Introduction
Patients and methods
Patients and tissues
Chromogenic in situ hybridization (CISH)
RNA extraction and mRNA investigations
Immunohistochemistry
Protein | Antibody [clone, source] | Ab dilution | Pretreatment/EU | IT | IHC Staining Detection System/Chromogen | Scoring System | Cut-off (%) | Staining Pattern | Reference |
---|---|---|---|---|---|---|---|---|---|
Ki67 | MIB1 (1) | 1:70 | 15'/EU2 | 20' | Polymer HRP/DAB | SQ | ≥ 14%* | Nuclear | [28] |
HER2 | PL (1) | 1:200 | 25'/EU1 | 30' | Polymer HRP/DAB | 0-3 | > 30%# | Membranous | [23] |
TopoIIa | KiS1 (1) | 1:200 | 15'/EU2 | 30' | EnVision/DAB | SQ | > 5%** | Nuclear | [27] |
ER | 6F11 (2) | 1:70 | 20'/EU1 | 20' | Polymer HRP/DAB | H-Score | ≥ 1% | Nuclear | [29] |
PgR | 1A6 (2) | 1:70 | 20'/EU1 | 20' | Polymer HRP/DAB | H-Score | ≥ 1% | Nuclear | [29] |
Statistical analysis
Results
Patient and Tumor Characteristics
E-T-CMF | E-CMF | Analyzed cohort | ||||
---|---|---|---|---|---|---|
(N = 159) | (N = 193) | (N = 352) | ||||
Median | Range | Median | Range | Median | Range | |
Age (in years) | 50.2 | 23.8-75.9 | 50.6 | 22.5-78.0 | 50.6 | 22.5-78.0 |
Number of nodes removed
| 19 | 5-59 | 19 | 4-53 | 19 | 4-59 |
Number of positive nodes
| 7 | 0-54 | 6 | 0-49 | 6 | 0-54 |
N
|
%
|
N
|
%
|
N
|
%
| |
Nodal involvement (n = 352)
| ||||||
0-3 nodes | 34 | 21.4 | 54 | 28.0 | 88 | 25.0 |
≥ 4 nodes | 125 | 78.6 | 139 | 72.0 | 264 | 75.0 |
Menopausal status (n = 352)
| ||||||
Premenopausal | 83 | 52.2 | 104 | 53.9 | 187 | 53.1 |
Postmenopausal | 76 | 47.8 | 89 | 46.1 | 165 | 46.9 |
Type of operation (n = 352)
| ||||||
Modified radical mastectomy | 122 | 76.7 | 151 | 78.2 | 273 | 77.6 |
Breast conserving surgery | 37 | 23.3 | 42 | 21.8 | 79 | 22.4 |
Interval from operation (n = 352)
| ||||||
< 2 weeks | 19 | 11.9 | 26 | 13.5 | 45 | 12.8 |
2-4 weeks | 81 | 50.9 | 84 | 43.5 | 165 | 46.9 |
> 4 weeks | 59 | 37.1 | 83 | 43.0 | 142 | 40.3 |
Tumor grade* (n = 351)
| ||||||
I-II | 64 | 40.5 | 112 | 58.0 | 176 | 50.1 |
III-Undifferentiated | 94 | 59.5 | 81 | 42.0 | 175 | 49.9 |
Tumor size (n = 352)
| ||||||
< 2 cm | 45 | 28.3 | 67 | 34.7 | 112 | 31.8 |
2-5 cm | 88 | 55.3 | 91 | 47.2 | 179 | 50.9 |
> 5 cm | 26 | 16.4 | 35 | 18.1 | 61 | 17.3 |
