Introduction
Lung cancer is the leading cause of cancer-related deaths worldwide, and especially in China. The majority of these deaths are due to non-small cell lung cancer (NSCLC), which is the most common histologic type of lung cancer (1). It is current practice to treat advanced NSCLC with platinum based chemotherapy, although treatment outcomes are particularly poor (2,3). Therefore, target therapy for patients with advanced NSCLC are currently being evaluated.
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (TK) that is frequently overexpressed and plays a central role in the development of NSCLC (4,5). Abnormal activation of EGFR can promote tumor cell proliferation, differentiation and migration. EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, which target EGFR, have demonstrated promising outcomes in the treatment of NSCLC patients (6–8). The efficacy of EGFR-TKIs is associated with Asian race, shows gender specificity to women, non-smokers and adenocarcinoma histology (9). Furthermore, an association between mutations in the EGFR TK domain and sensitivity to EGFR-TKIs has been previously reported (6,10).
EGFR mutations are located in EGFR exons 18 to 21 (9) and most mutations are observed as in-frame deletions in exon 19 and a point mutation L858R in exon 21 (11). Thus, testing for EGFR mutations may be prognostically important to identify potential responders who would benefit from treatment with EGFR-TKIs. This is particularly true for Chinese NSCLC patients with high EGFR mutation rates (12). The samples used for EGFR mutations are usually from resected tumor tissues, which could be stably and easily detected. It is difficult to obtain sufficient tumor tissues with advanced NSCLC, thus alternative specimens need to be established for testing EGFR mutations.
Malignant pleural effusion is a common complication of lung cancer. It is present in ~15% of lung cancer patients and in ~10–50% of patients at the time of diagnosis (13). In about half of NSCLC patients with a pleural effusion, most effusions are determined to be malignant consistent with the progress of the disease. As sampling of pleural effusion fluid is usually a standard and uncomplicated procedure, which is also non-invasive and repeatable, we hypothesized that genetic alterations in the pleural effusion fluid of NSCLC patients could provide useful guidelines with regard the response to EGFR-TKIs therapy.
In the present study, we used two approaches to detect major EGFR mutations in malignant pleural effusions from 24 patients presenting with advanced NSCLC and compared the acquired results. The relationship between EGFR mutations with the efficacy of gefitinib was also evaluated.
Patients and methods
Patients
Cytologically or pathologically confirmed pleural effusions were obtained from 24 Chinese patients presenting with advanced NSCLC. Jinan General Hospital of PLA approved this study, and written informed consent was obtained from all participants. Eligibility criteria included patients with stage IIIB–IV, ECOG performance status (PS) of 0–3, and a life expectancy of at least 3 months. The records of all patients consisted of age, gender, smoking habit, histological type of NSCLC and treatment. The response of the patients to treatment with gefitinib was evaluated in accordance with the ‘Response Evaluation Criteria in Solid Tumors (RECIST)’ guidelines (14). No research results were entered into the records of any of the patients whatsoever or released to the patient or the physician of the patient. Each specimen was only labeled by a serial number without any identification.
Collection of pleural effusion fluid and DNA extraction
Pleural effusion fluid was collected from patients in heparinized tubes between 20th February and 22nd June 2012. No particular collection method was used. A 30 ml volume sample of the fluid was centrifuged at 250 × g for 10 min at room temperature, and the cell pellets were stored at −80°C until used. Genomic DNA in the cell pellets was extracted by DNeasy tissue kits (Qiagen, Germany), and according to the manufacturer's protocol. The concentration and purity of extracted DNA were assessed by spectrophotometry (Nanodrop, ADx, China).
Polymerase chain reaction amplification and direct sequencing
Exons 19, 20 and 21 of the EGFR gene were amplified by polymerase chain reaction (PCR). The primers specific for EGFR were designed using Primer Designer Software (primer premier 5.0). The sequences of primers for EGFR exon 19 to 21 are described in Table I. Each 50 μl reaction specimen contained 2 μl of template DNA, 0.25 μl of Ampli Taq Gold DNA polymerace (Roche, USA), 5 μl of 10X PCR buffer, and 10 μM of forward and reverse primer. The same PCR program was used for all amplicons: 95°C for 3 min; 32 cycles of 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec; 72°C for 10 min. After PCR assay had completed, the resultant amplicons were further purified by QIAquick PCR purification kit (Qiagen), and subjected to sequencing analysis in both sense and antisense directions.
ADx-ARMS for the detection of EGFR mutations
We used an EGFR Gene 4 Mutations Diagnostic kit (ADx, Xiamen, China), which combines the two technologies of ARMS and Bi-loop Probe, to detect mutations in real-time PCR reactions. All reactions proceeded in 25 μl volumes according to the manufacturer's protocol. Real-time PCR was performed using the Mx3000P™ real-time PCR system (Agilent, Germany) under the following conditions: initial denaturation at 95°C for 5 min, 15 cycles of 95°C for 25 sec, 64°C for 20 sec, 72°C for 20 sec, and 31 cycles of 95°C for 25 sec, 60°C for 35 sec (with fluorescence collection, set to FAM and HEX), and finally 72°C for 20 sec. Data were analyzed using Stratagene Mxpro software. The threshold cycle (Ct) was defined as the cycle at the highest peak of the curve, which represents the point of maximum curvature of the growth curve. Positive results were defined as Ct <26. Analysis of each sample was carried out in duplicate, and the whole test process required only 90 min. The EGFR mutation kit is intended for detection of the major somatic mutations in EGFR.
Statistical analysis
SPSS statistical software (version 13.0) was used for statistical analysis. The Chi-square test was used to compare the sensitivity between direct sequencing and ADx-ARMS. Two-sided P-values of <0.05 were considered statistically significant.
