Background
Materials and methods
Patient selection
Tissue microarray
Immunohistochemistry
Interpretation of immunohistochemical staining
FISH analysis
Tumor phenotype classification
Sarcosine metabolism phenotype
Statistical analysis
Results
Patient clinicopathologic characteristics
Parameter | Total (n =721) (%) | Luminal A (n =303) (%) | Luminal B (n =169) (%) | HER-2 (n =71) (%) | TNBC (n =178) (%) |
P-value |
---|---|---|---|---|---|---|
Age (years) |
0.012
| |||||
≤50 | 428 (59.4) | 173 (57.1) | 113 (66.9) | 32 (45.1) | 110 (61.8) | |
>50 | 293 (40.6) | 130 (42.9) | 56 (33.1) | 39 (54.9) | 68 (38.2) | |
Histologic grade |
<0.001
| |||||
I/II | 485 (67.3) | 275 (90.8) | 112 (66.3) | 37 (52.1) | 61 (34.3) | |
III | 236 (32.7) | 28 (9.2) | 57 (33.7) | 34 (47.9) | 117 (65.7) | |
Tumor stage |
0.002
| |||||
T1 | 355 (49.2) | 169 (55.8) | 86 (50.9) | 32 (45.1) | 68 (38.2) | |
T2/T3 | 366 (50.8) | 134 (44.2) | 83 (49.1) | 39 (54.9) | 110 (61.8) | |
Nodal metastasis | 0.162 | |||||
Absent | 427 (59.2) | 173 (57.1) | 93 (55.0) | 44 (62.0) | 117 (65.7) | |
Present | 294 (40.8) | 130 (42.9) | 76 (45.0) | 27 (38.0) | 61 (34.3) | |
Estrogen receptor status |
<0.001
| |||||
Negative | 259 (35.9) | 5 (1.7) | 5 (3.0) | 71 (100.0) | 178 (100.0) | |
Positive | 462 (64.1) | 298 (98.3) | 164 (97.0) | 0 (0.0) | 0 (0.0) | |
Progesterone receptor status |
<0.001
| |||||
Negative | 347 (48.1) | 50 (16.5) | 48 (28.4) | 71 (100.0) | 178 (100.0) | |
Positive | 374 (51.9) | 253 (83.5) | 121 (71.6) | 0 (0.0) | 0 (0.0) | |
HER-2 status |
<0.001
| |||||
Negative | 565 (78.4) | 303 (100.0) | 84 (49.5) | 0 (0.0) | 178 (100.0) | |
Positive | 156 (21.6) | 0 (0.0) | 85 (50.3) | 71 (100.0) | 0 (0.0) | |
Ki-67 LI (%) |
<0.001
| |||||
≤14 | 409 (56.7) | 303 (100.0) | 49 (29.0) | 29 (40.8) | 28 (15.7) | |
>14 | 312 (43.3) | 0 (0.0) | 120 (71.0) | 42 (59.2) | 150 (84.3) | |
Tumor recurrence | 63 (8.7) | 15 (5.0) | 13 (7.7) | 11 (15.5) | 24 (13.5) |
0.002
|
No. of patient deaths | 61 (8.5) | 13 (4.3) | 13 (7.7) | 11 (15.5) | 24 (13.5) |
0.001
|
Duration of clinical follow-up (months, mean ± SD) | 69.9 ± 31.2 | 71.9 ± 29.0 | 70.0 ± 30.1 | 66.1 ± 34.9 | 68.1 ± 34.1 | 0.419 |
Expression of sarcosine metabolism-related proteins according to tumor phenotype
Parameter | Total (n =721) (%) | Luminal A (n =303) (%) | Luminal B (n =169) (%) | HER-2 (n =71) (%) | TNBC (n =178) (%) |
P-value |
---|---|---|---|---|---|---|
GNMT |
0.005
| |||||
Negative | 664 (92.1) | 273 (90.1) | 153 (90.5) | 63 (88.7) | 175 (98.3) | |
Positive | 57 (7.9) | 30 (9.9) | 16 (9.5) | 8 (11.3) | 3 (1.7) | |
