Elsevier

Lung Cancer

Volume 60, Issue 2, May 2008, Pages 175-182
Lung Cancer

Detection and comparison of epidermal growth factor receptor mutations in cells and fluid of malignant pleural effusion in non-small cell lung cancer

https://doi.org/10.1016/j.lungcan.2007.10.011Get rights and content

Summary

Cells or cell-free fluid of malignant pleural effusion could be important clinical specimen for epidermal growth factor receptor (EGFR) mutation screening in advanced non-small cell lung cancer (NSCLC) patients. However, their usefulness in mutation detection has not been well compared. In this study we recruited 26 East Asian NSCLC patients with malignant pleural effusion, determined the mutation status of EGFR in both cells and matched cell-free fluid with the use of sequencing and mutant-enriched PCR. After comparing the mutation spectrums, we found both the cells and cell-free pleural fluid may be feasible clinical specimen for EGFR mutation detection in unresectable NSCLC given sensitive genotyping assays employed. Direct sequencing could miss a significant portion of mutations in these heterogeneous specimens. More sensitive methods, such as mutant-enriched PCR and gene scan, could provide more reliable mutational information.

Introduction

Lung cancer is the leading cause of cancer-related death in both men and women and non-small cell lung cancer (NSCLC) accounts for 80–85% of cases. Malignant pleural effusion is a frequent complication in advanced NSCLC. It typically signals an advanced cancer stage and poor prognosis. Chemotherapy is usually recommended as the primary treatment choice rather than surgery or radiation [1]. Although platinum-based regimen has become well established more and more molecular target-based drugs have been developed. Specific epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib (ZD1839, Iressa) and erlotibib (OSI-774, Tarceva) have been used as monotherapy for patients with locally advanced or metastatic NSCLC who have failed chemotherapy. Both agents adversely compete with ATP for the critical ATP-binding site so that phosphorylation of the receptors and activation of downstream molecules are inhibited. Clinically, the two drugs are well tolerated and have meaningful antitumor activity [2], [3]. However, the treatment response is mostly restricted to certain subgroup of patients who harbor somatic EGFR mutations [4], [5]. Patients with in-frame deletion in exon 19 (amino acids 746–753) and missense mutation in exon 21 (L858R) are highly sensitive to EGFR-TKIs. Due to the strong association with dramatic treatment response, EGFR somatic mutations have been considered the best predictive marker for EGFR-TKI treatment. Individual genotype-based therapeutic strategy has been strongly suggested.

Determining mutation status of EGFR is challenging in patients with advanced NSCLC because most of these patients are inoperable, which makes the tumor sample difficult to obtain. Given this situation the availability of noninvasive diagnostic specimens is of great importance. Malignant pleural effusion is present in approximately 50% of advanced NSCLC patients and most frequently in lung adenocarcinoma [6], [7]. Patients with pleural effusion usually feel short of breath, chest pain and cough, which makes thoracentasis or chest tube drainage necessary at most of the time. Pleural effusion is a convenient clinical sample with important clinical diagnostic significance. The cells in pleural effusion are critical for cytological analysis, whereas the cell-free pleural fluid is often used for the measurement of cytokine and soluble protein. In recent years with the advancement of molecular technologies, pleural effusion has become a useful and practical specimen for detection of molecular aberrations of diseases as well. The presence of tumor cells makes it a good source of tumor genomic DNA, whereas the little amount of soluble DNA in cell-free pleural fluid has been proved to be sufficient for most molecular analysis. Several studies have reported their determination of methylated DNA spectrum and somatic mutations of the genes of K-ras, Rho, P53 and FHIT in cell-free pleural fluid in patients with various neoplastic malignancies [8], [9], [10]. All above collectively demonstrate the importance of pleural effusion in genetic and epigenetic studies.

Somatic EGFR mutations have also been successfully determined with the use of either pleural effusion cells [11], [12], [13] or cell-free pleural fluid [11], [14], [15] in advanced NSCLC patients. However, the usefulness of pleural effusion cells and cell-free pleural fluid for mutation screening has not been well compared. It still remains unknown whether cell-free pleural fluid can provide the same mutational information as pleural effusion cells. If the answer is positive, mutation screening would become much easier and feasible to most advanced patients with pleural effusion because in good proportion of patients (30–40%) the yield of malignant cells from thoracentasis is inadequate for these cytological and molecular diagnostic testing.

In this study, we examined EGFR mutation status with the use of both DNA extracted from pleural effusion cells and DNA extracted from cell-free pleural fluid from same patients by direct DNA sequencing and mutant-enriched PCR and gene scan analysis with the primary objective to compare the concordance of mutation spectrum obtained from these two matched clinical specimens.

Section snippets

Patients and DNA extraction

Pathologically confirmed malignant pleural effusion specimens were obtained from 26 East Asian patients with advanced NSCLC. After centrifugation cell-free supernatant and cell pellets were stored at −80 °C separately until use. Genomic DNA in cell pellet (containing 5–10 × 105 cells) and 0.8–2.0 ml of cell-free pleural fluid was extracted respectively with the use of DNeasy tissue kits (Qiagen) and QIAamp blood midi kits (Qiagen) according to the manufacturer's protocols. The concentration and

Results

The concentration of DNA extracted from pleural effusion cell was approximately 40–260 ng/μl. Low amount of soluble DNA was obtained from cell-free pleural fluid, which could not be quantified by spectrophotometry. However, all the PCR reactions were successfully performed in all the specimens.

Discussion

Somatic EGFR mutations have been demonstrated as the most important predictive biomarker for clinical outcome among NSCLC patients treated with EGFR-TKIs. Determining the mutation status of each individual patient would greatly facilitate the selection of their therapeutic strategy. This genetic diagnostic analysis appears particularly important to East Asians because mutation frequency is significantly higher comparing with Caucasians (30–40 vs.10%) [16].

Lung cancer is the leading cause of

Conflict of interest

There is no any actual or potential conflict of interest declared.

Acknowledgement

The study was funded by Johns Hopkins Singapore Research Fund.

References (24)

  • V.B. Antony et al.

    Management of malignant pleural effusions

    Eur Respir J

    (2001)
  • S. Benlloch et al.

    Potential diagnostic value of methylation profile in pleural fluid and serum from cancer patients with pleural effusion

    Cancer

    (2006)
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