Results
Patients
Sixteen male and 8 female patients were enrolled for the study. The median age was 58 years. Fourteen patients had no history of cigarette smoking; the ten current smokers were all male (Table).
Results of direct sequencing analysis
EGFR mutations were observed in 10 samples by direct sequencing of DNA, 4 deletions in exon 19, and 6 L858R mutations in exon 21 (Fig. 1). We did not detect any mutations in exon 20 (data not shown).
ADx-ARMS analysis
ADx-ARMS analysis of EGFR mutations are shown in Fig. 2. The wild-type showed one increased curve, which was the positive control, and the mutant type showed two increased curves, which were the mutant and positive control curves, respectively. Using the EGFR Mutations Diagnostic kit, 6 deletion mutations in exon 19, and 8 L858R mutations in exon 21 of EGFR were detected. We confirmed that there was no mutation in exon 20 (not shown).
Comparison between direct sequencing and ADx-ARMS
We found gene mutations in EGFR in only 10 patients by the direct sequencing assay. Thus, direct gene sequencing was less sensitive than ADx-ARMS analysis. In 24 patients, EGFR mutations were detected in 14 samples (58.3%) by ADx-ARMS, while 10 mutations (41.7%) were detected by direct sequencing. However, no significant difference was seen between these approaches (χ2=1.333, P=0.248). Among the test results of 24 patients, there was an 83.3% concordance between direct sequencing and ADx-ARMS. Four EGFR mutation-negative samples found by direct sequencing were mutation-positive by ADx-ARMS.
Correlation between EGFR mutation and clinical response
For patients treated with gefitinib, EGFR mutations were detected in cells from malignant pleural effusions in ten of the 18 patients (Table III). Among those 10 EGFR mutant samples, 8 patients achieved partial response, and 2 presented with stable disease after 28 days of gefitinib therapy. In the 8 patients who partially responded, 6 of them showed decreased levels of pleural effusion, and reduced size of the tumor (Fig. 3). Six of the eight patients who had no demonstrable EGFR mutations progressed to develop the disease. While defining a patient with partial response as a responder, the frequency of EGFR mutations was significantly higher in gefitinib responders (8/9) than was found in non-responders (2/9, P=0.02).
Discussion
In this study, we demonstrated the feasibility of using DNA from malignant pleural effusion as an alternative to tumor samples for the detection of EGFR mutations from advanced NSCLC patients.
We used the pleural effusion samples to detect EGFR mutation status and compared two methods: i) gene sequencing and, ii) ADx-ARMS. We also showed that patients with mutant EGFR had a better response to treatment with EGFR-TKIs. In our study, the response rate was 80% (8 of 10 patients achieved partial response) in EGFR mutation patients, while EGFR wild-type patients had only a 12.5% response rate (1 of 8 patients achieved partial response). Patients with mutant EGFR, had a response rate which was significantly higher than patients with wild-type EGFR (P<0.05). The data are in agreement to other previously reported studies (15–17).
Direct gene sequencing has been regarded as a gold-standard method for gene mutation analysis in the last decades. Direct sequencing usually requires sufficient tumor tissue as the testing sample with a sensitivity of ~30% (18,19). However, it is challenging to obtain sufficient tissue for gene sequencing in advanced NSCLC. In addition, gene sequencing is both time-consuming and technically demanding (17). Many studies have shown that gene sequencing is unable to provide satisfactory data for the detection of pleural effusion fluid samples that contain mixtures of DNA from normal cells (20,21), thus it cannot be widely used in clinical practice. Therefore, alternative clinical samples with more sensitive methodological approaches are urgently needed for individualized therapy of EGFR-TKIs.
Pleural effusion fluid, which has DNA from tumor cell pellets or the free DNA from the tumor provide a good alternative (17,20,22). The advantage of collecting free DNA or cell pellets is that it is a relatively simple approach, it is non-invasive and a repeatable technique. Thus, it could dynamically guide clinical approaches. Due to different methods and the selectivity of lung cancer patients with pleural effusion fluid, the frequency of mutant EGFR is in the range of 12.5–73% (17,20,21,23–25).
In our study, the frequency of EGFR mutations (deletion mutations and L858R mutations) detected by sequencing and by ADx-ARMS was found to be 41.7% and 58.3%, respectively. ADx-ARMS appeared to be the more sensitive approach as compared with direct sequencing in this study. The mutations detected by ADx-ARMS consisted of an in-frame deletion in exon 19 (E746_A750 del: 2235_2249del15 and 2236_2250del15), an insertion mutation in exon 20 (T790M), and a point mutation in exon 21 (L858R). Other deletion patterns in exon 19 and other mutations in the tyrosine kinase domain of EGFR could not be detected by this assay.
Among the 24 patients, there was 83.3% concordance between direct sequencing and ADx-ARMS. Our findings of a correlation between EGFR mutations and tumor response to therapy with TKIs was consistent with previous studies (15,16). Due to the small number of our samples, the EGFR mutation rate showed no significant difference between these two methods (χ2=1.333, P=0.248). At this point, it is worthwhile mentioning two limitations of our study. One is that we did not compare EGFR mutations between effusion cells and primary tumors, the main reason being that some tumor samples were not available. In addition, our results need further study based on the relationship between EGFR mutations and progressive-free survival and overall survival.
In summary, the clinical responses of NSCLC to EGFR-targeted therapy are closely associated with EGFR sensitive mutations. Screening of EGFR mutations by the ADx-ARMS approach using malignant pleural effusion as the source specimen is more sensitive and faster as compared with traditional gene sequencing approaches. These observations support the utility of this technology in routine clinical practice, an approach that can benefit patients presenting with advanced NSCLC.