Background
Methods
Patients and samples
RNA extraction
Mutation screening
Real-time quantitative RT-PCR
Immunohistochemistry
Statistical analysis
Results
PIK3CA, PIK3R1 and AKT1mutational analysis
mRNA expression
Genes | Median Ct of normal breast tissue (n = 10) | Normal breast tissue (n = 10) | Breast tumors n = 458 | Percentage of underexpressed tumors (Ntarget ≤0.5) | Percentage of normal expressed tumors | Percentage of overexpressed tumors (Ntarget ≥2) | Percentage of overexpressed tumors (Ntarget ≥5) |
---|---|---|---|---|---|---|---|
EGFR
| 30.2 (29.3-31.5)a
| 1.0 (0.7 -1.3)b
| 0.2 (0.0-112.9)b
|
84.9%
c
|
13.3%
c
| 1.8%c
| 0.7%c
|
PIK3CA
| 29.7 (28.4-31.0) | 1.0 (0.7-1.3) | 0.9 (0.2-33.4) | 8.7% |
87.4%
| 3.9% | 0.7% |
PIK3R1
| 26.8 (25.8-28.1) | 1.0 (0.7-1.5) | 0.4 (0.0-5.2) |
61.8%
|
36.0%
| 2.2% | 0.2% |
PDK1
| 31.8 (29.7-33.5) | 1.0 (0.5-1.9) | 1.0 (0.0-14.7) |
13.3%
|
69.0%
|
17.7%
| 2.2% |
PTEN
| 26.4 (25.3-31.3) | 1.1 (0.7-2.0) | 0.8 (0.1-9.0) |
17.0%
|
81.0%
| 2.0% | 0.4% |
AKT1
| 28.7 (27.5-30.1) | 1.0 (0.7-1.5) | 1.5 (0.0-11.1) | 1.3% |
73.4%
|
25.3%
| 2.8% |
AKT2
| 26.7 (25.4-29.7) | 1.0 (0.7-2.0) | 1.7 (0.5-12.2)d
| 0.0% |
64.0%
|
36.0%
| 3.3% |
AKT3
| 26.0 (23.8-28.4) | 1.0 (0.6-1.9) | 0.4 (0.0-7.5)d
|
67.1%
|
31.1%
| 1.8% | 0.2% |
GOLPH3
| 27.9 (26.4-29.0) | 1.0 (0.8-1.6) | 1.4 (0.3-6.7) | 0.7% |
79.9%
|
19.4%
| 0.9% |
P70S6K
| 31.2 (29.9-32.7) | 1.0 (0.7-1.8) | 1.2 (0.0-19.6) | 2.2% |
79.7%
|
18.1%
| 3.7% |
WEE1
| 28.4 (26.1-29.8) | 1.0 (0.5-1.6) | 0.8 (0.2-6.9) |
18.3%
|
77.3%
| 4.4% | 0.2% |
All tumors
|
Tumor subtypes
| |||||
---|---|---|---|---|---|---|
HR-ERBB2-
|
HR-ERBB2+
|
HR + ERBB2-
|
HR + ERBB2+
|
P-value
a
| ||
n = 458
|
n = 69
|
n = 45
|
n = 290
|
n = 54
| ||
PIK3CA values: median [range]
|
0.9 (0.2-33.4)
|
0.9 (0.3-33.4)
|
0.7 (0.3-1.7)
|
0.9 (0.2-5.9)
|
1.0 (0.4-5.6)
|
NS
|
Underexpressed tumors (%)
|
40 (8.7)
|
6 (8.7)
|
5 (11.1)
|
25 (8.6)
|
4 (7.4)
| |
Non-underexpressed tumors (%)
| 418 (91.3) |
63 (91.3)
|
40 (88.9)
|
265 (91.4)
|
50 (92.6)
| |
PIK3R1 values: median [range]
|
0.4 (0.0-5.2)
|
0.2 (0.0-2.2)
|
0.3 (0.1-1.5)
|
0.5 (0.1-4.4)
|
0.4 (0.1-5.2)
|
<0.0000001
|
Underexpressed tumors (%)
|
283 (61.8)
|
61 (88.4)
|
40 (88.9)
|
150 (51.7)
|
32 (59.3)
| |
Non-underexpressed tumors (%)
| 175 (38.2) |
8 (11.6)
|
5 (11.1)
|
140 (48.3)
|
22 (40.7)
| |
PDK1 values: median [range]
|
1.0 (0.0-14.7)
|
2.4 (0.5-14.7)
|
1.7 (0.4-6.2)
|
0.9 (0.0-3.3)
|
0.9 (0.1-2.4)
|
<0.0000001
|