SARDH |
0.012
| |||||
Negative | 597 (82.8) | 261 (86.1) | 130 (76.9) | 53 (74.6) | 153 (86.0) | |
Positive | 124 (17.2) | 42 (13.9) | 39 (23.1) | 18 (25.4) | 25 (14.0) | |
PIPOX (T) |
0.008
| |||||
Negative | 570 (79.1) | 225 (74.3) | 143 (84.6) | 52 (73.2) | 150 (84.3) | |
Positive | 151 (20.9) | 78 (25.7) | 26 (15.4) | 19 (26.8) | 28 (15.7) | |
PIPOX (S) |
<0.001
| |||||
Negative | 671 (93.1) | 287 (94.7) | 154 (91.1) | 58 (81.7) | 172 (96.6) | |
Positive | 50 (6.9) | 16 (5.3) | 15 (8.9) | 13 (18.3) | 6 (3.4) |
Parameter | Total (N =721) (%) | Luminal A (n =303) (%) | Luminal B (n =169) (%) | HER-2 (n =71) (%) | TNBC (n =178) (%) |
P-value |
---|---|---|---|---|---|---|
Sarcosine metabolic type
|
0.003
| |||||
High sarcosine type | 36 (5.0) | 16 (5.3) | 12 (7.1) | 7 (9.9) | 1 (0.6) | |
Intermediate sarcosine type | 21 (2.9) | 14 (4.6) | 4 (2.4) | 1 (1.4) | 2 (1.1) | |
Low sarcosine type | 219 (30.4) | 93 (30.7) | 54 (32.0) | 26 (36.6) | 46 (25.8) | |
Null type | 445 (61.7) | 180 (59.4) | 99 (58.6) | 37 (52.1) | 129 (72.5) |
Parameter | Total N =721 (%) | High sarcosine type n = 36 (%) | Intermediate sarcosine type n = 21 (%) | Low sarcosine type n = 219 (%) | Null type n = 445 (%) |
P-value |
---|---|---|---|---|---|---|
Age (years) | 0.200 | |||||
≤50 | 428 (59.4) | 26 (72.2) | 13 (61.9) | 120 (54.8) | 269 (60.4) | |
>50 | 293 (40.6) | 10 (27.8) | 8 (38.1) | 99 (45.2) | 176 (39.6) | |
Histologic grade | 0.351 | |||||
I/II | 485 (67.3) | 29 (80.6) | 13 (61.9) | 147 (67.1) | 296 (66.5) | |
III | 236 (32.7) | 7 (19.4) | 8 (38.1) | 72 (32.9) | 149 (33.5) | |
Tumor stage | 0.082 | |||||
T1 | 355 (49.2) | 12 (33.3) | 10 (47.6) | 120 (54.8) | 213 (47.9) | |
T2/T3 | 366 (50.8) | 24 (66.7) | 11 (52.4) | 99 (45.2) | 232 (52.1) | |
Nodal metastasis | 0.799 | |||||
Absent | 427 (59.2) | 22 (61.1) | 14 (66.7) | 125 (57.1) | 266 (59.8) | |
Present | 294 (40.8) | 14 (38.9) | 7 (33.3) | 94 (42.9) | 179 (40.2) | |
Estrogen receptor status |
0.049
| |||||
Negative | 259 (35.9) | 9 (25.0) | 3 (14.3) | 75 (34.2) | 172 (38.7) | |
Positive | 462 (64.1) | 27 (75.0) | 18 (85.7) | 144 (65.8) | 273 (61.3) | |
Progesterone receptor status |
0.011
| |||||
Negative | 347 (48.1) | 13 (36.1) | 4 (19.0) | 102 (46.6) | 228 (51.2) | |
Positive | 374 (51.9) | 23 (63.9) | 17 (81.0) | 117 (53.4) | 217 (48.8) | |
HER-2 status | 0.072 | |||||
Negative | 565 (78.4) | 24 (66.7) | 17 (81.0) | 163 (74.4) | 361 (81.1) | |
Positive | 156 (21.6) | 12 (33.3) | 4 (19.0) | 56 (25.6) | 84 (18.9) | |
Ki-67 LI (%) |
0.007
| |||||
≤14 | 409 (56.7) | 21 (58.3) | 17 (81.0) | 138 (63.0) | 233 (52.4) | |