Adjuvant RT (n = 350)
| ||||||
No | 27 | 17.1 | 39 | 20.3 | 66 | 18.9 |
Yes | 131 | 82.9 | 153 | 79.7 | 284 | 81.1 |
Adjuvant HT (n = 351)
| ||||||
No | 12 | 7.6 | 21 | 10.9 | 33 | 9.4 |
Yes | 146 | 92.4 | 172 | 89.1 | 318 | 90.6 |
ER protein status (n = 306)
| ||||||
Negative | 40 | 28.2 | 41 | 25.0 | 81 | 26.5 |
Positive | 102 | 71.8 | 123 | 75.0 | 225 | 73.5 |
PgR protein status (n = 305)
| ||||||
Negative | 52 | 36.6 | 54 | 33.1 | 106 | 34.8 |
Positive | 90 | 63.4 | 109 | 66.9 | 199 | 65.2 |
Hormone receptor status (n = 307)
| ||||||
Negative | 36 | 25.4 | 29 | 17.6 | 65 | 21.2 |
Positive | 106 | 74.6 | 136 | 82.4 | 242 | 78.8 |
Ki67 (n = 309)
| ||||||
Low (< 14%) | 25 | 17.4 | 30 | 18.2 | 55 | 17.8 |
High (≥ 14%) | 119 | 82.6 | 135 | 81.8 | 254 | 82.2 |
HER2 CISH amplification (n = 285)
| ||||||
Non-amplified | 100 | 75.2 | 121 | 79.6 | 221 | 77.5 |
Amplified | 33 | 24.8 | 31 | 20.4 | 64 | 22.5 |
HER2 mRNA expression (n = 314)
| ||||||
Low (< 75th percentile) | 108 | 75.0 | 128 | 75.3 | 236 | 75.2 |
High (≥ 75th percentile) | 36 | 25.0 | 42 | 24.7 | 78 | 24.8 |
HER2 protein expression (n = 297)**
| ||||||
0-1+ | 86 | 63.2 | 125 | 77.6 | 211 | 71.0 |
2+ | 19 | 14.0 | 15 | 9.3 | 34 | 11.4 |
3+ | 31 | 22.8 | 21 | 13.0 | 52 | 17.5 |
TOP2A CISH amplification (n = 266)
| ||||||
Non-amplified | 110 | 90.9 | 137 | 94.5 | 247 | 92.9 |
Amplified | 11 | 9.1 | 8 | 5.5 | 19 | 7.1 |
TOP2A mRNA expression (n = 314)
| ||||||
Low (< median) | 72 | 50.0 | 85 | 50.0 | 157 | 50.0 |
High (≥ median) | 72 | 50.0 | 85 | 50.0 | 157 | 50.0 |
TopoIIa protein status (n = 273)
| ||||||
Negative | 43 | 35.2 | 56 | 37.1 | 99 | 36.3 |
Positive | 79 | 64.8 | 95 | 62.9 | 174 | 63.7 |
HER2 and TOP2A gene status, mRNA and protein expression
Association between HER2 and TOP2A parameters
Association of HER2 and TOP2A with clinicopathological parameters
ER status | PgR status | Ki67 status | |||||||
---|---|---|---|---|---|---|---|---|---|
Negative | Positive | p | Negative | Positive | p | Low | High | p | |
N (%) | N (%) | N (%) | N (%) | N (%) | N (%) | ||||
HER2 (CISH)
| < 0.001 | 0.001 | 0.035 | ||||||
Non-amplified | 47 (61.8) | 173 (83.2) | 63 (65.6) | 156 (83.4) | 42 (89.4) | 176 (75.2) | |||