Underexpressed tumors (%)
|
61 (13.3)
|
0
|
1 (2.2)
|
51 (7.9)
|
9 (16.6)
| |
Normally expressed tumors (%)
| 316 (69.0) |
25 (36.2)
|
27 (60.0)
|
221 (90.0)
|
43 (79.6)
| |
Overexpressed tumors (%)
|
81 (17.7)
|
44 (63.8)
|
17 (37.8)
|
18 (2.1)
|
2 (3.7)
| |
PTEN values: median [range]
|
0.8 (0.1-9.0)
|
0.6 (0.1-1.5)
|
0.8 (0.3-1.9)
|
0.8 (0.1-9.0)
|
0.9 (0.4-3.3)
|
0.0000066
|
Underexpressed tumors (%)
|
78 (17.0)
|
27 (39.1)
|
6 (13.3)
|
39 (13.4)
|
6 (11.1)
| |
Non-underexpressed tumors (%)
| 380 (83.0) |
42 (60.9)
|
39 (86.7)
|
251 (86.6)
|
48 (88.9)
| |
AKT1 values: median [range]
|
1.5 (0.0-11.1)
|
1.1 (0.0-11.1)
|
2.0 (0.6-10.0)
|
1.4 (0.4-6.1)
|
1.8 (0.6-9.9)
|
0.00019
|
Non-overexpressed tumors (%)
| 342 (74.7) |
55 (79.7)
|
24 (53.3)
|
230 (79.3)
|
33 (61.1)
| |
Overexpressed tumors (%)
|
116 (25.3)
|
14 (20.3)
|
21 (46.7)
|
60 (20.7)
|
21 (38.9)
| |
AKT2 values: median [range]
b
|
1.7 (0.5-12.2)
|
1.7 (0.7-12.2)
|
1.4 (0.8-8.7)
|
1.8 (0.5-10.6)
|
1.8 (0.5-7.0)
|
0.0097
|
Non-overexpressed tumors (%)
| 293 (64.3) |
46 (67.6)
|
38 (84.4)
|
180 (62.3)
|
29 (53.7)
| |
Overexpressed tumors (%)
|
163 (35.7)
|
22 (32.4)
|
7 (15.6)
|
109 (37.7)
|
25 (46.3)
| |
AKT3 values: median [range]
b
|
0.4 (0.0-7.5)
|
0.5 (0.0-2.2)
|
0.3 (0.1-0.9)
|
0.4 (0.0-7.5)
|
0.4 (0.1-2.3)
|
NS
|
Underexpressed tumors (%)
|
306 (67.1)
|
38 (55.9)
|
32 (71.1)
|
198 (68.5)
|
38 (70.4)
| |
Non-underexpressed tumors (%)
| 150 (32.9) |
30 (44.1)
|
13 (28.9)
|
91 (31.5)
|
16 (29.6)
| |
GOLPH3 values: median [range]
|
1.4 (0.3-6.7)
|
1.2 (0.6-3.3)
|
1.4 (0.7-5.0)
|
1.3 (0.3-6.7)
|
1.7 (0.8-5.4)
|
NS
|
Non-overexpressed tumors (%)
| 369 (80.6) |
54 (78.3)
|
36 (80.0)
|
241 (83.1)
|
38 (70.4)
| |
Overexpressed tumors (%)
|
89 (19.4)
|
15 (21.7)
|
9 (20.0)
|
49 (16.9)
|
16 (29.6)
| |
P70S6K values: median [range]
|
1.2 (0.0-19.6)
|
1.0 (0.0-5.4)
|
1.9 (0.6-9.9)
|
1.2 (0.3-8.0)
|
1.4 (0.4-19.6)
|
<0.0000001
|
Non-overexpressed tumors (%)
| 375 (81.9) |
64 (92.8)
|
24 (53.3)
|
250 (86.2)
|
37 (68.5)
| |
Overexpressed tumors (%)
|
83 (18.1)
|
5 (7.2)
|
21 (46.7)
|
40 (13.8)
|
17 (31.5)
| |
WEE1 values: median [range]
|
0.8 (0.2-6.9)
|
0.7 (0.2-6.9)
|
0.9 (0.3-2.8)
|
0.8 (0.2-3.9)
|
0.8 (0.3-4.1)
|
0.0014
|
Underexpressed tumors (%)
|
84 (18.3)
|
24 (34.8)
|
6 (13.3)
|
43 (14.8)
|
11 (20.4)
| |
Non-underexpressed tumors (%)
| 374 (81.7) |
45 (65.2)
|
39 (86.7)
|
247 (85.2)
|
43 (79.6)
|
Number of patients (%) | ||||
---|---|---|---|---|
Total population (%) |
