Detection of EGFR Gene Mutation in 3665 Patients with NSCLC by ARMS-PCR
Among 3665 NSCLC patients (2042 male and 1623 female) using ARMS-PCR, 1744 cases had EGFR gene mutation, and the total mutation rate was 47.6%. Among 2042 NSCLC male patients, 679 cases had EGFR gene mutations, and the mutation rate was 33.3%. 1065 out of 1623 NSCLC female patients were identified to have EGFR activating mutations, with the mutation rate of 65.6%. Among 3665 NSCLC patients, there was statistically significant (P=0) difference between the female and male cases in the mutation rates of EGFR. And patients, whose age ranged from 45 to 59 years old, had the highest EGFR gene mutation rate among patients of all ages, and the gene mutation rate was 50.9% (611/1200), with statistically significant difference among other different age groups (P=0.005) (There was no statistical significance because patients over 90 years old had fewer cases and a large margin of error.) Sample types of all cases included biopsies of diseased tissues, metastatic tissues, cytological examinations, sputum fluids, whole blood and the mutation rates of EGFR gene were respectively 48.7% (1032/2118), 48.5% (126/260), 48.4% (522/1078), 28.8% (40/139) and 34.3% (24/70), also with statistically significant difference between different sample types (P=0). The characteristics of patients and EGFR mutation rate were shown in Table 1.
Table 1
NCSLC patients EGFR mutation rate and clinical information analysis by ARMS-PCR
Overall patients
|
Case(n)
|
EGFR
|
Positive rate
|
χ²
|
P
|
|
|
Mutated type
|
Wild type
|
|
|
|
Sex
|
|
|
|
|
|
|
Male
|
2042
|
679
|
1363
|
33.3%
|
379.833
|
0.00
|
Female
|
1623
|
1065
|
558
|
65.6%
|
|
|
Age
|
|
|
|
|
|
|
<45
|
200
|
91
|
109
|
45.5%
|
14.302
|
0.005
|
45~59
|
1200
|
611
|
589
|
50.9%
|
|
|
60~74
|
1813
|
852
|
961
|
47.0%
|
|
|
75~89
|
447
|
186
|
261
|
41.6%
|
|
|
>90
|
5
|
4
|
1
|
80.0%
|
|
|
Sample type
|
|
|
|
|
|
|
Tumor Tissue
|
2118
|
1032
|
1086
|
48.7%
|
26.166
|
0.00
|
Metastatic Tissue
|
260
|
126
|
134
|
48.5%
|
|
|
Cytology
|
1078
|
522
|
556
|
48.4%
|
|
|
Sputum
|
139
|
40
|
99
|
28.8%
|
|
|
Blood
|
70
|
24
|
46
|
34.3%
|
|
|
Detection of EGFR Gene Mutation in 296 Patients with Advanced NSCLC by NGS
Totally, 802 NSCLC patients (468 male and 334 female) were further detected by NGS. Among them, 340 cases had EGFR gene mutation, and the total mutation rate was 42.2% (340/802). There were 141 male and 199 female in the EGFR gene mutation cases, and the gene mutation rate was 30.1% (141/468) and 59.6% (199/334) respectively which had statistically significant (P=0) difference. And patients, whose age ranged from 45 to 59 years old, had the highest EGFR gene mutation rate among patients of different ages, and the gene mutation rate was 48% (123/256), without statistically significant difference among different age groups (P=0.13). Sample types of all cases contained biopsies of diseased tissues, metastatic tissues, cytological examinations, sputum fluids, whole blood and the mutation rates of EGFR gene were respectively 46.6%(206/442), 50%(1/2), 63.3%(31/49), 50%(4/8), 32.6%(98/301). And the difference was statistically significant among different sample types(P=0). All characteristics of patients and EGFR mutation rate were shown in Table 2.
