tissues, such as, but not limited to, blood, brain, thymus, pancreas, kidney, etc. In some cases, each of the methylation sites that are hypermethylated in colon tissue may have a methylation density of more than 50% in colon tissue, and less than 20% in other tissues, such as, but not limited to, blood cells, lung, esophagus, stomach, small intestine, liver, pancreas, bladder, heart, and brain.

[0041]

[0052] In some cases, colon-specific markers as provided herein include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 50 methylation sites that are hypomethylated in colon tissue but hypermethylated in other tissues. In some cases, colon-specific markers as provided herein include at least 5 methylation sites that are hypomethylated in colon tissue but hypermethylated in other tissues. Each of the methylation sites that are hypomethylated in colon tissue may have a methylation density of up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, or up to 5% in colon tissue, and at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% in other tissues, such as, but not limited to, blood, brain, thymus, pancreas, kidney. In some cases, each of the methylation sites that are hypomethylated in colon tissue may have a methylation density of less than 50% in colon tissue, and greater than 80% in other tissues, such as, but not limited to, blood cells, lung, esophagus, stomach, small intestine, liver, pancreas, bladder, heart, and brain.

[0042]

[0053] Further, a liver-specific marker for identifying liver-derived DNA molecules is provided herein. The liver-specific marker may be located within an exon region of the protein tyrosine kinase 2 beta (PTK2B) gene on chromosome 8. Eight CpG sites in the liver-specific DMR may be hypermethylated in the liver, but hypomethylated in other tissues and blood cells. The liver-specific marker as provided herein may comprise a polynucleotide sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:1. The liver-specific marker as provided herein may comprise SEQ ID NO:1.

[0043]

[0054] Further provided herein is a colon-specific marker for identifying colon-derived DNA molecules. The colon-specific marker may be located within the exon region of the Sestrin 3 (SESN3) gene on chromosome 11. All six CpG sites located in the colon-specific DMR may be hypermethylated in the colon, but hypomethylated in other tissues and blood cells. The colon-specific marker may comprise a polynucleotide sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:8.

[0044]

[0055] As used herein, the term "identity" or "percent identity" between two or more nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison (e.g., gaps can be introduced into the sequence of the first sequence). The nucleotides at corresponding positions can then be compared, and the percent identity between the two sequences can be a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100). For example, a position in a first sequence can be occupied by the same nucleotide as the corresponding position in a second sequence, where the molecules are identical at that position. The percent identity between the two sequences can be a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. In some cases, the length of the sequences aligned for comparison purposes is at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 95% of the length of the reference sequence. A BLAST® search can be used to determine the homology between two sequences. The homology can be between the full length of the two sequences or between a portion of the full length of the two sequences. The two sequences can be genes, nucleotide sequences, protein sequences, peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm may be that described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90- 5873-5877 (1993). Such an algorithm may be incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, any of the relevant parameters of the respective programs (e.g., NBLAST) may be used.For example, parameters for sequence comparison can be set at score=100, word length=12, or can be varied (e.g., W=5 or W=20). Other examples include the Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA algorithms.

[0045]

[0056] Further provided herein is a method for identifying tissue-specific markers. The method may include comparing methylation status across genomes in different tissue samples. Publicly available databases, such as those from the RoadMap Epigenomics Project (Roadmap Epigenomics Consortium et al. Nature 2015;518:317-30) and the BLUEPRINT project (Martens et al. Haematologica 2013;98:1487-9), are available for bioinformatics analysis to screen for potential tissue-specific markers. In some cases, experimental validation is desirable. For example, to validate the methylation status in different tissues, methylation-aware sequencing, such as bisulfite sequencing, can be performed. In some cases, methylation-specific amplification can be used for relatively more targeted validation.

[0046]

[0057] II. Methods for analyzing cell-free DNA molecules from tissues

[0058] Provided herein is a method for analyzing a biological sample of an organism. The method as provided herein can determine the absolute amount of cell-free nucleic acid molecules, such as cell-free DNA molecules, from a tissue of the organism. The method can include identifying a cell-free DNA molecule from the biological sample as a cell-free DNA molecule from a first tissue of the organism when the cell-free DNA molecule comprises a first tissue-specific marker having a first methylation state. In some cases, the first tissue-specific marker comprises a predetermined sequence having one or more differential methylation sites. In some cases, the first tissue-specific marker can have a first methylation state in the first tissue and a second methylation state in another tissue of the organism, and the first methylation state and the second methylation state can be different.

[0047]

[0059] As provided herein, the method may include assessing the methylation status of the cell-free DNA molecule. Optionally, the method may further include bisulfite conversion of unmethylated cytosine residues to uracil before amplification. Optionally, the method may include conversion of either methylated or unmethylated cytosine residues by any other method, so that the converted residues can be distinguished by a subsequent detection method, such as primer-based amplification. Optionally, the cell-free DNA molecule may be digested with a methylation-sensitive enzyme that digests the DNA molecule at one or more specific methylation sites when the methylation site is methylated or unmethylated. Thus, the methylation-sensitive enzyme may be used to distinguish between methylated and unmethylated DNA molecules. Non-limiting examples of methylation-sensitive enzymes that can be used in the methods provided herein include Aat II, Acc II, Aor13H I, Aor51H I, BspT104 I, BssH II, Cfr10 I, Cla I, Cpo I, Eco52 I, Hae II, Hap II, Hha I, Mlu I, Nae I, Not I, Nru I, Nsb I, PmaC I, Psp1406 I, Pvu I, Sac II, Sal I, Sma I, SnaB I, and any combination thereof.

[0048]

[0060] The method as provided herein may include amplifying a first tissue-specific marker in cell-free DNA molecules from a biological sample based on the methylation status of the first tissue-specific marker. The method may further include identifying the tissue of origin in the cell-free DNA molecules by detecting the amplification of the first tissue-specific marker. The method may further include determining the absolute amount of cell-free DNA molecules from the biological sample derived from the first tissue of the organism. An amplification reaction may refer to a process for replicating a nucleic acid one or more times. In some cases, the amplification method includes, but is not limited to, polymerase chain reaction (PCR), self-sustained sequencing reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Qβ phage amplification, strand displacement amplification, or splice overlap extension polymerase chain reaction. In some cases, a single molecule of nucleic acid is amplified, for example, by digital PCR.

[0049]

[0061] In some cases, the amplification includes the use of a methylation-specific primer that is complementary to the first tissue-specific marker and anneals to at least a portion of one or more methylation sites. A methylation-specific primer may refer to a primer that can distinguish between methylated and unmethylated target sequences. For a given target sequence that has a methylation site, the methylation-specific primer may be designed to cover at least a portion of the methylation site. In some cases, when bisulfite conversion is performed before amplification, when detecting a methylated target sequence, the nucleotide residue on the methylation-specific primer that corresponds to a given methylation site may be designed to be complementary to the unconverted cytosine residue of the site, while when detecting an unmethylated target sequence, the nucleotide residue on the methylation-specific primer may be designed to be complementary to the converted residue (e.g., uracil residue) of the site.

[0050]

[0062] In some cases, the method may further include isolating the cell-free DNA molecules from the biological sample from other DNA molecules before amplification. By physically isolating the cell-free DNA molecules, a "digital" assay of the cell-free DNA molecules from the biological sample can be performed, such as, but not limited to, digital PCR, such as droplet digital PCR. The isolation method can be any method known to those skilled in the art, such as, but not limited to, isolation by microwell plate, capillary, oil emulsion, and array of small chambers. Digital PCR reaction can be performed using any technique known in the art, such as, for example, microfluidics-based or emulsion-based, such as BEAMing (Dressman et al. Proc Natl Acad Sci USA 2003; 100: 8817-8822).

