Introduction
Materials and methods
Patients
Breast cancer samples
DNA and RNA extraction
Gene expression profiling with DNA microarrays
Gene expression data analysis
Tissue microarrays construction and immunohistochemistry
Protein (Clone) | Antibody | Origin | Clone | Pre-treatment | Dilution | Location of staining | Normal |
---|---|---|---|---|---|---|---|
Androgen receptor
(AR)
| mmb | Dako | 4AR441 | PH9 Target retrieval solution (98°C, 40 min) | 1/50° | nucleus | + |
BCL2
| mmb | Dako | 124 | Citrate* | 1/100° | cytoplasm | + |
CyclinD1
CCND1
| mmb | Labvision | SP4 | EDTA* | prediluted | nucleus | + |
ERBB2
| mmb | Dako Herceptest Ltd | AO485 | Target retrieval * | 1/500 | membrane | - |
Estrogen receptor (ER)
| mmb | Novocastra laboratories Ltd | 6F11.2 | Target retrieval * | 1/60 | nucleus | + |
FOXA1
| mmb | AbNova | 2D7 | Citrate* | 1/250 | nucleus | + |
GATA3
| mmb | Santacruz | HG3-31 | Citrate* | 1/100° | nucleus | + |
P53
| mmb | Beckmann | DO-1 | Citrate* | 1/4 | nucleus | - |
Progesterone
receptor (PR)
| mmb | Dako | PFR 636 | Target retrival * | 1/80 | nucleus | + |
Ki67
| mmb | Dako | KI-67 | Target retrieval solution * | 1/100 | nuclear | + |
Statistical analysis
Results
Correlation between molecular subtype and oestrogen receptor immunohistochemical status
Spearman correlation test | AR | BCL2 | CCND1 | ERBB2 | FOXA1 | GATA3 | KI67 | P53 | ER | PR |
---|---|---|---|---|---|---|---|---|---|---|
Rho * | 0.49 | 0.56 | 0.43 | 0.28 | 0.58 | 0.64 | 0.32 | 0.30 | 0.73 | 0.69 |
P | < 0.0001 | < 0.0001 | < 0.0001 | 0.00067 | < 0.0001 | < 0.0001 | 0.00018 | 0.00023 | < 0.0001 | < 0.0001 |
Subtype N = 135 | Basal N = 36 (27%) | ERBB2 N = 17 (12.5%) | LuminalAa N = 45 (33%) | LuminalAb N = 21 (15.5%) | LuminalB N = 3 (2%) | Normal-like N = 13 (10%) |
---|---|---|---|---|---|---|
ER-positive N = 81 | 3 (8%) | 4 (23.5%) | 43 (95.5%) | 21 (100%) | 3 (100%) | 7 (53.8%) |
ER-negative N = 54 | 33 (91.6%) | 13 (76.4%) | 2 (4.5%) | 0 | 0 | 6 (46.2%) |
P-value | 4.5910-13 | 0.0029 | 9.6 10-11 | 4.9 10-6 | NS | NS |
Univariate and multivariate analyses of survival
Variable | NE (%) | Classes | N (%) | Hazard Ratio [IC95] | p-value |
---|---|---|---|---|---|
Hormonal therapy
| 0 (0%) | no | 361 (43%) | 1 | 0.000209 |
yes | 471 (57%) | 0.558 [0.408–0.763] | |||
Age
| 1 (0%) | < 45 years | 104 (13%) | 1 | 0.673 |
>= 45 years | 727 (87%) | 1.1 [0.711–1.696] | |||
< 45 | 104 (13%) | 1 | 0.145 | ||
Tumor Size
| 7 (1%) | < 25 mm | 381 (46%) | 1 | < 0.0001 |
>= 25 mm | 444 (54%) | 2.47 [1.719–3.562] | |||
Scarff-Bloom-Richardson grade
