Introduction
Materials and methods
Patients
Variables | Discovery Cohort A
N = 84 | Validation Cohort B
N = 75 |
p
|
---|---|---|---|
Age at diagnosis | |||
Mean | 50 | 52 | 0.07 |
Range | 29–75 | 28–71 | |
Age at brain metastases | |||
Mean | 47.6 | 51.5 |
0.03
|
Range | 30–64 | 33–69 |
N
| % |
N
| % | ||
---|---|---|---|---|---|
Brain metastases | |||||
No | 36 | 43 | 34 | 45 | 0.75 |
Yes | 48 | 57 | 41 | 55 | |
Menopausal status | |||||
Premenopausal | 43 | 51 | 28 | 37 | 0.07 |
Postmenopausal | 40 | 48 | 47 | 63 | |
Histology | |||||
Ductal | 70 | 83 | 58 | 77 |
0.02
|
Lobular | 6 | 7 | 5 | 7 | |
Other | 1 | 1 | 3 | 4 | |
Uncertain | 0 | 0 | 7 | 9 | |
Ductal and lobular | 6 | 7 | 2 | 3 | |
Grades | |||||
2 | 35 | 46 | 34 | 49 | 0.69 |
3 | 41 | 54 | 35 | 51 | |
ER (IHC) | |||||
Negative | 52 | 63 | 43 | 57 | 0.49 |
Positive | 31 | 37 | 32 | 43 | |
PR (IHC) | |||||
Negative | 59 | 71 | 54 | 72 | 0.89 |
Positive | 24 | 29 | 21 | 28 | |
Breast cancer surgery | |||||
No | 16 | 19 | 15 | 20 | 0.91 |
Yes | 67 | 81 | 60 | 80 | |
Radiotherapy | |||||
No | 38 | 46 | 25 | 34 | 0.12 |
Adjuvant | 27 | 32 | 28 | 37 | |
Definitive | 0 | 0 | 1 | 1 | |
Palliative | 13 | 17 | 10 | 13 | |
Combination thereof | 4 | 5 | 11 | 15 | |
Chemotherapy induction | |||||
No | 49 | 59 | 55 | 73 | 0.06 |
Yes | 34 | 41 | 20 | 27 | |
Chemotherapy | |||||
No | 2 | 2 | 1 | 1 | 0.22 |
Adjuvant | 11 | 13 | 19 | 25 | |
For metastatic disease | 15 | 18 | 9 | 12 | |
Combination thereof | 56 | 67 | 46 | 61 | |
Trastuzumab therapy | |||||
No | 13 | 16 | 12 | 16 | 0.72 |
Adjuvant | 2 | 2 | 2 | 3 | |
For metastatic disease | 68 | 81 | 58 | 77 | |
Combination thereof | 1 | 1 | 3 | 4 | |
Anti-HER2 tyrosine kinase inhibitors | |||||
No | 72 | 86 | 51 | 68 |
0.02
|
Adjuvant | 0 | 0 | 2 | 3 | |
For metastatic disease | 12 | 14 | 22 | 29 | |
Endocrine therapy | |||||
No | 44 | 52 | 41 | 55 | 0.85 |
Adjuvant/neoadjuvant | 21 | 25 | 21 | 28 | |
For metastatic disease | 8 | 10 | 6 | 8 | |
Combination thereof | 11 | 13 | 7 | 9 | |
Type of first progression | |||||
Local | 2 | 2 | 10 | 13 |
0.02
|
Regional | 3 | 4 | 3 | 4 | |
Distant | 77 | 92 | 56 | 75 | |
Local/regional and/or distant | 2 | 2 | 6 | 8 | |
Dominant site of metastatic disease | |||||
Soft tissue | 4 | 5 | 16 | 22 |
0.001
|
Bone | 3 | 4 | 7 | 10 | |
Visceral | 77 | 91 | 50 | 68 | |
Location of first extracranial visceral relapse | |||||
Lung | 23 | 28 | 25 | 35 | 0.19 |
Liver | 34 | 41 | 26 | 36 | |
Other | 16 | 19 | 15 | 21 | |
Lung and liver | 8 | 10 | 2 | 3 | |
Lung and other | 0 | 0 | 3 | 4 | |
Liver and other | 2 | 2 | 1 | 1 | |
Brain as first relapse | |||||
No | 77 | 93 | 67 | 89 | 0.45 |
Yes | 6 | 7 | 8 | 11 |
Pathology review
RNA extraction
DASL analysis
Generation of the 13-gene signature
Real-time qRT-PCR analysis
Gene symbols | Gene names | Human assay ID | Amplicon length (bp) |
---|---|---|---|
CDK4
| Cyclin dependent kinase 4 | Hs00175935_m1 | 65 |
CCNC
| Cyclin C | Hs00193177_m1 | 78 |
PTK2
| Focal adhesion kinase (protein tyrosine kinase 2) | Hs00178587_m1 | 68 |
MYC
| v-myc avian myelocytomatosis viral oncogene homolog | Hs99999003_m1 | 65 |
BARD1
|
BRCA1 associated RING domain 1 | Hs00184427_m1 | 73 |
RAD51
| RAD51 homolog | Hs00153418_m1 | 58 |
FANCG
| Fanconi anemia group G | Hs00184947_m1 | 116 |
PCNA
| Proliferating cell nuclear antigen | Hs00696862_m1 | 95 |
PRCC
| Papillary renal cell carcinoma-translocation associated | Hs00410541_m1 | 77 |
TPR
| Translocated promoter region | Hs00162918_m1 | 82 |