[0051]

[0063] In some cases, the first tissue comprises liver tissue, and the method comprises the use of a liver-specific marker as discussed above. In some cases, the liver-specific marker, e.g., SEQ ID NO: 1, comprises a site that is hypermethylated in the liver but hypomethylated in other tissues. In these cases, the method may comprise a primer for detecting a methylated liver tissue-specific marker ("methylation assay primer"). For example, the methylation assay primer may comprise a primer comprising SEQ ID NO: 2, a primer comprising SEQ ID NO: 3, or both. In some cases, the primers provided herein are used in an amplification reaction after bisulfite conversion. Alternatively, or cumulatively, the method may comprise the use of a detectably labeled probe comprising SEQ ID NO: 4 for the detection of a methylated liver tissue-specific marker. Optionally, the method may further comprise the use of a primer for detecting a non-methylated liver tissue-specific marker ("non-methylation assay primer"). The non-methylation assay primer may comprise a primer comprising SEQ ID NO: 5, a primer comprising SEQ ID NO: 6, or both. Alternatively, or cumulatively, the method may comprise the use of a detectably labeled probe comprising SEQ ID NO: 7 for the detection of a non-methylated liver tissue-specific marker.

[0052]

[0064] Primer, probe, or oligonucleotide may be used interchangeably herein and may refer to a polymer of two or more nucleotides, e.g., 2, 4, 6, 8, 10, 14, 18, 20, or 40 nucleotides or chemically modified nucleotides linked together via phosphodiester bonds. A primer may contain about 20 to about 30 nucleotides. A primer may contain at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 nucleotides. Chemical modifications may be introduced into the constituent nucleotides of a primer to modify certain properties of the primer, e.g., to enhance its stability, enhance hybridization specificity, and label it with a detectable signal. Chemical modifications that may be used in the compositions provided herein may include covalent modifications, attachment chemistries, and modifications of nucleotide bases. Chemical modifications can include phosphorylation, biotin, cholesteryl-TEG, amino modifiers (eg, C6, C12, or dT), azides, alkynes, thiol modifiers, fluorophores, dark quenchers, and addition of spacers. Primers may contain one or more phosphorothioate linkages, or one or more modified bases, such as 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyl dC, deoxyinoside, super T®, super G®, locked nucleic acid, 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, iso-dC, iso-dG, fluoro bases (e.g., fluoro C, U, A, G, T), and 2'-O-methoxy-ethyl bases (e.g., 2-methoxyethoxy A, G, U, C, T). Detectably labeled probes may contain any detectable label known to those of skill in the art, such as any suitable fluorophore. The PCR reaction (eg, digital PCR or real-time PCR) may be monitored through any suitable optical, magnetic, electronic, or any other technique available to one of skill in the art.

[0053]

[0065] In some cases, the first tissue comprises colon tissue, and the method comprises the use of a colon-specific marker as discussed above. In some cases, the colon-specific marker, e.g., SEQ ID NO: 8, comprises a site that is hypermethylated in the colon but hypomethylated in other tissues. In these cases, the primer for the methylation assay may comprise a primer comprising SEQ ID NO: 9, a primer comprising SEQ ID NO: 10, or both. In some cases, the primers provided herein are used in the amplification reaction after bisulfite conversion. Alternatively, or cumulatively, the method may comprise the use of a detectably labeled probe comprising SEQ ID NO: 11 for the detection of the methylated colon-specific marker. Optionally, the method may further comprise the use of a primer for detecting the unmethylated colon-specific marker ("unmethylated assay primer"). The unmethylated assay primer may comprise a primer comprising SEQ ID NO: 12, a primer comprising SEQ ID NO: 13, or both. Alternatively, or cumulatively, the method may comprise the use of a detectably labeled probe comprising SEQ ID NO: 14 for the detection of the unmethylated colon-specific marker.

[0054]

[0066] The method provided herein, for example, the use of digital PCR technology, can allow the direct determination of the actual number of target DNA molecules without the need for standards.Other techniques, such as specific sequencing-based methods, including but not limited to bisulfite sequencing and non-bisulfite-based methylation recognition sequencing using the PacBio sequencing platform, can determine the relative or fractional concentration of DNA from target tissue to other tissues.Absolute amount can refer to the absolute number of DNA molecules, or in some cases, the concentration of DNA molecules, for example, number, moles, or weight / volume, for example, copies / mL, moles / L, or mg / L.The analysis of absolute amount as provided herein can be useful in scenarios when DNA is released in increasing amounts from two or more tissue types. On the other hand, deconvolution analysis of methylation based on sequencing of cell-free nucleic acid molecules, e.g., as disclosed in U.S. Patent Application No. 14 / 803,692, can provide a readout in the form of fractional contribution of tissue of origin of cell-free nucleic acid, e.g., a first tissue contributes A% of cell-free nucleic acid from a biological sample and a second tissue contributes B% of cell-free nucleic acid from the same biological sample.

[0055]

[0067] In some cases, the methods, compositions and systems provided herein can also use technologies such as real-time PCR, sequencing and microarrays for methylation analysis of cell-free nucleic acids. In some cases, the absolute number of cell-free nucleic acids harboring tissue-specific markers, for example when counting positive reactions in digital PCR assays, may not be derived directly from methylation analysis by some technologies. However, such absolute number can be calculated indirectly based on the concentration (relative or fractional) of cell-free nucleic acids harboring tissue-specific markers, for example by considering the total number or concentration of cell-free nucleic acids in a given volume of biological sample. In some cases, the sequencing that can be used in the methods provided herein can include chain termination sequencing, hybridization sequencing, Illumina sequencing (e.g., using reversible terminator dyes), Ion Torrent semiconductor sequencing, mass spectrophotometric sequencing, massively parallel signature sequencing (MPSS), Maxam-Gilbert sequencing, nanopore sequencing, polony sequencing, pyrosequencing, shotgun sequencing, single molecule real-time (SMRT) sequencing, SOLiD sequencing (hybridization with four fluorescently labeled dibase probes), universal sequencing, or any combination thereof. Microarrays with probes targeting methylation sites can also be used to analyze the methylation status of cell-free DNA molecules in the methods provided herein.

[0056]

[0068] In some cases, the method provided herein further comprises determining the amount of cell-free DNA molecules from a biological sample derived from a second tissue based on the methylation pattern of a second tissue-specific marker, where the first tissue and the second tissue are different.The second tissue can belong to the same organism.The second tissue can also be obtained from a different organism, for example, a fetus in a pregnant woman.

[0057]

[0069] III. Methods for Diagnosing, Monitoring, and Prognosing Cancer

[0070] Provided herein is a method for diagnosing, monitoring, or prognosing cancer. The method may include determining an absolute amount of cell-free DNA molecules from a first tissue as discussed above, and diagnosing, monitoring, or prognosing cancer in the first tissue based on the absolute amount of cell-free DNA molecules from a biological sample derived from the first tissue of an organism.

[0058]

[0071] The absolute amount of cell-free DNA molecules from a first tissue can be correlated with the state of the first tissue.For example, the amount of plasma DNA molecules from liver can increase due to the increased release of DNA molecules from liver tissue as a result of tumor growth.Otherwise, the increased cell turnover, for example as a result of organ transplantation, can also lead to an increase in plasma DNA released from tissue with graft.

[0059]

[0072] The method provided herein may include determining the size of a tumor in a first tissue based on the absolute amount of cell-free DNA molecules from a biological sample derived from the first tissue of an organism. A predetermined comparison chart that correlates the amount of target cell-free DNA molecules with tumor size can be used to determine tumor size. Detection of tumor size can aid in the diagnosis, monitoring, and prognosis of cancer.

[0060]

[0073] The methods provided herein may include determining whether a cancer has metastasized to a first tissue based on the absolute amount of cell-free DNA molecules from a biological sample derived from the first tissue of an organism. The absolute amount of the target DNA molecules determined by the methods provided herein, compared to the fractional amount of the target DNA molecules, may provide a desirable distinction between cancer patients with metastasis and cancer patients without metastasis.

