DNA methylation diagnostic test for breast cancer

1. A method of detecting methylation or unmethylation of a ductal carcinoma in situ (DCIS) cell proliferation deoxyribonucleic acid (DNA) molecule of a human subject, the method comprising:

(i) contacting an isolated DCIS cancer cell proliferation DNA molecule from a breast tissue sample of said subject with a bisulfate salt thereby forming a reacted DCIS cancer cell proliferation DNA molecule and contacting said reacted DCIS cancer cell proliferation DNA molecule with a probe or primer complementary to a sequence at or within 1000 nucleotides of a plurality of methylation sites; and

(ii) detecting the presence or absence of uracil in said reacted DCIS cancer cell proliferation DNA molecule at said plurality of methylation sites, wherein said plurality of methylation sites is at Chromosome 1 (Chr1) position 4714314, Chromosome 3 (Chr3) position 121903470, Chromosome 4 (Chr4) position 44449864, Chromosome 7 (Chr7) position 157477232, Chromosome 12 (Chr12) position 95941925, Chr12 position 129338355, Chromosome 19 (Chr19) 30017283, and Chromosome 20 (Chr20) position 23016002 with respect to human genome assembly hg19,

thereby detecting methylation or unmethylation of said DCIS cancer cell proliferation DNA molecule of said subject.

2. The method of claim 1 , further comprising detecting the presence or absence of uracil in a plurality of reacted DCIS cancer cell proliferation DNA molecules at one or more additional methylation sites, wherein said one or more additional methylation sites is at Chr1 position 11413742, Chr1 position 39957798, Chr1 position 46951513, Chr1 position 47904912, Chr1 position 62660691, Chr1 position 63785800, Chr1 position 67600465, Chr1 position 91183172, Chr1 position 166853786, Chr1 position 179545096, Chr1 position 207669851, Chr1 position 237205704, Chr1 position 237205705, Chr1 position 240161215, Chr1 position 240934954, Chromosome 2 (Chr2) position 20870821, Chr2 position 45156764, Chr2 position 74743346, Chr2 position 80549703, Chr2 position 95989474, Chr2 position 105471544, Chr2 position 115919663, Chr2 position 115920004, Chr2 position 118982006, Chr2 position 177001540, Chr3 position 14852857, Chr3 position 170303393, Chr3 position 170303422, Chr3 position 170303423, Chr3 position 170303424, Chr3 position 170303425, Chr4 position 54976099, Chr4 position 56023880, Chromosome 5 (Chr5) positon 71014951, Chr5 position 72677229, Chr5 position 87981177, Chr5 position 140743998, Chr5 position 178421786, Chromosome 6 (Chr6) position 41337153, Chr6 position 85484102, Chr6 position 157557787, Chr6 position 160769248, Chr7 position 1282082, Chr7 position 32467637, Chr7 position 71801896, Chr7 position 71801905, Chr7 position 100946148, Chr7 position 100946151, Chr7 position 121957003, Chr7 position 150038502, Chr7 position 157477399, Chr7 position 157477401, Chromosome 8 (Chr8) position 9764011, Chr8 position 11566080, Chr8 position 11566102, Chr8 position 11566125, Chr8 position 56015232, Chr8 position 65281933, Chr8 position 145105472, Chromosome 9 (Chr9) position 126780185, Chr9 position 127239956, Chr9 position 140772369, Chromosome 10 (Chr10) position 8076277, Chr10 position 50818610, Chr10 position 77157527, Chr10 position 123778639, Chr10 position 123778640, Chr10 position 124902829, Chr10 position 124909545, Chr10 position 130085373, Chr10 position 134598235, Chr10 position 134599080, Chromosome 11 (Chr11) position 1215978, Chr11 position 9025912, Chr11 position 15963013, Chr11 position 66187593, Chr11 position 71318977, Chr11 position 101453451, Chr12 position 49726711, Chr12 position 50297756, Chr12 position 50297763, Chr12 position 50297768, Chr12 position 50297774, Chr12 position 50297776, Chr12 position 50444766, Chr12 position 75601447, Chr12 position 128750309, Chr12 position 129338471, Chromosome 13 (Chr13) position 28502190, Chr13 position 79181509, Chr13 position 92051154, Chr13 position 95363553, Chr13 position 95363592, Chromosome 14 (Chr14) position 29236052, Chr14 position 29236065, Chr14 position 101543886, Chromosome 15 (Chr15) position 29407958, Chr15 position 45403826, Chr15 position 76630094, Chr15 position 89951787, Chromosome 16 position 1255253, Chromosome 17 (Chr17) position 3211643, Chr17 position 30244229, Chr17 position 35294171, Chr17 position 64831307, Chr17 position 74136562, Chr17 position 74865566, Chromosome 18 (Chr18) position 19745047, Chr18 position 19745054, Chr18 position 19747206, Chr18 position 44774403, Chr18 position 55103840, Chr18 position 55106910, Chr18 position 70534832, Chr18 position 72880039, Chr18 position 77547934, Chr19 position 30016170, Chr19 position 30717013, Chr19 position 30719659, Chr20 position 1294019, Chr20 position 3073503, Chr20 position 10198305, Chr20 position 23015989, Chr20 position 26189258, Chr20 position 48626669, Chr20 position 53092916, Chr20 position 59827619, Chr20 position 59828325, Chromosome 21 (Chr21) position 9825842, Chr21 position 9826150, Chr21 position 9826934, or Chromosome 22 position 43807517 with respect to human genome assembly hg19.

