Original Article

Yonsei Med J 2018 Jan;59(1):13-19

https://doi.org/10.3349/ymj.2018.59.1.13 pISSN: 0513-5796 · eISSN: 1976-2437

Detection of Rare Mutations in

EGFR-ARMS-PCR-Negative Lung Adenocarcinoma
by Sanger Sequencing
Chaoyue Liang3*, Zhuolin Wu4*, Xiaohong Gan5, Yuanbin Liu1,2, You You1,2, Chenxian Liu3,
Chengzhi Zhou1,2, Ying Liang1, Haiyun Mo7, Allen M. Chen5,6, and Jiexia Zhang1,2
1
Guangzhou Institute of Respiratory Disease, Guangzhou, China;
2
Department of Internal Medicine, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China;
3
Department of Pulmonary Medicine, The Brain Hospital of Guangxi Zhuang Autonomous Region, Liuzhou, China;
4
Department of Biomedical Engineering, University of Minnesota, Twin Cities, Minneapolis, USA;
5
Guangzhou Life Technologies Daan Diagnostics Co., Ltd., Guangzhou, China;
6
Mendel Genes, Inc., Manhattan Beach, CA, USA;
7
Department of Health Care, Maternal and Child Health Hospital of Haizhu District, Guangzhou, China.

Purpose: This study aimed to identify potential epidermal growth factor receptor (EGFR) gene mutations in non-small cell lung
cancer that went undetected by amplification refractory mutation system-Scorpion real-time PCR (ARMS-PCR).
Materials and Methods: A total of 200 specimens were obtained from the First Affiliated Hospital of Guangzhou Medical University
from August 2014 to August 2015. In total, 100 ARMS-negative and 100 ARMS-positive specimens were evaluated for EGFR gene
mutations by Sanger sequencing. The methodology and sensitivity of each method and the outcomes of EGFR-tyrosine kinase
inhibitor (TKI) therapy were analyzed.
Results: Among the 100 ARMS-PCR-positive samples, 90 were positive by Sanger sequencing, while 10 cases were considered
negative, because the mutation abundance was less than 10%. Among the 100 negative cases, three were positive for a rare EGFR
mutation by Sanger sequencing. In the curative effect analysis of EGFR-TKIs, the progression-free survival (PFS) analysis based
on ARMS and Sanger sequencing results showed no difference. However, the PFS of patients with a high abundance of EGFR mu-
tation was 12.4 months [95% confidence interval (CI), 11.6−12.4 months], which was significantly higher than that of patients with
a low abundance of mutations detected by Sanger sequencing (95% CI, 10.7−11.3 months) (p<0.001).
Conclusion: The ARMS method demonstrated higher sensitivity than Sanger sequencing, but was prone to missing mutations
due to primer design. Sanger sequencing was able to detect rare EGFR mutations and deemed applicable for confirming EGFR
status. A clinical trial evaluating the efficacy of EGFR-TKIs in patients with rare EGFR mutations is needed.

Key Words: Non-small cell lung cancer, EGFR mutation, Sanger sequencing, ARMS

Received: February 20, 2017 Revised: September 2, 2017 Accepted: September 7, 2017
Co-corresponding authors: Dr. Jiexia Zhang, Guangzhou Institute of Respiratory Disease, Department of Internal Medicine, The First Affiliated Hospital of Guangzhou
Medical University, 151 Yanjiang Road, Guangzhou 510120, Guangdong, China.
Tel: 86-20-83337792, Fax: 86-20-83350363, E-mail: [email protected] and
Dr. Allen M. Chen, Mendel Genes, Inc., Manhattan Beach, CA 90266, USA and Guangzhou Life Technologies Daan Diagnostics Co., Ltd., Guangzhou, China.
Tel: 86-138-2333-7216, Fax: 86-20-83350363, E-mail: [email protected]
*Chaoyue Liang and Zhuolin Wu contributed equally to this work.
•The authors have no financial conflicts of interest.
© Copyright: Yonsei University College of Medicine 2018
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0)
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