[0061]

[0074] Cancer types to which the methods, compositions, and systems provided herein are applicable may include bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, hematopoietic malignancies, 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, gastric cancer, or thyroid cancer. Metastatic tissues assessed by the methods provided herein may include bladder, bone, brain, breast, cervix, colon, esophagus, gastrointestinal tract, blood, head, neck, liver, lung, lymph nodes, nose, nasopharynx, mouth, oropharyngeal, ovary, prostate, skin, stomach, or thyroid.

[0062]

[0075] As one skilled in the art would readily appreciate, cancer cells may spread locally by migrating to nearby normal tissues, may spread locally to nearby lymph nodes, tissues, or organs, and may also spread to distant parts of the body. The spread of cancer from an initial first tissue to a second tissue may be referred to as metastasis, and thus such cancers may be referred to as metastatic cancers. Representative types of cancer metastasis to which the methods, compositions, and systems provided herein are applicable may include metastasis occurring at the sites listed in Table 1.

[0063] [Table 1]

[0064]

[0076] In some cases, the methods, compositions, and systems as provided herein, when combined with other techniques available to one of skill in the art, may be applied to the diagnosis, monitoring, and prognosis of cancer. In some cases, detection of other molecular markers, such as copy number aberrations (CNAs), single nucleotide polymorphisms (SNPs), gene mutations, germline mutations, somatic mutations, nucleic acids from pathogens, such as viruses, such as Epstein-Barr virus, cell-free nucleic acid size, and cell-free nucleic acid fragmentation patterns, may also be applied in combination with the methods, compositions, and systems as provided herein. The combination of techniques may help facilitate detection of the level of cancer, including, but not limited to, whether cancer is present, the stage of cancer, the size of the tumor, how many deletions or amplifications of chromosomal regions are involved (e.g., duplex or triplex), and / or other measures of the severity of the cancer. The level of cancer may be some other characteristic. The level may be 0. The level of cancer may also include premalignant or precancerous conditions associated with deletions or amplifications.

[0065]

[0077] In some cases, detection of copy number aberrations (CNAs), such as the method disclosed in U.S. Patent No. 8,741,811, may be combined with the methods provided herein. As discussed above, in some cases, the methods provided herein may determine whether cancer has metastasized to a first tissue based on the absolute amount of cell-free nucleic acid from a first tissue. On the other hand, detection of CNAs may help identify the origin of metastatic cancer cells in a first tissue. In some cases, analysis of cell-free nucleic acid fragmentation patterns, such as the method disclosed in U.S. Patent Application No. 15 / 218,497, may be combined with the methods, compositions, and systems provided herein. In some cases, the subject methods, compositions, and systems may be combined with any available technique for detecting, monitoring, or prognosing cancer in a subject. In addition to the above detection methods, any appropriate tests may be performed, such as tumor biomarker tests (e.g., alpha-fetoprotein (AFP) for liver cancer, ALK gene for non-small cell lung cancer, prostate specific antigen (PSA) for prostate cancer, and thyroglobulin for thyroid cancer), physical examination, radiographic imaging (e.g., computed tomography scan, magnetic resonance imaging, positron emission tomography (PET)), ultrasound, endoscopy, biopsy, or cytology tests.

[0066]

[0078] In some cases, the subject method, composition, or system can be used to monitor cancer in a subject on a regular, semi-regular, or non-regular schedule.For example, a subject can undergo cancer monitoring examinations using the subject method, composition, or system on a weekly, monthly, quarterly, or annual basis.In some cases, a subject can undergo such examinations about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more than 12 months.In some cases, the interval between two consecutive examinations can be determined based on the results of the most recent examination, for example, according to a doctor's order or medical advice in some cases.

[0067]

[0079] IV. Methods for Evaluating Organ Transplants

[0080] Provided herein is a method for evaluating organ transplantation based on determining the absolute amount of cell-free DNA molecules derived from transplanted tissue. Transplanted tissue as described herein is considered to be the tissue of interest.

[0068]

[0081] The methods as provided herein can utilize the correlation between the amount of cell-free DNA molecules derived from the transplanted tissue and the cell turnover rate in the transplanted tissue, whereby the cell turnover rate can be used as a criterion for evaluating organ transplantation.

[0069]

[0082] V. Compositions for Analyzing Cell-Free DNA Molecules from Tissues

[0083] Additionally, provided herein are compositions for analyzing cell-free DNA molecules from specific tissues, such as bone, liver, lung, brain, peritoneum, adrenal gland, skin, muscle, vagina, colon, bladder, breast, kidney, melanoma, ovary, pancreas, prostate, rectum, stomach, thyroid, or uterus.

[0070]

[0084] The composition for determining the amount of cell-free DNA molecules from a biological sample derived from the liver of an organism may comprise a primer pair for amplifying a liver-specific marker based on the methylation status of the liver-specific marker. The liver-specific marker may comprise a polynucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:1. In some cases, the primer pair comprises a primer comprising SEQ ID NO:2 and a primer comprising SEQ ID NO:3. In some cases, the composition further comprises a detectable-labeled probe comprising SEQ ID NO:4 for detecting the liver-specific marker. In some cases, the composition further comprises a primer comprising SEQ ID NO:5 and a primer comprising SEQ ID NO:6. In some cases, the composition further comprises a detectable-labeled probe comprising SEQ ID NO:7 for detecting the liver-specific marker.

[0071]

[0085] The compositions provided herein may include a primer pair for amplification of a liver-specific marker based on the methylation status of the colon-specific marker. The colon-specific marker may include a polynucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:8. In some cases, the primer pair includes a primer including SEQ ID NO:9 and a primer including SEQ ID NO:10. In some cases, the composition further includes a detectably labeled probe including SEQ ID NO:11 for detection of the colon-specific marker. In some cases, the composition further includes a primer including SEQ ID NO:12 and a primer including SEQ ID NO:13. In some cases, the composition further includes a detectably labeled probe including SEQ ID NO:14 for detection of the colon-specific marker.

[0072]

[0086] VI. Biological Samples

[0087] The biological sample used in the methods provided herein may include any tissue or material derived from a living or dead subject. The biological sample may be a cell-free sample. The biological sample may include nucleic acid (e.g., DNA, such as genomic or mitochondrial DNA, or RNA) or fragments thereof. The nucleic acid in the sample may be cell-free nucleic acid. The sample may be a liquid sample or a solid sample (e.g., a cell or tissue sample). The biological sample may be a body fluid, such as blood, plasma, serum, urine, vaginal fluid, hydrocele fluid (e.g., from testes), vaginal washings, pleural fluid, peritoneal fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, nipple discharge, aspirates from different parts of the body (e.g., thyroid, breast), etc. Stool samples may also be used. In various embodiments, the majority of the DNA in a biological sample (e.g., a plasma sample obtained through a centrifugation protocol) that has been enriched for cell-free DNA may be cell-free (e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the DNA may be cell-free). The biological sample may be treated to physically disrupt tissue or cellular structures (e.g., centrifugation and / or cell lysis), thereby releasing intracellular components into solutions that may further contain enzymes, buffers, salts, detergents, etc. used to prepare the sample for analysis.

[0073]

[0088] The methods, compositions, and systems provided herein can be used to analyze nucleic acid molecules in biological samples. The nucleic acid molecules can be cellular nucleic acid molecules, cell-free nucleic acid molecules, or both. The cell-free nucleic acids used by the methods as provided herein can be nucleic acid molecules outside of cells in biological samples. Cell-free nucleic acid molecules can be present in various body fluids, such as blood, saliva, semen, and urine. Cell-free DNA molecules can be generated due to cell death in various tissues, which can be caused by health conditions and / or diseases, such as tumor invasion or growth, immune rejection after organ transplantation.

[0074]

[0089] The cell-free nucleic acid molecules, e.g., cell-free DNA, used in the methods as provided herein may be present in plasma, urine, saliva, or serum. Cell-free DNA may occur naturally in the form of short fragments. Fragmentation of cell-free DNA may refer to the process in which high molecular weight DNA (such as DNA in the nucleus of a cell) is cut, broken, or digested into short fragments when cell-free DNA molecules are produced or released. The methods, compositions, and systems provided herein can be used to analyze cellular nucleic acid molecules, in some cases cellular DNA from tumor tissue, or cellular DNA from white blood cells, for example, when a patient has leukemia, lymphoma, or myeloma. Samples obtained from tumor tissue can be subject to assays and analyses according to some examples of the present disclosure.