3. The method of claim 2 , wherein said plurality of methylation sites comprises at least about 10 additional methylation sites with respect to human genome assembly hg19.

4. The method of claim 2 , wherein the uracil level of at least one methylation site of said plurality of methylation sites is above a threshold as set forth in Table 2, wherein Table 2 is as follows:

TABLE 2

Uracil level in

invasive

competent DCIS

Chromosomal

is about above

Chromosome

position

indicated level*

chr1

11413742

76.09

chr1

240934954

78.57

chr10

123778639

74.49

chr10

123778640

79.31

chr11

1215978

74.47

chr12

128750309

 83.33.

5. The method of claim 1 , wherein said DCIS cancer cell proliferation comprises cancer cells isolated from a sample obtained by biopsy, by laser capture microdissection, or by surgical resection of DCIS tissue from said subject.

6. The method of claim 1 , wherein said subject has undergone lumpectomy, mastectomy, radiation therapy, and/or administration of an active agent.

7. The method of claim 6 , wherein said active agent comprises trastuzumab, trastuzumab emtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole, letrozole, doxorubicin, epirubicin, cyclophosphamide, docetaxel, paclitaxel, nab paclitaxel, eribulin, everolimus, palbociclib, capecitabine, ixabepilone, methotrexate, or fluorouracil.

8. The method of claim 1 , wherein said subject (a) is a woman; (b) is about 30 to about 75 years old; (c) has at least one mutant breast cancer 1 (BRCA1), breast cancer 2 (BRCA2), Partner and localizer of BRCA2 (PALB2), phosphatase and tensin homolog (PTEN), or p53 allele; (d) has a parent, sibling, or child who has been diagnosed with breast cancer; (e) has had atypical ductal hyperplasia or lobular carcinoma in situ; (f) has had previous radiation treatment to the chest or a breast before the age of 30; (g) has received a combination hormone therapy with estrogen and progestin for at least five years; and/or (h) has or has had breast cancer.

9. The method of claim 2 , further comprising:

(i) detecting a level of uracil of at least one methylation site of said plurality of methylation sites that is equal to or less than the corresponding threshold level set forth in Table 3; and

(ii) administering to said subject a treatment to treat or prevent invasive ductal carcinoma (IDC), wherein the treatment comprises (a) lumpectomy, mastectomy, radiation therapy, chemotherapy, or hormone therapy; or (b) administering an active agent comprising trastuzumab, trastuzumab emtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole, letrozole, doxorubicin, epirubicin, cyclophosphamide, docetaxel, paclitaxel, nab paclitaxel, eribulin, everolimus, palbociclib, capecitabine, ixabepilone, methotrexate, or fluorouracil;

wherein Table 3 is as follows:

TABLE 3

Uracil level in

Chromosomal

invasive-competent DCIS is

Chromosome

position

about below indicated level*

chr1

4714314

74.32

chr1

39957798

63.81

chr1

46951513

85.11

chr1

47904912

73.08

chr1

62660691

71.20

chr1

63785800

46.94

chr1

67600465

42.86

chr1

91183172

48.28

chr1

166853786

64.29

chr1

179545096

88.89

chr1

207669851

89.29

chr1

237205704

82.61

chr1

237205705

78.57

chr1

240161215

60.00

chr10

8076277

75.00

chr10

50818610

76.30

chr10

77157527

50.43

chr10

124902829

67.35

chr10

124909545

80.00

chr10

130085373

76.74

chr10

134598235

34.43

chr10

134599080

79.31

chr11

9025912

65.85

chr11

15963013

51.72

chr11

66187593

80.95

chr11

71318977

51.52

chr11

101453451

57.89

chr12

49726711

60.00

chr12

50297756

69.81

chr12

50297763

82.98

chr12

50297768

81.13

chr12

50297774

75.47

chr12

50297776

77.36

chr12

50444766

54.17

chr12

75601447

52.00

chr12

95941925

36.84

chr12

129338355

57.14

chr12

129338471

60.71

chr13

28502190

65.85

chr13

79181509

79.84

chr13

92051154

70.70

chr13

95363553

73.91

chr13

95363592

78.95

chr14

29236052

72.36

chr14

29236065

62.60

chr14

101543886

92.45

chr15

29407958

69.84

chr15

45403826

73.68

chr15

76630094

62.30

chr15

89951787

39.68

chr17

35294171

71.43

chr17

64831307

56.52

chr17

74136562

43.48

chr17

74865566

62.79

chr18

19745047

67.01

chr18

19745054

64.10

chr18

19747206

80.00

chr18

44774403

68.29

chr18

55103840

66.20

chr18

55106910

50.00

chr18

70534832

66.67

chr18

77547934

33.33

chr19

30016170

38.89

chr19

30017283

60.00

chr19

30717013

54.24

chr19

30719659

66.67

chr2

20870821

12.20

chr2

45156764

34.25

chr2

74743346

34.69

chr2

80549703

28.21

chr2

105471544

63.64

chr2

115919663

64.44

chr2

115920004

79.31

chr2

118982006

47.06

chr2

177001540

63.16

chr20

1294019

55.43

chr20

3073503

62.96

chr20

10198305

62.44

chr20

23015989

80.70

chr20

23016002

65.59

chr20

26189258

55.56

chr20

48626669

36.00

chr20

53092916

72.13

chr20

59827619

58.82

chr20

59828325

76.47

chr21

9825842

37.32

chr21

9826150

50.00

chr21

9826934

53.36

chr22

43807517

40.68

chr3

14852857

57.54

chr3

121903470

71.96

chr3

170303393

68.63

chr3

170303422

67.65

chr3

170303423

81.82

chr3

170303424

56.52

chr3

170303425

69.77

chr4

44449864

78.38

chr4

54976099

72.00

chr4

56023880

55.10

chr5

71014951

65.00

chr5

72677229

80.82

chr5

87981177

55.26

chr5

140743998

71.83

chr5

178421786

56.25

chr6

41337153

57.25

chr6

85484102

68.18

chr6

157557787

55.56

chr6

160769248

75.51

chr7

1282082

70.45

chr7

32467637

31.03

chr7

71801896

63.89

chr7

71801905

63.89

chr7

100946148

55.17

chr7

100946151

43.66

chr7

121957003

75.00

chr7

150038502

84.83

chr7

157477232

26.45

chr7

157477399

71.43

chr7

157477401

92.59

chr8

9764011

70.91

chr8

11566080

85.51

chr8

11566102

65.22

chr8

11566125

65.22

chr8

56015232

53.85

chr8

65281933

95.00

chr8

145105472

44.93

chr9

126780185

85.19

chr9

127239956

66.96

chr9

140772369

84.62.

10. The method of claim 9 , wherein said treatment comprises chemotherapy, or hormone therapy.

11. The method of claim 9 , wherein treatment comprises administering to said subject the active agent comprising trastuzumab, trastuzumab emtansine, lapatinib, pertuzumab, bevacizumab, tamoxifen, exemestane, anastrozole, letrozole, doxorubicin, epirubicin, cyclophosphamide, docetaxel, paclitaxel, nab paclitaxel, eribulin, everolimus, palbociclib, capecitabine, ixabepilone, methotrexate, or fluorouracil.

12. The method of claim 9 , wherein said treatment comprises a lumpectomy, mastectomy, and/or radiation therapy.