Introduction
Materials and methods
Patients
Patients enrolled | AdnaTest |
CK19
| Multiplex | |||||||
---|---|---|---|---|---|---|---|---|---|---|
All number (%) | Positive number (%) | Negative number (%) |
P
a
| Positive number (%) | Negative number (%) |
P
a
| Positive number (%) | Negative number (%) |
P
a
| |
254 (100) | 42 (16.5) | 212 (83.5) | 36 (14.2) | 218 (85.8) | 58 (22.8) | 196 (77.2) | ||||
Age, years | 0.398 | 0.383 | 0.614 | |||||||
≥ 61 | 130 (51.2) | 24 (18.5) | 106 (81.5) | 16 (12.3) | 114 (87.7) | 28 (21.5) | 102 (78.5) | |||
< 61 | 124 (48.8) | 18 (14.5) | 106 (85.5) | 20 (16.1) | 104 (83.9) | 30 (24.2) | 94 (75.8) | |||
Staging | 0.827 | 0.343 | ||||||||
Tx | 1 (0.39) | 0 (0) | 1 (100) | 0 (0) | 1 (100) | 0 (0) | 1 (100) | |||
is | 8 (3.15) | 2 (25.0) | 6 (75.2) | 0 (0) | 8 (100) | 1 (12.5) | 7 (87.5) | |||
To | 6 (2.36) | 1 (16.7) | 5 (83.3) | 0 (0) | 6 (100) | 2 (33.3) | 4 (66.7) | |||
I/II | 206 (81.1) | 29 (14.1) | 177 (85.9) | 34 (16.5) | 172 (83.5) | 48 (23.3) | 158 (76.7) | |||
III/IV | 19 (7.48) | 4 (21.1) | 15 (78.9) | 1 (5.26) | 18 (94.7) | 4 (21.0) | 15 (79.0) | |||
Unknown | 14 (5.51) | |||||||||
Grading | 0.386 | 0.822 | ||||||||
1 | 45 (17.7) | 5 (11.1) | 40 (88.9) | 6 (13.3) | 39 (86.7) | 12 (26.7) | 33 (73.3) | 0.816 | ||
2 | 105 (41.3) | 19 (18.1) | 86 (81.9) | 17 (16.2) | 88 (83.8) | 23 (21.9) | 82 (78.1) | |||
3 | 83 (32.7) | 10 (12.1) | 73 (88.0) | 11 (13.2) | 72 (86.8) | 19 (22.9) | 64 (77.1) | |||
Unknown | 21 (8.37) | |||||||||
Lymph nodes | ||||||||||
N0 | 130 (51.2) | 17 (13.1) | 113 (86.9) | 0.099 | 23 (17.7) | 107 (82.3) | 0.450 | 31 (23.8) | 99 (76.2) | 0.871 |
N1 | 54 (21.3) | 9 (16.7) | 45 (83.3) | 6 (11.1) | 48 (88.9) | 15 (27.8) | 39 (72.2) | |||
N2 | 15 (5.91) | 3 (20.0) | 12 (80.0) | 2 (13.3) | 13 (86.7) | 3 (20.0) | 12 (80.0) | |||
N3 | 6 (2.36) | 3 (50.0) | 3 (50.0) | 2 (33.3) | 4 (66.7) | 1 (16.7) | 5 (83.3) | |||
Unknown | 49 (19.3) | |||||||||
Histology | ||||||||||
Ductal | 175 (68.9) | 23 (13.1) | 152 (86.9) | 0.251 | 23 (13.1) | 152 (86.9) | 0.201 | 39 (22.3) | 136 (77.7) | |
Lobular | 35 (13.8) | 7 (20.0) | 28 (80.0) | 9 (25.7) | 26 (74.3) | 12 (34.3) | 23 (65.7) | |||
Ductal/Lobular | 5 (1.97) | 2 (40.0) | 3 (60.0) | 1 (20.0) | 4 (80.0) | 0 (0) | 5 (100) | |||
Other | 24 (9.45) | 5 (20.8) | 19 (79.2) | 2 (8.3) | 22 (91.7) | 4 (16.7) | 20 (83.3) | |||
Unknown | 15 (5.91) | |||||||||
ER | 0.553 | 0.275 | 0.581 | |||||||
Positive | 179 (70.5) | 28 (15.6) | 151 (84.4) | 29 (16.2) | 150 (83.8) | 40 (22.3) | 139 (77.7) | |||
Negative | 58 (22.8) | 11 (19.0) | 47 (81.0) | 6 (10.3) | 52 (89.7) | 15 (25.9) | 43 (74.1) | |||
Unknown | 17 (6.7) | |||||||||
PR | 0.474 | 0.184 | 0.361 | |||||||
Positive | 175 (68.9) | 27 (15.4) | 148 (84.6) | 29 (16.6) | 146 (83.4) | 38 (21.7) | 137 (78.3) | |||
Negative | 62 (24.4) | 12 (19.4) | 50 (80.6) | 6 (9.7) | 56 (90.3) | 17 (27.4) | 45 (72.6) | |||
Unknown | 17 (6.7) | |||||||||
HER2 | 0.725 | 0.397 | ||||||||
Positive | 38 (15.0) | 7 (18.4) | 31 (81.6) | 0.639 | 5 (13.2) | 33 (86.8) | 11 (29.0) | 27 (71.0) | ||
Negative | 195 (76.8) | 30 (15.4) | 165 (84.6) | 30 (15.4) | 165 (84.6) | 44 (22.6) | 151 (77.4) | |||
Unknown | 21 (8.27) |
CTC isolation
RNA isolation from CTC and cDNA synthesis
CTC detection and molecular characterization