[0075]

[0090] VII. Target

[0091] The methods, compositions, and systems provided herein can be used to analyze samples from subjects, such as organisms, such as host organisms. The subject can be any human patient, such as a cancer patient, a patient at risk of cancer, or a patient with a family or personal history of cancer. In some cases, the subject is at a certain stage of cancer treatment. In some cases, the subject may have cancer or be suspected of having cancer. In some cases, it is unknown whether the subject has cancer.

[0076]

[0092] The subject may have any type of cancer or tumor. In one example, the subject may have colon cancer, or cancer of the large intestine. In another example, the subject may have colorectal cancer, or cancer of the colon and rectum. In another example, the subject may have liver cancer, such as hepatocellular carcinoma. Non-limiting examples of cancer include, but are not limited to, adrenal gland cancer, anal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, cardiovascular system cancer, cervical cancer, colon cancer, colorectal cancer, digestive system cancer, endocrine system cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal tumors, hepatocellular carcinoma, renal cancer, hematopoietic malignancies, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, muscle system cancer, bone Examples of cancer include myelodysplastic syndrome (MDS), myeloma, nasal cavity cancer, nasopharyngeal cancer, cancer of the nervous system, cancer of the lymphatic system, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, rectal cancer, renal pelvis cancer, cancer of the reproductive system, cancer of the respiratory system, sarcoma, salivary gland cancer, skeletal system cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, tumor, cancer of the urinary system, uterine cancer, vaginal cancer, or vulvar cancer. The lymphoma can be any type of lymphoma, including B cell lymphoma (e.g., diffuse large B cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system lymphoma) or T cell lymphoma (e.g., precursor T lymphoblastic lymphoma, or peripheral T cell lymphoma). The leukemia can be any type of leukemia, including acute leukemia or chronic leukemia. Types of leukemia include acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, acute anaplastic leukemia, or chronic lymphocytic leukemia. In some cases, a cancer patient does not have a specific type of cancer. For example, in some cases, a patient can have a cancer that is not breast cancer.

[0077]

[0093] Examples of cancers include cancers that cause solid tumors and cancers that do not cause solid tumors. Furthermore, any of the cancers referred to herein may be a primary cancer (e.g., a cancer named for the part of the body in which it first began to grow) or a secondary or metastatic cancer (e.g., a cancer that has originated in another part of the body).

[0078]

[0094] A subject diagnosed by any of the methods described herein can be of any age and can be an adult, an infant, or a child. In some cases, the subject is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, The age of the patient may be 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 years old, or within a range therebetween (e.g., between 2 and 20 years old, between 20 and 40 years old, or between 40 and 90 years old). A particular class of patients that may be of benefit may be patients over 40 years old. Another particular class of patients that may be of benefit may be pediatric patients. Additionally, the subject diagnosed by any of the methods or compositions described herein may be male or female.

[0079]

[0095] Any of the methods disclosed herein can also be performed on non-human subjects, such as laboratory or livestock animals, or cell samples from organisms disclosed herein. Non-limiting examples of non-human subjects include dogs, goats, guinea pigs, hamsters, mice, pigs, non-human primates (e.g., gorillas, apes, orangutans, lemurs, or baboons), rats, sheep, cows, or zebrafish.

[0080]

[0096] As discussed above, the subject methods, compositions, and kits can be used for subjects at various stages of cancer treatment. Results from the analysis of cell-free nucleic acids in a subject's biological sample using the subject methods, compositions, and kits can be used to guide a treatment plan for the subject. In some cases, a drug or therapy may be required to treat or cure the cancer in the subject. Representative treatment options may include chemotherapy, radiation therapy, surgical removal of tumor tissue, immunotherapy, targeted therapy, hormone therapy, and stem cell therapy. In some cases, guidance may be provided for the selection of different types of treatment options. In some non-limiting examples, a patient may have finished treatment for an early stage cancer, for example, surgical removal of tumor tissue in an affected liver lobe, and the patient may undergo routine monitoring tests using the subject methods, compositions, or kits to check for recurrence of liver cancer or metastases. In these cases, the test results can be used to provide guidance as to whether the patient requires further treatment for cancer, whether recurrence of liver cancer or metastases to other tissues has occurred, and which treatment options are applicable. In some cases, guidance may be provided for a specific dose or administration schedule of treatment. For example, the amount of cell-free nucleic acid from a particular tissue may correlate with the dosage to be administered to the patient, or the frequency / interval of drug administration (e.g., daily, weekly, biweekly, or monthly). In some cases, the results from the final analysis can be used as a basis for evaluating and designing treatment options and subsequent monitoring analyses.

[0081]

[0097] VIII. Computer Systems

[0098] Any of the methods disclosed herein can be implemented and / or controlled by one or more computer systems. In some examples, any steps of the methods disclosed herein can be implemented and / or controlled, either in whole, individually, or over time, by one or more computer systems. Any of the computer systems referred to herein can utilize any suitable number of subsystems. In some embodiments, a computer system includes a single computer device, where the subsystems may be components of the computer device. In other embodiments, a computer system may include multiple computer devices (each of which is a subsystem) along with internal components. Computer systems may include desktop and laptop computers, tablets, mobile phones, and other mobile devices.

[0082]

[0099] The subsystems may be interconnected via a system bus. Additional subsystems include a printer, keyboard, storage device, and monitor connected to a display adapter. Peripherals and input / output (I / O) devices are connected to the I / O controller and may be connected to the computer system by any number of connections known in the art, such as input / output (I / O) ports (e.g., USB, FireWire). For example, an I / O port or external interface (e.g., Ethernet, Wi-Fi, etc.) may be used to connect the computer system to a wide area network such as the Internet, a mouse input device, or a scanner. The interconnection via the system bus allows a central processor to communicate with each subsystem and control the execution of instructions from a system memory or storage device (e.g., a fixed disk, such as a hard drive, or an optical disk) and the exchange of information between the subsystems. The system memory and / or storage device may utilize a computer readable medium. Another subsystem is a data collection device, such as a camera, microphone, accelerometer, etc. Any of the data mentioned herein may be output from one component to another component and may be output to a user.

[0083]

[0100] A computer system may include multiple identical components or subsystems, connected together, for example, by external or internal interfaces. In some embodiments, computer systems, subsystems, or devices may communicate across a network. In such an example, one computer may be considered a client and another computer a server, where each may be part of the same computer system. The client and server may each include multiple systems, subsystems, or components.

[0084]

[0101] The present disclosure provides a computer control system programmed to carry out the disclosed methods. Figure 10 shows a computer system 101 programmed or otherwise configured to determine the absolute amount of cell-free nucleic acid molecules from a biological tissue as described herein. The computer system 101 can execute and / or regulate various aspects of the methods provided in the present disclosure, including, for example, controlling the sequencing of nucleic acid molecules from a biological sample, performing various steps of bioinformatics analysis of the sequencing data as described herein, incorporating data collection, analysis and results reporting, and data management. The computer system 101 can be a computer system located in the user's electronic device or remotely relative to the electronic device. The electronic device can be a portable electronic device.

[0085]

[0102] The computer system 101 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 105, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 101 also includes memory or memory locations 110 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 115 (e.g., hard disk), a communication interface 120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 125, such as cache, other memory, data storage, and / or electronic display adapters. The memory 110, the storage unit 115, the interface 120, and the peripheral devices 125 communicate with the CPU 105 via a communication bus (solid lines), e.g., a motherboard. The storage unit 115 may be a data storage unit (or data repository) for storing data. The computer system 101 may be operatively connected to a computer network ("network") 130 with the aid of the communication interface 120. Network 130 may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. Network 130 may be a telecommunications and / or data network in some cases. Network 130 may include one or more computer servers, which may enable distributed computing, such as cloud computing. Network 130 may implement a peer-to-peer network, which may enable devices connected to computer system 101 to act as clients or servers, possibly with the assistance of computer system 101.

