NON-INVASIVE DETERMINATION OF METHYLOME OF TUMOR FROM PLASMA

• US 20150011403A1
• Filed: 09/24/2014
• Published: 01/08/2015
• Est. Priority Date: 09/20/2012
• Status: Active Grant

• Current Assignee: Chinese University of Hong Kong
• Original Assignee: Chinese University of Hong Kong
• Inventors: Lo, Yuk-Ming Dennis, Chiu, Rossa Wai Kwun, Chan, Kwan Chee, Lun, Miu Fan, Chan, Wai Man, Jiang, Peiyong
• Granted Patent: US 10,392,666 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Systems, methods, and apparatuses can determine and use methylation profiles of various tissues and samples. Examples are provided. A methylation profile can be deduced for fetal/tumor tissue based on a comparison of plasma methylation (or other sample with cell-free DNA) to a methylation profile of the mother/patient. A methylation profile can be determined for fetal/tumor tissue using tissue-specific alleles to identify DNA from the fetus/tumor when the sample has a mixture of DNA. A methylation profile can be used to determine copy number variations in genome of a fetus/tumor. Methylation markers for a fetus have been identified via various techniques. The methylation profile can be determined by determining a size parameter of a size distribution of DNA fragments, where reference values for the size parameter can be used to determine methylation levels. Additionally, a methylation level can be used to determine a level of cancer.

Claims

1. A method of analyzing a biological sample of an organism, the biological sample including nucleic acid molecules originating from normal cells and potentially from cells associated with cancer, wherein at least some of the nucleic acid molecules are cell-free in the biological sample, the method comprising:

analyzing a plurality of DNA molecules from the biological sample, wherein analyzing a DNA molecule includes;

determining a location of the DNA molecule in a genome of the organism;

determining, by a computer system, whether the DNA molecule is methylated at one or more sites;

for each of a plurality of sites;

determining, by the computer system, a respective number of DNA molecules that are methylated at the site;

calculating, by the computer system, a first methylation level based on the respective numbers of DNA molecules methylated at the plurality of sites;

comparing the first methylation level to a first cutoff value; and

determining a first classification of a level of cancer based on the comparison.

2. The method of claim 1, wherein determining whether the DNA molecule is methylated at one or more sites includes:

performing methylation-aware sequencing.

3. The method of claim 2, wherein performing methylation-aware sequencing includes:

treating the DNA molecules with sodium bisulfite; and

performing sequencing of the treated DNA molecules.

4. The method of claim 3, wherein the treatment of the cell-free DNA molecules with sodium bisulfite is part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for the detection of 5-hydroxymethylcytosine.

5. The method of claim 2, wherein the respective numbers of DNA molecules for each region are determined by aligning sequence reads obtained from the methylation-aware sequencing.

6. The method of claim 1, wherein determining whether the DNA molecule is methylated at one or more sites includes using methylation-sensitive restriction enzymes digestion, methylation-specific PCR, methylation-dependent DNA precipitation, methylated DNA binding protein/peptide or single molecule sequencing without sodium bisulfate treatment.

7. The method of claim 1, further comprising:

for each of a first plurality of regions of the genome;

determining a respective number of DNA molecules as being from the region;

calculating a respective normalized value from the respective number;

comparing the respective normalized value to a reference value to determine whether the respective region exhibits a deletion or an amplification;

determining a first amount of regions that are determined to exhibit a deletion or an amplification;

comparing the first amount to a first threshold value to determine a second classification of a level of cancer; and

using the first classification and the second classification to determine a third classification of a level of cancer.

8. The method of claim 7, wherein the first threshold value is a percentage of the first plurality of regions that are determined to exhibit a deletion or an amplification.

9. The method of claim 7, wherein the third classification is positive for cancer only when both the first classification and the second classification indicate cancer.

10. The method of claim 7, wherein the third classification is positive for cancer when either the first classification or the second classification indicate cancer.

11. The method of claim 1, wherein the first classification indicates that cancer exists for the organism, the method further comprising:

identifying a type of cancer associated with the organism by comparing the first methylation level to a corresponding value determined from other organisms, wherein at least two of the other organisms are identified as having different types of cancer.

12. The method of claim 11, wherein the third classification indicates that cancer exists for the organism, the method further comprising:

identifying the type of cancer associated with the organism by comparing the first amount of regions to corresponding values determined from the other organisms.

