Background
Methods
Patient material
BRCA1-C |
BRCA2-C | Sporadic-C |
BRCA1-N |
BRCA2-N | Non-BRCA- related-N |
p ValueA
| |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
n
| % |
n
| % |
n
| % |
n
| % |
n
| % |
n
| % | ||
Number of samples (%) | 39 | 23.5 | 33 | 19.9 | 80 | 48.2 | 5 | 3 | 4 | 2.4 | 5 | 3 | |
Age, years | 9.56°10−9*ǂ | ||||||||||||
Median | 43 | N/A | 46 | N/A | 58 | N/A | 31 | N/A | 36.5 | N/A | 22 | N/A | 0.549B
|
Range | 30–80 | N/A | 21–69 | N/A | 29–86 | N/A | 29–33 | N/A | 35–38 | N/A | 18–52 | N/A | |
GradeC
| 0.00007*ǂǂ
| ||||||||||||
1 | 0 | 0 | 1 | 3 | 20 | 25.3 | |||||||
2 | 9 | 23.1 | 10 | 30.3 | 25 | 31.6 | |||||||
3 | 30 | 76.9 | 22 | 66.7 | 34 | 43 | |||||||
Tumor type | 0.230ǂǂ
| ||||||||||||
Ductal | 34 | 87.2 | 29 | 87.9 | 69 | 86.3 | |||||||
Lobular | 2 | 5.1 | 2 | 6.1 | 10 | 12.5 | |||||||
Other | 3 | 7.7 | 2 | 6.1 | 1 | 1.3 | |||||||
ER | 0.0004*ǂǂǂ
| ||||||||||||
Negative | 26 | 66.7 | 8 | 24.2 | 23 | 28.8 | |||||||
Positive | 13 | 33.3 | 25 | 75.8 | 57 | 71.3 | |||||||
PR | 0.001*ǂǂǂ
| ||||||||||||
Negative | 29 | 74.4 | 17 | 51.5 | 30 | 37.5 | |||||||
Positive | 10 | 25.6 | 16 | 48.5 | 50 | 62.5 | |||||||
HER2 | 0.68ǂǂ
| ||||||||||||
Negative | 38 | 97.4 | 32 | 97 | 75 | 93.8 | |||||||
Positive | 1 | 2.6 | 1 | 3 | 5 | 6.3 |
DNA isolation
Methylation analysis
Gene | Probe ID | Length | Chromosome position | 5′ Probe | 3′ Probe | Start | End | CpG site | CpG loci ID |
---|---|---|---|---|---|---|---|---|---|
BRCA1
| BRCA1.1 | 165 | 17q21.31 | CCTCTGAGAGGCTGCTGCTTAGCGGTAGCCCCTT | GGTTTCCGTGGCAACGGAAAAGCGCGGGAATTACAGA | 41277415 | 41277483 | 41277429 | cg04110421 |
BRCA1
| BRCA1.2 | 230 | 17q21.31 | CATGCATCTGAGAAACCCCACAGCCTGTCCCCCGTCCAGGAA | GTCTCAGCGAGCTCACGCCGCGCAGTCGCAGTTT | 41277407 | 41321886 | 41277395 | cg16630982 |
BRCA1
| BRCA1.3 | 252 | 17q21.31 | GTGGGGTTTCTCAGATAACTGGGCCCCTGC | GCTCAGGAGGCCTTCACCCTCTGCTCTGGGTAAAGGT | 41277286 | 41277352 | 41277323 | cg08993267 |
BRCA2
| BRCA2.1 | 130 | 13q13.1 | CCATCTTGTGGCGCGAGCTTCTGAAACTA | GGCGGCAGAGGCGGAGCCGCTGTGGCACTGCT | 32889614 | 32889670 | 32889621 | N/A |
BRCA2
| BRCA2.2 | 149 | 13q13.1 | TGCGGGTTAGTGGTGGTGGTAGTGGGTT | GGGACGAGCGCGTCTTCCGCAGTCCCAGTCCAGCGTGG | 32889801 | 32889865 | 32889836 | N/A |
BRCA2
| BRCA2.3 | 160 | 13q13.1 | CCTCTGAGAGGCTGCTGCTTAGCGGTAGCCCCTT | GGTTTCCGTGGCAACGGAAAAGCGCGGGAATTACAGA | 32889665 | 32889746 | 32889672 + 32889683 | N/A |
BRCA2
| BRCA2.4 | 217 | 13q13.1 | CTTCCGGGTGGTGCGTGTGCTGCGTGTCGC | GTCACGGCGTCACGTGGCCAGCGCGGGCTTGT | 32889557 | 32889618 | 2889608 | N/A |
Statistical analysis
