Fig. 1Part of a direct sequencing chromatogram showing codon 12 to codon 14. No secondary peaks are identified in case no. 92 or case no. 133 compared with case no. 55 and case no. 76, which have missense mutations in codon 12.
Fig. 2(A) Proportion of KRAS mutation genotypes in this study (n=23). All mutatons are located in codon 12. (B) COSMIC data on the proportion of lung cancer KRAS mutations (n=3,743). Proportion of each genotype in this study is similar to COSMIC data. (C) COSMIC data on the proportion of large intestine cancer KRAS mutation (n=17,853). Proportion of genotype is also similar between lung cancer and large intestine cancer.
Fig. 3Hematoxylin and eosin staining. (A) Case no. 92. Heavy inflammatory infiltrates are seen around the tumor glands. DNA of inflammatory cells can lower the proportion of tumor DNA. (B) Case no. 133. Tumor cells are sparsely distributed within the fibrous stroma. Tumor volume is reduced after neoadjuvant chemoradiotherapy.
Fig. 4Comparison of conventional next-generation sequencing (NGS) and mutant-enriched NGS. While more than 10% of mutant DNA is needed for conventional NGS to detect mutations, mutant-enriched NGS can detect as low as 0.05% of mutant DNA.
Table 1.Next-generation sequencing results
|
Sample
|
Depth |
Mutant (%)
|
Character |
|
n |
Application |
G12C |
G12S |
G12V |
1 |
2 |
Insight |
9,215 |
0 |
0 |
0 |
Wild |
2 |
2 |
Normal |
17,592 |
0 |
0 |
0 |
|
3 |
92 |
Insight |
8,714 |
0 |
0 |
90.37 |
G12V |
4 |
92 |
Normal |
16,983 |
0 |
0 |
2.07 |
|
5 |
133 |
Insight |
10,016 |
89.34 |
0 |
0 |
G12C |
6 |
133 |
Normal |
17,339 |
4 |
0 |
0 |
|
7 |
A549 |
Insight |
11,220 |
0 |
95.38 |
0 |
G12S |
8 |
Hela |
Insight |
12,227 |
0 |
0 |
0 |
Wild |
Table 2.Clinicopathologic data of KRAS-mutated adenocarcinoma cases
Case No. |
Result |
Sex |
Age (yr) |
Pattern |
Size (cm) |
T stage |
N stage |
M stage |
Stage |
Smoking |
Chemotherapy |
1 |
c.35G>C (p.G12A) |
F |
61 |
A&S |
1.8 |
2a |
2 |
1b |
IV |
Never |
Yes |
5 |
c.35G>T (p.G12V) |
M |
77 |
NA |
2.5 |
1b |
2 |
1b |
IV |
Former |
Yes |
6 |
c.35G>A (p.G12D) |
F |
67 |
S |
3.5 |
2a |
1 |
0 |
IIA |
Never |
No |
11 |
c.35G>C (p.G12A) |
M |
59 |
NA |
4.3 |
2a |
1 |
1b |
IV |
Former |
No |
16 |
c.35G>A (p.G12D) |
M |
61 |
S |
6 |
2b |
0 |
0 |
IIA |
Current |
No |
26 |
c.35G>A (p.G12D) |
F |
52 |
NA |
8 |
3 |
0 |
1a |
IV |
Never |
Yes |
29 |
c.35G>A (p.G12D) |
M |
74 |
A |
3.7 |
4 |
0 |
1a |
IV |
Current |
Yes |
38 |
c.35G>A (p.G12D) |
M |
52 |
A |
2.2 |
1b |
2 |
0 |
IIIA |
Current |
No |
49 |
c.35G>T (p.G12V) |
M |
67 |
A |
3.4 |
2a |
0 |
1b |
IV |
Former |
No |
51 |
c.34G>T (p.G12C) |
M |
56 |
A&S |
3 |
1b |
1 |
1b |
IV |
Current |
No |
52 |
c.35G>A (p.G12D) |
M |
58 |
P |
5.5 |
3 |
3 |
1b |
IV |
Current |
No |
54 |
c.34G>T (p.G12C) |
M |
66 |
A |
5.9 |
4 |
3 |
1b |
IV |
Current |
No |
55 |
c.34G>T (p.G12C) |
M |
55 |
S |
7.8 |
3 |
0 |
0 |
IIB |
Former |
No |
61 |
c.35G>T (p.G12V) |
M |
70 |
NA |
4.1 |
2a |
2 |
1a |
IV |
Current |
No |
72 |
c.35G>A (p.G12D) |
M |
73 |
A&P |
2.4 |
1b |
1 |