PIK3R1underexpression |
PIK3R1non-underexpression |
P-value
a
| |
Total
|
458 (100.0)
|
283 (61.8)
|
175 (38.2)
| |
EGFR values: median [range]
|
0.2 (0.0-112.9)
|
0.1 (0.0-7.3)
|
0.2 (0.0-112.9)
| |
Underexpressed tumors (%) |
389 (84.9)
| 250 (88.3) | 139 (79.4) |
0.0096
|
Non-underexpressed tumors (%) | 69 (15.1) | 33 (11.7) | 36 (20.6) | |
PDK1 values: median [range]
|
1.0 (0.0-14.7)
|
1.2 (0.1-14.7)
|
0.9 (0.0-6.2)
| |
Underexpressed tumors (%) |
61 (13.3)
| 26 (9.2) | 35 (20.0) |
0.000004
|
Normally expressed tumors (%) | 316 (69.0) | 189 (66.8) | 127 (72.6) | |
Overexpressed tumors (%) |
81 (17.7)
| 68 (24.0) | 13 (7.4) | |
AKT1 values: median [range]
|
1.5 (0.0-11.1)
|
1.4 (0.4-10.0)
|
1.6 (0.0-11.1)
| |
Non-overexpressed tumors (%) | 342 (74.7) | 216 (76.3) | 126 (72.0) |
NS
|
Overexpressed tumors (%) |
116 (25.3)
| 67 (23.7) | 49 (28.0) | |
AKT2 values: median [range]
b
|
1.0 (0.7-2.3)
|
1.6 (0.5-10.6)
|
1.8 (0.5-12.2)
| |
Non-overexpressed tumors (%) | 293 (64.3) | 189 (67.0) | 104 (59.8) |
NS
|
Overexpressed tumors (%) |
163 (35.7)
| 93 (33.0) | 70 (40.2) | |
AKT3 values: median [range]
b
|
1.0 (0.4-1.9)
|
0.3 (0.0-2.4)
|
0.5 (0.1-7.5)
| |
Underexpressed tumors (%) |
306 (67.1)
| 215 (76.2) | 91 (52.3) |
0.00000013
|
Non-underexpressed tumors (%) | 150 (32.9) | 67 (23.8) | 83 (47.7) | |
GOLPH3 values: median [range]
|
1.4 (0.3-6.7)
|
1.3 (0.3-5.2)
|
1.7 (0.7-6.7)
| |
Non-overexpressed tumors (%) | 369 (80.6) | 242 (85.5) | 127 (72.6) |
0.00067
|
Overexpressed tumors (%) |
89 (19.4)
| 41 (14.5) | 48 (27.4) | |
P70S6K values: median [range]
|
1.2 (0.0-19.6)
|
1.2 (0.4-19.6)
|
1.2 (0.0-8.9)
| |
Non-overexpressed tumors (%) | 375 (81.9) | 226 (79.9) | 149 (85.1) |
NS
|
Overexpressed tumors (%) |
83 (18.1)
| 57 (20.1) | 26 (14.9) | |
WEE1 values: median [range]
|
0.8 (0.2-6.9)
|
0.7 (0.2-4.1)
|
0.9 (0.2-6.9)
| |
Underexpressed tumors (%) |
84 (18.3)
| 68 (24.0) | 16 (9.1) |
0.000063
|
Non-underexpressed tumors (%) | 374 (81.7) | 215 (76.0) | 159 (90.9) | |
PTEN values: median [range]
|
0.8 (0.1-9.0)
|
0.7 (0.1-9.0)
|
1.0 (0.4-5.8)
| |
Underexpressed tumors (%) |
78 (17.0)
| 71 (25.1) | 7 (4.0) |
< 0.0000001
|
Non-underexpressed tumors (%) | 380 (83.0) | 212 (74.9) | 168 (96.0) | |
PIK3CA
| ||||
Wild-type (%) | 307 (67.0) |
205 (72.4)
|
102 (58.3)
|
0.0017
|
Mutation (%) |
151 (33.0)
|
78 (27.6)
|
73 (41.7)
| |
PIK3R1
c
| ||||
Wild-type (%) | 444 (97.8) |
276 (98.6)
|
168 (96.6)
|
NS
|
Mutation (%) |
10 (2.2)
|
4 (1.4)
