Background
Reference (year) | No. of patients | Method | Mutation frequencies | Region | Prognostic value |
---|---|---|---|---|---|
[2] Gao J., et al. (2011) | 273 | Direct sequencing |
KRAS (38.5%); BRAF (5.1%) | Chinese | |
[24] Li H.T., et al. (2011) | 200 | Pyrosequencing |
KRAS (31.5%); BRAF (7.0%); PIK3CA (12.5%) | Chinese |
KRAS and PIK3CA bi-mutations were more likely to develop liver metastases. |
[25] Shen H., et al. (2011) | 118 | Pyrosequencing |
KRAS (34.7%); BRAF (1.7%) | Chinese | |
[26] Liou J.M., et al. (2011) | 314 | Direct sequencing |
KRAS (20.7%); BRAF (3.8%) | Taiwan |
BRAF mutation was associated with worse overall survival. |
[27] Mao C., et al. (2012) | 69 | Direct sequencing |
KRAS (43.9%); BRAF (25.4%); PIK3CA (8.2%) | Chinese | |
[28] Hsieh L.L., et al. (2012) | 182 | Direct sequencing & HRM |
KRAS (33.5%); BRAF (1.1%); PIK3CA (7.1%) | Taiwan | |
[29] Zhu Y.F., et al. (2012) | 60 | Direct sequencing |
PIK3CA (21.6%) | Chinese | High PI3K expression was associated with CRC metastases. |
[30] Li Z., et al. (2012) | 78 | Direct sequencing |
KRAS (33.3%) | Chinese |
KRAS mutations were associated with poor survival and liver metastasis. |
[31] Shen Y., et al. (2013) | 676 | Direct sequencing |
KRAS (35.9%); BRAF (6.96%); PIK3CA (9.9%) | Chinese | |
[32] Pu X., et al. (2013) | 115 | Direct sequencing |
KRAS (32.2%); BRAF (3.5%) | Chinese | |
[33] Wang J., et al. (2013) | 574 | Direct sequencing | KRAS (34.2%) | Chinese | |
[34] Chang Y.S., et al. (2013) | 165 | HRM |
KRAS (36.97%); BRAF (4.24%) | Taiwan |
KRAS mutation was associated with poor survival. |
Methods
Patients and tumor samples
KRAS exon 2 | BRAF exon 15 | PIK3CA exon 9&20* | PI3K/RAS-RAF pathway* | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
No. patients (214/212) | No (%) | Yes (%) | P | No (%) | Yes (%) | P | No (%) | Yes (%) | P | No (%) | Yes (%) | P | ||
Sex
| male | 127 (126) | 73 (61.9) | 54 (56.3) | 0.406a
| 122 (59.5) | 5 (55.6) | 1.000b
| 109 (58.6) | 17 (65.4) | 0.509a
| 60 (61.2) | 66 (57.9) | 0.623a
|
female | 87 (86) | 45 (38.1) | 42 (43.8) | 83 (40.5) | 4 (44.4) | 77 (41.4) | 9 (34.6) | 38 (38.8) | 48 (42.1) | |||||
Age
| 68.0 | 67.1 | 69.1 | 0.286d
| 68.0 | 66.9 | 0.801d
| 68.7 | 64.6 | 0.133d
| 67.8 | 68.4 | 0.728d
| |
Location
| colon | 126 (124) | 73 (61.9) | 53 (55.2) | 0.325a
| 117 (57.1) | 9 (100.0) |
0.011
b
| 103 (55.4) | 21 (80.8) |
0.014
a
| 54 (55.1) | 70 (61.4) | 0.353a
|
rectum | 88 (88) | 45 (38.1) | 43 (44.8) | 88 (42.9) | 0 (0) | 83 (44.6) | 5 (19.2) | 44 (44.9) | 44 (38.6) | |||||
Differentiation
| well | 29 (29) | 19 (16.1) | 10 (10.4) | 0.912c
