Methods and systems for tumor detection
Methods are provided to improve the positive predictive value for cancer detection using cell-free nucleic acid samples. Various embodiments are directed to applications (e.g., diagnostic applications) of the analysis of the fragmentation patterns and size of cell-free DNA, e.g., plasma DNA and serum DNA, including nucleic acids from pathogens, including viruses. Embodiments of one application can determine if a subject has a particular condition. For example, a method of present disclosure can determine if a subject has cancer or a tumor, or other pathology. Embodiments of another application can be used to assess the stage of a condition, or the progression of a condition over time. For example, a method of the present disclosure may be used to determine a stage of cancer in a subject, or the progression of cancer in a subject over time (e.g., using samples obtained from a subject at different times).
1 . A method of non-invasively detecting a tumor in a subject, comprising:
obtaining, through a non-invasive procedure, a first biological sample from the subject, wherein the first biological sample comprises substantially cell-free DNA (cfDNA) derived from normal cells and comprises or is suspected of comprising tumor-derived cfDNA;
performing a first assay using the first biological sample, wherein the first assay has a sensitivity for a first set of markers indicative of the tumor, and wherein the first assay comprises:
measuring a methylation status of the tumor-derived cfDNA in the first biological sample; and
determining if the first biological sample includes the first set of markers indicative of the tumor based on a comparison of the methylation status of the tumor-derived cfDNA in the first biological sample to at least one first calibration value;
responsive to determining that the first biological sample includes the first set of markers indicative of the tumor, obtaining, through a non-invasive procedure, a second biological sample from the subject, wherein the second biological sample comprises cfDNA derived from normal cells and comprises or is suspected of comprising tumor-derived cfDNA;
only when the first biological sample includes the first set of markers indicative of the tumor, performing a second assay using the second biological sample, wherein the second assay has a specificity for a second set of markers indicative of the tumor, and wherein the second assay comprises:
measuring a methylation status of the tumor-derived cfDNA in the second biological sample;
identifying the tumor in the subject based on the comparison of the methylation status of the tumor-derived cfDNA in the second biological sample to at least one second calibration value; and
outputting a report indicative of the tumor in the subject.
2 . The method of claim 1 , wherein the second biological sample is obtained between 1 day and 12 weeks after obtaining the first biological sample.
3 . The method of claim 1 , wherein a positive predictive value of the method of non-invasively detecting a tumor in a subject is greater than a positive predictive value of the first assay or second assay alone.
4 . The method of claim 3 , wherein the positive predictive value of the method of non-invasively detecting a tumor in a subject is at least 1.5-fold, at least 2-fold, at least 4-fold, at least 10-fold, or at least 20-fold greater than the positive predictive value of the first assay or second assay alone.
5 . The method of claim 1 , wherein the sensitivity of the method of non-invasively detecting a tumor in a subject is same or greater than the sensitivity of the first assay alone, and wherein the specificity of the method is same or greater than a specificity of the first assay alone.
6 . The method of claim 1 , wherein the first set of markers and the second set of markers are the same set of markers.
7 . The method of claim 1 , wherein the sensitivity of the first assay for the first set of markers is at least about 35%.
8 . The method of claim 1 , wherein the specificity of the second assay for the second set of markers is at least about 95%.
9 . The method of claim 1 , wherein the tumor is selected from a group consisting of liver cancer, nasopharyngeal cancer, oropharyngeal cancer, oral cancer, bladder cancer, bone cancer, a brain tumor, breast cancer, esophageal cancer, gastrointestinal cancer, hematopoietic malignancy, leukemia, lung cancer, lymphoma, myeloma, ovarian cancer, prostate cancer, sarcoma, stomach cancer, and thyroid cancer.
10 . The method of claim 1 , wherein at least one of the first biological sample and the second biological sample are independently selected from a group consisting of whole blood, blood plasma, blood serum, urine, buffy coat, and a combination thereof.
11 . The method of claim 1 , wherein the first assay and the second assay further comprise using methylation-aware sequencing to detect the methylation status of tumor-derived cfDNA in the first biological sample and second biological sample, respectively.
12 . The method of claim 11 , wherein the methylation-aware sequencing comprises using bisulfite DNA conversion with the tumor-derived cfDNA prior to sequencing.
13 . The method of claim 1 , further comprising performing an imaging examination on the subject, wherein the imaging examination is responsive to the first assay and/or the second assay.