13. The method of claim 1, wherein calculating the first methylation level includes:

identifying a second plurality of regions of the genome;

identifying one or more sites within each of the regions;

calculating a region methylation level for each region, wherein the first methylation level is for a first region,

the method further comprising;

comparing each of the region methylation levels to a respective region cutoff value, including comparing the first methylation level to the first cutoff value;

determining a second amount of regions that are determined to have a region methylation level exceeding the respective region cutoff value; and

comparing the second amount of regions to a second threshold value to determine the first classification.

14. The method of claim 13, wherein the regions that are determined to have a region methylation level exceeding the respective region cutoff value correspond to a first set of regions, the method further comprising:

comparing region methylation levels of the first set of regions to corresponding region methylation levels of other organisms for the first set of regions, the other organisms having at least two of;

a first type of cancer, absence of cancer, and a second type of cancer; and

determining whether the organism has the first type of cancer, absence of cancer, or the second type of cancer based on the comparison.

15. The method of claim 14, further comprising:

clustering the other organisms based on the region methylation levels of the first set of regions, wherein two of the clusters correspond to any two of;

the first type of cancer, absence of cancer, and the second type of cancer,

wherein the comparison of the region methylations levels determines which cluster the organism belongs.

16. The method of claim 15, wherein the clustering of the other organisms uses the region methylation levels of the organism.

17. The method of claim 15, wherein the clusters include a first cluster corresponding to the first type of cancer, a second cluster corresponding to the second type of cancer, and a third cluster corresponding to absence of cancer.

18. The method of claim 15, wherein the clustering of the other organisms is further based on respective normalized values of a second set of regions for the other organisms, the second set of regions correspond to regions that are determined to exhibit a deletion or an amplification, wherein the respective normalized value for a region is determined from a respective number of DNA molecules from the region, the method further comprising:

for each of the second set of regions;

determining a respective number of DNA molecules as being from the region;

calculating a respective normalized value from the respective number; and

comparing the respective normalized values of the second set of regions for the organism to the respective normalized values of the other organisms as part of determining which cluster the organism belongs.

19. The method of claim 18, wherein the clustering of the other organisms is further based on respective methylation densities of hypermethylated CpG islands, the method further comprising:

for each of the hypermethylated CpG islands;

determining a respective methylation density,

comparing the respective methylation densities of the hypermethylated CpG islands for the organism to the methylation densities of the other organisms as part of determining which cluster the organism belongs.

20. The method of claim 13, further comprising:

for each of the second plurality of regions;

computing a respective difference between the region methylation level and the respective region cutoff value; and

calculating a respective probability corresponding to the respective difference;

wherein determining the second amount of regions includes;

calculating a cumulative score including the respective probabilities.

21. The method of claim 20, wherein calculating the cumulative score includes:

taking a logarithm of the respective probability to obtain a respective logarithm result; and

calculating a sum including the respective logarithm results.

22. The method of claim 21, wherein the cumulative score is a negative of the sum of the respective logarithm results.

23. The method of claim 20, wherein each difference is normalized with a standard deviation associated with the respective region cutoff value.

24. The method of claim 20, wherein the respective probability corresponds to a probability for the respective difference according to a statistical distribution.

25. The method of claim 20, wherein the second threshold value corresponds to a highest cumulative score from a reference group.

26. The method of claim 13, further comprising:

for each of the first plurality of regions;

computing a respective difference between the respective normalized value and the reference value; and

calculating a respective probability corresponding to the respective difference;

wherein determining the first amount of regions includes;

calculating a first sum including the respective probabilities.

27. The method of claim 13, wherein the respective region cutoff values are a specified amount from a reference methylation level.

28. The method of claim 13, wherein the second threshold value is a percentage, and wherein comparing the second amount of regions to a second threshold value includes:

dividing the second amount of regions by a second number of the second plurality of regions before comparing to the second threshold value.

29. The method of claim 28, wherein the second number corresponds to all of the second plurality of regions.

30. The method of claim 13, wherein the first plurality of regions are the same as the second plurality of regions, and wherein the respective region cutoff values are dependent on whether the respective region exhibits a deletion or an amplification.

31. The method of claim 30, wherein a respective region cutoff value has a larger magnitude when the respective region exhibits an amplification than when no amplification is exhibited, and wherein a respective region cutoff value has a smaller magnitude when the respective region exhibits a deletion than when no deletion is exhibited.