>14 | 312 (43.3) | 15 (41.7) | 4 (19.0) | 81 (37.0) | 212 (47.6) | |
Tumor recurrence | 63 (8.7) | 8 (22.2) | 1 (4.8) | 15 (6.8) | 39 (8.8) |
0.022
|
No. of patient deaths | 61 (8.5) | 5 (13.9) | 1 (4.8) | 20 (9.1) | 35 (7.9) | 0.558 |
Correlations between expression of sarcosine metabolism-related proteins and clinicopathologic factors
Parameter | GNMT | SARDH | PIPOX in tumor | PIPOX in stroma | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Negative n =664 (%) | Positive n = 57 (%) |
P-value* | Negative n =597 (%) | Positive n = 124 (%) |
P-value* | Negative n = 570 (%) | Positive n = 151 (%) |
P-value* | Negative n = 671 (%) | Positive n =50 (%) |
P-value* | |
Age (years) | 0.588 | 0.336 | 0.620 | 1.088 | ||||||||
≤50 | 389 (58.6) | 39 (68.4) | 363 (60.8) | 65 (52.4) | 346 (60.7) | 82 (54.3) | 402 (59.9) | 26 (52.0) | ||||
>50 | 275 (41.4) | 18 (31.6) | 234 (39.2) | 59 (47.6) | 224 (39.3) | 69 (45.7) | 269 (40.1) | 24 (48.0) | ||||
Histologic grade | 1.128 | 3.068 | 3.036 | 1.024 | ||||||||
I/II | 443 (66.7) | 42 (73.7) | 403 (67.5) | 82 (66.1) | 385 (67.5) | 100 (66.2) | 455 (67.8) | 30 (60.0) | ||||
III | 221 (33.3) | 15 (26.3) | 194 (32.5) | 42 (33.9) | 185 (32.5) | 51 (33.8) | 216 (32.2) | 20 (40.0) | ||||
Tumor stage | 0.376 | 1.316 | 0.308 | 3.644 | ||||||||
T1 | 333 (50.2) | 22 (38.6) | 289 (48.4) | 66 (53.2) | 271 (47.5) | 84 (55.6) | 330 (49.2) | 25 (50.0) | ||||
T2/T3 | 331 (49.8) | 35 (61.4) | 308 (51.6) | 58 (46.8) | 299 (52.5) | 67 (44.4) | 341 (50.8) | 25 (50.0) | ||||
Nodal metastasis | 2.116 | 3.640 | 2.604 | 2.524 | ||||||||
Absent | 391 (58.9) | 36 (63.2) | 353 (59.1) | 74 (59.7) | 340 (59.6) | 87 (57.6) | 399 (59.5) | 28 (56.0) | ||||
Present | 273 (41.1) | 21 (36.8) | 244 (40.9) | 50 (40.3) | 230 (40.4) | 64 (42.4) | 272 (40.5) | 22 (44.0) | ||||
Estrogen receptor status | 0.060 | 3.644 | 1.672 | 2.132 | ||||||||
Negative | 247 (37.2) | 12 (21.1) | 215 (36.0) | 44 (35.5) | 209 (36.7) | 50 (33.1) | 239 (35.6) | 20 (40.0) | ||||
Positive | 417 (62.8) | 45 (78.9) | 382 (64.0) | 80 (64.5) | 361 (63.3) | 101 (66.9) | 432 (64.4) | 30 (60.0) | ||||
Progesterone eceptor status |
0.004
| 1.572 | 0.204 | 3.020 | ||||||||
Negative | 330 (49.7) | 17 (29.8) | 283 (47.4) | 64 (51.6) | 285 (50.0) | 62 (41.1) | 324 (48.3) | 23 (46.0) | ||||
Positive | 334 (50.3) | 40 (70.2) | 314 (52.6) | 60 (48.4) | 285 (50.0) | 89 (58.9) | 347 (51.7) | 27 (54.0) | ||||
HER-2 status | 0.876 |
0.004
| 3.072 |
0.001
| ||||||||
Negative | 524 (78.9) | 41 (71.9) | 480 (80.4) | 85 (68.5) | 448 (78.6) | 117 (77.5) | 535 (79.7) | 30 (60.0) | ||||