www.eymj.org 13
 Novel Rare EGFR Mutations Detected by Sanger Sequencing

INTRODUCTION cally and pathologically confirmed NSCLC and patient con-

sent. The other inclusion criteria were no previous chemotherapy
Lung cancer is a leading malignancy in thoracic oncology that or radiotherapy and no other severe systemic disease. We also
causes a majority of deaths both in China and worldwide.1 The included patients with stage I–III NSCLC who were EGFR
prevalence of epidermal growth factor receptor (EGFR) muta- ARMS-positive and self-medicated with an EGFR-TKI after re-
tions ranges from 5−10% in Caucasians to 60−70% in never- fusing adjuvant chemotherapy and radiotherapy. There were
smoking Asian adenocarcinoma patients, indicating that EGFR 108 male and 92 female patients ranging in age from 48−87
mutation-positive non-small cell lung cancer (NSCLC) may years included in this study. Samples were obtained by CT-
have a unique disease course.2 In fact, NSCLC patients with sen- guided fine-needle aspiration (n=35) or surgery (n=165). All
sitive EGFR mutations are highly responsive to EGFR inhibi- samples were confirmed to be adenocarcinoma. There were 113
tors, including gefitinib and erlotinib, compared with standard stage I, 52 stage II, 29 stage III, and six stage IV cases.
chemotherapy.3,4 Because of inevitable EGFR-tyrosine kinase
inhibitor (TKI) resistance, next-generation EGFR-TKIs have DNA isolation
been developed, and clinical trials have demonstrated a higher DNA was extracted from formalin-fixed, paraffin-embedded
response rate and longer progression-free survival (PFS) and tumor tissue using the QIAamp DNA FFPE Tissue Kit (Qiagen,
overall survival (OS) among previously treated patients with Hilsen, Germany) according to the manufacturer’s recommen-
EGFR-mutant NSCLC.5,6 Therefore, the precise detection of dations. Genomic DNA was stored at -20±5°C after measuring
EGFR mutations plays a key role in the clinical management of the concentration (ng/mL) thereof and absorbance (A260/280
EGFR mutation-positive NSCLC patients. ratio) using a NanoDrop 1000 Spectrophotometer (Thermo
Currently, the methods for detecting EGFR mutations include Fisher Scientific, Cleveland, OH, USA).
Sanger sequencing,7 amplification refractory mutation system
(ARMS),8 pyrosequencing,9 high resolution melting analysis,10 Sanger sequencing
and genome sequencing.11 Sanger sequencing remains the Genomic DNA was amplified with four primer pairs targeting
gold standard for EGFR mutation detection in clinical practice exons 18 to 21 and labeled using the EGFR Mutation Detection
and may detect unknown EGFR mutations. The ARMS method, Kit (Guangzhou Life Technologies Daan Diagnostics Co., Ltd.,
which has also been approved by the China Food and Drug Ad- Guangzhou, China). Sequencing and data collection were per-
ministration (CFDA), is a highly sensitive and reliable method formed using an ABI 3100 Genetic Analyzer (Applied Biosys-
for detecting EGFR mutations. Due to limitations regarding la- tems). All sequence variations were confirmed by multiple in-
bor, time, and expertise requirements, as well as low sensitivity, dependent PCR amplifications and repeat sequencing as
other methods, such as pyrosequencing, high resolution melt- previously described.12 The difference between high and low
ing analysis, and whole genome sequencing, were excluded mutation abundance was as previously defined.13
from the current clinical EGFR mutation analysis.
In this article, we compared patient outcomes based on ARMS qPCR
EGFR mutation analysis by Sanger sequencing and ARMS in Common EGFR mutations (Del19, L858R and L861Q in exon
small specimens: both assays have been approved by the CFDA. 21, G719X in exon 18, S768I in exon 20, and three insertions in
Upon investigation of the survival data, we found that the cu- exon 20) were detected using an ADx-ARMS EGFR 21 Detec-
rative effect of EGFR-TKIs may be better in lung cancer pa- tion Kit (Amoy Diagnostics Co., Ltd., Xiamen, China). qRT-PCR
tients with a high abundance of EGFR mutations than in those was performed in a StepOneTM PCR System (Thermo Fisher
with a low mutation abundance. Sanger sequencing could be Scientific) according to the manufacturer’s instructions.14
useful for EGFR mutation detection, and our data support the
implementation of secondary genetic testing of EGFR muta- Treatment and assessment
tion-negative NSCLC patients with a promising response to Treatment with EGFR-TKIs included oral administration of 250
EGFR-TKI treatment. mg/d gefitinib or 150 mg/d erlotinib, and efficacy was evaluat-
ed after treatment by chest CT of the thoracic lesion according
to standard clinical practice. Patients with stage I–IIIA disease
MATERIALS AND METHODS who self-purchased the targeted drugs after initial disease pro-
gression were included in our analysis. According to Response
Samples collection Evaluation Criteria in Solid Tumors, the effects were defined
A total of 200 NSCLC patients with an equal number of EGFR and categorized as complete response, partial response, stable
ARMS-positive and ARMS-negative cases at The First Affiliated disease, or progressive disease. OS and PFS were defined as the
Hospital of Guangzhou Medical University from August 2014 time interval from the beginning of treatment to documented
to August 2015 were selected as study participants (IRB num- disease progression or death from any cause censored at the
ber: 2016-29). The two main eligibility criteria were radiologi- last follow-up.15

14 https://doi.org/10.3349/ymj.2018.59.1.13
Chaoyue Liang, et al.

