Introduction
Materials and methods
Patient population
Tissue microarray construction
Immunohistochemistry
Interpretation of the immunohistochemistry results
Fluorescence in situ hybridization
RNA isolation from formalin-fixed paraffin-embedded tissue and quantitative reverse transcription-polymerase chain reaction assessment
Statistical analysis
Results
Patient and tumor characteristics
E-T-CMF | E-CMF | All patients | ||||
---|---|---|---|---|---|---|
Number | 141 | 167 | 308 | |||
Age (years) | ||||||
Median | 50 | 50 | 50 | |||
Range | 24 to 76 | 22 to 78 | 22 to 78 | |||
Number of nodes removed | ||||||
Median | 19 | 20 | 20 | |||
Range | 5 to 59 | 4 to 53 | 4 to 59 | |||
Number of positive nodes | ||||||
Median | 7 | 6 | 6 | |||
Range | 0 to 54 | 0 to 49 | 0 to 54 | |||
N
|
%
|
N
|
%
|
N
|
%
| |
0 to 3 nodes | 30 | 21.3 | 45 | 26.9 | 75 | 24.4 |
≥4 | 111 | 78.7 | 122 | 73.1 | 233 | 75.6 |
Menopausal status | ||||||
Premenopausal | 76 | 53.9 | 89 | 53.3 | 165 | 53.6 |
Postmenopausal | 65 | 46.1 | 78 | 46.7 | 143 | 46.4 |
Type of operation | ||||||
Modified radical mastectomy | 111 | 78.7 | 132 | 79.0 | 243 | 78.9 |
Breast conserving surgery | 30 | 21.3 | 35 | 21.0 | 65 | 21.1 |
Interval from operation | ||||||
<2 weeks | 17 | 12.1 | 24 | 14.4 | 41 | 13.3 |
2 to 4 weeks | 72 | 51.1 | 70 | 41.9 | 142 | 46.1 |
>4 weeks | 52 | 36.9 | 73 | 43.7 | 125 | 40.6 |
Tumor size | ||||||
≤2cm | 40 | 28.4 | 52 | 31.1 | 92 | 29.9 |
2 to 5cm | 79 | 56.0 | 83 | 49.7 | 162 | 52.6 |
>5cm | 22 | 15.6 | 32 | 19.2 | 54 | 17.5 |
Histological gradea | ||||||
I-II | 60 | 42.6 | 97 | 58.1 | 157 | 51.0 |
III-IV | 81 | 57.4 | 70 | 41.9 | 151 | 49.0 |
ER/PgR status | ||||||
Negative | 28 | 19.9 | 29 | 17.4 | 57 | 18.5 |
Positive | 95 | 67.4 | 108 | 64.7 | 203 | 65.9 |
Missing data | 18 | 12.8 | 30 | 18.0 | 48 | 15.6 |
HER2 statusb | ||||||
Negative | 79 | 56.0 | 100 | 59.9 | 179 | 58.1 |
Positive | 35 | 24.8 | 32 | 19.2 | 67 | 21.8 |
Missing data | 27 | 19.1 | 35 | 21.0 | 62 | 20.1 |
Adjuvant RT | ||||||
No | 21 | 14.9 | 33 | 19.8 | 54 | 17.5 |
Yes | 119 | 84.4 | 133 | 79.6 | 252 | 81.8 |
Missing data | 1 | 0.7 | 1 | 0.6 | 2 | 0.6 |
Adjuvant HT | ||||||
No | 8 | 5.7 | 18 | 10.8 | 26 | 8.4 |
Yes | 133 | 94.3 | 149 | 89.2 | 282 | 91.6 |
Tamoxifen | 120 | 85.1 | 127 | 76.0 | 247 | 80.2 |
LH-RH agonist | 65 | 46.1 | 62 | 37.1 | 127 | 41.2 |
Aromatase inhibitors | 5 | 3.5 | 6 | 3.6 | 11 | 3.6 |
Other | 1 | 0.7 | 2 | 1.2 | 3 | 1.0 |
Normalized mRNA expression
Associations of vascular endothelial growth factor family gene expression with patient and tumor characteristics
VEGF-A mRNA expression | VEGF-B mRNA expression | VEGFR2 mRNA expression | VEGFR3 mRNA expression | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
(Number = 307) | (Number = 304) | (Number = 308) | (Number = 308) | ||||||||||
Median | Range | P value | Median | Range | P value | Median | Range | P value | Median | Range | P value | ||
Age | <50 | 34.8 | (32.4-38.3) |
0.001