| 22 (3%) | I | 266 (33%) | 1 | < 0.0001 |
II–III | 544 (67%) | 3.69 [2.244–6.083] | |||
VPI
| 4 (0%) | Absent | 536 (65%) | 1 | < 0.0001 |
present | 292 (35%) | 2.1 [1.519–2.897] | |||
Lymph node invasion
| 16 (2%) | N- | 442 (54%) | 1 | < 0.0001 |
N+ | 374 (46%) | 2.61 [1.693–4.013] | |||
AR
| 175 (21%) | 0 | 220 (33%) | 1 | 0.0122 |
> 0 | 437 (67%) | 0.65 [0.463–0.913] | |||
< 80 | 410 (62%) | 1 | 0.00422 | ||
>= 80 | 247 (38%) | 0.568 [0.383–0.841] | |||
BCL2
| 201 (24%) | 0 | 162 (26%) | 1 | 0.000944 |
> 0 | 469 (74%) | 0.548 [0.381–0.787] | |||
CCND1
| 142 (17%) | 0 | 284 (41%) | 1 | 0.562 |
> 0 | 406 (59%) | 0.905 [0.647–1.268] | |||
ERBB2
| 123 (15%) | 0 or 1 | 621 (88%) | 1 | 0.209 |
2 or 3 | 88 (12%) | 1.33 [0.852–2.071] | |||
FOXA1
| 187 (22%) | 0 | 40 (6%) | 1 | 0.324 |
> 0 | 605 (94%) | 1.43 [0.698–2.952] | |||
GATA3
| 188 (23%) | 0 | 247 (38%) | 1 | 0.00024 |
> 0 | 397 (62%) | 0.534 [0.38–0.75] | |||
Ki67
| 171 (21%) | < 20 | 560 (85%) | 1 | 0.000748 |
>= 20 | 101 (15%) | 1.96 [1.316–2.923] | |||
P53
| 140 (17%) | 0 | 525 (76%) | 1 | 0.00095 |
> 0 | 167 (24%) | 1.76 [1.253–2.47] | |||
ER
| 79 (9%) | 0 | 169 (22%) | 1 | 0.000276 |
> 0 | 584 (78%) | 0.54 [0.386–0.756] | |||
PR
| 120 (14%) | 0 | 263 (37%) | 1 | 0.0875 |
> 0 | 449 (63%) | 0.752 [0.542–1.044] |
N = 378 | Coefficient | HR | IC95 | p-value | |
---|---|---|---|---|---|
Hormonal therapy
| no | 1 | |||
yes | -0.897 | 0.408 | [0.257–0.647] | 0.00014 | |
Size
| < 25 mm | 1 | |||
>= 25 mm | 0.9 | 2.46 | [1.449–4.175] | 0.00085 | |
Scarff-Bloom-Richardson grade
| I | 1 | |||
II–III | 0.521 | 1.68 | [0.805–3.522] | 0.17 | |
VPI
| no | 1 | |||
yes | 0.657 | 1.93 | [1.213–3.068] | 0.0055 | |
Lymph node
| N- | 1 | |||
N+ | 0.68 | 1.97 | [1.208–3.222] | 0.0066 | |
BCL2
| < 160 | 1 | |||
>= 160 | -0.613 | 0.542 | [0.331–0.886] | 0.015 | |
Ki67
| < 20 | 1 | |||
>= 20 | 0.641 | 1.9 | [1.167–3.086] | 0.0098 | |
P53
| 0 | 1 | |||
> 0 | 0.369 | 1.45 | [0.928–2.254] | 0.1 |
Molecular subtype and oestrogen receptor positivity
Basal | ERBB2 | Luminal Aa | Luminal Ab | Luminal B | Normal-like | p-value | |
---|---|---|---|---|---|---|---|
ER-positive N = 84 | 3 | 4 | 43 (%)x | 21(%)x | 3 | 7 | 3 cases non evaluable |
Grade I | 1 | 0 |
14(32.5%)
|
0
| 0 | 5 |
4.710
-6
|
Grade II | 0 | 0 |
24(55.8%)
|
7(30%)
| 0 | 2 | |
Grade III | 2 | 4 |
5(11.6%)
|
14(66.6%)
| 3 | 0 | |
VPI positive | 0 | 1 | 20/42 (47.6%) | 10/21 (47.6%) | 3 | 1 | |
Positive lymph nodes | 1 | 2 | 26/43 (60.4%) | 15/21 (71.4%) | 2 | 2 | |
AR positive | 1 | 2 | 31/36(86%) | 15/21(71.4%) | 2 | 3 | |
