Background
Methods
Study populations
SNPs selection
DNA extraction and SNPs genotyping
Statistical analyses
Results
Patient characteristics and clinical features
Variables | Patients N = 1177 | iDFS | DDFS | BCSS | OS | ||||
---|---|---|---|---|---|---|---|---|---|
Events | LogRank P | Events | LogRank P | Events | LogRank P | Events | LogRank P | ||
Age at diagnosis | 0.021 | 0.087 | 0.420 | 0.402 | |||||
≤ 35 | 184 | 85 | 76 | 59 | 61 | ||||
> 35 | 993 | 361 | 334 | 274 | 282 | ||||
Tumor size (cm) | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |||||
≤ 2 | 403 | 88 | 80 | 67 | 70 | ||||
> 2 | 774 | 358 | 330 | 266 | 273 | ||||
Nodal status | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |||||
Negative | 510 | 116 | 101 | 69 | 75 | ||||
Positive | 667 | 330 | 309 | 264 | 268 | ||||
Clinical stage | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |||||
I | 257 | 40 | 35 | 29 | 31 | ||||
II + III | 920 | 406 | 375 | 304 | 312 | ||||
Gradea | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |||||
I + II | 904 | 310 | 286 | 228 | 236 | ||||
III | 271 | 134 | 122 | 103 | 105 | ||||
ER | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |||||
Negative | 378 | 177 | 165 | 149 | 150 | ||||
Positive | 799 | 269 | 245 | 184 | 193 |
Variables | Patients N = 1177 | iDFS | DDFS | BCSS | OS | iDFS | DDFS | BCSS | OS |
---|---|---|---|---|---|---|---|---|---|
Events | LogRank P | Events | LogRank P | Events | LogRank P | Events | LogRank P | ||
PR | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |||||
Negative | 367 | 171 | 159 | 144 | 145 | ||||
Positive | 810 | 275 | 251 | 189 | 198 | ||||
HER2 | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |||||
Negative | 860 | 292 | 268 | 214 | 222 | ||||
Positive | 317 | 154 | 142 | 119 | 121 | ||||
Subtype | < 0.001 | < 0.001 | < 0.001 | < 0.001 | |||||
Luminal A | 236 | 35 | 33 | 26 | 26 | ||||
Luminal B | 574 | 240 | 218 | 163 | 172 | ||||
HER2+ | 160 | 80 | 76 | 67 | 67 | ||||
Triple negative | 207 | 91 | 83 | 77 | 78 |
Effects of each polymorphism on survival of EBC
SNPs | Cases WH/H/VH | iDFS (LogRank P) | DDFS (LogRank P) | BCSS (logRank P) | OS (Log Rank P) | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Events WH/H/VH | DOM | REC | COD | Events WH/H/VH | DOM | REC | COD | Events WH/H/VH | DOM | REC | COD | Events WH/H/VH | DOM | REC | COD | ||
rs10069690 | 789/353/34 | 298/139/8 | 0.938 | 0.065 | 0.152 | 273/129/8 | 0.689 | 0.128 | 0.221 | 218/107/7 | 0.510 | 0.230 | 0.291 | 225/110/7 | 0.533 | 0.191 | 0.257 |
