Background
Methods
Patient material
Clinicopathological factor | N(%) |
---|---|
Tumorsize
| |
T0
|
26(11%)
|
T1
|
67(28.3%)
|
T2
|
35(14.8%)
|
T3
|
64(27%)
|
T4
|
45(18.9%)
|
Unknown
|
1
|
Stage
| |
DCIS
|
26(11.5%)
|
I
|
65(28.9%)
|
II
|
20(8.9%)
|
III
|
92(40.9%)
|
IV
|
22(9.8%)
|
Unknown
|
13
|
Lymphnode status
| |
N0
|
114(50.7%)
|
N1
|
76(34.9%)
|
N2
|
32(14.4%)
|
Unknown
|
16
|
Grade
| |
G1
|
42(17.7%)
|
G2
|
128(54%)
|
G3
|
67(28.3%)
|
Unknown
|
1
|
HER2 status
| |
Negative
|
60(57.1%)
|
Positive
|
45(42.9%)
|
Unknown
|
133
|
Progesteron receptor
| |
Negative
|
69(29.2%)
|
Positive
|
167(70.8%)
|
Unknown
|
2
|
Estrogene receptor
| |
Negative
|
53(22.7%)
|
Positive
|
180(77.3%)
|
Unknown
|
5
|
TP53 mutation
| |
Wild type
|
174(73.4%)
|
Mutated
|
63(26.6%)
|
Unknown
|
1
|
Distant metastasis
| |
No distant metastasis
|
214(90.3%)
|
Distant metastasis
|
23(9.7%)
|
Unknown
|
1
|
Molecular subtypes
| |
Luminal A
|
52(30.8%)
|
Luminal B
|
34(20.1%)
|
ERRB2
|
29(17.2%)
|
Basal
|
37(21.9%)
|
Normal
|
17(10%)
|
Unknown
|
69
|
Methylation assays
Statistical analysis
Gene | Mean of samples | SD of samples | Mean of normal | SD of normal |
---|---|---|---|---|
BRCA1
| 85.36 | 11.32 | 85.48 | 6.15 |
RASSF1A
| 30.92 | 17.34 | 3.37 | 0.99 |
PTEN
| 5.15 | 4.07 | 2.83 | 1.28 |
PPP2R2B
| 10.83 | 9.80 | 3.18 | 1.17 |
CDKN2A
| 4.68 | 4.64 | 3.58 | 1.08 |
MLH1
| 2.94 | 3.85 | 2.72 | 0.47 |
MGMT
| 4.21 | 3.53 | 4.48 | 1.11 |
ABCB1
| 16.18 | 17.25 | 2.63 | 1.23 |
IGF2
| 38.28 | 9.13 | 40.7 | 2.41 |
GSTP1
| 17.18 | 17.27 | 3.73 | 1.01 |
FOXC1
| 13.41 | 13.23 | 3.6 | 1.98 |
ESR1
| 2.92 | 2.50 | 4.43 | 2.94 |
Results
Methylation analysis and correlation with clinico-pathological parameters: stage and grade
Normal | DCIS | Invasive | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
I | II | III | IV | ||||||||||
GENE
|
Methylation status
|
N
|
%
|
Sample number
|
%
|
Sample number
|
%
|
Sample number
|
%
|
Sample number
|
%
|
Sample number
|
%
|
ABCB1
|
Hypomethylated
|
0
|
0
|
0
|
0
|
0
|
0
|
0
| |||||
Normal like
|
6
|
100
|
15
|
57.7
|
33
|
52.4
|
6
|
30
|
31
|
36
|
6
|
30
| |
Hypermethylated
|
0
|
11
|
42.3
|
30
|
47.6
|
14
|
70
|
57
|
64
|
14
|
70
| ||
BRCA1
|
Hypomethylated
|
0
|
0
|
0
|
0
|
2
|
3
