Background
Methods
Patients
DNA and RNA extraction
Immunohistochemical analysis
Interpretation of immunohistochemical staining
Genetic analysis of EGFR
Real-Time Reverse Transcription (RT)-PCR
Statistical analyses
Results
Patient Characteristics and Immunohistochemical results
Variables | Total | Triple negative | Non-triple negative | P value | |||
---|---|---|---|---|---|---|---|
N = 486 (100%)
|
N = 71 (15%)
|
N = 415 (85%)
| |||||
No.
|
%
|
No.
|
%
|
No.
|
%
| ||
Age, years | 0.49 | ||||||
≤50 | 198 | 41 | 32 | 44 | 166 | 40 | |
>50 | 288 | 59 | 39 | 56 | 249 | 60 | |
Size | 0.23 | ||||||
≤2 cm | 181 | 37 | 21 | 30 | 160 | 39 | |
>2 cm | 288 | 59 | 46 | 65 | 242 | 58 | |
Unknown | 17 | 4 | 5 | 13 | 3 | ||
Nodal status | 0.02 | ||||||
Negative | 275 | 57 | 48 | 68 | 227 | 55 | |
Positive | 192 | 40 | 19 | 27 | 173 | 42 | |
Unknown | 19 | 3 | 4 | 5 | 15 | 3 | |
Histology | 0.14 | ||||||
Ductal | 454 | 94 | 66 | 93 | 388 | 93 | |
Lobular | 16 | 3 | 0 | 0 | 16 | 4 | |
Other | 16 | 3 | 5 | 7 | 11 | 3 | |
Grade | <0.0001 | ||||||
1 | 92 | 19 | 4 | 6 | 88 | 21 | |
2 | 273 | 56 | 32 | 45 | 241 | 58 | |
3 | 101 | 21 | 34 | 48 | 67 | 16 | |
Unknown | 20 | 4 | 1 | 1 | 19 | 5 | |
TP53 expression | <0.0001 | ||||||
Negative | 392 | 81 | 43 | 61 | 349 | 84 | |
Positive | 92 | 19 | 28 | 39 | 64 | 15 | |
Unknown | 2 | 0 | 0 | 0 | 2 | 1 | |
Ki67 expression | <0.0001 | ||||||
Low | 333 | 69 | 34 | 48 | 299 | 72 | |
High | 150 | 31 | 37 | 52 | 113 | 27 | |
Unknown | 3 | 1 | 0 | 0 | 3 | 1 |
Variables | Triple negative (N = 110) | |
---|---|---|
No.
|
%
| |
EGFR
| ||
Negative | 69 | 62 |
Positive | 33 | 31 |
Unknown | 8 | 7 |
Cytokeratin 5/6
| ||
Negative | 47 | 43 |
Positive | 57 | 52 |
Unknown | 6 | 5 |
Cytokeratin 14
| ||
Negative | 43 | 40 |
Positive | 61 | 55 |
Unknown | 6 | 5 |
Cytokeratin 5/6/14
| ||
Negative | 32 | 30 |
Posotive | 72 | 65 |
Unknown | 6 | 5 |
Basal-like phenotype
| ||
Negative | 24 | 22 |
Positive | 77 | 70 |
Unknown | 9 | 8 |
Genomic analysis of EGFR
Mutation No. | Probe name | Nucleotide | Amino Acid | Primer sequence (forward) |
---|---|---|---|---|
WT1 | CCCAGAAGGTGAGAAAGTTAAAATTC | |||
1 | Del 1a | 2235-2249del | E746-A750del | (same as above) |
2 | Del 1b | 2236-2250del | (same as above) | |
3 | Del 2 | 2254-2277del | S752-I759del | (same as above) |
WT2 | (same as above) | |||
4 | Del 3 | 2239-2247del, 2248G>C | L747-E749del, A750P | (same as above) |
5 | Del 4 | 2240-2257del | L747-S752del, P753S | (same as above) |
6 | Del 5 | 2238-2255del, 2237A>T | L747-A750del, E746V | (same as above) |
7 | Del 6 | 2240-2251del | L747-A750del, T747S | (same as above) |