[0086]

[0103] CPU 105 can execute a series of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a memory location, such as memory 110. The instructions can be directed to CPU 105, which can then be programmed or otherwise configured to execute the methods of the present disclosure. Examples of operations performed by CPU 105 can include fetch, decode, execute, and writeback.

[0087]

[0104] The CPU 105 may be part of a circuit, such as an integrated circuit. One or more other components of the system 101 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0088]

[0105] The storage unit 115 can store files, such as drivers, libraries, and saved programs. The storage unit 115 can store user data, such as user preferences and user programs. The computer system 101 may optionally include one or more additional data storage units, external to the computer system 101, for example located on a remote server that communicates with the computer system 101 via an intranet or the Internet.

[0089]

[0106] The computer system 101 can communicate with one or more remote computer systems via the network 130. For example, the computer system 101 can communicate with a user's remote computer system (e.g., a smart phone with an application installed that receives and displays the results of sample analysis sent from the computer system 101). Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., Apple® iPad®, Samsung® Galaxy Tab), a phone, a smart phone (e.g., Apple® iPhone®, Android-powered device, Blackberry®), or a personal digital assistant. A user can access the computer system 101 via the network 130.

[0090]

[0107] Methods as described herein may be implemented through machine (e.g., computer processor) executable code stored in electronic storage locations of computer system 101, such as memory 110 or electronic storage unit 115. Machine executable or machine readable code may be provided in the form of software. In use, the code is executable by processor 105. In some cases, the code may be retrieved from storage unit 115 and stored in memory 110 for ready access by processor 105. In some circumstances, electronic storage unit 115 may be omitted and machine executable instructions are stored in memory 110.

[0091]

[0108] The code may be pre-compiled and configured for use on a machine having a suitable processor to execute the code, or may be compiled during run-time. The code may be provided in a selectable programming language such that the code can be executed in a pre-compiled or as-compiled manner.

[0092]

[0109] Aspects of the systems and methods provided herein, such as the computer system 101, can be embodied in programming. Various aspects of the technology can be considered as a "product" or "article of manufacture" in the form of machine (or processor) executable code and / or associated data, typically embodied or operated on some type of machine-readable medium. The machine executable code can be stored in an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium can include some or all of the tangible memory of a computer, processor, etc., or their associated modules, such as various semiconductor memories, tape drives, disk drives, etc., that can provide optional non-transitory storage in software programming. All or parts of the software can sometimes be communicated over the Internet or various other information and communication networks. Such communication can, for example, enable loading of the software from one computer or processor to another, such as from a management server or host computer to a computer platform of an application server. Thus, other types of media that may bear software elements include light waves, radio waves, and electromagnetic waves used across physical interfaces between local devices, for example, through wired and optical terrestrial networks, and across various air links. The physical elements that carry such waves, e.g., wired or wireless links, optical links, etc., may also be considered media bearing the software. As used herein, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution, unless limited to non-transitory, tangible "storage" media.

[0093]

[0110] Thus, machine-readable media, e.g., computer executable code, can take many forms, including, but not limited to, tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, e.g., optical or magnetic disks, e.g., any of the storage devices within any computer, e.g., that can be used to execute, e.g., the illustrated databases, etc. Volatile storage media include dynamic memory, e.g., the main memory of such a computer platform. Tangible transmission media include, e.g., coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier wave transmission media can take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or a DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium having a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link which transports such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0094]

[0111] The computer system 101 may include or be in communication with an electronic display 135 that includes a user interface (UI) 140 for providing results of the sample analysis including, for example, but not limited to, a graphical display of relative and / or absolute amounts of cell-free nucleic acids from different tissues, a control or reference amount of cell-free nucleic acid from a particular tissue, a comparison between the detected amount and the reference amount, and a readout of the presence or absence of cancer metastasis. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0095]

[0112] The methods and systems of the present disclosure can be implemented through one or more algorithms. The algorithms can be implemented through software when executed by the central processing unit 105. The algorithms can, for example, control the sequencing of nucleic acid molecules from a sample, the direct collection of sequencing data, the analysis of the sequencing data, or the determination of a pathology classification based on the analysis of the sequencing data.

[0096]

[0113] In some cases, as shown in FIG. 11, a sample 202 can be obtained from a subject 201, e.g., a human subject. The sample 202 can be subjected to one or more methods as described herein, such as performing an assay. In some cases, the assay can include hybridization, amplification, sequencing, labeling, epigenetic modification of bases, or any combination thereof. One or more results from the method can be input to a processor 204. One or more input parameters, such as sample identification, subject identification, sample type, reference, or other information, can be input to the processor 204. One or more metrics from the assay can be input to the processor 204, so that the processor can generate a result, e.g., a classification of a pathology (e.g., a diagnosis) or a recommended treatment. The processor can send the result, the input parameters, the metrics, the reference, or any combination thereof to a display 205, e.g., a visual display or a graphical user interface. The processor 204 may (i) send results, input parameters, metrics, or any combination thereof to the server 207; (ii) receive results, input parameters, metrics, or any combination thereof from the server 207; or (iii) or any combination thereof.

[0097]

[0114] Aspects of the present disclosure may be implemented using hardware (e.g., application-specific integrated circuits or field programmable gate arrays) in the form of control logic and / or using computer software in a processor that is generally programmable in a modular or integrated fashion. As used herein, a processor includes a single-core processor, a multi-core processor on the same integrated chip, or multiple processing units on a single circuit or network board. Based on the disclosure and teachings provided herein, those skilled in the art will recognize and understand other ways and / or methods for implementing the embodiments described herein using hardware and combinations of hardware and software.

[0098]

[0115] Any of the software components or functions described herein can be implemented as software code to be executed by a processor using any suitable computer language, such as Java, C, C++, C#, Objective-C, Swift, or scripting languages, such as Perl or Python, using, for example, conventional or object-oriented techniques. The software code can be stored as a set of instructions or commands on a computer-readable medium for storage and / or transmission. Suitable non-transitory computer-readable media can include random access memory (RAM), read-only memory (ROM), magnetic media such as hard drives or floppy disks, or optical media such as compact disks (CDs) or DVDs (digital versatile disks), flash memory, and the like. The computer-readable medium can be any combination of such storage or transmission devices.

[0099]

[0116] Such programs may also be encoded and transmitted using carrier signals suitable for transmission over wired, optical, and / or wireless networks conforming to various protocols, including the Internet. As such, computer-readable media may be created using data signals encoded with such programs. Computer-readable media encoded with program code may be packaged with a compatible device or provided separately from other devices (e.g., via Internet download). Any such computer-readable medium may be external or internal to a single computer product (e.g., a hard drive, CD, or an entire computer system), or may be external or internal to different computer products within a system or network. A computer system may include a monitor, printer, or other suitable display for providing a user with any of the results mentioned herein.

[0100]

[0117] Any of the methods described herein may be implemented in whole or in part in a computer system including one or more processors that can be configured to perform the steps. Thus, embodiments may be directed to a computer system configured to perform the steps of any of the methods described herein having different components performing each step or each group of steps. The steps of the methods herein are presented as numbered steps, but may be performed simultaneously or in different orders. In addition, portions of the steps may be combined with portions of other steps from other methods. Furthermore, all or portions of the steps may be optional. In addition, any of the steps of any of the methods may be implemented using modules, units, circuits, or other techniques for performing those steps. EXAMPLES

[0101] Working Example

[0118] The following examples further illustrate the described embodiments without limiting the scope of the disclosure.

[0102]

[0119] Example 1. Materials and Methods

[0120] This example describes some of the methods utilized in Examples 2-5.