Amplified | 29 (38.2) | 35 (16.8) | 33 (34.4) | 31 (16.6) | 5 (10.6) | 58 (24.8) | |||
HER2 (mRNA)
| < 0.001 | < 0.001 | 0.086 | ||||||
Low (< 75th perc) | 38 (55.9) | 166 (82.6) | 53 (59.6) | 151 (84.4) | 40 (87.0) | 168 (74.0) | |||
High (≥ 75th perc) | 30 (44.1) | 35 (17.4) | 36 (40.4) | 28 (15.6) | 6 (13.0) | 59 (26.0) | |||
HER2 (IHC)
| 0.003 | 0.002 | 0.016 | ||||||
0-1+ | 47 (58.8) | 163 (75.5) | 61 (58.7) | 148 (77.5) | 43 (87.8) | 163 (67.4) | |||
2+ | 9 (11.3) | 25 (11.6) | 15 (14.4) | 19 (9.9) | 3 (6.1) | 31 (12.8) | |||
3+ | 24 (30.0) | 28 (13.0) | 28 (26.9) | 24 (12.6) | 3 (6.1) | 48 (19.8) | |||
TOP2A (CISH)
| 0.29 | 0.46 | 0.33 | ||||||
Non-amplified | 64 (90.1) | 182 (93.8) | 83 (91.2) | 162 (93.6) | 41 (97.6) | 202 (91.8) | |||
Amplified | 7 (9.9) | 12 (6.2) | 8 (8.6) | 11 (6.4) | 1 (2.4) | 18 (8.2) | |||
TOP2A (mRNA)
| 0.99 | 0.20 | 0.003 | ||||||
Low (< median) | 34 (50.0) | 100 (49.8) | 39 (43.8) | 94 (52.5) | 33 (71.7) | 107 (47.1) | |||
High (≥ median) | 34 (50.0) | 101 (50.2) | 50 (56.2) | 85 (47.5) | 13 (28.3) | 120 (52.9) | |||
TopoIIa (IHC)
| 0.39 | 0.99 | < 0.001 | ||||||
Negative | 29 (40.3) | 69 (34.5) | 33 (35.9) | 64 (35.8) | 32 (78.0) | 66 (28.8) | |||
Positive | 43 (59.7) | 131 (65.5) | 59 (64.1) | 115 (64.2) | 9 (22.0) | 163 (71.2) |
Prognostic/predictive significance of HER2 and TOP2A
Disease-free survival | Overall survival | |||||
---|---|---|---|---|---|---|
HR | 95% C.I. | Wald's p | HR | 95% C.I. | Wald's p | |
HER2 (CISH)
| ||||||
Non-amplified | 1 | 1 | ||||
Amplified | 1.55 | 1.01-2.39 |
0.045
| 1.62 | 0.98-2.66 | 0.060 |
HER2 (mRNA)
| ||||||
Low | 1 | 1 | ||||
High | 1.89 | 1.29-2.79 |
0.001
| 1.91 | 1.21-3.02 |
0.005
|
HER2 (IHC)
| ||||||
0-1+ | 1 | 1 | ||||
2+ | 1.61 | 0.93-2.78 | 0.091 | 2.13 | 1.18-3.84 |
0.012
|
3+ | 1.75 | 1.10-2.79 |
0.019
| 1.78 | 1.02-3.08 |
0.041
|
HER2 status
| ||||||
Negative | 1 | 1 | ||||
Positive* | 1.57 | 1.04-2.39 |
0.033
| 1.68 | 1.04-2.72 |
0.034
|
TOP2A (CISH)
| ||||||
Non-amplified | 1 | 1 | ||||
Amplified | 0.35 | 0.11-1.10 | 0.072 | 0.34 | 0.08-1.38 | 0.131 |
TOP2A (mRNA)
| ||||||
Low | 1 | 1 | ||||
High | 1.45 | 1.01-2.10 |
0.046
| 2.34 | 1.46-3.73 |
< 0.001
|
TopoIIa (IHC)
| ||||||
Negative | 1 | 1 | ||||