| 28 (13.7) | 1 (11.1) | 0.131c
| 22 (11.8) | 7 (26.9) | 0.215c
| 15 (15.3) | 14 (12.3) | 0.521c
|
moderate | 163 (161) | 83 (70.3) | 80 (83.3) | 159 (77.6) | 4 (44.4) | 145 (78.0) | 16 (61.5) | 73 (74.5) | 88 (77.2) | |||||
poor | 7 (7) | 7 (5.9) | 0 (0) | 5 (2.4) | 2 (22.2) | 5 (2.7) | 2 (7.7) | 3 (3.1) | 4 (3.5) | |||||
missing | 15 (15) | 9 (7.6) | 6 (6.3) | 13 (6.3) | 2 (22.2) | 14 (7.5) | 1 (3.8) | 7 (7.1) | 8 (7.0) | |||||
Tumor diameter
| <5 cm | 103 (102) | 53 (44.9) | 50 (52.1) | 0.254c
| 101 (49.3) | 2 (22.2) | 0.171b
| 93 (50.0) | 9 (34.6) | 0.172a
| 46 (46.9) | 56 (49.1) | 0.710a
|
> = 5 cm | 108 (107) | 64 (54.2) | 44 (45.8) | 101 (49.3) | 7 (77.8) | 91 (48.9) | 16 (61.5) | 51 (52.0) | 56 (49.150.0) | |||||
missing | 3 (3) | 1 (0.8) | 2 (2.1) | 3 (1.5) | 0 (0) | 2 (1.1) | 1 (3.8) | 1 (1.0) | 2 (1.8) | |||||
TNM-stage
| I | 32 (32) | 15 (12.7) | 17 (17.7) | 0.828c
| 32 (15.6) | 0 (0) |
0.007
c
| 26 (14.0) | 6 (23.1) | 0.433c
| 13 (13.3) | 19 (16.7) | 0.231c
|
II | 78 (77) | 50 (42.4) | 28 (29.2) | 76 (37.1) | 2 (22.2) | 69 (37.1) | 8 (30.8) | 44 (44.9) | 33 (28.9) | |||||
III | 82 (81) | 38 (32.2) | 44 (45.8) | 79 (38.5) | 3 (33.3) | 70 (37.6) | 11 (42.3) | 31 (31.6) | 50 (43.9) | |||||
IV | 19 (19) | 13 (11.0) | 6 (6.3) | 15 (7.3) | 4 (44,4) | 18 (9.7) | 1 (3.8) | 8 (8.2) | 11 (9.6) | |||||
missing | 3 (3) | 2 (1.7) | 1 (1.0) | 3 (1.5) | 0 (0) | 3 (1.6) | 0 (0) | 2 (2.0) | 1 (0.9) | |||||
T
| T1 | 5 (5) | 4 (3.4) | 1 (1.0) | 0.236c
| 5 (2.4) | 0 (0) | 0.057c
| 5 (2.7) | 0 (0) | 0.808c
| 4 (4.1) | 1 (0.9) | 0.724c
|
T2 | 35 (35) | 15 (12.7) | 20 (20.8) | 35 (17.1) | 0 (0) | 29 (15.6) | 6 (23.1) | 13 (13.3) | 22 (19.3) | |||||
T3 | 167 (166) | 93 (78.8) | 74 (77.1) | 159 (77.6) | 8 (88.9) | 147 (79.0) | 19 (73.1) | 77 (78.6) | 89 (78.1) | |||||
T4 | 5 (4) | 4 (3.4) | 1 (1.0) | 4 (2.0) | 1 (11.1) | 3 (1.6) | 1 (3.8) | 2 (2.0) | 2 (1.8) | |||||
missing | 2 (2) | 2 (1.7) | 0 (0) | 2 (1.0) | 0 (0) | 2 (1.1) | 0 (0) | 2 (2.0) | 0 (0) | |||||
N
| N(−) | 115 (114) | 70 (59.3) | 45 (46.9) |
0.050
a
| 113 (55.1) | 2 (22.2) | 0.083b
| 99 (53.2) | 15 (57.7) | 0.710a
| 61 (62.2) | 53 (46.5) |
0.013
a
|
N(+) | 97 (96) | 46 (39.0) | 51 (53.1) | 90 (43.9) | 7 (77.8) | 85 (45.7) | 11 (42.3) | 35 (35.7) | 61 (53.5) | |||||
missing | 2 (2) | 2 (1.7) | 0 (0) | 2 (1.0) | 0 (0) | 2 (1.1) | 0 (0) | 2 (2.0) | 0 (0) | |||||
Metastases
| M(−) | 163 (161) | 88 (74.6) | 75 (78.1) | 0.367a
| 159 (77.6) | 4 (44.4) |
0.037
b
| 144 (77.4) | 17 (65.4) | 1.808a
| 77 (78.6) | 84 (73.7) | 0.689a
|
M(+) | 51 (51) | 30 (25.4) | 21 (21.9) | 46 (22.4) | 5 (55.6) | 42 (22.6) | 9 (34.6) | 21 (21.4) | 30 (26.3) | |||||