[0103]

[0121] subject

[0122] Patients who had previously undergone liver transplantation were recruited during their visit to the Liver Transplant Clinic from the Department of Surgery of the Prince of Wales Hospital, Hong Kong. Patients with chronic hepatitis B virus (HBV) infection and cirrhosis were recruited from the Department of Medicine and Therapeutics of the Prince of Wales Hospital, Hong Kong. Patients with hepatocellular carcinoma (HCC) and CRC were recruited from the Department of Surgery and the Department of Clinical Oncology of the Prince of Wales Hospital, Hong Kong. The demographics of the recruited subjects are shown in Table 2. All recruited subjects provided signed consent. The study was approved by the Joint Hospital Authority New Territories East Cluster - The Chinese University of Hong Kong Clinical Research Ethics Committee.

[0104] [Table 2]

[0105]

[0123] Sample preparation

[0124] For each subject, 10 mL of peripheral blood was collected in EDTA-containing tubes. Blood samples were processed within 6 h of collection for separation of plasma and buffy coat. DNA was extracted from plasma using a QIAamp DSP DNA Mini Kit (Qiagen) according to the manufacturer's protocol. DNA extracted from 2–4 mL of plasma was subjected to two rounds of bisulfite treatment using an Epitect Plus Bisulfite Kit (Qiagen). Bisulfite-converted DNA was eluted in 50 μL of water for downstream analysis.

[0106]

[0125] Identification of liver- and colon-specific methylation markers

[0126] To search for tissue-specific methylation markers, the methylation profile of the tissue of interest (i.e., liver or colon) was compared with that of other blood cells and tissues. The methylation profiles of different cell types were retrieved from the RoadMap Epigenomics Project database for lung, esophagus, small intestine, colon, pancreas, bladder, heart, and liver, and from the BLUEPRINT Project database for erythroblasts, neutrophils, B lymphocytes, and T lymphocytes.

[0107]

[0127] For the methylation markers, the following criteria were established:

[0128] 1. A CpG site was defined as hypermethylated in a target tissue (i.e., liver or colon) if the methylation density of the CpG site was >50% in the target tissue and <20% in other blood cells and tissues.

[0129] 2. A stretch of at least five hypermethylated CpG sites was found to lie within a differentially methylated region (DMR), improving the latter signal-to-noise ratio and analytical specificity of the methylation marker.

[0130] 3. Since the majority of circulating DNA molecules are short fragments, with a peak size of 166 bp, the DMR must be shorter than 166 bp.

[0108]

[0131] DMR in the liver and colon

[0132] Using the above criteria, one liver-specific marker and one colon-specific marker were identified. The liver-specific DMR was located within the exon region of the protein tyrosine kinase 2 beta (PTK2B) gene on chromosome 8. Eight CpG sites within the liver-specific DMR were hypermethylated in the liver but hypomethylated in other tissues and blood cells (Figure 7). The PTK2B gene is located on chromosome 8, and the genomic coordinates of the CpG sites are shown on the X-axis in Figure 7. All eight CpG sites located within the DMR (region between the two vertical dotted lines) are hypermethylated in the liver compared to other tissues. The region highlighted in yellow has three CpG sites within the fluorescent probe of the digital PCR assay (see Table 3). The other CpG sites on each side of the highlighted region within the DMR were covered by the primers of the digital PCR assay. The colon-specific DMR was located within the exon region of the sestrin 3 (SESN3) gene on chromosome 11. All six CpG sites within the colon-specific DMR were hypermethylated in colon tissue (Figure 8), but hypomethylated in other tissues. SESN3 is located on chromosome 11, and the genomic coordinates of the CpG sites are shown on the X-axis in Figure 8. All six CpG sites located within the DMR (the region between the two vertical dotted lines) are hypermethylated in colon compared to other tissues. Three CpG sites located within the yellow highlighted region were covered by the fluorescent probe of the colon-specific methylation assay (see Table 3). The other CpG sites on each side of the highlighted region within the DMR were covered by the primers of the digital PCR assay. For illustrative purposes, the individual results in Figures 7 and 8 for liver, bladder, esophagus, heart, lung, pancreas, and small intestine are not shown, but their average values ​​are represented by "other tissues".

[0109]

[0133] Design and performance of droplet digital PCR

[0134] Two droplet digital PCR assays were developed to quantify methylated and unmethylated DNA molecules in liver-specific and colon-specific methylation markers, respectively. The primer and probe sequences for the assays are listed in Table 3 (nucleotides underlined in the primers and probes were differentially methylated cytosines at CpG sites). The two droplet digital PCR assays can quantify methylation (from target tissues) and unmethylation (from non-target tissues) using FAM- and VIC-labeled probes, respectively. The liver-specific marker was the PTK2B gene marker site (chr8:27,183,116-27,183,176), and the colon-specific marker was the SESN3 gene marker site (chr11:94,965,508-94,965,567).

[0110]

[0135] Digital PCR analysis was performed in duplicate for each sample. A reaction mixture containing 8 μL bisulfite converted DNA, 450 nM final concentration of each of forward and reverse primers, 250 nM of unmethylated specific probe, and 350 nM (liver assay) or 250 nM (colon assay) of methylated specific probe was prepared for the colon assay in a total volume of 20 μL. The reaction mixture was subjected to droplet generation using a BioRad QX200 ddPCR droplet generator prior to the PCR reaction. Universally methylated DNA (CpGenome Human Methylated DNA from EMD Millipore) and universally unmethylated DNA (EpiTect Unmethylated Human Control DNA from Qiagen) were run on each plate as positive and negative controls. The thermal profile was 95°C for 10 min, followed by 45 cycles of 94°C for 15 s and 60°C (liver assay) or 56°C (colon assay) for 1 min, with a final incubation of 98°C for 10 min. After PCR, droplets from each sample were analyzed by a QX200 droplet reader and the results were interpreted using QuantaSoft (version 1.7) software. The cut-off value for positive fluorescent signal was determined by reference to the control. The number of methylated and unmethylated DNA sequences in each sample was calculated using the number of combinations from duplicate wells followed by Poisson correction. Calculation of the concentration of methylated or unmethylated DNA sequences in plasma is as follows:

number

[0111] [Table 3]

[0112]

[0136] Analysis of DNA from different types of samples

[0137] Formalin-fixed paraffin-embedded (FFPE) samples of 10 types of tissues (e.g., liver, lung, esophagus, stomach, small intestine, colon, pancreas, bladder, heart, and brain) were retrieved from the Department of Anatomical and Cellular Pathology of the Prince of Wales Hospital, Hong Kong. These tissues were confirmed to be normal upon histological examination. Buffy coat samples were collected from healthy subjects. DNA was extracted from FFPE tissues using a QIAamp DNA Mini Kit (Qiagen). DNA from buffy coats was extracted using a QIAamp DNA Blood Mini Kit (Qiagen). Bisulfite conversion was performed using 1 μg of cellular DNA. The converted DNA was eluted in 20 μL of water and then diluted 50-fold for downstream analysis.

[0113]

[0138] Measurement of donor-derived DNA in plasma of liver transplant recipients

[0139] DNA extracted from donor liver tissue and recipient buffy coat was analyzed using the Illumina iScan system to determine genotype information for donors and recipients. DNA extracted from 4 mL of plasma in each recipient was used for sequencing library preparation. Plasma DNA sequencing libraries were prepared using the KAPA Library Preparation Kit (KAPA Biosystems) according to the manufacturer's instructions. The index libraries were then multiplexed and sequenced using the Illumina HiSeq 2500 platform (75 × 2 cycles). At least 20 million paired-end reads were obtained for each sample. Paired-end reads were aligned to the non-repeat masked human reference genome (GRCh 37 / hg 19) using Short Oligonucleotide Alignment Program 2 (SOAP2). Only paired-end reads with both ends aligned to the same chromosome in the correct orientation and aligned to a single location in the human genome were included. Paired-end reads spanning insert sizes ≦600 bp were collected for analysis. If two or more read pairs mapped to the same genomic location (i.e., double reads), only one read pair was retained for subsequent analysis. A maximum of two nucleotide mismatches were allowed for either member of the paired end read. The fractional concentration of circulating donor-specific DNA was determined by counting sequencing reads with single nucleotide polymorphism (SNP) alleles that were homozygous in the recipient and heterozygous in the donor.