rs13281615 | 293/575/308 | 126/196/124 | 0.043 | 0.397 | 0.035 | 112/186/112 | 0.178 | 0.619 | 0.241 | 86/154/93 | 0.592 | 0.402 | 0.482 | 89/157/97 | 0.531 | 0.320 | 0.362 |
rs13387042 | 932/234/11 | 351/91/4 | 0.803 | 1.000 | 0.968 | 322/84/4 | 0.767 | 0.830 | 0.944 | 264/66/3 | 0.891 | 0.891 | 0.977 | 274/66/3 | 0.664 | 0.934 | 0.898 |
rs1562430 | 801/344/32 | 297/136/13 | 0.419 | 0.787 | 0.720 | 272/125/13 | 0.363 | 0.516 | 0.600 | 228/97/8 | 0.840 | 0.738 | 0.938 | 234/100/9 | 0.940 | 0.955 | 0.996 |
rs2046210 | 361/602/214 | 142/220/84 | 0.327 | 0.873 | 0.611 | 134/198/78 | 0.180 | 0.964 | 0.361 | 107/162/64 | 0.359 | 0.970 | 0.633 | 112/166/65 | 0.231 | 0.829 | 0.481 |
rs2180341 | 715/394/68 | 270/147/29 | 0.858 | 0.381 | 0.679 | 245/136/29 | 0.556 | 0.136 | 0.326 | 198/115/20 | 0.554 | 0.783 | 0.836 | 204/118/21 | 0.547 | 0.676 | 0.809 |
rs2981582 | 493/545/139 | 187/204/55 | 0.891 | 0.459 | 0.708 | 173/189/48 | 0.843 | 0.843 | 0.945 | 143/149/41 | 0.491 | 0.554 | 0.556 | 146/154/43 | 0.581 | 0.459 | 0.547 |
rs3112612 | 776/354/46 | 290/140/15 | 0.541 | 0.563 | 0.610 | 263/132/14 | 0.263 | 0.660 | 0.391 | 210/110/12 | 0.213 | 0.818 | 0.393 | 218/111/13 | 0.290 | 1.000 | 0.545 |
rs3803662 | 532/512/133 | 214/185/47 | 0.102 | 0.472 | 0.258 | 193/172/45 | 0.309 | 0.795 | 0.594 | 157/138/38 | 0.284 | 0.957 | 0.537 | 165/139/39 | 0.141 | 0.946 | 0.309 |
rs4415084 | 392/558/226 | 144/204/98 | 0.332 | 0.043 | 0.124 | 130/189/91 | 0.256 | 0.038 | 0.106 | 105/152/76 | 0.245 | 0.039 | 0.107 | 110/156/77 | 0.345 | 0.059 | 0.160 |
rs4784227 | 550/513/113 | 191/211/44 | 0.035 | 0.714 | 0.104 | 177/195/38 | 0.077 | 0.905 | 0.164 | 146/155/32 | 0.173 | 0.793 | 0.389 | 148/162/33 | 0.091 | 0.773 | 0.235 |
rs889312 | 346/631/200 | 130/252/64 | 0.770 | 0.059 | 0.111 | 124/235/51 | 0.823 | 0.003 | 0.010 | 98/189/46 | 0.840 | 0.070 | 0.142 | 101/196/46 | 0.841 | 0.038 | 0.080 |
rs9485372 | 388/588/200 | 136/227/82 | 0.122 | 0.177 | 0.200 | 127/208/74 | 0.230 | 0.360 | 0.415 | 104/169/59 | 0.334 | 0.529 | 0.592 | 107/173/62 | 0.320 | 0.382 | 0.513 |
rs10474352 | 374/572/230 | 158/214/74 | 0.052 | 0.029 | 0.041 | 143/199/68 | 0.142 | 0.049 | 0.101 | 115/161/57 | 0.285 | 0.156 | 0.301 | 119/165/59 | 0.241 | 0.160 | 0.284 |
rs10816625 | 350/595/231 | 145/213/88 | 0.047 | 0.825 | 0.127 | 136/196/78 | 0.022 | 0.468 | 0.073 | 114/156/63 | 0.017 | 0.559 | 0.056 | 118/160/65 | 0.012 | 0.567 | 0.041 |
rs12922061 | 539/529/108 | 199/206/41 | 0.590 | 0.905 | 0.865 | 185/188/37 | 0.799 | 0.926 | 0.953 | 156/147/30 | 0.613 | 0.943 | 0.877 | 158/154/31 | 0.847 | 0.970 | 0.981 |