Positive | 1.37 | 0.90-2.10 | 0.145 | 1.55 | 0.93-2.60 | 0.094 |
HER2/TOP2A CISH
| ||||||
All other | 1 | 1 | ||||
Both amplified | 0.17 | 0.2-1.20 | 0.075 | 0.25 | 0.04-1.80 | 0.17 |
HER2/TOP2A mRNA
| ||||||
All other | 1 | 1 | ||||
Both high | 2.15 | 1.37-3.38 |
0.001
| 2.51 | 1.51-4.18 |
< 0.001
|
HER2/TopoIIa IHC
| ||||||
All other | 1 | 1 | ||||
Both positive | 1.92 | 1.14-3.25 |
0.015
| 1.89 | 1.04-3.44 |
0.038
|
Disease-free survival | Overall survival | ||||||
---|---|---|---|---|---|---|---|
HR
| 95% C.I. | Wald's p | HR | 95% C.I. | Wald's p | ||
A)
|
Group
| ||||||
E-CMF vs E-T-CMF | 1.07 | 0.72-1.59 | 0.75 | 1.58 | 0.96-2.60 | 0.071 | |
Tumor size
| |||||||
2-5 cm vs < 2 cm | 1.62 | 0.99-2.67 | 0.056 | ||||
> 5 cm vs < 2 cm | 1.73 | 0.92-3.22 | 0.087 | ||||
Adjuvant HT
| |||||||
Yes vs No | 0.50 | 0.27-0.94 |
0.031
| ||||
Nodal involvement
| |||||||
≥ 4 vs 0-3 | 2.16 | 1.19-3.93 |
0.011
| 1.83 | 0.92-3.61 | 0.082 | |
Hormone receptor status
| |||||||
Positive vs Negative | 0.54 | 0.30-0.94 |
0.030
| ||||
Ki67
| |||||||
High vs Low | 1.69 | 0.91-3.15 | 0.096 | 3.54 | 1.27-9.84 |
0.015
| |
HER2 (mRNA)
| |||||||
High vs Low | 1.68 | 1.10-2.58 |
0.016
| 1.77 | 1.04-3.02 |
0.035
| |
B)
|
Group
| ||||||
E-CMF vs E-T-CMF | 1.20 | 0.79-1.82 | 0.40 | 1.58 | 0.96-2.60 | 0.071 | |
Nodal involvement
| |||||||
≥ 4 vs 0-3 | 3.03 | 1.56-5.85 |
0.001
| 2.21 | 1.11-4.38 |
0.024
| |
Hormone receptor status
| |||||||
Positive vs Negative | 0.49 | 0.31-0.78 |
0.003
| 0.42 | 0.24-0.71 |
0.001
| |
Ki67
| |||||||
High vs Low | 2.04 | 1.05-3.97 |
0.035
| 3.50 | 1.25-9.76 |
0.017
| |
TOP2A (CISH)
| |||||||
Amplified vs Non-amplified | 0.29 | 0.09-0.93 |
0.038
| ||||
TOP2A (mRNA)
| |||||||
High vs Low | 2.08 | 1.24-3.47 |
0.005
| ||||
C)
|
Group
| ||||||
E-CMF vs E-T-CMF | 1.17 | 0.74-1.84 | 0.51 | 1.60 | 0.97-2.64 | 0.065 | |
Nodal involvement
| |||||||
≥ 4 vs 0-3 | 2.82 | 1.39-5.71 |
0.004
| 2.09 | 1.05-4.16 |
0.036
| |
Hormone receptor status
| |||||||
Positive vs Negative | 0.56 | 0.33-0.96 |
0.035
| 0.49 | 0.28-0.86 |
0.014
| |
Ki67
| |||||||
High vs Low | 2.24 | 1.01-4.98 |
0.048
| 3.18 | 1.13-8.92 |
0.028
| |
HER2 (mRNA)
| |||||||
High vs Low | 1.80 | 1.10-2.93 |
0.019
| 1.73 | 1.02-2.93 |
0.040
| |
TOP2A (CISH)
| |||||||
