Detection of Epidermal Growth Factor Receptor (EGFR) Gene Mutation in Formalin Fixed Paraffin Embedded Tissue by Polymerase Chain Reaction-Single Strand Conformational Polymorphism (PCR-SSCP) in Non-Small Cell Lung Cancer in the Northeastern Region of Thailand

Abstract

Background: The use of targeted specific genes in therapeutic and treatment decisions has been considered for lung cancer. The epidermal growth factor receptor (EGFR) gene, which is over expressed in non-small cell lung cancer (NSCLC), was considered as one of the targeted specific genes. EGFR mutations in exons 18–21, which encode a portion of the EGFR kinase domain, were found in NSCLC patients and were associated with the response of EGFRtyrosine kinase inhibitors (EGFR-TKIs). Therefore, a molecular technique for EGFR mutation detection has important benefits for therapy in NSCLC patients. This study aims to determine the EGFR mutations in patients with NSCLC using polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) in exons 18-21. Methods: DNA samples were extracted from formalin fixed paraffin embedded tissues of NSCLC patients who attended hospital. The extracted DNA was used as a template for the EGFR gene amplification. Results: Occurrence of EGFR mutations were found in 29 out of 50 cases (58%).The frequency of EGFR mutations by first PCR at exon 18, 19, 20 and 21 were 6 (12%), 19 (38%) 20 (40%) and at 21 (42%), respectively. By PCR-SSCP, the frequencies of EGFR mutations at exon 18, 19, 20 and 21 were 3(6%), 18(36%), 23(46%) and 13(26%), respectively. All of the mutations found were in agreement with DNA sequencings. Conclusion: The high frequency of EGFR mutations in NSCLC suggests that PCR-SSCP is a efficient screening method and useful for treatment plan.

Keywords: Epidermal growth factor receptor; polymerase chain reaction; single strand conformational polymorphism.

Figure 1

Optimized Condition for PCR of Control and Exons 18 - 21. Lane M for marker, lanes 1, 3, 5, 7 and 9 for blank and lane 2 for control (β-actin, 295 bp.). Lanes 4, 6, 8 and 10 were exon 18 (277 bp.), 19 (192 bp.), 20 (123 bp.) and 21 (212 bp.) respectively. The annealing temperature was different for each exon, for exon 18 (58.5 °C), 19 (57.5 °C), 20 (55.5 °C) and 21 (59.5 °C)

Figure 2

Age Ranges of NSCLC Patients.

Figure 3

PCR-SSCP Patterns on 11% PAG for Exon 19 at 150 v for 150 mins. Lane C, control and NSCLC cases 32, 33, 35, 37 and 38 in lanes 1 - 5 respectively

Figure 4

PCR-SSCP Pattern on 10% PAG for Exon 20 at 150 v for 150 mins. Lane C: control and NSCLC cases 4, 6, 7 and 12 in lanes 1- 4 respectively

Figure 5

DNA Sequencing of Control Normal EGFR Gene Exon 19, 100% Identity

Figure 6

Histogram and Data Base of EGFR Exon 19 (Case No. 38) That had 15 bp Deletion at 2235_2249 Delggaattaagagaagc, Amino Acid Change (E746-A750del.), Detected by DNA Sequencing

Figure 7

DNA Sequencing of Control Normal EGFR Gene Exon 20,100% Identity

Figure 8

DNA Sequencing of Insertion at 2313-ACC-2314; Overlapping Peaks of Normal and Mutant, Insertion of Three Base Nucleotides -GGT- at 2313-ACC-2314, Producing Amino Acid Histamine Insertion at Amino Acid Position 772