|
6 (3.4)
| |
AKT1
d
| ||||
Wild-type (%) | 442 (96.7) | 272 (96.5) | 170 (97.1) |
NS
|
Mutation (%) |
15 (3.3)
| 10 (3.5) | 5 (2.9) |
Number of patients (%) | ||||
---|---|---|---|---|
Total population (%) |
PIK3CAwild-type |
PIK3CA-mutated |
P-value
a
| |
Total
|
458 (100.0)
|
307 (67.0)
|
151 (33.0)
| |
EGFR values: median [range]
|
0.2 (0.0-112.9)
|
0.2 (0.0-112.9)
|
0.2 (0.0-7.3)
| |
Underexpressed tumors (%) |
389 (84.9)
| 256 (83.4) | 133 (88.1) |
NS
|
Non-underexpressed tumors (%) | 69 (15.1) | 51 (16.6) | 18 (11.9) | |
PIK3R1 values: median [range]
|
0.4 (0.0-5.2)
|
0.3 (0.0-4.4)
|
0.5 (0.1-5.2)
| |
Underexpressed tumors (%) |
283 (61.8)
| 205 (66.8) | 78 (51.7) |
0.0017
|
Non-underexpressed tumors (%) | 175 (38.2) | 102 (33.2) | 73 (48.3) | |
PDK1 values: median [range]
|
1.0 (0.0-14.7)
|
1.1 (0.0-14.7)
|
0.8 (0.1-4.5)
| |
Underexpressed tumors (%) |
61 (13.3)
| 35 (11.4) | 26 (17.2) |
0.0011
|
Normally expressed tumors (%) | 316 (69.0) | 204 (66.5) | 112 (74.2) | |
Overexpressed tumors (%) |
81 (17.7)
| 68 (22.1) | 13 (8.6) | |
PTEN values: median [range]
|
0.8 (0.1-9.0)
|
0.8 (0.1-5.8)
|
0.9 (0.1-9.0)
| |
Underexpressed tumors (%) |
78 (17.0)
| 64 (20.8) | 14 (9.3) |
0.0019
|
Non-underexpressed tumors (%) | 380 (83.0) | 243 (79.2) | 137 (90.7) | |
AKT1 values: median [range]
|
1.5 (0.0-11.1)
|
1.5 (0.0-11.1)
|
1.5 (0.4-9.9)
| |
Non-overexpressed tumors (%) | 342 (74.7) | 230 (74.9) | 112 (74.2) |
NS
|
Overexpressed tumors (%) |
116 (25.3)
| 77 (25.1) | 39 (25.8) | |
AKT2 values: median [range]
b
|
1.0 (0.7-2.3)
|
1.7 (0.5-12.2)
|
1.6 (0.5-10.6)
| |
Non-overexpressed tumors (%) | 293 (64.3) | 190 (62.3) | 103 (68.2) |
NS
|
Overexpressed tumors (%) |
163 (35.7)
| 115 (37.7) | 48 (31.8) | |
AKT3 values: median [range]
b
|
1.0 (0.4-1.9)
| 0.4 (0.0-3.6) |
0.4 (0.1-7.5)
| |
Underexpressed tumors (%) |
306 (67.1)
| 206 (67.5) | 100 (66.2) |
NS
|
Non-underexpressed tumors (%) | 150 (32.9) | 99 (32.5) | 51 (33.8) | |
GOLPH3 values: median [range]
|
1.4 (0.3-6.7)
|
1.4 (0.5-6.7)
|
1.3 (0.3-5.4)
| |
Non-overexpressed tumors (%) | 369 (80.6) | 242 (78.8) | 127 (84.1) |
NS
|
Overexpressed tumors (%) |
89 (19.4)
| 65 (21.2) | 24 (15.9) | |
P70S6K values: median [range]
|
1.2 (0.0-19.6)
|
1.2 (0.0-19.6)
|
1.1 (0.4-8.0)
| |
Non-overexpressed tumors (%) | 375 (81.9) | 233 (75.9) | 142 (94.0) |
0.0000022
|
Overexpressed tumors (%) |
83 (18.1)
| 74 (24.1) | 9 (6.0) | |
WEE1 values: median [range]
|
0.8 (0.2-6.9)
|
0.8 (0.2-6.9)