Correlation between BRCA1/2 methylation and messenger RNA expression
Results
BRCA1 promoter methylation in BRCA1/2-related and sporadic breast carcinomas
Probe |
BRCA1C-C, median % (range) |
BRCA2-C, median % (range) | Sporadic-C, median % (range) | Test statistica
|
p Value |
---|---|---|---|---|---|
BRCA1.1 | 3 (0–11) | 3 (0–91) | 3 (0–80) | 2494.500 | 0.140 |
BRCA1.2 | 21 (11–34) | 17 (11–100) | 15 (9–85) | 1795.500 | 0.00006* |
BRCA1.3 | 11 (5–18) | 9 (5–100) | 7 (3–71) | 1760.600 | 0.00003* |
BRCA2.1 | 24 (14–56) | 21 (8–100) | 15 (8–43) | 947.500 | 9.85°10−13* |
BRCA2.2 | 11 (5–27) | 10 (5–100) | 5 (3–15) | 596.000 | 1.00°10−13* |
BRCA2.3 | 17 (9–33) | 14 (8–100) | 9 (5–18) | 536.500 | 1.00°10−13* |
BRCA2.4 | 9 (0–18) | 8 (0–100) | 5 (2–12) | 984.500 | 1.95°10−12* |
Probe |
BRCA1-C, total n = 39 (%) |
BRCA2-C, total n = 33 (%) | Sporadic-C, total n = 80 (%) | Test statistica
|
P-value | Total (%) | Cutoffb
|
---|---|---|---|---|---|---|---|
BRCA1.1 | 0 (0) | 2 (6.1) | 11 (13.8) | 5.833c
| 0.019* | 13 (8.6) | >15% |
BRCA1.2 | 32 (82.1) | 14 (42.4) | 22 (27.5) | 20.296c
| 0.000009* | 68 (44.7) | >17% |
BRCA1.3 | 5 (12.8) | 2 (6.1) | 11 (13.8) | 0.589c
| 0.465 | 18 (11.8) | >15% |
BRCA1 totald
| 32 (82.1) | 14 (42.4) | 22 (27.5) | 20.296c
| 0.000009* | 68 (44.7) | |
BRCA2.1 | 30 (76.9) | 18 (54.5) | 10 (12.5) | 47.117c
| 2.93°10−12* | 58 (38.2) | >19% |
BRCA2.2 | 4 (10.3) | 3 (9.1) | 0 (0) | 8.153e
| 0.005* | 7 (4.6) | >15% |
BRCA2.3 | 22 (56.4) | 14 (42.4) | 2 (2.5) | 45.600c
| 1.65°10−12* | 38 (25) | >15% |
BRCA2.4 | 3 (7.7) | 4 (12.1) | 0 (0.0) | 8.153e
| 0.005* | 7 (4.6) | >15% |
BRCA2 totald
| 30 (76.9) | 20 (60.6) | 10 (12.5) | 51.432c
| 0.029* | 60 (39.5) |
Probe | Sensitivity | 95% CI | Specificity | 95% CI |
---|---|---|---|---|
BRCA1.1a
| 70 of 72 (97.2%) | 90.3–99.6% | 11 of 80 (13.8%) | 7.1–23.3% |
BRCA1.2b
| 46 of 72 (63.9%) | 51.7–74.9% | 58 of 80 (72.5%) | 61.4–81.9% |
BRCA1.3a
| 65 of 72 (90.3%) | 90.0–96.0% | 11 of 80 (13.8%) | 7.1–23.3% |
BRCA1 totalc
|
46 of 72 (63.9%)
|
51.7–74.9%
|
58 of 80 (72.5%)
|
61.4–81.9%
|
BRCA2.1b
| 48 of 72 (66.7% | 54.6–77.3% | 70 of 80 (87.5%) | 78.2–93.8% |
BRCA2.2b
| 7 of 72 (9.7%) | 4.0–19.0% | 80 of 80 (100%) | 95.5–100% |
BRCA2.3b
| 36 of 72 (50.0%) | 38.0–62.0% | 78 of 80 (97.5%) | 91.3–99.7% |
BRCA2.4b
| 7 of 72 (9.7%) | 4.0–19.0% | 80 of 80 (100%) | 95.5–100% |
BRCA2 totalc
|
50 of 72 (69.4%)
|
57.5–79.8%
|
70 of 80 (87.5%)
|
78.2–93.8%
|
BRCA2 promoter methylation in BRCA1/2-related and sporadic breast carcinomas
Correlation with clinicopathological variables