Table 2
NCSLC patients EGFR mutation rate and clinical information analysis by NGS
Overall patients
|
Case(n)
|
EGFR
|
Positive rate
|
χ²
|
P
|
|
|
Mutated type
|
Wild type
|
|
|
|
Sex
|
|
|
|
|
|
|
Male
|
468
|
141
|
327
|
30.1%
|
69.23
|
0.00
|
Female
|
334
|
199
|
135
|
59.6%
|
|
|
Age
|
|
|
|
|
|
|
<45
|
32
|
12
|
20
|
37.5%
|
12.087
|
0.13
|
45~59
|
256
|
123
|
133
|
48.0%
|
|
|
60~74
|
379
|
163
|
216
|
43.0%
|
|
|
75~89
|
131
|
42
|
89
|
32.1%
|
|
|
>90
|
4
|
0
|
4
|
0.0%
|
|
|
Sample type
|
|
|
|
|
|
|
Tumor Tissue
|
442
|
206
|
236
|
46.6%
|
24.109
|
0.00
|
Metastatic Tissue
|
2
|
1
|
2
|
50.0%
|
|
|
Cytology
|
49
|
31
|
18
|
63.3%
|
|
|
Sputum
|
8
|
4
|
4
|
50.0%
|
|
|
Blood
|
301
|
98
|
202
|
32.6%
|
|
|
EGFR Gene Mutation unit types detected by ARMS-PCR in 3665 Advanced NSCLC patients
3665 NSCLC patients were detected EGFR Gene Mutation unit types using ARMS-PCR, 1744 cases had mutation units, which including single site mutation in 1654 patients and multiple locus mutation in 90 patients. Total mutation rate was 47.6%. The total number of positive EGFR mutation units was 1838, due to multiple mutation. The total mutation rates of exon 18, 19, 20 and 21 were 1.7%, 20.7%, 3.1% and 24.7% respectively, and their composition ratios were 3.4%, 41.2%, 6.2% and 49.3%. Among seven mutation types, unit points mutation rate was G719X (1.7%), 19 del (20.7%), S768I (0.7%), T790M (1.1%), 20ins (1.3%), L858R (23.4%), L861Q (1.3%) which has been presented in table 3. Among these NSCLC patients using ARMS-PCR, exon 19 deletion and exon 21 mutations were the main EGFR mutation types which accounted for 90.5% of total mutation cases. It is of great significance for guiding targeting therapy with TKI in clinic to those patients with advanced NSCLC.
Table 3
Characteristics of mutation units types of EGFR in 3665 NSCLC patients analysis by ARMS-PCR
Exons
|
Sites
|
Mutations
|
Amino acid sequence changes
|
Unit positive Case (n)
|
Unit Point mutation rate (%)
|
Total mutation rate of exon (%)
|
Composition ratio (%)
|
18
|
3
|
G719X
G719A
G719S
G719C
|
2156G>C
2155G>A
2155G>T
|
62
|
1.7%
|
1.7%
|
3.4%
|
19
|
19
|
19del-
|
|
757
|
20.7%
|
20,7%
|
41.2%
|
E746-A750del
|
2236-2250del
|
|
|
|
|
S752-I759del
|
2254-2277del
|
|
|
|
|
The rest are not listed
|
|
|
|
|
|
20
|
5
|
T790M
|
2369C>T
|
39
|
1.1%
|
3.1%
|
6.2%
|
S768I
|
2303G>T
|
26
|
0.7%
|
|
|
20Ins
|
|
49
|
1.3%
|
|
|
H773-V774insH
D770-N771insG
V769-D770insASV
|
2319-2320insCAC
2310-2311insGGT
2307-2308insgccagcgtg
|
|
|
|
|
21
|
2
|
L858R
|
2573T>G
|
858
|
23.4%
|
24.7%
|
49.3%
|
L861Q
|
2582T>A
|
47
|
1.3%
|
|
|
NSCLC: non-small cell lung cancer; EGFR: epidermal growth factor receptor; §: include 1654 single site mutation patients and 90 multiple locus mutation patients
Note: The mutation of EGFR gene in patients with advanced NSCLC was detected by ARMS-PCR. Unit point mutation rate=the number of mutations per unit point/total number of mutations; total mutation rate of exons=the number of exon mutations/total number of mutations; composition ratio=the number of mutations/ total number of mutations
|
EGFR Gene Mutation unit types detected by NGS in 802 Advanced NSCLC patients
A total 802 advanced NSCLC patients were further detected for EGFR Gene Mutation units types by NGS. Among them, there were 209 single site mutation patients and 131 multiple locus mutation patients. The total mutation rate was 42.4%, and the single site and multiple locus mutation rate were 26.1% and 16.3% respectively. The total mutation rates of exon 18, 19, 20, 21 and gene amplification were 1.9%, 15.5%, 6.9%, 20.9% and 7.2% respectively, and their composition ratios were 3.5%, 29.2%, 13.0%, 49.3% and 13.7%. Among them, unit point mutation rate was G719X (1.4%), E709 (0.5%), 19del (15.5%), S768I (0.5%), T790M (3.9%), 20Ins (2.0%), L858R (19.5%), L861Q (1.4%) which has been presented in table 4. Otherwise, there were 5 rare mutations detected including p.R547*, p.R677C, p.E1079K, p.C624Y and EGFR-PPP1R17. In patients with advanced NSCLC detected by NGS, exon 19 deletion and exon 21 mutation were still the main types of EGFR gene mutations, while mutation rates of exon 19 and 21 detected by NGS were significantly lower than which detected by ARMS-PCR. However, the detection of multiple locus mutation was much higher than that of ARMS-PCR which had important clinical significance for better selection of targeted diagnosis and therapy in advanced NSCLC.