Synchronous metastases
| M(−) | 193 (191) | 104 (88.1) | 89 (92.7) | 0.224a
| 188 (91.7) | 5 (55.6) |
0.004
b
| 166 (89.2) | 25 (96.2) | 0.479b
| 89 (90.8) | 102 (89.5) | 0.708a
|
M(+) | 19 (19) | 13 (11.0) | 6 (6.3) | 15 (7.3) | 4 (44.4) | 18 (9.7) | 1 (3.8) | 8 (8.2) | 11 (9.6) | |||||
missing | 2 (2) | 1 (0.8) | 1 (1.0) | 2 (1.0) | 0 (0) | 2 (1.1) | 0 (0) | 1 (1.0) | 1 (1.0) | |||||
Metachronous metastases
| M(−) | 176 (174) | 95 (80.5) | 81 (84.4) | 0.462a
| 171 (83.4) | 5 (55.6) | 0.055b
| 156 (83.9) | 18 (69.2) | 0.097b
| 82 (83.7) | 92 (80.7) | 0.574a
|
M(+) | 38 (38) | 23 (19.5) | 15 (15.6) | 34 (16.6) | 4 (44.4) | 30 (16.1) | 8 (30.8) | 16 (16.3) | 22 (19.3) |
DNA extraction and mutation analysis
Genes | Primers (sequence 5’-- > 3’) |
---|---|
KRAS
| |
Exon 2
|
F: TTAACCTTATGTGTGACATGTTCTAA |
R: ATCAAAGAATGGTCCTGCAC | |
BRAF
| |
Exon 15
|
F: CTTTACTTACTACACCTCAG |
R: TAACTCAGCAGCATCTCAGG | |
PIK3CA
| |
Exon 9
|
F: AGTAACAGACTAGCTAGAGACAAT |
R: CATGCTGAGATCAGCCAAAT | |
Exon 20
|
F: ATGATGCTTGGCTCTGGAAT |
R: TGTGGAATCCAGAGTGAGCTT |
Statistical analysis
Results
Frequency and distribution of KRAS, BRAF and PIK3CAmutations
Nucleotide | Amino acid | Case (total) | % | ||
---|---|---|---|---|---|
KRAS
| 96 (214) | 44.9 | |||
exon 2 | 34G > A | G12S | 2 | ||
34G > C | G12R | 1 | |||
34G > T | G12C | 5 | |||
35G > A | G12D | 34 | |||
35G > C | G12A | 8 | |||
35G > T | G12V | 20 | |||
35G > T & 35G > A | G12V & G12D | 1 | |||
37G > T | G13C | 2 | |||
38G > A | G13D | 23 | |||
BRAF
| 9 (214) | 4.2 | |||
exon 15 | 1799 T > A | V600E | 8 | ||
1801A > G | K601E | 1 | |||
PIK3CA
| 26 (212)* | 12.3 | |||
exon 9 | 12 | 5.7 | |||
1624G > A | E542K | 1 | |||
1633G > A | E545K | 7 | |||
1634A > C | E545A | 1 | |||
1636C > A | Q546K | 2 | |||
1637A > G | Q546R | 1 | |||
exon 20 | 14 | 6.6 | |||
3062A > T | Y1021F | 2 | |||
3139C > T | H1047Y | 1 | |||
3140A > G | H1047R | 8 | |||
3140A > T | H1047L | 1 | |||
3145G > C | G1049R | 1 | |||
3155C > A | T1052K | 1 |
PI3K/RAS-RAF pathway mutations and clinicopathological characteristics
BRAF codon 600 mutation | ||||
---|---|---|---|---|
No (%) | Yes (%) | P | ||
Sex
| male | 71 (60.2) | 5 (62.5) | 1.000b
|
female | 47 (39.8) | 3 (37.5) | ||
Age
| 69.4 | 65.4 | 0.664d
| |
Differentiation
| well | 15 (12.7) | 1 (12.5) | 0.192c
|
moderate | 91 (77.1) | 3 (37.5) | ||
poor | 5 (4.2) | 2 (25.0) | ||
missing | 7 (5.9) | 2 (25.0) | ||
Tumor diameter
| <5 cm | 49 (41.5) | 2 (25.0) | 0.469b
|
> = 5 cm | 67 (56.8) | 6 (75.0) | ||
missing | 2 (1.7) | 0 (0) | ||
TNM-stage