rs2290203 | 270/587/319 | 96/229/121 | 0.464 | 0.891 | 0.760 | 89/211/110 | 0.519 | 0.962 | 0.800 | 67/174/92 | 0.218 | 0.689 | 0.468 | 69/179/95 | 0.206 | 0.647 | 0.449 |
rs2296067 | 418/567/191 | 160/215/71 | 0.869 | 0.814 | 0.968 | 144/200/66 | 0.774 | 0.923 | 0.940 | 116/166/51 | 0.649 | 0.751 | 0.798 | 119/172/52 | 0.590 | 0.668 | 0.704 |
rs2981578 | 416/548/212 | 150/219/77 | 0.465 | 0.619 | 0.556 | 132/208/70 | 0.148 | 0.512 | 0.650 | 105/172/56 | 0.158 | 0.488 | 0.166 | 110/176/57 | 0.232 | 0.421 | 0.210 |
rs4951011 | 522/528/126 | 204/191/51 | 0.350 | 0.421 | 0.340 | 186/178/46 | 0.516 | 0.475 | 0.516 | 150/142/41 | 0.597 | 0.163 | 0.233 | 157/145/41 | 0.388 | 0.246 | 0.230 |
rs9693444 | 572/486/118 | 215/179/52 | 0.762 | 0.154 | 0.357 | 196/164/50 | 0.619 | 0.068 | 0.188 | 156/141/36 | 0.379 | 0.483 | 0.616 | 160/144/39 | 0.329 | 0.259 | 0.428 |
SNPs | Cases | iDFS | DDFS | BCSS | OS | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Events | Adjusted HR (95% CI)a | P value | Events | Adjusted HR (95% CI)a | P value | Events | Adjusted HR (95% CI)a | P value | Events | Adjusted HR (95% CI)a | P value | ||
All cases | |||||||||||||
rs889312 | |||||||||||||
CC | 346 | 130 | 1 (reference) | 124 | 1 (reference) | 98 | 1 (reference) | 101 | 1 (reference) | ||||
CA | 631 | 252 | 1.089 (0.880–1.347) | 0.433 | 235 | 1.065 (0.856–1.326) | 0.569 | 189 | 1.087 (0.850–1.389) | 0.507 | 196 | 1.094 (0.859–1.393) | 0.465 |
AA | 200 | 64 | 0.804 (0.595–1.087) | 0.157 | 51 | 0.658 (0.474–0.913) | 0.012 | 46 | 0.814 (0.573–1.158) | 0.253 | 46 | 0.782 (0.510–1.111) | 0.170 |
DOM | 1.017 (0.828–1.248) | 0.876 | 0.960 (0.777–1.187) | 0.706 | 1.020 (0.804–1.293) | 0.872 | 1.017 (0.805–1.285) | 0.887 | |||||
REC | 0.761 (0.583–0.994) | 0.045 | 0.631 (0.470–0.848) | 0.002 | 0.772 (0.564–1.055) | 0.105 | 0.738 (0.540–1.009) | 0.057 | |||||
rs2046210 | |||||||||||||
GG | 361 | 142 | 1 (reference) | 134 | 1 (reference) | 107 | 1 (reference) | 112 | 1 (reference) | ||||
GA | 602 | 220 | 0.796 (0.644–0.985) | 0.035 | 198 | 0.761 (0.610–0.949) | 0.015 | 162 | 0.775 (0.606–0.991) | 0.042 | 166 | 0.762 (0.598–0.970) | 0.027 |
AA | 214 | 84 | 0.948 (0.722–1.244) | 0.700 | 78 | 0.963 (0.727–1.275) | 0.792 | 64 | 0.951 (0.696–1.299) | 0.752 | 65 | 0.919 (0.675–1.250) | 0.589 |
DOM | 0.833 (0.682–1.018) | 0.074 | 0.809 (0.658–0.996) | 0.045 | 0.818 (0.649–1.031) | 0.090 | 0.800 (0.638–1.005) | 0.055 | |||||
REC | 1.094 (0.861–1.391) | 0.462 | 1.142 (0.890–1.464) | 0.296 | 1.116 (0.847–1.469) | 0.436 | 1.089 (0.829–1.430) | 0.541 | |||||
OVE | 0.812 (0.673–0.980) | 0.030 | 0.771 (0.635–0.938) | 0.009 | 0.790 (0.636–0.981) | 0.033 | 0.786 (0.635–0.934) | 0.028 | |||||