Sporadic carcinomas | Grades 1–2 vs. 3 | Ductal vs. lobular tumors | ER-positive vs. ER-negative | PR-positive vs. PR-negative | HER2-positive vs. HER2-negative | |||||
---|---|---|---|---|---|---|---|---|---|---|
Probe | Test statistica
|
p Value | Test statistica
|
p Value | Test statistica
|
p Value | Test statistica
|
p Value | Test statistica
|
p Value |
BRCA1.1 | 12.230b
| 0.001* | 1.852b
| 0.342 | 7.577b
| 0.011* | 6.753b
| 0.016* | 0.176b
| 0.533 |
BRCA1.2 | 8.190c
| 0.006* | 0.026b
| 1.000 | 6.689c
| 0.014* | 3.762c
| 0.071 | 2.825b
| 0.125 |
BRCA1.3 | 12.230b
| 0.001* | 1.852b
| 0.342 | 7.577b
| 0.011* | 6.753b
| 0.016* | 0.176b
| 0.533 |
BRCA1 totalc
| 8.190c
| 0.006* | 0.026b
| 1.000 | 6.689c
| 0.014* | 3.762c
| 0.071 | 2.825b
| 0.125 |
BRCA2.1 | 6.577b
| 0.015* | 1.659b
| 0.345 | 0.706b
| 0.462 | 0.274b
| 0.736 | 3.688b
| 0.115 |
BRCA2.2 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
BRCA2.3 | 2.775b
| 0.178 | 0.297b
| 1.000 | 5.084b
| 0.080 | 0.137b
| 1.000 | 0.137b
| 1.000 |
BRCA2.4 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
BRCA2 totalc
| 6.577b
| 0.015* | 1.659c
| 0.345 | 0.706c
| 0.462 | 0.274c
| 0.736 | 3.688c
| 0.115 |
BRCA1-related carcinomas | Grades 1–2 vs. 3 | Ductal vs. lobular tumors | ER-positive vs. ER-negative | PR-positive vs. PR-negative | HER2-positive vs. HER2-negative | |||||
---|---|---|---|---|---|---|---|---|---|---|
Probe | Test statistica
|
p Value | Test statistica
|
p Value | Test statistica
|
p Value | Test statistica
|
p Value | Test statistica
|
p Value |
BRCA1.1 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
BRCA1.2 | 0.145b
| 0.653 | 1.262b
| 0.356 | 0.087b
| 1.000 | 0.038b
| 1.000 | 0.225b
| 1.000 |
BRCA1.3 | 0.031b
| 1.000 | 0.342b
| 1.000 | 0.115b
| 1.000 | 0.096b
| 1.000 | 0.151b
| 1.000 |
BRCA1 totalc
| 0.145b
| 0.653 | 1.262b
| 0.356 | 0.087b
| 1.000 | 0.038b
| 1.000 | 0.225b
| 1.000 |
BRCA2.1 | 0.005b
| 1.000 | 1.694b
| 0.310 | 0.000b
| 1.000 | 0.363b
| 0.669 | 0.308b
| 1.000 |
BRCA2.2 | 0.009b
| 1.000 | 0.265b
| 1.000 | 3.482b
| 0.099 | 0.001b
| 1.000 | 0.117b
| 1.000 |
BRCA2.3 | 0.003b
| 1.000 | 3.328b
| 0.144 | 0.209bb
| 0.740 | 0.070b
| 1.000 | 0.793b
| 1.000 |
BRCA2.4 | 0.975b
| 1.000 | 0.193b
| 1.000 | 1.625b
| 0.253 | 1.121b
| 0.556 | 0.086b
| 1.000 |
BRCA2 totalc
| 0.005b
| 1.000 | 1.694b
| 0.310 | 0.000b
| 1.000 | 0.363b
| 0.669 | 0.308b
| 1.000 |
BRCA2-related carcinomas | Grades 1–2 vs. 3 | Ductal vs. lobular tumors | ER-positive vs. ER-negative | PR-positive vs. PR-negative | HER2-positive vs. HER2-negative | |||||
---|---|---|---|---|---|---|---|---|---|---|
Probe | Test statistica
|
p Value | Test statistica