|
2
|
10
|
10
|
12.8
|
1
|
5.9
|
Normal like
|
6
|
100
|
26
|
100
|
63
|
97
|
18
|
90
|
68
|
87.2
|
16
|
94.1
| |
Hypermethylated
|
0
|
0
|
0
|
0
|
0
|
0
|
0
| ||||||
CDKN2A
|
Hypomethylated
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
| ||||
Normal like
|
6
|
100
|
26
|
100
|
60
|
92.3
|
18
|
90
|
61
|
67.8
|
14
|
70
| |
Hypermethylated
|
0
|
0
|
5
|
7.7
|
2
|
10
|
29
|
32.2
|
6
|
30
| |||
ESR1
|
Hypomethylated
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
| |||
Normal like
|
6
|
100
|
26
|
100
|
63
|
97
|
19
|
95
|
63
|
98.4
|
19
|
100
| |
Hypermethylated
|
0
|
0
|
2
|
3
|
1
|
5
|
1
|
1.6
|
0
| ||||
FOXC1
|
Hypomethylated
|
0
|
0
|
0
|
0
|
0
|
0
|
0
| |||||
Normal like
|
6
|
100
|
6
|
23
|
34
|
52.3
|
7
|
35
|
40
|
46.5
|
10
|
47.6
| |
Hypermethylated
|
0
|
20
|
77
|
31
|
47.7
|
13
|
65
|
46
|
53.5
|
11
|
52.4
| ||
GSTP1
|
Hypomethylated
|
0
|
0
|
1
|
3.8
|
0
|
0
|
0
|
0
| ||||
Normal like
|
6
|
100
|
10
|
38.5
|
27
|
41.5
|
5
|
25
|
29
|
33
|
5
|
25
| |
Hypermethylated
|
0
|
15
|
57.7
|
38
|
58.5
|
15
|
75
|
59
|
67
|
15
|
75
| ||
IGF2
|
Hypomethylated
|
0
|
0
|
11
|
42.3
|
25
|
38.4
|
6
|
30
|
34
|
39.1
|
9
|
47.4
|
Normal like
|
6
|
100
|
14
|
53.8
|
32
|
49.2
|
9
|
45
|
32
|
36.8
|
5
|
26.3
| |
Hypermethylated
|
0
|
0
|
1
|
3.9
|
8
|
12.4
|
5
|
25
|
21
|
24.1
|
5
|
26.3
| |
MGMT
|
Hypomethylated
|
0
|
0
|
0
|
13
|
20.3
|
13
|
65
|
16
|
24.2
|
8
|
44.4
| |
Normal like
|
6
|
100
|
25
|
96.1
|
50
|
78.1
|
5
|
25
|
35
|
53.1
|
5
|
27.7
| |
Hypermethylated
|
0
|
1
|
3.9
|
1
|
11.6
|
2
|
10
|
15
|
22.7
|
5
|
27.7
| ||
MLH1
|
Hypomethylated
|
0
|
0
|
4
|
15.4
|
12
|
18.5
|
6
|
32
|
4
|
4.5
|
3
|
17.6
|
Normal like
|
6
|
100
|
21
|
80.8
|
47
|
72.3
|
10
|
53
|
68
|
77.3
|
13
|
76.5
| |
Hypermethylated
|
0
|
1
|
3.8
|
6
|
9.2
|
3
|
16
|
16
|
18.2
|
1
|
5.9
| ||
PPP2R2B
|
Hypomethylated
|
0
|
0
|
0
|
0
|
0
|
0
|
0
| |||||
Normal like
|
6
|
100
|
7
|
26.9
|
15
|
24.6
|
6
|
35
|
40
|
30.8
|
3
|
16.7
| |
Hypermethylated
|
0
|
19
|
73.1
|
46
|
75.4
|
11
|
65
|
45
|
69.2
|
15
|
83.3
| ||
PTEN
|
Hypomethylated
|
0
|
0
|
0
|
0
|
0
|
0
|
0
| |||||
Normal like
|
6
|
100
|
21
|
80.8
|
56
|
86.2
|
13
|
65
|
23
|
27.7
|
9
|
45
| |
Hypermethylated
|
0
|
5
|
19.2
|
9
|
13.8
|
7
|
35
|
60