CK-19 RT-qPCR
Multiplex RT-qPCR
AdnaTest BreastCancerDetect
Statistical analysis
Results
CK-19 RT-qPCR
Molecular assay | CK19RT-qPCR | Multiplex- RT-qPCR | Total | ||
---|---|---|---|---|---|
Early breast cancer, (number = 254) | |||||
AdnaTest |
Positive
|
Negative
|
Positive
|
Negative
|
Total (%)
|
Positive | 4 | 38 | 5 | 37 | 42 (16.5) |
Negative | 32 | 180 | 53 | 159 | 212 (83.5) |
Total (%) | 36 (14.2) | 218 (85.8) | 58 (22.8) | 196 (77.2) | 254 |
Kappa test | poor agreement: Kappa = -0.059 (P = 0.344) | poor agreement: Kapp = -0.114 (P = 0.065) | |||
Concordance (%) | 184 (72.4), Pa = 0.344 | 164 (64.6), P
a
= 0.065 | |||
Verified metastasis, (number = 51)
| |||||
AdnaTest |
Positive
|
Negative
|
Positive
|
Negative
|
Total (%)
|
Positive | 17 | 11 | 16 | 12 | 28 (54.9) |
Negative | 4 | 19 | 4 | 19 | 23 (45.1) |
Total (%) | 21 (41.2) | 30 (58.8) | 20 (39.2) | 31 (60.8) | 51 |
Kappa test | Moderate agreement: Kappa = 0.422 (P = 0.002), | Fair agreement: Kappa = 0.386 (P = 0.004) | |||
Concordance (%) | 36 (70.6), Pa = 0.002 | 35 (68.6), Pa = 0.004 |
Multiplex RT-qPCR
Multiplex RT-qPCR | Singleplex RT-qPCR | ||||||||
---|---|---|---|---|---|---|---|---|---|
AdnaTest/ |
CK19
|
HER-2
|
MAGE A3
|
CK19
| |||||
Pos. | Neg. | Pos. | Neg. | Pos. | Neg. | Pos. | Neg. | ||
Early breast cancer, (number = 254) | |||||||||
GA733-2
| Positive | 1 | 8 | 0 | 9 | 0 | 9 | 2 | 7 |
Negative | 24 | 221 | 28 | 217 | 12 | 233 | 34 | 211 | |
Concordance (%) | 87.4 | Pa = 0.897 | 85.4 | Pa = 0.284 | 91.7 | Pa = 0.496 | 83.9 | Pa = 0.481 | |
MUC-1
| Positive | 0 | 14 | 1 | 13 | 0 | 14 | 1 | 13 |
Negative | 24 | 215 | 27 | 213 | 12 | 228 | 35 | 205 | |
Concordance (%) | 84.6 | Pa = 0.213 | 84.3 | Pa = 0.633 | 89.8 | Pa = 0.391 | 81.1 | Pa = 0.438 | |
HER-2
| Positive | 2 | 24 | 2 | 24 | 0 | 26 | 2 | 24 |
Negative | 23 | 205 | 26 | 202 | 12 | 216 | 34 | 194 | |
Concordance (%) | 81.5 | Pa = 0.698 | 80.3 | Pa = 0.567 | 85 | Pa = 0.231 | 77.2 | Pa = 0.317 | |
Verified metastasis, (number = 51)
| |||||||||
GA733-2
| Positive |
14
|
6
| 6 | 14 | 3 | 17 |
15
|
5
|
Negative |
4
|
27
| 2 | 29 | 1 | 30 |
6
|
25
| |
Concordance (%) |
80.4
|
P
b
< 0.001
| 68.6 | Pb = 0.045 | 64.7 | Pb = 0.287 |
78.4
|
P
b
= 0.000
| |
MUC-1
| Positive |
14
|
9
| 6 | 17 | 3 | 20 |
14
|
9
|
Negative |
4
|
24
| 2 | 26 | 1 | 27 |
7
|
21
| |
Concordance (%) |
74.5
|
P
b
= 0.001
| 62.7 | Pb = 0.119 | 58.8 | Pb = 0.316 |
68.6
|
P
b
= 0.012
| |
HER-2
| Positive |
11
|
7
| 5 | 13 | 1 | 17 |
13
|
5
|
Negative |
7
|
26
| 3 | 30 | 3 | 30 |
8
|
25
| |
Concordance (%) |
72.6
|
P
b
= 0.006
| 68.6 | Pb = 0.112 | 60.8 | Pb = 1 |
74.5
|
P
b
= 0.001
|
AdnaTest BreastCancer™
Comparison between the three molecular assays for CTC detection
Molecular assay | CK-19RT-qPCR | |||
---|---|---|---|---|
CK-19 multiplexRT-qPCR | Early breast cancer, (number = 254) | Verified metastasis, (number = 51) | ||
Positive | Negative | Positive | Negative | |
Positive | 15 | 10 | 18 | 0 |
Negative | 21 | 208 | 3 | 30 |
Total (%) | 36 (14.2) | 218 (85.8) | 21 (41.2) | 30 (58.8) |
Kappa test | Moderate agreement: Kappa = 0.425 (P = 0.000) Pa = 0.000P = = Pp(P = 0.000) | Almost perfect agreement: Kappa = 0.876 (P = 0.000) | ||
Concordance (%) | 223 (87.8), P
a
= 0.000 | 48 (94.1), P
a
= 0.000 |