1a |
IV |
Former |
Yes |
76 |
c.35G>A (p.G12D) |
M |
66 |
A |
2.5 |
1b |
1 |
0 |
IIA |
Current |
No |
80 |
c.35G>A (p.G12D) |
M |
73 |
A&S |
4 |
4 |
3 |
1b |
IV |
Former |
No |
81 |
c.35G>T (p.G12V) |
M |
75 |
S |
3.5 |
4 |
3 |
1a |
IV |
Former |
No |
92a
|
c.35G>T (p.G12V) |
F |
69 |
A |
1 |
1a |
0 |
0 |
IA |
Never |
No |
96 |
c.35G>C (p.G12A) |
M |
55 |
A |
4.3 |
2a |
0 |
1a |
IV |
Current |
No |
113 |
c.35G>T (p.G12V) |
M |
70 |
P |
1.9 |
1a |
3 |
1b |
IV |
Former |
No |
133a
|
c.34G>T (p.G12C) |
M |
55 |
A |
1.8 |
2a |
2 |
0 |
IIIA |
Current |
Yes |
134 |
c.34G>A (p.G12S) |
F |
58 |
A |
6.9 |
2a |
3 |
1b |
IV |
Never |
No |
Table 3.Statistical differences between KRAS-mutated and wild-type cases
|
|
Mutation |
Wild type |
p-value |
Gender |
Male |
18 (78) |
60 (54) |
.032 |
|
Female |
5 (22) |
51 (46) |
|
Age (yr) |
Mean |
63.9 |
62.6 |
.517 |
|
≤ 60 |
9 (39) |
44 (40) |
.964 |
|
>60 |
14 (61) |
67 (60) |
|
Smoking |
Never |
5 (22) |
56 (50) |
.011 |
|
Former |
8 (35) |
35 (32) |
|
|
Current |
10 (43) |
20 (18) |
|
Type |
Adenocarcinoma |
22 (96) |
101 (91) |
.644 |
|
Mucinous adenocarcinoma |
1 (4) |
3 (3) |
|
|
Squamous cell carcinoma |
0 (0) |
3 (3) |
|
|
Others |
0 (0) |
4 (4) |
|
Size (cm) |
≤ 3 |
9 (39) |
47 (42) |
.766 |
|
>3 |
14 (61) |
64 (58) |
|
T stage |
0-1 |
7 (30) |
42 (38) |
.921 |
|
2 |
9 (39) |
42 (38) |
|
|
3 |
3 (13) |
14 (13) |
|
|
4 |
4 (17) |
13 (12) |
|
N stage |
0 |
7 (30) |
51 (46) |
.446 |
|
1 |
5 (22) |
13 (12) |
|
|
2 |
5 (22) |
23 (21) |
|
|
3 |
6 (26) |
24 (22) |
|
M stage |
0 |
7 (30) |
61 (55) |
.032 |
|
1 |
16 (70) |
50 (45) |
|
Table 4.Recent studies examining the relationship between survival outcomes and KRAS mutation in non-small cell lung cancer
|
Country |
KRAS(+) (%) |
No, of cases |
Method |
Patients |
Survival relation |
Guan et al. (2013) [18] |
China |
5 |
1,928 |
DS or high resolution melting analysis |
Operable and inoperable |
Yes |
Marchetti et al. (2009) [24] |
Italy |
36 |
83 |
Mutant-enriched sequencing |
Adenocarcinoma |
Yes |
Treated with EGFR-TKI |
Sun et al. (2013) [25] |
Korea |
8 |
484 |
DS |
Advanced stage |
Yes |
Johnson et al. (2013) [26] |
USA |
23 |
1,036 |
DS or mass-spectrometry-based genotyping |
Advanced stage |
Yes |
Adenocarcinoma |
Sonobe et al. (2012) [22] |
Japan |
18 |
180 |
RFLP |
Resection |
No |
Adenocarcinoma |
Cadranel et al. (2012) [17] |
France |
14 |
522 |
DS or nested sequencing |
Advanced stage |
Yes |
Treated with EGFR-TKI |
Kerner et al. (2013) [27] |
The Netherlands |
30 |
442 |
DS |
Operable and inoperable |
No |
Kim et al. (2012) [19] |
Korea |
4 |
229 |
DS |
Never-smoker |
Yes |
Ragusa et al. (2013) [21] |
Italy |
17 |
230 |
DS |
Resection |
No |
Kosaka et al. (2009) [20] |
Japan |
13 |
397 |
DS |
Resection |
No |
Adenocarcinoma |
Lim et al. (2009) [28] |
Singapore |
6 |
88 |
Whole genome amplification and DS |
Advanced stage |
Yes |
Liu et al. (2010) [29] |
Taiwan |
5 |
73 |
DS |
Resection |
No |
Scoccianti et al. (2012) [30] |
Europe |
19 |
250 |
Mutant-enriched sequencing |
Resection |
No |