BCL2 positive | 2 | 2 | 36/39 (92.3%) | 14/15 (93%) | 2 | 6/6 (100%) | |
CCND1 Positive | 2 | 0 | 26/41 (63,4%) | 13/19 (68%) | 2 | 2/5 | |
ERBB2 (2+/3+) | 0 | 3 (3+) | 3/43 (6.9%) | 0 | 1(2+) | 0 | |
FOX A1 positive | 1 | 3 | 38/39 (97.4%) | 19/19 (100% | 3 | 4/5 | |
GATA3 Positive | 0 | 1 | 29/36 (80.5%) | 12/18 (66.6%) | 3 | 4/5 | |
Ki67 > 20% | 1 | 0 |
2/40(5%)
|
5/18(27.7%)
| 0 | 0 |
0.02
|
P53 positive | 2 | 2 | 8/42 (19%) | 4/20 (20%) | 1 | 0/6 | |
PR positive | 0 | 0 | 37/43 (86%) | 16/20 (80%) | 2 | 5/6 |
Markers and survival
Variable | NE (%) | Classes | N (%) | Hazard Ratio [IC95] | p-value × |
---|---|---|---|---|---|
Age
| 0 (0%) | < 45 years | 29 (8%) | 1 | 0.964 |
>= 45 years | 355 (92%) | 1.02 [0.401–2.605] | |||
< 45 | 29 (8%) | 1 | 0.315 | ||
45–60 | 155 (40%) | 0.834 [0.312–2.226] | |||
> 60 | 200 (52%) | 1.32 [0.493–3.522] | |||
Tumoral size
| 2 (1%) | < 25 mm | 141 (37%) | 1 | 0.0222 |
>= 25 mm | 241 (63%) | 2.16 [1.1–4.23] | |||
Scarff-Bloom-Richardson grade
| 2 (1%) | I | 141 (37%) | 1 | 0.000487 |
II–III | 241 (63%) | 4.1 [1.74–9.644] | |||
VPI
| 3 (1%) | absent | 237 (62%) | 1 | 0.00363 |
present | 144 (38%) | 2.36 [1.305–4.268] | |||
N
| 3 (1%) | N- | 174 (46%) | 1 | 0.0495 |
N+ | 207 (54%) | 2.22 [1–4.921] | |||
N < 3 | 288 (76%) | 1 | < 0.0001 | ||
N >= 3 | 93 (24%) | 3.81 [2.113–6.883] | |||
AR
| 66 (17%) | 0 | 74 (23%) | 1 | 0.306 |
> 0 | 244 (77%) | 0.712 [0.371–1.369] | |||
BCL2
| 66 (17%) | 0 | 38 (12%) | 1 | 0.0676 |
> 0 | 280 (88%) | 0.492 [0.226–1.069] | |||
CCND1
| 47 (12%) | 0 | 97 (29%) | 1 | 0.971 |
> 0 | 240 (71%) | 1.01 [0.542–1.886] | |||
ERBB2
| 36 (9%) | 1 or 2 | 319 (92%) | 1 | 0.558 |
2 or 3 | 29 (8%) | 1.32 [0.519–3.359] | |||
FOXA1
| 78 (20%) | 0 | 3 (1%) | 1 | 0.406 |
> 0 | 303 (99%) | 9220000 [0-Inf] | |||
GATA3
| 66 (17%) | 0 | 70 (22%) | 1 | 0.0952 |
> 0 | 248 (78%) | 0.599 [0.326–1.1] | |||
Ki67
| 60 (16%) | < 20 | 299 (92%) | 1 | 0.000304 |
>= 20 | 25 (8%) | 3.58 [1.709–7.488] | |||
P53
| 55 (14%) | 0 | 272 (83%) | 1 | < 0001 |
> 0 | 57 (17%) | 3.13 [1.745–5.601] | |||
ER
| 0 (0%) | < 120 | 153 (40%) | 1 | 0.227 |
>= 120 | 231 (60%) | 0.71 [0.406–1.24] | |||
PR
| 44 (11%) | 0 | 76 (22%) | 1 | 0.941 |
> 0 | 264 (78%) | 0.975 [0.499–1.904] |
N = 240 | Coefficient | HR | IC95 | p-value | |
---|---|---|---|---|---|
VPI
| No | 1 | |||
yes | 0.903 | 2.47 | [1.148–5.297] | 0.021 | |
GATA3
| 0 | 1 | |||
> 0 | -0.655 | 0.519 | [0.251–1.076] | 0.078 | |
Ki67
| < 20 | 1 | |||
>= 20 | 1.06 | 2.9 | [1.272–6.604] | 0.011 | |
P53
| 0 | 1 | |||
> 0 | 1.08 | 2.94 | [1.505–5.76] | 0.0016 |