Table 4
Characteristics of mutation units types of EGFR in 802 NSCLC patients analysis by NGS
Exons
|
Sites
|
Mutations
|
Amino acid sequence changes
|
Unit positive Case (n)
|
Unit Point mutation rate (%)
|
Total mutation rate of exon (%)
|
Composition ratio (%)
|
18
|
3
|
G719X
|
G719A
|
G719S
|
G719C
|
E709
|
|
2156G>C
2155G>A
2155G>T
|
11
4
|
1.4%
0.5%
|
1.9%
|
3.5%
|
19
|
19
|
19del-
|
|
124
|
15.5%
|
15.5%
|
29.2%
|
E746-A750del
|
2236-2250del
|
|
|
|
|
S752-I759del
|
2254-2277del
|
|
|
|
|
The rest are not listed
|
|
|
|
|
|
20
|
5
|
T790M
|
2369C>T
|
31
|
3.9%
|
6.9%
|
13.0%
|
S768I
|
2303G>T
|
4
|
0.5%
|
|
|
20Ins
|
|
16
|
2.0%
|
|
|
The rest are not listed
|
|
4
|
0.5%
|
|
|
21
|
2
|
L858R
|
2573T>G
|
156
|
19.5%
|
20.9%
|
49.1%
|
L861Q
|
2582T>A
|
11
|
1.4%
|
|
|
Rare mutation
|
5
|
|
|
5
|
0.6%
|
|
1.2%
|
5
|
|
p.R547*
|
|
|
|
|
|
17
|
|
p.R677C
|
|
|
|
|
|
27
|
|
p.E1079K
|
|
|
|
|
|
15
|
|
p.C624Y
|
|
|
|
|
|
Exon28_Intron4
|
|
EGFR-PPP1R17
|
|
|
|
|
|
Gene amplification
|
|
|
|
58
|
7.2%
|
|
13.7%
|
NSCLC: non-small cell lung cancer; EGFR: epidermal growth factor receptor; §: include 209 single site mutation patients and 131 multiple locus mutation patients
Note: The mutation of EGFR gene in patients with advanced NSCLC was detected by ARMS-PCR. Unit point mutation rate=the number of mutations per unit point/total number of mutations
|
Comparison and analysis of the two methods for detecting mutation rates of EGFR gene
By detecting NSCLC patients with two different ways, ARMS-PCR and NGS, we had compared and analysed the mutation rates of the two methods. Surprisingly, the result had showed that the difference between the EGFR mutation rate in patients determined by ARMS-PCR and those by NGS was significant. Although ARMS-PCR and NGS showed exon 19 deletion and exon 21 mutation were both the main mutation types, the difference of mutation rates was statistically significant. Then we compared the mutation rates of exon 18, 19, 20, 21 and multiple locus mutations by row X list chi-square test. The results showed that the total mutation rates detected by ARMS-PCR were higher than those by NGS (47.6% vs. 42.4%, P<0.05), however, the multiple locus mutation rate detected by ARMS-PCR was much lower than those by NGS (2.7% vs. 16.3%, P<0.001) (table 5). Except for the difference in exon 18 mutation rate which was similar between the two methods, the mutation rate in exon 19, 20 and 21 had statistical difference. This results suggested that ARMS-PCR had higher detection rate and higher sensitivity than NGS in single site gene mutation of EGFR, which could meet the needs of clinicians to select the EGFR-TKI targeted drugs quickly and accurately. In contrast, NGS had significant advantages in detecting multiple and rare mutation, which could meet the needs of personalized medicine better.
Table 5
Comparison of the two methods for detecting mutation rates of EGFR gene
Relevant factors
|
Method
|
Wild type
|
Mutant type
|
Mutation rate
|
χ²
|
P
|
Exon 18
|
ARMS-PCR
NGS
|
3603
787
|
62
15
|
1.8%
1.9%
|
0.124
|
0.725
|
Exon 19
|
ARMS-PCR
|
2908
|
757
|
20.7%
|
11.21
|
0.001
|
NGS
|
678
|
124
|
15.5%
|
|
|
Exon 20
|
ARMS-PCR
|
3551
|
114
|
3.1%
|
25.384
|
0.000
|
NGS
|
747
|
55
|
6.9%
|
|
|
Exon 21
|
ARMS-PCR
|
2760
|
905
|
24.7%
|
5.403
|
0.020
|
NGS
|
635
|
167
|
39.4%
|
|
|
Multiple locus mutations
|
ARMS-PCR
|
|
90
|
2.7%
|
269.319
|
0.000
|
NGS
|
|
131
|
16.3%
|
|
|
Total
|
ARMS-PCR
|
1921
|
1744
|
47.6%
|
7.125
|
0.008
|
NGS
|
462
|
340
|
42.4%
|
|
|
Note: Comparison of mutation rates of exon-related mutation sites detected by the two methods. The mutation rates were compared by row X listchi-square test, and P. |