32. The method of claim 31, wherein a respective region cutoff values test for hypomethylation of the second plurality of regions, wherein a respective region cutoff value has a larger negative value when the respective region exhibits an amplification than when no amplification is exhibited, and wherein a respective region cutoff value has a smaller negative value when the respective region exhibits a deletion than when no deletion is exhibited.

33. The method of claim 13, wherein the biological sample is taken prior to treatment, the method further comprising:

repeating the method of claim 13 for another biological sample taken after treatment to obtain;

a subsequent first amount of regions that are determined to exhibit a deletion or an amplification; and

a subsequent second amount of regions that are determined to have a region methylation level exceeding the respective region cutoff value;

comparing the first amount to the subsequent first amount and the second amount to the subsequent second amount to determine a prognosis of the organism.

34. The method of claim 33, wherein comparing the first amount to the subsequent first amount and the second amount to the subsequent second amount to determine the prognosis of the organism includes:

determining a first difference between the first amount and the subsequent first amount;

comparing the first difference to one or more first difference thresholds;

determining a second difference between the second amount and the subsequent second amount; and

comparing the second difference to one or more second difference thresholds.

35. The method of claim 34, wherein the prognosis is predicted to be worse when the first difference is below one first difference threshold than when the first difference is above the one first difference threshold, and wherein the prognosis is predicted to be worse when the second difference is below one second difference threshold than when the second difference is above the one second difference threshold.

36. The method of claim 35, wherein the one first difference threshold and the one second difference threshold are zero.

37. The method of claim 33, wherein the treatment is immunotherapy, surgery, radiotherapy, chemotherapy, antibody-based therapy, epigenetic therapy or targeted therapy.

38. The method of claim 1, wherein the first cutoff value is a specified distance from a reference methylation level established from a biological sample obtained from a healthy organism.

39. The method of claim 38, wherein the specified distance is a specified number of standard deviations from the reference methylation level.

40. The method of claim 1, wherein the first cutoff value is established from a reference methylation level determined from a previous biological sample of the organism obtained previous to the biological sample being tested.

41. The method of claim 1, wherein comparing the first methylation level to the first cutoff value includes:

determining a difference between the first methylation level and a reference methylation level; and

comparing the difference to a threshold corresponding to the first cutoff value.

42. The method of claim 1, further comprising:

determining a fractional concentration of tumor DNA in the biological sample;

calculating the first cutoff value based on the fractional concentration.

43. The method of claim 1, further comprising:

determining whether a fractional concentration of tumor DNA in the biological sample is greater than a minimum value; and

if the fractional concentration is not greater than the minimum value, flagging the biological sample.

44. The method of claim 43, wherein the minimum value is determined based on an expected difference in methylation levels for a tumor relative to a reference methylation level.

45. The method of claim 1, further comprising:

measuring a size of DNA molecules at the plurality of sites; and

before comparing the first methylation level to the first cutoff value, normalizing the first methylation level based on the measured sizes of the DNA molecules.

46. The method of claim 45, wherein normalizing the first methylation level based on the measured sizes includes:

selecting DNA molecules having a first size;

using the selected DNA molecules to calculate the first methylation level, the first cutoff value corresponding to the first size.

47. The method of claim 46, wherein the first size is a range of lengths.

48. The method of claim 46, wherein the DNA molecules are selected based on a physical separation that is dependent on size.

49. The method of claim 46, wherein selecting DNA molecules having a first size includes:

performing paired-end massively parallel sequencing of the plurality of DNA molecules to obtain pairs of sequences for each of the DNA molecules;

determining a size of a DNA molecule by comparing the pair of sequences to a reference genome; and

selecting DNA molecules having the first size.

50. The method of claim 45, wherein normalizing the first methylation level based on the measured sizes includes:

obtaining a functional relationship between size and methylation levels; and

using the functional relationship to normalize the first methylation level.

51. The method of claim 50, wherein the functional relationship provides scaling values corresponding to respective sizes.

52. The method of claim 51, further comprising:

computing an average size corresponding to DNA molecules used to calculate the first methylation level; and

multiplying the first methylation level by the corresponding scaling value.