|
p Value | Test statistica
|
p Value | Test statistica
|
p Value | Test statistica
|
p Value |
BRCA1.1 | 1.065b
| 0.542 | 0.147b
| 1.000 | 0.769b
| 0.432 | 0.002b
| 1.000 | 0.067b
| 1.000 |
BRCA1.2 | 0.062b
| 1.000 | 0.057b
| 1.000 | 0.248b
| 0.695 | 0.730c
| 0.491 | 0.760b
| 1.000 |
BRCA1.3 | 1.065b
| 0.542 | 0.147b
| 1.000 | 0.769b
| 0.432 | 0.002b
| 1.000 | 0.067b
| 1.000 |
BRCA1 totalc
| 0.062b
| 1.000 | 0.057b
| 1.000 | 0.248b
| 0.695 | 0.730bb
| 0.491 | 0.760b
| 1.000 |
BRCA2.1 | 2.200c
| 0.266 | 0.002b
| 1.000 | 0.088b
| 1.000 | 2.528c
| 0.166 | 1.238b
| 0.455 |
BRCA2.2 | 1.650b
| 0.534 | 0.229b
| 1.000 | 0.149b
| 1.000 | 0.437b
| 0.601 | 0.103b
| 1.000 |
BRCA2.3 | 3.039b
| 0.136 | 0.115b
| 1.000 | 1.742b
| 0.238 | 0.022c
| 1.000 | 0.760b
| 1.000 |
BRCA2.4 | 0.569b
| 0.586 | 0.317b
| 1.000 | 0.001b
| 1.000 | 0.004b
| 1.000 | 0.142b
| 1.000 |
BRCA2 totalc
| 3.110b
| 0.132 | 0.057b
| 1.000 | 0.916b
| 0.431 | 0.863c
| 0.481 | 1.587b
| 0.394 |
Age | CMI for BRCA1
| CMI for BRCA2
| CMI for BRCA1 + BRCA2
| |||
---|---|---|---|---|---|---|
Spearman’s rho |
p Value | Spearman’s rho |
p Value | Spearman’s rho |
p Value | |
BRCA1-C | −0.297 | 0.066 | −0.287 | 0.077 | −0.275 | 0.090 |
BRCA2-C | −0.019 | 0.918 | 0.003 | 0.989 | −0.035 | 0.846 |
Sporadic-C | −0.153 | 0.175 | 0.003 | 0.982 | −0.120 | 0.289 |
BRCA1/2-N | −0.252 | 0.513 | −0.467 | 0.205 | −0.417 | 0.265 |
Non-BRCA-related-N | 0.300 | 0.624 | 0.100 | 0.873 | 0.100 | 0.873 |
Correlation between BRCA1/2 methylation and mRNA expression
BRCA promoter methylation in BRCA1/2-related and non-BRCA-related normal breast tissue
Probe |
BRCA1/2-N, median % (range) | Non-BRCA-related-N, median % (range) | Test statistica
|
p Value |
---|---|---|---|---|
BRCA1.1 | 2 (0–3) | 3 (0–5) | 15.500 | 0.325 |
BRCA1.2 | 17 (13–26) | 13 (13–17) | 8.500 | 0.057 |
BRCA1.3 | 9 (6–16) | 7 (5–8) | 8.000 | 0.050 |
BRCA2.1 | 19 (15–26) | 16 (14–19) | 8.500 | 0.060 |
BRCA2.2 | 8 (6–16) | 6 (5–12) | 12.000 | 0.158 |
BRCA2.3 | 13 (10–28) | 9 (8–14) | 6.500 | 0.031* |
BRCA2.4 | 8 (6–9) | 5 (5–6) | 2.000 | 0.005* |
Probe |
BRCA1, total n = 5 (%) |
BRCA2, total n = 4 (%) | Total, n = 9 (%) | Cutoffa
|
---|---|---|---|---|
BRCA1.1 | 0 (0) | 0 (0) | 0 (0) | >15% |
BRCA1.2 | 2 (40) | 1 (25) | 3 (30) | >17% |
BRCA1.3 | 1 (20) | 0 (0) | 1 (11.1) | >15% |
BRCA1 totalb
| 2 (40) | 1 (25) | 3 (30) | |
BRCA2.1 | 2 (40) | 2 (50) | 4 (44.4) | >19% |
BRCA2.2 | 1 (20) | 0 (0) | 1 (11.1) | >15% |
BRCA2.3 | 2 (50) | 0 (0) | 2 (22.2) | >15% |
BRCA2.4 | 0 (0) | 0 (0) | 0 (0) | >15% |
BRCA2 totalb
| 3 (60) | 2 (50) | 5 (55.5) |