|
0.7 (0.3-3.4)
| |
Underexpressed tumors (%) |
84 (18.3)
| 61 (19.9) | 23 (15.2) |
NS
|
Non-underexpressed tumors (%) | 374 (81.7) | 246 (80.1) | 128 (84.8) | |
PIK3R1
c
| ||||
Wild-type (%) | 444 (97.8) | 294 (97.0) | 150 (99.3) |
NS
|
Mutation (%) |
10 (2.2)
| 9 (3.0) | 1 (0.7) | |
AKT1
d
| ||||
Wild-type (%) | 442 (96.7) | 292 (95.4) | 150 (99.3) |
NS
|
Mutation (%) |
15 (3.3)
| 14 (4.6) | 1 (0.7) |
Immunohistochemistry
Survival analysis
Variables | Total population | ER-positive patients | ER-negative patients | PR-positive patients | PR-negative patients | ERBB2-positive patients | ERBB2-negative patients | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
HR (95% CI)a
|
P-valueb
| HR (95% CI)a
|
P-valueb
| HR (95% CI)a
|
P-valueb
| HR (95% CI)a
|
P-valueb
| HR (95% CI)a
|
P-valueb
| HR (95% CI)a
|
P-valueb
| HR (95% CI)a
|
P-valueb
| |
SBR
| ||||||||||||||
I | 1 |
0.041
| 1 |
0.018
| NS | 1 |
0.00068
| NS | NS | 1 |
0.0034
| |||
II | 1.27 (1.01-1.61) | 1.42 (1.06-1.90) | 1.87 (1.30-2.68) | 1.49 (1.14-1.95) | ||||||||||
III | 1.62 (1.02-2.59) | 2.02 (1.13-3.61) | 3.49 (1.70-7.17) | 2.23 (1.31-3.82) | ||||||||||
pN
| ||||||||||||||
0 | 1 |
0.00021
| 1 |
0.0024
| 1 |
0.02
| 1 |
0.0097
| 1 |
0.0055
| 1 |
0.0000076
| NS | |
1-3 | 1.54 (1.22-1.93) | 1.53 (1.16-2.01) | 1.61 (1.08-2.40) | 1.57 (1.12-2.22) | 1.53 (1.13-2.07) | 2.91 (1.82-4.64) | ||||||||
>3 | 2.36 (1.50-3.72) | 2.34 (1.35-4.04) | 2.58 (1.16-5.74) | 2.47 (1.24-4.91) | 2.34 (1.28-4.28) | 8.47 (3.32-21.57) | ||||||||
pT
| ||||||||||||||
≤25 mm | 1 |
0.004
| 1 |
0.00014
| NS | 1 |
0.00096
| NS | NS | 1 |
0.00034
| |||
>25 mm | 1.62 (1.17-2.24) | 2.16 (1.45-3.20) | 2.22 (1.38-3.55) | 1.97 (1.36-2.86) | ||||||||||
ER
| - | - | ||||||||||||
Negative
| NS | NS | NS | NS | NS | |||||||||
Positive
| ||||||||||||||
PR
| - | - | ||||||||||||
Negative
| NS | NS | NS | NS | NS | |||||||||
Positive
| ||||||||||||||
ERBB2
| - | - | ||||||||||||
Negative
| NS | NS | NS | NS | NS | |||||||||
Positive
| ||||||||||||||
PIK3R1
| ||||||||||||||
Underexpression
| 1 |
0.0013
| 1 |
0.0076
| NS | 1 |
0.043
| NS | 1 |
0.018
| 1 |
0.024
| ||
Normal
| 0.59 (0.43-0.81) | 0.62 (0.44-0.88) | 0.65 (0.43-0.99) | 0.40 (0.19-0.85) | 0.66 (0.46-0.95) | |||||||||
Overexpression
| 0.35 (0.18-0.66) | 0.38 (0.19-0.77) | 0.42 (0.18-0.97) | 0.16 (0.03-0.73) | 0.44 (0.21-0.89) | |||||||||
PI3KCA
| ||||||||||||||
Normal
| NS | NS | NS | NS | NS | 1 |
0.051
| NS | ||||||
Mutated
| 0.39 (0.15-1.00) |