[0114]

[0140] Example 2. Tissue specificity of liver and colon markers

[0141] For both liver-specific and colon-specific markers, DNA molecules from target tissues were hypermethylated and those from non-target tissues were hypomethylated. Thus, the percentage of total molecules was expressed as L% methylation in the liver assay, and the percentage of total molecules was expressed as C% methylation in the colon assay. To confirm the specificity of liver and colon markers, DNA extracted from buffy coat samples and 10 types of normal tissues were analyzed using these two sets of digital PCR assays. For each type of tissue, four samples from different individuals were included.

[0115]

[0142] The mean L% in liver tissue was 67% (range: 57%-76%), and the mean L% in other tissue types was 0.6% (range: 0.0%-2.2%). The results for each tissue type are summarized in Table 4. These results suggested that the liver assay can specifically detect liver-derived DNA.

[0116] [Table 4]

[0117]

[0143] The mean C% in colon tissue was 22% (range: 17%-33%). The mean C% in all other tissues was 1.2% (range: 0.1%-4.1%), indicating the specificity of the methylated sequences when derived from the colon. The relatively low C% in colon tissue was probably due to the non-homogeneous cellular composition of colon tissue. This relatively low C% in colon tissue did not significantly interfere with its clinical application when using the same assay to compare levels in subjects with different disease states.

[0118]

[0144] Example 3. Plasma concentrations of liver-derived DNA in liver transplant recipients

[0145] The quantitative accuracy of the liver-specific assay was verified through the analysis of plasma from liver transplant recipients. In these subjects, the fractional concentration of transplanted liver-derived DNA could be accurately determined from the proportion of plasma DNA molecules carrying donor-specific alleles using next-generation sequencing. Fourteen plasma samples collected from 13 patients who had undergone liver transplantation were analyzed by both liver-specific methylation markers and sequencing. A positive linear relationship was observed between the concentrations measured by these two methods (R = 0.99, P < 0.0001, Pearson correlation, Figure 2), indicating that the liver-specific methylation markers can accurately reflect the concentration of liver-derived DNA in plasma.

[0119]

[0146] As shown here, measurements of the percentage contribution of liver DNA concentration by liver-specific methylation markers correlated well with results based on measurements of donor-specific alleles. These results confirmed the accuracy of liver-specific markers in reflecting the concentration of liver-derived DNA in plasma.

[0120]

[0147] Example 4. Increase in liver-derived DNA in plasma of HCC patients

[0148] Absolute and fractional concentrations of liver-derived DNA were measured by digital PCR targeting sequences with liver-specific methylation patterns in 40 HCC patients, 9 cirrhosis patients, 20 chronic HBV carriers, and 30 healthy subjects.

[0121]

[0149] The median concentrations of liver-derived methylated sequences in healthy subjects, chronic HBV carriers, cirrhotic patients, and HCC patients were 40 copies / mL (interquartile range (IQR): 18–86), 122 copies / mL (IQR: 47–185), 118 copies / mL (IQR: 86–159), and 487 (IQR: 138–1151), respectively (Figure 3A). The concentrations were significantly different across the four groups (P<0.001, Kruskal-Wallis test). In post hoc analyses, plasma concentrations of liver-derived DNA in HCC patients were significantly higher than those in healthy subjects (P<0.001, Dunn test) and chronic HBV carriers (P=0.015, Dunn test), but not in the cirrhotic patient group (P=0.248, Dunn test). Concentrations in healthy subjects, chronic HBV carriers and cirrhotic patients were not statistically different (P>0.05, Dunn's test).

[0122]

[0150] The median fractional concentrations of liver-derived DNA in plasma in healthy subjects, chronic HBV carriers, cirrhotic patients, and HCC patients were 1.4% (IQR: 0.94%-3.2%), 4.6% (IQR: 1.7%-6.0%), 3.0% (interquartile range: 1.8%-7.3%), and 9.4% (IQR: 4.1%-16.0%), respectively (Figure 3B). The fractional concentrations were significantly different across the four groups (P<0.001, Kruskal-Wallis test). In post hoc analyses, plasma concentrations of liver-derived DNA in HCC patients were significantly higher than in healthy subjects (P<0.001, Dunn's test), but not in chronic HBV carriers (P=0.129, Dunn's test) and cirrhotic patients (P=0.592, Dunn's test). The concentrations in healthy subjects, chronic HBV carriers, and cirrhotic patients were not statistically different.

[0123]

[0151] These results showed that analysis of both absolute and fractional concentrations of liver-derived DNA in plasma could distinguish HCC patients from non-HCC subjects (including healthy subjects, chronic HBV carriers, and cirrhosis patients). To further determine whether absolute or fractional concentrations were superior in distinguishing HCC subjects from non-HCC subjects, receiver operating characteristic (ROC) curve analysis was performed (Figure 9). The areas under the curve (AUC) for absolute and fractional concentrations were 0.82 and 0.78, respectively. The difference in AUC was statistically significant (P=0.022, Delong test).

[0124]

[0152] We further analyzed the correlation between plasma liver-derived DNA concentrations (absolute and fractional) and the maximum tumor dimensions (measured by computed tomography scan or after tumor resection) in HCC patients. Interestingly, the maximum tumor dimensions showed a stronger positive correlation with the absolute concentration (R = 0.74, P < 0.0001, Spearman correlation) than with the fractional concentration (R = 0.56, P = 0.0002, Spearman correlation) (Figures 4A and 4B). In HCC patients, the concentration of liver-derived DNA was positively correlated with the maximum tumor dimensions, indicating that the amount of DNA released from the liver reflects the tumor burden.

[0125]

[0153] Example 5. Analysis of DNA from liver and colon in CRC patients with and without liver metastases

[0154] Plasma concentrations of liver- and colon-derived DNA were measured in 30 healthy subjects, 35 CRC patients without liver metastasis, and 27 CRC patients with liver metastasis. The median plasma concentrations of colon-derived DNA in the three groups were 0 copies / mL (IQR: 0-0), 4 copies / mL (IQR: 0-31), and 138 copies / mL (IQR: 0-6850), respectively (Figure 5A). The concentrations were significantly different among the three groups (P<0.001, Kruskal-Wallis test). In post-hoc analysis, concentrations in CRC patients with and without liver metastasis were significantly higher than in healthy subjects (P<0.001 and P=0.042, respectively, Dunn's test). The difference between CRC patients with and without liver metastasis was not statistically significant (P=0.079, Dunn's test).

[0126]

[0155] The median fractional concentrations of colon-derived DNA in plasma in healthy control subjects, CRC patients without liver metastases, and CRC patients with liver metastases were 0% (IQR: 0%-0%), 0.09% (IQR: 0%-1.1%), and 0.84% ​​(IQR: 0%-49.5%), respectively (Figure 5B). The fractional concentrations were significantly different among the three groups (P<0.001, Kruskal-Wallis test). In post-hoc analysis, the concentrations in CRC patients with and without liver metastases were significantly higher than in healthy subjects (P<0.001 and P=0.041, respectively, Dunn's test). The difference between CRC patients with and without liver metastases was not statistically significant (P=0.084, Dunn's test).

[0127]

[0156] The median concentrations of liver-derived DNA in plasma in healthy control subjects, CRC patients without liver metastases, and CRC patients with liver metastases were 40 copies / mL (IQR: 18–86), 23 copies / mL (IQR: 13–108), and 233 copies / mL (IQR: 56–2290), respectively (Figure 5C). The fractional concentrations were significantly different among the three groups (P<0.001; Kruskal-Wallis test). In post-hoc analysis, the concentrations in CRC patients with liver metastases were significantly higher than those in CRC patients without liver metastases and healthy controls (P<0.001 and P<0.001, respectively; Dunn's test). Interestingly, no significant differences were found between patients without liver metastases and healthy controls (P=1.0; Dunn's test).