WT3 | TGAGGATCTTGAAGGAAACTGAATTC | |||
8 | G719C | 2155G>T | G719C | (same as above) |
9 | G719S | 2155G>A | G719S | (same as above) |
WT4 | CCGCAGCATGTCAAGATCAC | |||
10 | L858R | 2573T>G | L858R | (same as above) |
WT5 | (same as above) | |||
11 | L861Q | 2582T>A | L861Q | (same as above) |
WT6 | CCCAGAAGGTGAGAAAGTTAAAATTC | |||
12 | L747Del | 2239-2262del | L747-K754del | (same as above) |
13 | N756Ins | 2265-2267InsCAA | N756Ins | (same as above) |
WT7 | GGAGGACCGTCGCTTGGT | |||
14 | L838P | 2513T>C | L838P | (same as above) |
TaqMan probe
|
Primer sequence (reverse)
| |||
VIC-ATTAAGAGAAGCAACATCT | CCCACACAGCAAAGCAGAAA | |||
FAM-CGCTATCAAAACATCT | (same as above) | |||
FAM-CTATCAAGACATCTCC | (same as above) | |||
FAM-AGAAGCAACACTCGAT | (same as above) | |||
VIC-CGAAAGCCAACAAG | (same as above) | |||
FAM-CAAGGAACCAACATC | (same as above) | |||
FAM-AAGGAATCGAAAGCC | (same as above) | |||
FAM-CAAGGTTCCGAAAGC | (same as above) | |||
FAM-TCAAGGAATCATCTCC | (same as above) | |||
VIC-AAGTGCTGGGCTCC | TGCCAGGGACCTTACCTTATACA | |||
FAM-AAAGTGCTGTGCTCC | (same as above) | |||
FAM-AAAGTGCTGTGCTCC | (same as above) | |||
VIC-TTGGGCTGGCCAAA | TCCTTCTGCATGGTATTCTTTCTCT | |||
FAM-TTGGGCGGGCCAA | (same as above) | |||
VIC-CCAAACTGCTGGGTG | (same as above) | |||
FAM-CCAAACAGCTGGGTG | (same as above) | |||
VIC-ATTAAGAGAAGCAACATCT | CCCACACAGCAAAGCAGAAA | |||
FAM-CTATCAAGGAAGCCAACAA-MGB | (same as above) | |||
FAM-CCAACAACAAGGAAAT-MGB | (same as above) | |||
VIC-CGCGACCTGGCAG-MGB | CCCAAAATCTGTGATCTTGACATG | |||
FAM-CGCGACCCGGCAG-MGB | (same as above) |
Increased EGFR gene copy No. | No increased EGFR gene copy No. | |
---|---|---|
EGFR IHC
| ||
0 | 4 | 14 |
1 | 6 | 22 |
2 | 2 | 8 |
3 | 0 | 2 |
BRCA1mRNA expression analysis
Clinical outcome by treatment epoch
Clinical outcome according to tumor subtype
Variables | P value | Relative Risk of recurrence (95% CI) |
---|---|---|
Size | ||
≤2 cm | 1.0 (reference) | |
>2 cm | 0.92 | 1.26 (0.44 to 3.65) |
Nodal status | ||
Negative | 1.0 (reference) | |
Positive | 0.02 | 2.60 (1.14 to 6.96) |
Grade | ||
1 | 1.0 (reference) | |
2, 3 | 0.22 | 2.46 (0.68 to 10.39) |
TP53 expression | ||
Negative | 1.0 (reference) | |
Positive | 0.61 | 1.12 (0.72 to 1.77) |
Ki67 expression | ||
Low | 1.0 (reference) | |
High | 0.55 | 0.88 (1.59 to 1.34) |
EGFR IHC | ||
Negative | 1.0 (reference) | |
Positive | 0.88 | 1.07 (0.48 to 2.36) |
EGFR gene copy number | ||
<3 | 1.0 (reference) | |
≥3 | 0.2 | 0.27 (0.03 to 2.05) |
Cytokeratins | ||
Negative | 1.0 (reference) | |
Positive | 0.42 | 0.72 (0.33 to 1.68) |
Basal like type | ||
+ | 1.0 (reference) | |
- | 0.67 | 0.79 (0.34 to 1.80) |