Positive | 140 (21.1) | 16 (28.1) | 117 (19.6) | 39 (31.5) | 122 (21.4) | 34 (22.5) | 136 (20.3) | 20 (40.0) | ||||
Ki-67 LI (%) | 0.456 | 3.156 |
<0.001
| 1.280 | ||||||||
≤14 | 371 (55.9) | 38 (66.7) | 340 (57.0) | 69 (55.6) | 303 (53.2) | 106 (70.2) | 384 (57.2) | 25 (50.0) | ||||
>14 | 293 (44.1) | 19 (33.3) | 257 (43.0) | 55 (44.4) | 267 (46.8) | 45 (29.8) | 287 (42.8) | 25 (50.0) |
Impact of expression of sarcosine metabolism-related proteins on patient prognosis
Parameter | Number of patients/recurrence/death | Disease-free survival | Overall survival | ||
---|---|---|---|---|---|
Mean survival (95% CI) months |
P-value | Mean survival (95% CI) months |
P-value | ||
GNMT |
0.042
| 0.498 | |||
Negative | 664/54/55 | 127 (123–130) | 129 (127–132) | ||
Positive | 57/9/6 | 98 (91–106) | 126 (116–135) | ||
SARDH | 0.767 | 0.264 | |||
Negative | 597/53/47 | 126 (122–130) | 130 (127–133) | ||
Positive | 124/10/14 | 121 (115–127) | 124 (117–132) | ||
PIPOX (T) |
0.039
| 0.361 | |||
Negative | 570/57/52 | 126 (123–129) | 129 (126–131) | ||
Positive | 151/6/9 | 123 (117–129) | 131 (126–135) | ||
PIPOX (S) | 0.927 | 0.636 | |||
Negative | 671/59/58 | 126 (122–130) | 129 (126–132) | ||
Positive | 50/4/3 | 122 (114–130) | 126 (119–132) | ||
Sarcosine metabolic type
|
0.021
| 0.503 | |||
High sarcosine type | 36/8/5 | 90 (79–101) | 118 (106–130) | ||
Intermediate sarcosine type | 21/1/1 | 108 (102–114) | 132 (122–143) | ||
Low sarcosine type | 219/15/20 | 120 (115–125) | 126 (121–132) | ||
Null type | 445/39/35 | 127 (124–131) | 130 (127–133) |
Included parameters | Disease-free survival | Overall survival | ||||
---|---|---|---|---|---|---|
Hazard ratio | 95% CI |
P-value | Hazard ratio | 95% CI |
P-value | |
T stage |
0.014
|
0.040
| ||||
T1 versus T2-3 | 2.123 | 1.167-3.861 | 1.829 | 1.028-3.255 | ||
N stage |
0.001
|
0.011
| ||||
N0 versus N1-3 | 2.344 | 1.389-3.956 | 1.971 | 1.166-3.333 | ||
Histologic grade | 0.632 | 0.545 | ||||
I/II versus III | 1.143 | 0.661-1.977 | 0.839 | 0.475-1.482 | ||
ER status | 0.111 | 0.079 | ||||
Negative versus Positive | 1.795 | 0.874-3.686 | 1.888 | 0.929-3.836 | ||
PR status | 0.198 | 0.070 | ||||
Negative versus Positive | 1.638 | 0.773-3.473 | 2.036 | 0.944-4.393 | ||
HER-2 status | 0.469 | 0.508 | ||||
Negative versus Positive | 1.235 | 0.697-2.188 | 1.214 | 0.683-2.159 | ||
GNMT |
0.019
| 0.272 | ||||
Negative versus Positive | 2.408 | 1.154-5.024 | 1.622 | 0.684-3.849 | ||
PIPOX (T) | 0.069 | 0.563 | ||||
Negative versus Positive | 0.457 | 0.196-1.064 | 0.810 | 0.397-1.652 |