The clinical significance and function of EGFR mutation in TKI treatments of NSCLC patients

2.1 Patients

This study obtained approval from the Ethics Committee of Affiliated People’s Hospital of Jiangsu University. A total of 150 NSCLC patients were included in this study from 2013 to 2015. Among the enrolled patients, a total of 118 patients diagnosed with EGFR mutant-positive advanced or recurrent NSCLC received first/second-generation EGFR-TKI treatments, and another 32 NSCLC patients were confirmed as EGFR wild-type. The clinical information of all patients was completed and available. A 5-year follow-up survey was conducted to obtain the survival information of the participants. Serum samples were collected from each patient and stored in liquid nitrogen at - 80 ∘ C for the following analyses.

2.2 EGFR mutation analysis

The EGFR mutations were detected by a Scorpion Amplification Refractory Mutation System, a highly sensitive PCR-based test system covering 29 EGFR mutations from exon 18 to exon 21. DNA was extracted from collected samples with the help of Qiagen QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany). According to the manufacturer’s protocol, the assay was performed with 80 ng DNA and the EGFR 29 Mutation Detection Kit (Amoy Diagnostics, China).

2.3 Cell culture and cell transfection

A549 (EGFR wild-type), PC9 (exon 19 deletion mutation), H1975 (exon 20 T790M mutation), and H3255 (exon 21 L858R point mutation) cell lines were obtained from American Type Culture Collection. All cells were cultured in RPMI 1640 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) at 37 ∘ C with 5% CO 2 .

Cells were transfected with pcDNA 3.1-EGFR or EGFR-siRNA using Lipofectamine 2000 Reagent (Thermo Fisher Scientific, Inc.) following the manufacturer’s instructions, to overexpress or silence EGFR, respectively.

2.4 Erlotinib resistance assessment

Cells (A549, PC9, H1975, and H3225 cells) were seeded into 96-well plates supplemented with FBS containing the culture medium supplemented with FBS and exposed to various concentrations of erlotinib. After incubation for 0, 24, 48, and 72 h, CCK8 reagent (Dojindo, Japan) was added to each well, followed by a 1-h incubation. Thereafter, the absorbance at 450 nm of each well was detected using a microplate reader. The IC 50 values of erlotinib for the cells were obtained by incubating with 0–10 μ M erlotinib for 72 h.

The transfected cells were collected in the PBS buffer solution and lysed with RIPA buffer (Life Technologies, USA). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane for 2 h and then blocked with 5% nonfat milk for 1 h. Thereafter, the membrane was incubated with the primary antibody at 4 ∘ C overnight. After washing with PBS solution thrice, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Finally, the membrane was mixed with an electrochemiluminescence chromogenic kit (Beyotime Institute of Biotechnology, China) and analyzed by Bio-Rad ChemiDoc Touch Imaging System.

2.6 Statistical analysis

All data are represented as mean value ± SD and analyzed by GraphPad Prism 7.0 Software (Graphpad, USA) and SPSS 20.0 Software (IBM, USA). Differences among multiple groups were assessed by one-way ANOVA followed by the Tukey’s post-hoc test. The survival of patients with NSCLC with different EGFR mutations was analyzed by the Kaplan-Meier method followed by a log-rank test. 0.05 was considered to indicate statistical significance.

OPEN IN VIEWER

OPEN IN VIEWER

3.1 EGFR mutations were closely associated with the degree of differentiation and the histological subtypes of NSCLC cases

The clinical features of the recruited NSCLC patients are summarized in Table 1. Of the recruited 150 patients, 118 found to possess EGFR mutations and 32 without EGFR mutations. The EGFR mutantion subtypes were exon 19 deletion ( n = 56), exon 20 T790M mutation ( n = 19), and exon 21 L858R point mutation ( n = 43). Notably, these mutations were significantly related to the differentiation degree ( P = 0.031) and adenocarcinoma histology ( P = 0.028) in the studied cases (Table 1).

OPEN IN VIEWER

OPEN IN VIEWER

The survival of the recruited patients has been depicted by the Kaplan-Meier curve. Patients without any EGFR mutation showed a relatively poorer survival rate than those with EGFR mutation (log-rank P = 0.027, Fig. 1). EGFR mutation was identified as an independent prognostic factor for patients with NSCLC with the HR value of 2.599, 95% CI of 1.073–6.291 ( P = 0.034, Table 2), however the prognostic value of other characteristics, including age, gender, differentiation degree, smoking, and histological subtypes was found to be insignificant (all $P>$ 0.05, Table 2). The significantly different prognosis of the recruited patients may reflect their resistance to TKI, implying the potential role of EGFR mutation in TKI resistance.

NSCLC cell lines with different EGFR mutation subtypes were employed to evaluate the function of EGFR mutation in erlotinib resistance. In the presence of various concentrations of erlotinib, the H1975 cell (exon 20 T790M mutation) showed a relatively higher IC 50 value of 0.14 μ M, while the IC 50 values of A549, PC9, and H3255 cells were 0.072, 0.084, and 0.096 μ M, respectively (Fig. 2). Consistently, the proliferation of A549, PC9, and H3255 cells was dramatically inhibited by erlotinib treatment, while H1975 was not affected by erlotinib (Fig. 3). These results imply that exon 20 T790M mutation might be involved in the resistance to erlotinib and the underlying mechanism was further evaluated.

3.4 EGFR is involved in erlotinib resistance through the PI3K/Akt signaling pathway

The knockdown of EGFR in H1975 cells showed a notable inhibitory effect on the expression level of PI3K and Akt proteins. In contrast, the overexpression of EGFR enhanced the PI3K and Akt levels (Fig. 4). Therefore, it was speculated that EGFR participated in the resistance to erlotinib through the PI3K/Akt signaling pathway.