Yonsei Med J.  2018 Jan;59(1):13-19. 10.3349/ymj.2018.59.1.13.

Detection of Rare Mutations in EGFR-ARMS-PCR-Negative Lung Adenocarcinoma by Sanger Sequencing

Affiliations
  • 1Guangzhou Institute of Respiratory Disease, Guangzhou, China. drzhangjx@126.com
  • 2Department of Internal Medicine, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
  • 3Department of Pulmonary Medicine, The Brain Hospital of Guangxi Zhuang Autonomous Region, Liuzhou, China.
  • 4Department of Biomedical Engineering, University of Minnesota, Twin Cities, Minneapolis, USA.
  • 5Guangzhou Life Technologies Daan Diagnostics Co., Ltd., Guangzhou, China. chen@mendel-genes.com
  • 6Mendel Genes, Inc., Manhattan Beach, CA, USA.
  • 7Department of Health Care, Maternal and Child Health Hospital of Haizhu District, Guangzhou, China.

Abstract

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 mutation 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.

Keyword

Non-small cell lung cancer; EGFR mutation; Sanger sequencing; ARMS

MeSH Terms

Adenocarcinoma/*genetics/pathology
Aged
Aged, 80 and over
Animals
Base Sequence
Disease-Free Survival
Female
Humans
Lung Neoplasms/*genetics/pathology
Male
Middle Aged
Mutation/*genetics
Mutation Rate
Real-Time Polymerase Chain Reaction/*methods
Receptor, Epidermal Growth Factor/*genetics
Sequence Analysis, DNA/*methods
Treatment Outcome
Receptor, Epidermal Growth Factor

Figure

  • 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 refractory mutation system. CDS, coding DNA sequence.

  • 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.


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