CTTN
| Cortactin | Hs00193322_m1 | 81 |
DSP
| Desmoplakin | Hs00189422_m1 | 74 |
HDGF
| Hepatoma-derived growth factor | Hs00610314_m1 | 110 |
ACTB
| Actin, beta, cytoplasmic | Hs00357333_g1 | 77 |
GAPDH
| Glyceraldehyde-3-phosphate dehydrogenase | Hs99999905_m1 | 122 |
TFRC
| Transferrin receptor | Hs99999911_m1 | 105 |
Statistical analysis
Results
Characteristics and outcomes of study cohorts
Discovery Cohort A
Validation Cohort B
Determinants of BMFS and OS
Variables | Univariate analysis | Multivariate analysis* | ||||
---|---|---|---|---|---|---|
HR | 95 % CI |
p
| HR | 95 % CI |
p
| |
Discovery Cohort A | ||||||
ER negative versus positive | 3.3 | 1.1–10.0 |
0.03
| 2.8 | 0.9–9.1 | 0.07 |
Visceral site of first distant relapse | 4.5 | 1.9–10.7 |
0.001
| 7.4 | 2.4–22.3 |
<0.001
|
13-Gene expression high versus low | 5.6 | 1.9–16.5 |
0.002
| 8.5 | 2.6–28.0 |
<0.001
|
3-Gene classifier high versus low | 3.7 | 1.3–11.1 |
0.01
| 5.3 | 1.6–16.7 |
0.005
|
Trastuzumab therapy for metastatic disease no versus yes | 3.3 | 1.1–10.0 |
0.02
| 5.0 | 1.4–10.0 |
0.009
|
Validation Cohort B | ||||||
ER negative versus positive | 2.5 | 0.9–10.0 | 0.09 | 5.0 | 1.1–10.0 |
0.04
|
Visceral site of first distant relapse | 5.9 | 1.8–19.7 |
0.003
| 6.1 | 1.5–25.6 |
0.01
|
3-Gene classifier high versus low | 1.2 | 0.3–20.0 | 0.8 | NC | ||
Grade high versus low | 3.3 | 1.1–14.3 |
0.03
| 3.8 | 0.9–16.7 | 0.07 |
Trastuzumab therapy for metastatic disease no versus yes | 2.5 | 1.0–10.0 | 0.06 | 10.0 | 2.0–100.0 |
0.008
|
Variables | 3-Gene classifier | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Discovery Cohort A (N = 83) | Validation Cohort B (N = 75) | |||||||||
Low (N = 34; 41 %) | High (N = 49; 59 %) | Low (N = 63; 84 %) | High (N = 12; 16 %) | |||||||
N
| % |
N
| % |
p
|
N
| % |
N
| % |
p
| |
Menopausal status | ||||||||||
Premenopausal | 20 | 60 | 23 | 47 | 24 | 38 | 4 | 33 | 0.75 | |
Postmenopausal | 13 | 40 | 26 | 53 | 0.22 | 39 | 62 | 8 | 67 | |
Primary tumor grades | ||||||||||
2 | 20 | 67 | 15 | 33 | 28 | 49 | 6 | 50 | 0.95 | |
3 | 10 | 33 | 30 | 67 |
0.05
| 29 | 51 | 6 | 50 | |
ER | ||||||||||
Negative | 16 | 47 | 36 | 75 | 35 | 56 | 8 | 67 | 0.47 | |
Positive | 18 | 53 | 12 | 25 |
0.01
| 28 | 44 | 4 | 33 | |
PR | ||||||||||
Negative | 23 | 68 | 36 | 75 | 45 | 71 | 9 | 75 | 0.80 | |
Positive | 11 | 32 | 12 | 25 | 0.46 | 18 | 29 | 3 | 25 | |
Induction chemotherapy | ||||||||||
No | 21 | 62 | 28 | 58 | 50 | 79 | 5 | 42 |
0.007
| |
Yes | 13 | 38 | 20 | 42 | 0.75 | 13 | 21 | 7 | 58 | |
Endocrine therapy | ||||||||||
No | 12 | 36 | 31 | 63 | 33 | 52 | 8 | 67 | 0.36 | |
Yes | 22 | 65 | 18 | 37 |
0.01
| 30 | 48 | 4 | 33 | |
Trastuzumab for metastatic disease | ||||||||||
No | 3 | 9 | 13 | 26 | 15 | 24 | 2 | 17 | 0.59 | |
Yes | 31 | 91 | 36 | 73 |
0.04
| 48 | 76 | 10 | 83 | |
Visceral location of first relapse | ||||||||||
No | 31 | 94 | 34 | 69 | 44 | 73 | 9 | 75 | 0.90 | |
Yes | 2 | 6 | 15 | 31 |
0.01
| 16 | 27 | 3 | 25 | |
First site of visceral metastasis | ||||||||||
Lung | 11 | 33 | 12 | 25 | 22 | 37 | 3 | 25 |
0.05
| |
Liver | 16 | 48 | 18 | 37 | 22 | 37 | 4 | 33 | ||
Other | 2 | 6 | 13 | 27 | 13 | 22 | 2 | 17 | ||
Lung and liver | 4 | 12 | 4 | 8 | 0 | 0 | 2 | 17 | ||
Lung and other | 0 | 0 | 0 | 0 | 2 | 3 | 1 | 8 | ||
Liver and other | 0 | 0 | 2 | 4 | 0.19 | 1 | 2 | 0 | 0 | |
Brain metastases | ||||||||||
No | 11 | 32 | 25 | 51 | 28 | 44 | 6 | 50 | ||
Yes | 23 | 67 | 24 | 49 | 0.09 | 35 | 55 | 6 | 50 | 0.72 |