Luminal A | |||||||||||||
rs9485372 | |||||||||||||
GG | 72 | 10 | 1 (reference) | 10 | 1 (reference) | 7 | 1 (reference) | 7 | 1 (reference) | ||||
GA | 124 | 16 | 0.833 (0.372–1.863) | 0.656 | 14 | 0.717 (0.313–1.644) | 0.432 | 11 | 0.890 (0.332–2.385) | 0.817 | 11 | 0.890 (0.332–2.385) | 0.817 |
AA | 40 | 9 | 2.201 (0.883–5.486) | 0.090 | 9 | 2.192 (0.880–5.459) | 0.092 | 8 | 3.280 (1.152–9.378) | 0.026 | 8 | 3.280 (1.152–9.378) | 0.026 |
DOM | 1.087 (0.518–2.283) | 0.825 | 0.995 (0.469–2.109) | 0.989 | 1.328 (0.546–3.229) | 0.532 | 1.328 (0.546–3.229) | 0.532 | |||||
REC | 2.465 (1.133–5.360) | 0.023 | 2.671 (1.214–5.875) | 0.015 | 3.522 (1.464–8.473) | 0.005 | 3.522 (1.464–8.473) | 0.005 | |||||
Triple negative | |||||||||||||
rs4415084 | |||||||||||||
TT | 59 | 24 | 1 (reference) | 20 | 1 (reference) | 20 | 1 (reference) | 20 | 1 (reference) | ||||
CT | 83 | 44 | 1.622 (0.979–2.688) | 0.061 | 42 | 1.799 (1.048–3.087) | 0.033 | 39 | 1.686 (0.975–2.917) | 0.062 | 40 | 1.736 (1.006–2.996) | 0.047 |
CC | 65 | 23 | 1.785 (0.996–3.201) | 0.052 | 21 | 1.813 (0.971–3.385) | 0.062 | 18 | 1.549 (0.809–2.969) | 0.187 | 18 | 1.551 (0.810–2.972) | 0.186 |
DOM | 1.674 (1.043–2.687) | 0.033 | 1.804 (1.084–3.002) | 0.023 | 1.640 (0.979–2.750) | 0.060 | 1.674 (1.000–2.803) | 0.049 | |||||
REC | 1.345 (0.827–2.187) | 0.232 | 1.274 (0.765–2.120) | 0.352 | 1.139 (0.661–1.962) | 0.639 | 1.119 (0.650–1.926) | 0.685 | |||||
Luminal B | |||||||||||||
rs4951011 | |||||||||||||
AA | 265 | 120 | 1 (reference) | 109 | 1 (reference) | 82 | 1 (reference) | 88 | 1 (reference) | ||||
GA | 253 | 92 | 0.682 (0.526–0.896) | 0.006 | 84 | 0.698 (0.524–0.929) | 0.014 | 59 | 0.652 (0.466–0.914) | 0.013 | 62 | 0.630 (0.454–0.874) | 0.006 |
GG | 55 | 28 | 0.883 (0.579–1.346) | 0.562 | 25 | 0.888 (0.568–1.386) | 0.645 | 22 | 1.025 (0.631–1.664) | 0.921 | 22 | 0.965 (0.597–1.560) | 0.885 |
DOM | 0.719 (0.557–0.928) | 0.011 | 0.734 (0.561–0.960) | 0.024 | 0.721 (0.528–0.984) | 0.039 | 0.690 (0.510–0.934) | 0.016 | |||||
REC | 1.068 (0.714–1.598) | 0.749 | 1.075 (0.703–1.645) | 0.738 | 1.259 (0.794–1.998) | 0.328 | 1.205 (0.762–1.908) | 0.425 | |||||
rs889312 | |||||||||||||
CC | 162 | 74 | 1 (reference) | 70 | 1 (reference) | 51 | 1 (reference) | 54 | 1 (reference) | ||||
CA | 308 | 135 | 1.304 (0.778–1.374) | 0.819 | 126 | 1.048 (0.782–1.406) | 0.753 | 94 | 1.113 (0.790–1.568) | 0.542 | 100 | 1.108 (0.794–1.546) | 0.545 |
AA | 104 | 31 | 0.570 (0.373–0.870) | 0.009 | 22 | 0.432 (0.266–0.701) | 0.001 | 18 | 0.534 (0.310–0.918) | 0.023 | 18 | 0.498 (0.290–0.853) | 0.011 |