| 35.3 | (33.1-37.5) |
<0.001
| 32.1 | (29.6-34.4) | 0.190 | 31.9 | (27.4-34.2) |
0.030
|
≥50 | 35.3 | (28.2-38.3) | 35.7 | (27.5-38.0) | 32.2 | (29.2-34.5) | 32.1 | (29.1-34.4) | |||||
Treatment group | E-T-CMF | 35.0 | (33.4-38.3) | 0.629 | 35.5 | (27.5-37.4) | 0.13 | 32.1 | (29.5-34.5) | 0.99 | 32.0 | (27.4-34.0) | 0.47 |
E-CMF | 35.1 | (28.2-38.3) | 35.5 | (32.4-38.0) | 32.1 | (29.2-34.4) | 32.1 | (29.1-34.4) | |||||
Menopausal status | Premenopausal | 34.8 | (32.4-38.3) |
0.001
| 35.4 | (33.1-37.5) |
0.002
| 32.1 | (29.6-34.4) | 0.17 | 31.9 | (27.4-34.2) | 0.069 |
Postmenopausal | 35.3 | (28.2-38.3) | 35.7 | (27.5-38.0) | 32.3 | (29.2-34.5) | 32.1 | (29.1-34.4) | |||||
ER/PgR status | Negative | 35.5 | (32.4-38.3) |
<0.001
| 35.4 | (27.5-38.0) |
0.023
| 32.3 | (29.6-34.4) | 0.310 | 32.1 | (27.4-34.4) | 0.288 |
Positive | 34.9 | (33.1-38.0) | 35.6 | (33.4-37.6) | 32.1 | (29.2-34.5) | 32.0 | (29.1-34.4) | |||||
HER2 statusa | Negative | 35.0 | (28.2-38.2) |
0.020
| 35.5 | (32.4-37.5) | 0.856 | 32.0 | (29.2-34.5) | 0.088 | 32.0 | (29.1-34.4) | 0.294 |
Positive | 35.4 | (33.4-38.3) | 35.6 | (27.5-38.0) | 32.4 | (29.5-34.4) | 32.4 | (27.4-34.4) | |||||
Positive nodes | 0 to 3 | 35.0 | (32.4-38.1) | 0.97 | 35.4 | (33.7-37.2) | 0.51 | 32.0 | (30.3-34.1) | 0.42 | 31.9 | (30.0-34.2) | 0.35 |
≥4 | 35.0 | (28.2-38.3) | 35.6 | (27.5-38.0) | 32.1 | (29.2-34.5) | 32.0 | (27.4-34.4) | |||||
Tumor size | ≤2 | 34.9 | (28.2-38.0) | 0.13 | 35.7 | (33.4-36.9) | 0.140 | 32.2 | (29.6-34.3) | 0.44 | 32.2 | (27.4-34.4) | 0.43 |
2-5 | 35.0 | (32.4-38.3) | 35.4 | (27.5-38.0) | 32.0 | (29.2-34.5) | 32.0 | (29.1-34.4) | |||||
>5 | 35.3 | (33.1-37.9) | 35.5 | (33.1-37.6) | 32.2 | (29.5-34.0) | 32.1 | (29.4-34.1) | |||||
Histological grade | I-II | 34.9 | (28.2-38.0) |
0.027
| 35.6 | (33.1-37.6) |
0.024
| 32.2 | (29.9-34.4) | 0.25 | 32.1 | (29.5-34.4) | 0.12 |
III-IV | 35.1 | (32.4-38.3) | 35.4 | (27.5-38.0) | 32.0 | (29.2-34.5) | 32.0 | (27.4-34.4) | |||||
Adjuvant HT | No | 35.8 | (33.1-37.9) |
0.003
| 35.5 | (33.8-36.8) | 0.59 | 32.1 | (30.1-33.9) | 0.85 | 31.8 | (29.9-33.6) | 0.62 |
Yes | 34.9 | (28.2-38.3) | 35.5 | (27.5-38.0) | 32.1 | (29.2-34.5) | 32.0 | (27.4-34.4) | |||||
Adjuvant RT | No | 35.4 | (32.4-37.9) | 0.17 | 35.4 | (33.4-37.2) | 0.72 | 32.0 | (30.1-34.1) | 0.52 | 31.9 | (29.4-33.9) | 0.48 |
Yes | 35.0 | (28.2-38.3) | 35.5 | (27.5-38.0) | 32.1 | (29.2-34.5) | 32.1 | (27.4-34.4) |
VEGF-C mRNA expression (Number = 305) | VEGFR1 mRNA expression (Number = 306) | ||||||
---|---|---|---|---|---|---|---|
Low (n= 229) Number (%) | High (n= 76) Number (%) | Pvalue | Low (n= 230) Number (%) | High (n= 76) Number (%) | Pvalue | ||
Age | <50 | 123 (53.9) | 29 (38.2) |
0.024
| 118 (51.5) | 35 (46.1) | 0.43 |
≥50 | 105 (46.1) | 47 (61.8) | 111 (48.5) | 41 (53.9) | |||
Treatment group | E-T-CMF | 113 (49.3) | 28 (36.8) | 0.064 | 110 (47.8) | 30 (39.5) | 0.23 |
E-CMF | 116 (50.7) | 48 (63.2) | 120 (52.2) | 46 (60.5) | |||