14 . The method of claim 1 , wherein the first assay and the second assay further comprise enriching the sample for targeted cfDNA fragments corresponding to one or more predetermined regions of a genome.
15 . The method of claim 1 , wherein the first biological sample or second biological sample further comprises virus-derived DNA fragments, the method further comprising detecting the virus-derived DNA, wherein detection of the virus-derived DNA is a negative control for tumor-derived DNA.
16 . A method of non-invasively detecting a tumor in a subject, comprising:
obtaining, through a non-invasive procedure, a first biological sample from the subject, wherein the first biological sample comprises substantially cell-free DNA (cfDNA) derived from normal cells and comprises or is suspected of comprising tumor-derived cfDNA;
performing a first assay using the first biological sample, wherein the first assay has a specificity for a first set of markers indicative of the tumor, and wherein the first assay comprises:
measuring a methylation status of the tumor-derived cfDNA in the first biological sample; and
determining if the first biological sample includes the first set of markers indicative of the tumor based on a comparison of the methylation status of the tumor-derived cfDNA in the first biological sample to at least one first calibration value;
responsive to determining that the first biological sample includes the first set of markers indicative of the tumor, obtaining, through a non-invasive procedure, a second biological sample from the subject, wherein the second biological sample comprises cfDNA derived from normal cells and comprises or is suspected of comprising tumor-derived cfDNA;
only when the first biological sample includes the first set of markers indicative of the tumor, performing a second assay using the second biological sample, wherein the second assay has a sensitivity for a second set of markers indicative of the tumor, and wherein the second assay comprises:
measuring a methylation status of the tumor-derived cfDNA in the second biological sample;
identifying the tumor in the subject based on the comparison of the methylation status of the tumor-derived cfDNA in the second biological sample to at least one second calibration value; and
outputting a report indicative of the tumor in the subject.
17 . The method of claim 16 , wherein the second biological sample is obtained between 1 day and 12 weeks after obtaining the first biological sample.
18 . The method of claim 16 , wherein a positive predictive value of the method of non-invasively detecting a tumor in a subject is greater than a positive predictive value of the first assay or second assay alone.
19 . The method of claim 18 , wherein the positive predictive value of the method of non-invasively detecting a tumor in a subject is at least 1.5-fold, at least 2-fold, at least 4-fold, at least 10-fold, or at least 20-fold greater than the positive predictive value of the first assay or second assay alone.
20 . The method of claim 16 , wherein the sensitivity of the method of non-invasively detecting a tumor in a subject is same or greater than the sensitivity of the second assay alone, and wherein the specificity of the method is same or greater than the specificity of the first assay alone.
21 . The method of claim 16 , wherein the first set of markers and the second set of markers are the same set of markers.
22 . The method of claim 16 , wherein the specificity of the first assay for the first set of markers is at least about 95%.
23 . The method of claim 16 , wherein the sensitivity of the second assay for the second set of markers is at least about 35%.
24 . The method of claim 16 , wherein the tumor is selected from a group consisting of liver cancer, nasopharyngeal cancer, oropharyngeal cancer, oral cancer, bladder cancer, bone cancer, a brain tumor, breast cancer, esophageal cancer, gastrointestinal cancer, hematopoietic malignancy, leukemia, lung cancer, lymphoma, myeloma, ovarian cancer, prostate cancer, sarcoma, stomach cancer, and thyroid cancer.
25 . The method of claim 16 , wherein at least one of the first biological sample and the second biological sample are independently selected from a group consisting of whole blood, blood plasma, blood serum, urine, buffy coat, and a combination thereof.
26 . The method of claim 16 , wherein the first assay and the second assay further comprise using methylation-aware sequencing to detect the methylation status of tumor-derived cfDNA in the first biological sample and second biological sample, respectively.
27 . The method of claim 26 , wherein the methylation-aware sequencing comprises using bisulfite DNA conversion with the tumor-derived cfDNA prior to sequencing.
28 . The method of claim 16 , further comprising performing an imaging examination on the subject, wherein the imaging examination is responsive to the first assay and/or the second assay.
29 . The method of claim 16 , wherein the first assay and the second assay further comprise enriching the sample for targeted cfDNA fragments corresponding to one or more predetermined regions of a genome.
30 . The method of claim 16 , wherein the first biological sample or second biological sample further comprises virus-derived DNA fragments, the method further comprising detecting the virus-derived DNA, wherein detection of the virus-derived DNA is a negative control for tumor-derived DNA.