| I | 9 (7.6) | 0 (0) |
0.014
c
|
II | 50 (42.4) | 2 (25.0) | ||
III | 49 (42.4) | 2 (25.0) | ||
IV | 7 (5.9) | 4 (50.0) | ||
missing | 2 (1.7) | 0 (0) | ||
T
| T1 | 0 (0) | 0 (0) | 0.106c
|
T2 | 9 (7.6) | 0 (0) | ||
T3 | 105 (89.0) | 7 (87.5) | ||
T4 | 2 (1.7) | 1 (12.5) | ||
missing | 2 (1.7) | 0 (0) | ||
N
| N(−) | 62 (52.5) | 2 (25.0) | 0.157b
|
N(+) | 55 (46.6) | 6 (75.0) | ||
missing | 1 (0.8) | 0 (0) | ||
Metastases
| M(−) | 91 (77.1) | 3 (37.5) |
0.025
b
|
M(+) | 27 (22.9) | 5 (62.5) | ||
Synchronous metastases
| M(−) | 110 (93.2) | 4 (50.0) |
0.002
b
|
M(+) | 7 (5.9) | 4 (50.0) | ||
missing | 1 (0.8) | 0 (0) | ||
Metachronous metastases
| M(−) | 96 (81.4) | 4 (50.0) | 0.056b
|
M(+) | 22 (18.6) | 4 (50.0) |
Prognostic value of BRAF and KARScodon 13 mutations
Univariate and multivariate analysis of outcome predictors
Variables | Univariate analysis | Multivariate analysis | ||
---|---|---|---|---|
HR (95% CI) | P | HR (95% CI) | P | |
Age | 0.995 | |||
<=65 | 1.0 | |||
>65 | 1.0 (0.5-2.108) | |||
Sex |
0.009
|
0.009
| ||
Female | 1.0 | 1.0 | ||
Male | 3.0 (1.3-7.0) | 3.3 (1.3-7.8) | ||
Differentiation | 0.160 | |||
well | 1.0 | |||
moderate | 0.5 (0.2-1.1) | 0.089 | ||
poor | 1.0 (0.3-3.9) | 0.962 | ||
Lymphnode examined | 0.052 | |||
>12 | 1.0 | |||
<=12 | 2.0 (1.0-4.1) | |||
Tumor diameter | 0.188 | |||
<5 cm | 1.0 | |||
> = 5 cm | 1.7 (0.8-3.5) | |||
TNM-stage |
<0.001
|
0.017
| ||
I | 1.0 | 1.0 | ||
II | 1.4 (0.2-10.8) | 0.749 | 1.3 (0.2-9.9) | |
III | 1.8 (0.2-13.9) | 0.568 | 2.1 (0.3-15.9) | |
IV | 9.7 (1.2-78.3) | 0.032 | 5.8 (0.7-47.8) | |
KRAS status | 0.795 | |||
wt | 1.0 | |||
mutant | 1.1 (0.5-2.2) | |||
BRAF V600E status |
<0.001
|
0.004
| ||
wt | 1.0 | 1.0 | ||
mutant | 5.1 (2.1-12.4) | 4.2 (1.6-11.0) | ||
PIK3CA status | 0.727 | |||
wt | 1.0 | |||
mutant | 1.2 (0.5-2.8) |
Univariate analysis | Multivariate analysis | ||||||
---|---|---|---|---|---|---|---|
KRAS
|
BRAF
| Total N | No. of events | HR (95% CI) | P | HR (95% CI) | P |
wt | wt | 109 | 22 | 1.0 | 1.0 | ||
All codon 12 mutants | wt | 71 | 20 | 1.6 (0.9-2.9) | 0.139 | 1.4 (0.8-2.6) | 0.247 |
All codon 13 mutants | wt | 25 | 10 | 2.5 (1.2-5.2) |
0.019
| 2.7 (1.3-5.7) |
0.011
|
Univariate analysis | Multivariate analysis | ||||||
---|---|---|---|---|---|---|---|
KRAS
|
BRAF
| Total N | No. of events | HR (95% CI) | P | HR (95% CI) | P |
wt | wt | 109 | 22 | 1 | 1 | ||
c.38G > A | wt | 23 | 9 | 2.4 (1.1-5.3) |
0.026
| 2.6 (1.2-5.8) |
0.016
|
c.35G > A | wt | 34 | 9 | 1.4 (0.6-3.0) | 0.425 | 1.1 (0.5-2.4) | 0.853 |
c.35G > T | wt | 20 | 5 | 1.4 (0.5-3.6) | 0.546 | 1.5 (0.6-4.0) | 0.408 |