DOM | 0.901 (0.684–1.187) | 0.459 | 0.871 (0.654–1.160) | 0.344 | 0.954 (0.682–1.333) | 0.781 | 0.940 (0.679–1.301) | 0.708 | |||||
REC | 0.558 (0.381–0.817) | 0.003 | 0.419 (0.269–0.653) | < 0.000 | 0.498 (0.304–0.815) | 0.006 | 0.465 (0.285–0.761) | 0.002 | |||||
Luminal B | |||||||||||||
rs9485372 | |||||||||||||
GG | 204 | 72 | 1 (reference) | 63 | 1 (reference) | 47 | 1 (reference) | 49 | 1 (reference) | ||||
GA | 275 | 125 | 1.439 (1.076–1.924) | 0.014 | 115 | 1.524 (1.121–2.073) | 0.007 | 89 | 1.517 (1.065–2.162) | 0.021 | 93 | 1.520 (1.075–2.149) | 0.018 |
AA | 95 | 43 | 1.622 (1.111–2.370) | 0.122 | 38 | 1.665 (1.116–2.485) | 0.013 | 27 | 1.463 (0.910–2.350) | 0.116 | 30 | 1.596 (1.012–2.516) | 0.044 |
DOM | 1.482 (1.124–1.954) | 0.005 | 1.557 (1.161–2.088) | 0.003 | 1.504 (1.071–2.112) | 0.018 | 1.538 (1.104–2.142) | 0.011 | |||||
REC | 1.307 (0.939–1.820) | 0.112 | 1.294 (0.914–1.831) | 0.146 | 1.137 (0.752–1.720) | 0.544 | 1.239 (0.835–1.839) | 0.288 |
Prognostic implication of risk variants in molecular subtypes
Combined analysis of three risk SNPs on survival of luminal B EBC
Number of risk genotypesa | Cases | iDFS | DDFS | BCSS | OS | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Events | Adjusted HR (95% CI)b | P value | Events | Adjusted HR (95% CI)b | P value | Events | Adjusted HR (95% CI)b | P value | Events | Adjusted HR (95% CI)b | P value | ||
0–1 | 165 | 49 | 1 (reference) | 42 | 1 (reference) | 32 | 1 (reference) | 33 | 1 (reference) | ||||
2 | 272 | 123 | 1.912 (1.369–2.670) | 1.44 × E−4 | 109 | 1.894 (1.324–2.711) | 4.74 × E−4 | 81 | 1.787 (1.184–2.697) | 5.70 × E−3 | 84 | 1.786 (1.192–6.678) | 4.97 × E−3 |
3 | 137 | 68 | 2.431 (1.679–3.519) | 2.52 × E−6 | 67 | 2.744 (1.862–4.043) | 3.53 × E−7 | 50 | 2.525 (1.617–3.943) | 4.61 × E−5 | 55 | 2.755 (1.786–4.251) | 4.59 × E−6 |
Trend P | 1.534 (1.288–1.827) | 1.63 × E−6 | 1.632 (1.356–1.964) | 2.18 × E−7 | 1.570 (1.267–1.944) | 3.66 × E−5 | 1.645 (1.334–2.029) | 3.25 × E−6 |
Stratification and interaction analysis
SNPs | Variables | iDFS | DDFS | BCSS | OS | ||||
---|---|---|---|---|---|---|---|---|---|
Adjusted HR (95% CI) | P valuea | Adjusted HR (95% CI) | P valuea | Adjusted HR (95% CI) | P valuea | Adjusted HR (95% CI) | P valuea | ||
rs4415084 | Age at diagnosis | ||||||||
≤ 35 | 1.792 (1.161–2.915) | 0.068 | 2.172 (1.310–3.602) | 0.014 | 2.250 (1.278–3.959) | 0.018 | 1.871 (0.988–3.544) | 0.009 | |
> 35 | 1.073 (0.830–1.386) | 1.056 (0.809–1.379) | 1.067 (0.796–1.431) | 0.743 (0.584–0.946) | |||||
rs2046210 | Tumor size (cm) | ||||||||