Amplified vs Non-amplified | 0.36 | 0.11-1.17 | 0.091 | ||||
TOP2A (mRNA)
| |||||||
High vs Low | 1.52 | 0.97-2.41 | 0.070 | 2.06 | 1.23-3.46 |
0.006
|
Disease-free survival | Overall survival | ||||||
---|---|---|---|---|---|---|---|
HR | 95% C.I. | Wald's p | HR | 95% C.I. | Wald's p | ||
A)
| E-CMF vs E-T-CMF | 1.26 | 0.85-1.88 | 0.25 | 1.67 | 1.03-1.72 |
0.038
|
Nodal involvement (≥ 4 vs 0-3) | 3.53 | 1.82-6.85 |
< 0.001
| 2.96 | 2.96 |
0.004
| |
Hormone receptor status (pos. vs neg.) | 0.48 | 0.31-0.77 |
0.002
| 0.40 | 0.24-0.68 |
0.018
| |
Ki-67 (high vs low) | 1.97 | 1.06-3.65 |
0.031
| 2.77 | 1.19-6.46 |
0.018
| |
HER2&TOP2A gene (both ampl. vs all other) | 0.12 | 0.02-0.88 |
0.037
| 0.16 | 0.02-1.14 | 0.067 | |
B)
| E-CMF vs E-T-CMF | 1.16 | 0.77-1.75 | 0.47 | 1.56 | 0.95-2.57 | 0.079 |
Nodal involvement (≥ 4 vs 0-3) | 2.67 | 1.44-4.93 |
0.002
| 2.07 | 1.05-4.09 |
0.035
| |
Hormone receptor status (pos. vs neg.) | 0.57 | 0.36-0.91 |
0.018
| 0.48 | 0.28-0.83 |
0.008
| |
Ki67 (high vs low) | 1.92 | 0.98-3.77 | 0.057 | 3.40 | 1.22-9.48 |
0.019
| |
HER2&TOP2A mRNA (high vs low) | 2.30 | 1.39-3.81 |
0.001
| 2.48 | 1.41-4.36 |
0.002
| |
C)
| E-CMF vs E-T-CMF | 1.18 | 0.80-1.74 | 0.39 | 1.62 | 1.02-2.57 |
0.042
|
Nodal involvement (≥ 4 vs 0-3) | 3.00 | 1.64-5.49 |
< 0.001
| 2.43 | 1.25-4.73 |
0.009
| |
Hormone receptor status (pos. vs neg.) | 0.64 | 0.41-0.99 |
0.047
| 0.51 | 0.31-0.85 |
0.009
| |
Ki67 (high vs low) | 1.68 | 0.93-3.05 | 0.086 | 2.27 | 1.03-5.00 |
0.041
| |
HER2&TopoIIa IHC (both pos. vs all other) | 1.77 | 1.04-3.05 |
0.034
| 1.58 | 0.86-2.91 | 0.14 | |
D)
| E-CMF vs E-T-CMF | 1.13 | 0.73-1.75 | 0.59 | 1.53 | 0.89-2.26 | 0.12 |
Nodal involvement (≥ 4 vs 0-3) | 3.62 | 1.78-7.34 |
< 0.001
| 2.87 | 1.29-6.40 |
0.010
| |
Hormone receptor status (pos. vs neg.) | 0.51 | 0.31-0.84 |
0.009
| 0.45 | 0.25-0.81 |
0.008
| |
Ki67 (high vs low) | 2.05 | 0.99-4.21 | 0.051 | 4.35 | 1.34-14.18 |
0.015
| |
HER2&TOP2A gene (both ampl. vs all other) | 0.13 | 0.02-0.96 |
0.046
| 0.23 | 0.03-1.74 | 0.15 | |
HER2&TOP2A mRNA (high vs low) | 2.41 | 1.31-4.42 |
0.005
| 2.83 | 1.42-5.63 |
0.003
| |
HER2&TopoIIa IHC (both pos. vs all other) | 1.36 | 0.68-2.70 | 0.38 | 0.92 | 0.42-2.18 | 0.92 |