[0128]

[0157] The median fractional concentrations of liver-derived DNA in plasma in healthy control subjects, CRC patients without liver metastases, and CRC patients with liver metastases were 0.8% (IQR: 0.3%-2.8%), 1.4% (IQR: 0.9%-3.3%), and 3.1% (IQR: 1.5%-5.3%), respectively (Figure 5D). The fractional concentrations were significantly different among the three groups (P<0.001, Kruskal-Wallis test). In post-hoc analysis, the concentrations in CRC patients with liver metastases were significantly higher than those in CRC patients without liver metastases (P<0.003, Dunn's test). The fractional concentrations in healthy subjects were not statistically significantly different from those in CRC patients with and without liver metastases (P=0.114 and P=0.717, respectively).

[0129]

[0158] Since the absolute and fractional concentrations of liver- and colon-derived DNA in plasma were significantly different between CRC patients with and without liver metastasis, receiver operating characteristic curve analysis was used to determine which parameters would be most useful in discriminating between the two groups. The AUCs for the absolute and fractional concentrations of liver-derived DNA were 0.85 and 0.75 (P = 0.01, Delong test), respectively, and the AUCs for the absolute and fractional concentrations of colon-derived DNA were 0.69 and 0.69 (P = 0.75, Delong test), respectively (Figure 6).

[0130]

[0159] Analysis of the absolute concentration of liver-derived DNA was superior to the fractional concentration in distinguishing CRC patients with and without liver metastasis in ROC analysis (AUC: 0.85 vs. 0.75, P=0.01, FIG. 6). Without being bound by theory, a likely explanation is that in some patients with metastatic CRC to the liver, the absolute concentrations of both liver- and colon-derived DNA appear to increase. In some patients, the absolute concentration of liver-derived DNA increased, but the fractional concentration of liver remained unchanged or decreased due to a further increase in colon-derived DNA. Similarly, it was also shown that the absolute concentration of liver-derived DNA correlated better with tumor size compared to the fractional concentration (R=0.74 vs. 0.56, Spearman correlation, FIG. 4).

[0131]

[0160] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the disclosed embodiments described herein can be used in implementing the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for assaying cell-free DNA molecules in a sample, comprising: (a) obtaining cell-free DNA molecules from a first biological sample of an organism having cancer, wherein said cancer comprises cells of a primary cancer of an initial tissue, said first biological sample comprising cell-free DNA from said cancer and cell-free DNA from a further tissue different from the initial tissue of said primary cancer; (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 comprising the target sequence originates from a further tissue of the organism; (c) measuring the absolute amount of cell-free DNA molecules from the first biological sample that contain the target sequence having the first methylation state; (d) determining whether cell-free DNA molecules from the additional tissue at a metastatic site are present in the sample by comparing the measured absolute amount with a reference amount, where (i) the reference amount is an absolute amount of cell-free DNA comprising a target sequence having the first methylation state for one or more control subjects without metastasis in the additional tissue, and (ii) a measured absolute amount exceeding the reference amount indicates the presence of the cell-free DNA molecules from the additional tissue at a metastatic site. A method comprising:

2. The assay comprising: (a) isolating the cell-free DNA molecule comprising the target sequence from the first biological sample; (b) isolating said cell-free DNA molecules containing said target sequence in an oil emulsion; (c) bisulfite conversion of unmethylated cytosine residues in said cell-free DNA molecules to uracil; or (d) performing methylation-aware sequencing of cell-free DNA molecules from said first biological sample. The method of claim 1 , comprising:

3. The method of claim 1 , wherein the assay comprises hybridizing the cell-free DNA molecule containing the target sequence to a probe.

4. (a) the affinity of hybridization of the probe to the target sequence is dependent on the first methylation state of the target sequence in the first biological sample; (b) the probe hybridizes to the target sequence when a methylation site of the target sequence is methylated in the first biological sample; (c) the probe hybridizes to the target sequence when a methylation site of the target sequence is unmethylated in the first biological sample; or (d) the assay comprises detecting hybridization of the probe to the target sequence; The method according to claim 3.

5. The method of claim 1 , wherein the assay comprises amplifying the cell-free DNA molecules.

6. (a) the amplification comprises the use of a primer pair; (b) the affinity of hybridization of at least one primer of a primer pair to said target sequence is dependent on said first methylation state of said target sequence; (c) at least one primer of the primer pair hybridizes to the target sequence when a methylation site of the target sequence is methylated in the first biological sample; or 6. The method of claim 5, wherein (d) at least one primer of the primer pair hybridizes to the target sequence when a methylation site of the target sequence is unmethylated in the first biological sample.

7. The method of claim 1 , wherein the target sequence comprises at least five methylation sites.

8. 2. The method of claim 1, wherein the first methylation state comprises a methylation density at individual sites within the target sequence, a distribution of methylated / unmethylated sites across adjacent regions within the target sequence, a pattern or level of methylation at each individual methylation site within the target sequence, or non-CpG methylation.

9. 2. The method of claim 1, wherein the target sequence comprises a higher methylation density in the further tissue than in the cancer.

10. The target sequence is (a) a methylation density in said further tissue of greater than 50%; (b) a methylation density in the cancer of up to 50%; or (c) a methylation density in the cancer of less than 20%.

10. The method of claim 9, comprising:

11. 2. The method of claim 1, wherein the target sequence comprises a lower methylation density in the further tissue than in the cancer.

12. The target sequence is (a) a methylation density in said further tissue of less than 50%; (b) a methylation density in at least 50% of the cancer; or (c) a methylation density in the cancer that is greater than 80%. The method of claim 11 , comprising:

13. 2. The method of claim 1, wherein the additional tissue comprises liver tissue and the target sequence comprises a polynucleotide sequence having at least 90% identity to SEQ ID NO:

1.

14. (a) the further tissue comprises liver tissue, and the assay comprises amplification using a primer comprising SEQ ID NO:2, a primer comprising SEQ ID NO:3, or both, or the use of a detectably labeled probe comprising SEQ ID NO:4 to detect the target sequence; The method of claim 13.

15. (b) the amplification further comprises the use of a primer comprising SEQ ID NO:5, a primer comprising SEQ ID NO:6, or both, or the use of a detectably labeled probe comprising SEQ ID NO:7 to detect the target sequence; The method of claim 14.

16. 2. The method of claim 1, wherein the additional tissue comprises colon tissue and the target sequence comprises a polynucleotide sequence having at least 90% identity to SEQ ID NO:

8.

17. the further tissue comprises colon tissue, and the assay comprises amplification using a primer comprising SEQ ID NO:9, a primer comprising SEQ ID NO:10, or both, or the use of a detectably labeled probe comprising SEQ ID NO:11 to detect the target sequence.

17. The method of claim 16.

18. the amplification further comprises the use of a primer comprising SEQ ID NO:12, a primer comprising SEQ ID NO:13, or both, or the use of a detectably labeled probe comprising SEQ ID NO:14 to detect the target sequence; 20. The method of claim 17.

19. 2. The method of claim 1, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, hematopoietic malignancies, 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, gastric cancer, melanoma, thyroid cancer, hepatocellular carcinoma, and colorectal cancer.

20. 10. The method of claim 1, further comprising determining a classification of the cancer in the additional tissue.

21. 21. The method of claim 20, wherein the determination of the classification of cancer in the further tissue comprises evaluating a cell-free nucleic acid molecule from a second biological sample from the organism.

22. The evaluation (a) determining a methylation profile, copy number variation, single nucleotide polymorphism (SNP) profile, or fragmentation pattern of the cell-free nucleic acid molecule from the second biological sample; or (b) determining the amount of cell-free nucleic acid molecules from the second biological sample derived from a pathogen.

22. The method of claim 21 , comprising:

23. A system configured to carry out the method according to any one of claims 1 to 22.

24. A non-transitory computer readable medium comprising a set of instructions for controlling a computer system to perform the method of any one of claims 1 to 22.

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