≤ 2 | 1.277 (0.791–2.061) | 0.052 | 1.277 (0.773–2.109) | 0.048 | 1.558 (0.874–2.780) | 0.015 | 1.522 (0.867–2.670) | 0.012 | |
> 2 | 0.757 (0.606–0.944) | 0.732 (0.582–0.919) | 0.713 (0.553–0.920) | 0.694 (0.540–0.992) | |||||
rs2981582 | Grade | ||||||||
I + II | 0.922 (0.642–1.323) | 0.048 | 0.791 (0.532–1.177) | 0.017 | 0.822 (0.529–1.278) | 0.023 | 0.872 (0.571–1.331) | 0.040 | |
III | 1.666 (1.051–2.639) | 1.682 (1.049–2.698) | 1.783 (1.080–2.944) | 1.732 (1.050–2.855) | |||||
rs3803662 | Grade | ||||||||
I + II | 1.017 (0.812–1.273) | 0.010 | 1.096 (0.866–1.387) | 0.005 | 1.151 (0.884–1.500) | 0.000 | 1.075 (0.830–1.392) | 0.001 | |
III | 0.588 (0.414–0.834) | 0.586 (0.407–0.845) | 0.479 (0.319–0.717) | 0.484 (0.324–0.722) |
SNPs | Variable | iDFS | DDFS | BCSS | OS | ||||
---|---|---|---|---|---|---|---|---|---|
Adjusted HRa | P value | Adjusted HRa | P value | Adjusted HRa | P value | Adjusted HRa | P value | ||
rs4415084 | Age at diagnosis | ||||||||
CC | ≤ 35 | 1 (reference) | 1 (reference) | 1 (reference) | 1 (reference) | ||||
CC | > 35 | 1.113 (0.739–1.676) | 0.609 | 1.270 (0.814–1.983) | 0.292 | 1.366 (0.829–2.249) | 0.221 | 1.346 (0.827–2.189) | 0.232 |
CT | ≤ 35 | 1.317 (0.797–2.176) | 0.282 | 1.421 (0.829–2.438) | 0.202 | 1.358 (0.733–2.516) | 0.331 | 1.271 (0.692–2.336) | 0.440 |
CT | > 35 | 1.090 (0.734–1.619) | 0.669 | 1.246 (0.810–1.917) | 0.316 | 1.373 (0.847–2.229) | 0.198 | 1.340 (0.835–2.148) | 0.225 |
TT | ≤ 35 | 2.013 (1.161–3.488) | 0.013 | 2.427 (1.357–4.339) | 0.003 | 2.505 (1.310–4.788) | 0.005 | 2.497 (1.328–4.693) | 0.004 |
TT | > 35 | 1.180 (0.767–1.815) | 0.452 | 1.332 (0.836–2.124) | 0.228 | 1.461 (0.868–2.460) | 0.153 | 1.378 (0.826–2.298) | 0.219 |
P for multiplicative interaction | 0.045 | 0.013 | 0.025 | 0.018 | |||||
rs3803662 | Grade | ||||||||
GG | I + II | 1 (reference) | 1 (reference) | 1 (reference) | 1 (reference) | ||||
GG | III | 1.858 (1.400–2.466) | 1.8E−5 | 1.877 (1.394–2.527) | 3.3E−5 | 2.134 (1.543–2.952) | 4.6E−6 | 2.018 (1.469–2.773) | 1.5E−5 |
GA | I + II | 1.031 (0.814–1.306) | 0.801 | 1.106 (0.864–1.416) | 0.425 | 1.139 (0.862–1.505) | 0.361 | 1.054 (0.801–1.385) | 0.709 |
GA | III | 1.043 (0.746–1.459) | 0.804 | 1.014 (0.711–1.446) | 0.939 | 0.979 (0.655–1.462) | 0.917 | 0.946 (0.639–1.403) | 0.784 |
AA | I + II | 0.994 (0.684–1.443) | 0.973 | 1.081 (0.735–1.592) | 0.691 | 1.246 (0.820–1.893) | 0.303 | 1.195 (0.793–1.800) | 0.394 |
AA | III | 1.085 (0.582–2.023) | 0.798 | 1.245 (0.665–2.331) | 0.493 | 1.043 (0.501–2.169) | 0.911 | 0.983 (0.474–2.041) | 0.964 |
P for multiplicative interaction | 0.011 | 0.001 | 4.7E−4 | 9.9E−4 |