Statistical analysis were well balanced among groups.

All the analyses were performed using SPSS software, version
22.0 (IBM Corp., Armonk, NY, USA). The Kaplan-Meier meth- Comparison of mutation detection rates by direct
od was used to compare median PFS after TKI therapy in the sequencing and ARMS
same follow-up group with different detection methods. p- The EGFR mutation statuses of all patients detected by the two
values less than 0.05 were considered statistically significant. methods are summarized in Table 2. Among the 100 ARMS-
positive EGFR samples, Sanger sequencing detected muta-
tions in 90 samples; the other 10 were negative. Among the
RESULTS 100 ARMS-negative samples, three were positive for a mutation
by the Sanger method, and 97 negative samples were con-
Patient characteristics and samples firmed. Based on the positive likelihood ratio (10.409) and the
From August 2014 to August 2015, 200 patients were screened positive predictive value (96.77%), the ARMS-PCR method
and met the enrollment criteria. The patient characteristics can detect EGFR mutations with high efficiency and specificity.
were as follows: 108 male and 92 female patients ranging in Thus, the EGFR mutation rate was higher using ARMS than
age from 48−87 years were included in this study. Samples direct sequencing. Notably, the ARMS method covers only 29
were obtained by CT-guided fine-needle aspiration (n=35) or EGFR mutation hotspots in exons 18–21, and Sanger sequenc-
surgery (n=165). All samples were confirmed to be adenocar- ing detected three coding DNA sequence (CDS) mutations in
cinoma. Disease specimens of TNM stage I to IV were included. ARMS-negative samples: c.2237_2251>TTC (complex),
All patients with an EGFR-sensitive mutation who received a c.2231_2232ins18 (insertion), and c.2515G>A (substitution, po-
first-generation EGFR-TKI were also included. The patient sition 2515, G→A) (Fig. 1).
characteristics are provided in Table 1. Age and TNM stage

Table 1. Clinicopathologic Features of Patients with Lung Adenocarcinoma

Number of patients Number of patients
Total p value
(EGFR positive) (EGFR negative)
Age 0.67
≥60 46 49 95
<60 54 51 105
Gender 0.00*
Male 43 65 108
Female 57 35 92
Smoking history 0.00*
Non-smoker 84 48 132
Smoker 16 52 68
Stage 0.131
I 77 36 113
II 17 35 52
III 4 25 29
IV 2 4 6
EGFR mutation 0.00*
19-del 54 0 -
L858R 46 0 -
EGFR, epidermal growth factor receptor; 19-del, exon 19 deletion; L858R, arginine for leucine substitution at residue 858.
*p<0.01.

Table 2. Mutation Rate with Different Methods in Our Clinic

Sanger sequencing
ARMS Total p value
Mutant Wild-type
Positive (n=100) 90 10 100 0.00*
Negative (n=100) 3 97 100
Total 93 107 200
ARMS, amplification refractory mutation system.
*p<0.05.

https://doi.org/10.3349/ymj.2018.59.1.13 15
 Novel Rare EGFR Mutations Detected by Sanger Sequencing

C
Fig. 1. Results of Sanger sequencing of ARMS-negative samples. (A) Patient 1 had a very rare complex inframe deletion: c.2237_2251>TTC (p.E746_
T751>VP), which was only reported once in the COSMIC database with mutation Id COSM18421. (B) Patient 2 had another complex inframe insertion:
c.2231_2232ins18 (p.K745_E746insIPVAIK, with 18-bp “taaaattcccgtcgctat” inserted), it was reported six times in the COSMIC database with mutation
Id COSM12423. (C) Patient 3 had a rare point mutation: c.2515G>A (p.A839T, COSM13430), which was reported four times. ARMS, amplification refrac-
tory mutation system. CDS, coding DNA sequence.

16 https://doi.org/10.3349/ymj.2018.59.1.13
 Chaoyue Liang, et al.