53. The method of claim 51, further comprising:

for each of the plurality of sites;

for each of the DNA molecules located at the site;

obtaining a respective size of the DNA molecule at the site; and

using the scaling value corresponding to the respective size to normalize a contribution of the DNA molecule to the respective number of DNA molecules that are methylated at the site.

54. The method of claim 1, wherein the plurality of sites includes CpG sites, wherein the CpG sites are organized into a plurality of CpG islands, each CpG island including one or more CpG sites, wherein the first methylation level corresponds to a first CpG island.

55. The method of claim 54, wherein the CpG islands have a mean methylation density of less than a first percentage in a reference group, and wherein the coefficient of variation for the methylation density in the reference group is less than a second percentage.

56. The method of claim 54, further comprising:

for each of the CpG islands;

determining whether the CpG island is hypermethylated relative to a reference group of samples of other organisms by comparing a methylation level of the CpG island to a respective cutoff value;

for each of the hypermethylated CpG islands;

determining a respective methylation density;

calculating a cumulative score from the respective methylation densities; and

comparing the cumulative score to a cumulative cutoff value to determine the first classification.

57. The method of claim 56, wherein calculating the cumulative score from the respective methylation densities includes:

for each of the hypermethylated CpG islands;

calculating a respective difference between the respective methylation density and a reference density; and

calculating a respective probability corresponding to the respective difference; and

using the respective probabilities to determine the cumulative score.

58. The method of claim 56, wherein the cumulative score is determined by:

taking a logarithm of the respective probability to obtain a respective logarithm result; and

calculating a sum including the respective logarithm results, the cumulative score is a negative of the sum.

59. The method of claim 56, wherein each respective difference is normalized with a standard deviation associated with the reference density.

60. The method of claim 56, wherein the cumulative cutoff value corresponds to a highest cumulative score from the reference group.

61. The method of claim 56, wherein determining whether the first CpG island is hypermethylated includes:

comparing the first methylation level to the first cutoff value and to a third cutoff value,

wherein the first cutoff value corresponds to a mean of methylation densities for the reference group plus a specified percentage, and wherein the third cutoff value corresponds to a specified number of standard deviations plus the mean of methylation densities for the reference group.

62. The method of claim 61, wherein the specified percentage is 2%.

63. The method of claim 61, wherein the specified number of standard deviations is three.

64. The method of claim 1, further comprising:

for each of a first plurality of regions of the genome;

determining a respective number of DNA molecules as being from the region;

calculating a respective normalized value from the respective number;

comparing the respective normalized value to a reference value to determine whether the respective region exhibits a deletion or an amplification;

determining a first set of regions that are determined to all exhibit one of;

a deletion, an amplification, or normal representation, wherein the first methylation level corresponds to the first set of regions;

determining a second set of regions that are determined to all exhibit a second of;

a deletion, an amplification, or normal representation;

calculating a second methylation level based on the respective numbers of DNA molecules methylated at sites in the second set of regions,

wherein comparing the first methylation level to the first cutoff value includes;

computing a parameter between the first methylation level and the second methylation; and

comparing the parameter to the first cutoff value.

65. The method of claim 64, wherein the first methylation level is a statistical value of region methylation levels calculated for each region of the first set of regions, and wherein the second methylation level is a statistical value of region methylation levels calculated for each region of the second set of regions.

66. The method of claim 65, wherein the statistical values are determined using Student'"'"'s t-test, analysis of variance (ANOVA) test, or Kruskal-Wallis test.

67. The method of claim 64, wherein the parameter includes a ratio or a difference.

68. The method of claim 67, wherein computing the parameter includes applying a probability distribution to the ratio or the difference.

69. A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to analyze a biological sample of an organism, the biological sample including nucleic acid molecules originating from normal cells and potentially from cells associated with cancer, wherein at least some of the nucleic acid molecules are cell-free in the biological sample, the instructions comprising:

analyzing a plurality of DNA molecules from the biological sample, wherein analyzing a DNA molecule includes;

determining a location of the DNA molecule in a genome of the organism;

determining whether the DNA molecule is methylated at one or more sites;

for each of a plurality of sites;

determining a respective number of DNA molecules that are methylated at the site;

calculating a first methylation level based on the respective numbers of DNA molecules methylated at the plurality of sites;

comparing the first methylation level to a first cutoff value;

determining a first classification of a level of cancer based on the comparison.