|
72.3
|
11
|
55
| ||
RASSF1A
|
Hypomethylated
|
0
|
0
|
2
|
7.7
|
2
|
3.1
|
1
|
0
|
0
| |||
Normal like
|
6
|
100
|
2
|
7.7
|
10
|
15.4
|
2
|
11
|
3
|
4.8
|
1
|
5.6
| |
Hypermethylated
|
0
|
22
|
84.6
|
53
|
81.5
|
17
|
90
|
60
|
95.2
|
17
|
94.4
|
Correlation with TP53mutations and hormone receptor status
GENE | TP53 wt/mut p-value | ER pos/neg p-value | HER2 pos/neg p-value | ||
---|---|---|---|---|---|
WT% meth | Mut% meth | p-value | |||
BRCA1
| 87.34 | 79.99 | 0.001 | ||
PTEN
| 5.34 | 4.67 | 0.031 | 3.6e-6 | |
PPP2R2B
| 11.33 | 9.47 | 0.002 | 0.012 | |
FOXC1
| 17.12 | 13.56 | 0.017 | 0.032 | |
ABCB1
| 14.52 | 10.28 | 0.010 | ||
RASSF1A
| 32.48 | 26.58 | 0.028 | 0.004 | 0.023 |
GSTP1
| 0.012 | ||||
CDKN2A
| 5.8e-9 | ||||
MGMT
| 1.1e-7 | ||||
ESR1
| 0.017 |
Survival analysis
Covariate | Baseline | Coefficient(bi) | HR(exp(bi) | p-value | 95,0%CI for Exp(B) |
---|---|---|---|---|---|
TP53
| TP53 wild type | −2.332 | 0.097 | 6.6e-5 | (0.030-0.305) |
ER | ER positive | 1.481 | 4.401 | 0.008 | (1.460-13.269) |
Stage II | Stage I | 2.260 | 9.584 | 0.013 | (1.593-57.667) |
Stage III | Stage I | −0.011 | 0.988 | 0.992 | (0.088-11.019) |
N1 | N0 | −6.362 | 0.529 | 0.338 | (0.143-1.947) |
N2 | N0 | 1.673 | 5.332 | 0.047 | (1.019-27.885) |
IGF2
| Normal like IGF2
| 1.597 | 4.940 | 0.009 | (1.472-16.581) |
GSTP1
| Normal like GSTP1
| −1.621 | 0.197 | 0.014 | (0.053-0.729) |
PPP2R2B
| Normal like PPP2R2B
| −0.972 | 0.378 | 0.085 | (0.125-1.143) |
CDKN2A
| Normal like CDKN2A
| −0.839 | 0.432 | 0.133 | (0.144-1.294) |
Covariate | Baseline | Coefficient (bi) | HR(exp(bi) | p-value | 95,0% CI for Exp(B) |
---|---|---|---|---|---|
GSTP1
| Normal like GSTP1
| −0.066 | 0.935 | 4e-4 | (0.902-0.970) |
IGF2
| Normal like IGF2
| 0.079 | 1.082 | 0.002 | (1.031-1.136) |
MLH1
| Normal like MLH1
| −0.552 | 0.575 | 0.135 | (0.278-1.187) |
ER | ER positive | 1.659 | 5.256 | 0.002 | (1.804-15.317) |
TP53
| TP53 wild type | −2.169 | 0.114 | 0.002 | (0.028-0.450) |
N 1 | N0 | 1.684 | 5.391 | 0.022 | (1.265-22.975) |
N 2 | N0 | 1.706 | 5.511 | 0.032 | (1.151-26.371) |
T 2 | T1 | 1.937 | 6.942 | 0.035 | (1.143-42.157) |
T3 | T1 | 0.050 | 1.051 | 0.960 | (0.143-7.716) |
T4 | T1 | 1.125 | 3.081 | 0.258 | (0.437-21.692) |