EGFR mutation status and clinical outcomes years, NSCLC has been managed according to molecular sub-
As a higher EGFR mutation abundance may yield better re- type. In EGFR-mutant NSCLC patients, EGFR-TKI treatment
sults with EGFR-TKI treatment,16 we compared patient out- has greatly increased survival compared to those with EGFR
comes after EGFR-TKI treatment based on ARMS and Sanger wild-type lung cancer.21,22 The predominant EGFR mutations
sequencing. In terms of EGFR-TKI treatment, the median are in exons 18 through 21 and serve as predictors of the effica-
PFSs among EGFR-positive patients detected by Sanger se- cy of EGFR-TKIs. Therefore, the identification of an EGFR mu-
quencing or ARMS were 11.1 months [95% confidence interval tation plays a critical role in NSCLC management.
(CI), 10.6–11.4 months] and 10.9 months (95% CI, 10.7−11.3 Although it has been well recognized that EGFR mutations
months), respectively; this difference was not significant. The are associated with the therapeutic effect of TKIs in NSCLC
PFS was 12.4 months (95% CI, 11.6−12.4 months) for patients patients, current methods do not provide the precision re-
with a high EGFR mutation abundance (n=35), which was quired for clinical practice. Currently, the two main detection
longer than that for patients with a low EGFR mutation abun- methods are ARMS and Sanger sequencing. Although Sanger
dance (95% CI, 10.7−11.3 months) (p<0.001) (Fig. 2). Interest- sequencing remains then gold standard, the ARMS method is
ingly, patients with the c.2237_2251>TTC (complex) or c.2231_ considered an alternative because of its high sensitivity in de-
2232ins18 (insertion) mutation who received EGFR-TKIs had tecting EGFR mutations;23,24 EGFR mutations can be detected
a PFS of 3 months and 6 months, respectively. One patient with in small samples using ARMS. The reason for the high sensitivi-
a c.2515G>A mutation (substitution, position 2515, G→A) was ty with ARMS is its special primer design. One pair of primers
lost to follow-up after 4 months of EGFR-TKI treatment. amplifies a conserved region, and another primer pair targets
the point mutation. ARMS is limited to the detection of known
mutations; each reaction system can only detect the pre-speci-
DISCUSSION fied gene mutation. Therefore, a large number of DNA samples
and primer pairs are needed, making this method expensive,
NSCLC accounts for over 80% of lung cancer cases and in- if an unknown region must be analyzed. Sanger sequencing
cludes adenocarcinoma, large cell carcinoma, and squamous can analyze unknown DNA sequences at relatively low cost;
cell carcinoma.17 Similar to our results, patients who are fe- the biggest problem is the low sensitivity. Mutations are diffi-
male, never smokers, of Asian origin, and present with adeno- cult to detect in specimens with a low content of tumor cells or
carcinoma have a higher EGFR mutation frequency,18,19 and mutant cells. Moreover, noise within peaks can affect calling
this EGFR mutation rate is higher than that in non-adenocar- EGFR mutations. Therefore, Sanger sequencing is suitable for
cinoma patients, who have a rate of less than 10%.20 In recent detecting EGFR mutations in surgical specimens with a high

1.0 1.0

0.8 0.8
Cumulative survival

Cumulative survival

0.6 0.6

p=0.793 p<0.001
0.4 0.4

0.2 Group 0.2 Group

Sanger Sequencing High EGFR mutation abundance
ARMS Low EGFR mutation abundance
0 0

0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
A Time (month) B Time (month)
Fig. 2. PFS curves for patients treated with EGFR-TKIs. (A) PFS of patients with EGFR mutation status detected by Sanger sequencing or ARMS
(p=0.793). (B) PFS of patients with high or low EGFR mutation abundance detected by Sanger sequencing (p<0.001). PFS, progression-free survival;
EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor; ARMS, amplification refractory mutation system.

https://doi.org/10.3349/ymj.2018.59.1.13 17
 Novel Rare EGFR Mutations Detected by Sanger Sequencing

proportion of tumor cells potentially harboring a mutation. The Science and Technology Planning Project of Guangdong Prov-
results of this study suggest that Sanger sequencing is recom- ince (20140212).
mended for EGFR redetection and for initial detection in sur- We thank Dr. Xiaoshun Shi from the Affiliated Cancer Hos-
gical specimens. pital & Institute of Guangzhou Medical University for expert
At least 90% of EGFR mutations occur in exons 19 and 21; medical advice and writing assistance.
the remaining 10% of mutations are in less common sites, and
these are called rare EGFR mutations. With the application of ORCID
EGFR sequencing technology, the discovery of mutations in
exons 18−21 is increasing.25 Few treatment strategies have been Chaoyue Liang https://orcid.org/0000-0002-2833-4139
reported for less common EGFR mutations. For example, first- Jiexia Zhang https://orcid.org/0000-0002-2254-862X
Allen M. Chen https://orcid.org/0000-0002-4914-8802
generation EGFR-TKIs could be used in patients with A763_
Y764insFQEA, an exon 20 insertion.26 In our study, we detect-
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