Menopausal status | Premenopausal | 130 (56.8) | 34 (44.7) | 0.084 | 128 (55.7) | 37 (48.7) | 0.35 |
Postmenopausal | 99 (43.2) | 42 (55.3) | 102 (44.3) | 39 (51.3) | |||
ER/PgR status | Negative | 34 (17.3) | 23 (37.7) |
0.001
| 37 (18.8) | 20 (33.3) |
0.021
|
Positive | 162 (82.7) | 38 (62.3) | 160 (81.2) | 40 (66.7) | |||
HER2 Statusa | Negative | 146 (78.5) | 31 (53.5) |
<0.001
| 142 (76.3) | 35 (60.3) |
0.028
|
Positive | 40 (21.5) | 27 (46.6) | 44 (23.7) | 23 (39.7) | |||
Positive nodes | 0-3 | 61 (26.6) | 14 (18.4) | 0.17 | 61 (26.5) | 14 (18.4) | 0.17 |
≥4 | 168 (73.4) | 62 (81.6) | 169 (73.5) | 62 (81.6) | |||
Tumor size | ≤2 | 61 (26.6) | 30 (39.5) | 0.093 | 64 (27.8) | 27 (35.5) | 0.40 |
2-5 | 127 (55.5) | 33 (43.4) | 123 (53.5) | 38 (50.0) | |||
>5 | 41 (17.9) | 13 (17.1) | 43 (18.7) | 11 (14.5) | |||
Histological grade | I-II | 113 (49.3) | 41 (53.9) | 0.51 | 118 (51.3) | 37 (48.7) | 0.79 |
III-IV | 116 (50.7) | 35 (46.1) | 112 (48.7) | 39 (51.3) | |||
Adjuvant HT | No | 17 (7.4) | 9 (11.8) | 0.24 | 20 (8.7) | 6 (7.9) | 0.99 |
Yes | 212 (92.6) | 67 (88.2) | 210 (91.3) | 70 (92.1) | |||
Adjuvant RT | No | 43 (18.9) | 11 (14.5) | 0.49 | 47 (20.5) | 7 (9.3) |
0.036
|
Yes | 184 (81.1) | 65 (85.5) | 182 (79.5) | 68 (90.7) |
Association of vascular endothelial growth factor ligands with survival
VEGF-A and VEGF-B
VEGF-C
Association of vascular endothelial growth factor receptors with survival
Interactions between ligands and receptors
Multivariate Cox regression model for overall survival and disease-free survival adjusting for clinical parameters
Overall survival | HR | 95% CI | Wald's P |
---|---|---|---|
Histological grade | |||
III-IV versus I-II | 1.94 | 1.21-3.11 | 0.006 |
Number of positive nodes | |||
≥4 versus 0 to 3 | 2.58 | 1.32-5.02 | 0.005 |
Adjuvant HT | |||
No versus Yes | 2.86 | 1.56-5.26 | 0.001 |
VEGF-B | |||
Continuous mRNA values | 0.82 | 0.69-0.97 | 0.019 |
VEGF-C/Treatment group Interaction | 3.84 | 1.24-11.84 | 0.019 |
Treatment group | |||
E-CMF versus E-T-CMF for VEGF-C low | 0.96 | 0.54-1.70 | 0.885 |
E-CMF versus E-T-CMF for VEGF-C high | 3.68 | 1.38-9.80 | 0.009 |
VEGF-C | |||
High versus Low for E-T-CMF | 0.74 | 0.28-1.96 | 0.547 |
High versus Low for E-CMF | 2.85 | 1.55-5.22 | <0.001 |
Disease-free survival
|
HR
|
95% CI
|
Wald's
P
|
Histological grade | |||
III-IV versus I-II | 1.83 | 1.24-2.71 | 0.002 |
Number of positive nodes | |||
≥4 versus 0 to 3 | 2.80 | 1.58-4.95 | <0.001 |
Adjuvant HT | |||
No versus Yes | 2.16 | 1.22-3.84 | 0.008 |
VEGF-B | |||
Continuous mRNA values | 0.86 | 0.72-1.02 | 0.084 |
VEGFR1 | |||
High versus Low | 1.58 | 0.98-2.55 | 0.060 |
VEGF-C/Treatment group Interaction | 2.56 | 1.04-6.31 | 0.041 |
Treatment group | |||
E-CMF versus E-T-CMF for VEGF-C low | 0.95 | 0.60-1.52 | 0.846 |
E-CMF versus E-T-CMF for VEGF-C high | 2.44 | 1.12-5.31 | 0.057 |
VEGF-C | |||
High versus Low for E-T-CMF | 0.68 | 0.31-1.48 | 0.327 |
High versus Low for E-CMF | 1.73 | 0.98-3.08 | 0.166 |