Introduction
Materials and methods
Patients
Categorization | Test set (na = 112) | % | Validation set (na = 138) | % | Pvalueb |
---|---|---|---|---|---|
Age at diagnosis | 0.327 | ||||
Median | 60 years (range, 36-87 years) | 63 years (range, 33-86 years) | |||
Menopausal status | 0.791 | ||||
Premenopausal | 22 | 19.6 | 21 | 15.2 | |
Postmenopausal | 90 | 80.4 | 117 | 84.8 | |
Tumor sizec | 0.337 | ||||
pT1 | 49 | 43.8 | 67 | 48.6 | |
pT2 | 51 | 45.5 | 59 | 42.8 | |
pT3 | 8 | 7.1 | 8 | 5.9 | |
pT4 | 2 | 1.8 | 2 | 1.4 | |
Unknown | 2 | 1.8 | 2 | 1.4 | |
Lymph node statusc | 0.997 | ||||
pN0 | 55 | 49.1 | 77 | 55.8 | |
pN1 | 38 | 33.9 | 42 | 30.4 | |
pN2 | 9 | 8.0 | 10 | 7.2 | |
pN3 | 7 | 6.3 | 6 | 4.3 | |
Unknown | 3 | 2.7 | 3 | 2.2 | |
Histologic type | 0.617 | ||||
Invasive ductal | 89 | 79.5 | 115 | 83.3 | |
Invasive lobular | 15 | 13.4 | 17 | 12.3 | |
Other | 8 | 7.2 | 6 | 4.2 | |
Tumor graded | 0.175 | ||||
G1 | 6 | 5.4 | 14 | 10.1 | |
G2 | 62 | 55.4 | 76 | 55.1 | |
G3 | 44 | 39.3 | 48 | 34.8 | |
Estrogen-receptor statuse | 0.293 | ||||
Negative (IRS0-2) | 22 | 19.7 | 27 | 19.5 | |
Positive (IRS3-12) | 84 | 75.0 | 109 | 78.9 | |
Unknown | 6 | 5.4 | 2 | 1.4 | |
Progesterone-receptor statuse | 0.112 | ||||
Negative (IRS0-2) | 30 | 26.8 | 39 | 28.2 | |
Positive (IRS3-12) | 76 | 68.0 | 96 | 69.3 | |
Unknown | 6 | 5.4 | 3 | 2.2 | |
HER2 statusf | 0.112 | ||||
Negative (0; 1+; 2+) | 90 | 80.4 | 114 | 82.6 | |
Positive (3+) | 14 | 12.5 | 20 | 14.9 | |
Unknown | 8 | 7.1 | 4 | 2.9 |
Nucleic acid extraction
DNA bisulfite modification
Methylation-specific polymerase chain reaction (MSP)
Quantitative methylation-specific polymerase chain reaction (qMSP)
Analytic performance of the blood-based qMSP assays
Statistical analysis
Results
Determination of the best-performing genes for a breast cancer blood test with qualitative MSP
Sensitivity in breast cancer serum | Sensitivity in breast cancer tissue | |||
---|---|---|---|---|
Gene | Methylation Positive | % | Methylation positive | % |
ITIH5
| 27 of 112 |
24
| 81 of 112 |
72
|
DKK3
| 37 of 112 |
33
| 97 of 112 |
87
|
WIF1
| 39 of 112 |
35
| 108 of 112 |
96
|
SFRP1
| 12 of 112 |
11
| 89 of 111 |
79
|
SFRP2
| 21 of 112 |
19
| 90 of 112 |
80
|
SFRP5
| 9 of 112 |
8
| 104 of 110 |
93
|
DKK3/ITIH5
a
| 46 of 112 |
41
| 105 of 112 |
94
|
Sensitivity in breast cancer | Specificity in healthy controls | Specificity in benign breast diseases | ||||
---|---|---|---|---|---|---|
Gene | Methylation positive | % | Methylation negative | % | Methylation negative | % |
ITIH5
| 27 of 112 |
24
| 96 of 102 |
94
| 20 of 20 |
100
|
DKK3
| 37 of 112 |
33
| 101 of 102 |
99
| 20 of 20 |
100
|
WIF1
| 39 of 112 |
35
| 97 of 102 |
95
| 15 of 20 |
75
|
SFRP1
| 12 of 112 |
11
| 92 of 102 |
90
| 17 of 20 |
85
|
SFRP2
| 21 of 112 |
19
| 90 of 102 |
88
| 18 of 20 |
90
|
SFRP5
| 9 of 112 |
8
| n.d. | n.d. | ||
DKK3/ITIH5
a
| 46 of 112 |
41
| 95 of 102 |
93
| 20 of 20 |
100
|
Specificity in colon cancer | ||||
---|---|---|---|---|
Test set | Validation set | |||
Gene | Methylation negative | % | Methylation negative | % |
DKK3
| 27 of 27 |
100
| 24 of 31 |
77
|
ITIH5
| 24 of 27 |
89
| 22 of 31 |
71
|
WIF1
| 23 of 27 |
85
| n.d. | |
SFRP1
| 16 of 22 |
73
| n.d. | |
SFRP2
| 22 of 26 |
85
| n.d. | |
SFRP5
| n.d. | n.d. | ||
RASSF1A
| n.d. | 13 of 31 |
42
| |
DKK3/ITIH5
a
| 24 of 27 |
88
| 16 of 31 |
52
|
Three-gene panela | n.d. | 6 of 31 |
19
|
Detection of breast cancer in an independent validation set by using ITIH5, DKK3, and RASSF1A
Sensitivity in breast cancer | Specificity in healthy and benign breast disease controls | Pvalueb | AUC (95% CI) | PPV | NPV | |||
---|---|---|---|---|---|---|---|---|
Genea | Methylation positive | % | Methylation negative | % | % | % | ||
RASSF1A
ITIH5
| 75 of 138 |
54
| 127 of 174 |
73
| 7.8 × e-6 | 0.647 (0.585-0.709) | 61 | 67 |
RASSF1A
DKK3
| 82 of 138 |
59
| 131 of 174 |
75
| 1.6 × e-8 | 0.686 (0.626-0.747) | 66 | 70 |
DKK3
ITIH5
| 55 of 138 |
40
| 164 of 174 |
94
| 1.6 × e-7 | 0.673 (0.611-0.735) | 85 | 66 |
RASSF1A
DKK3
ITIH5
|
92 of 138
|
67
|
125 of 174
|
72
|
1.3 × e
-10
|
0.712 (0.653-0.770)
| 65 | 73 |