Detection and comparison of EGFR mutations in matched tumor tissues, cell blocks, pleural effusions, and sera from patients with NSCLC with malignant pleural effusion, by PNA clamping and direct sequencing

doi:10.1016/j.lungcan.2013.04.023

Lung Cancer

Volume 81, Issue 2, August 2013, Pages 207-212

https://doi.org/10.1016/j.lungcan.2013.04.023 Get rights and content

Abstract

Peptide nucleic acid (PNA)-mediated real-time PCR clamping has higher sensitivity than conventional direct sequencing for detecting mutations. Pleural effusion and serum may provide good samples in which to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients.

We studied 37 NSCLC patients with malignant pleural effusion. EGFR mutations were assessed by PNA clamping and direct sequencing using tumor tissues, cell blocks, pleural effusion, and serum. Concordance between PNA clamping and direct sequencing results, and the diagnostic performance of pleural effusion were investigated.

The κ coefficients for the two methods were 0.68 (p = 0.0007), 0.91 (p < 0.0001), 0.75 (p < 0.0001) and −0.01 (p = 0.8639) for tissues, cell blocks, pleural effusion, and serum, respectively. The diagnostic performance of pleural effusion compared with the combination of tumor tissue and cell blocks showed 89% sensitivity, 100% specificity, positive predictive value of 100%, and negative predictive value of 95% by PNA clamping, and 67% sensitivity, 90% specificity, positive predictive value of 75%, and negative predictive value of 86% by directing sequencing. A patient in whom an EGFR mutation was identified in pleural effusion only by PNA clamping showed a significant response to EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment.

In contrast to the limited role of serum samples, pleural effusion had a diagnostic performance for the detection of EGFR mutations in NSCLC that was comparable to that of tumor tissues and cell blocks. The diagnostic performance of PNA clamping was good compared with that of direct sequencing. A more sensitive and accurate detection of EGFR mutations would benefit patients by allowing a better prediction of the response to EGFR-TKI treatment.

Introduction

Lung cancer is a leading cause of cancer deaths worldwide. The presence of activating epidermal growth factor receptor (EGFR) mutations is associated with a good response to EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment, making the detection of EGFR mutations clinically important for personalized therapy [1]. Among the variety of methods used to detect EGFR mutations in lung cancer specimens, direct DNA sequencing is the most commonly used and widely available [2], [3]. It can exactly detect all mutations. However, it is time-consuming and its sensitivity is suboptimal; mutant DNA must comprise ≥25% of the total amount of DNA for detections to be easily detected [2], [3], [4].

Peptide nucleic acid (PNA) is a synthetic DNA analog that binds strongly to its complementary DNA sequence. It has high sensitivity, specificity, and stability as a molecular probe. The PNA probe suppresses the PCR amplification of wild-type sequences, allowing greater amplification of mutant sequences [5]. The advantages of PNA-clamping PCR are high sensitivity, speed, and simplicity when applied to clinical samples, although this method cannot detect novel mutations [5], [6], [7].

Pleural effusion is observed in 8–15% of patients with lung cancer [8]. Approaches for examining molecular biomarkers in body fluids such as effusion or serum may be clinically helpful in predicting the response to EGFR-TKI treatment. The advantages of using pleural fluid are that it is easily accessible, can be sampled by relatively non-invasive methods, and can be repeatedly sampled. Moreover, pleural fluid samples are useful when tumor tissues are not available for mutation analysis. Previous reports have demonstrated the benefits of body fluids for detecting EGFR mutations [9], [10], [11], [12]. However, there are insufficient data to evaluate the usefulness of body fluids for determining overall mutation rates and EGFR-TKI response.

In the current study, we analyzed EGFR mutations in matched tumor tissues, cell blocks, pleural effusion samples, and serum samples by both PNA clamping and direct sequencing to investigate the diagnostic performance of PNA clamping and the usefulness of body fluids. To our knowledge, this is the first study to use PNA clamping to detect EGFR mutations in pleural effusion samples (cell-free fluid and cell-block cells) and to compare this technique with direct sequencing.

Section snippets

Patient Characteristics

We selected 37 consecutive NSCLC patients who presented with malignant pleural effusion at the time of diagnosis and who underwent diagnostic thoracentesis at the Division of Pulmonology of Seoul St. Mary's Hospital (Seoul, Korea) between December 2008 and September 2011. Malignant pleural effusion was confirmed by demonstration of malignant cells on cytological examination or by histologically proven primary lung cancer with the exclusion of other causes of pleural effusion. All subjects

Patient characteristics

Table 1 shows the demographic characteristics for the 37 enrolled patients. The mean age of the patients was 70.8 years, and 26 patients (70.3%) were male. The major histological type was adenocarcinoma (89.2%). All 37 patients were diagnosed as primary NSCLC with either biopsy specimens (n = 29) or cell block specimens of pleural effusion. Among them, 24 (64.9%) had malignant cells in their effusion specimens and 13 cases (35.1%) were diagnosed as malignant effusion with the exclusion of other

Discussion

The purpose of this study was to investigate the concordance of EGFR mutations detected by PNA clamping and direct sequencing in different samples, and to determine the diagnostic performance of pleural effusion and serum as DNA sources in lung cancer. We observed high concordance between the two methods in tumor tissues, cell blocks, and pleural effusion, and the diagnostic performance of pleural effusion was good. Moreover, case 6, who had an EGFR exon 21 L858R mutation that was detected in

Conflict of interest

None of the authors have financial or other potential conflicts of interest.

Acknowledgments

This research was supported by the Cancer Research Institute, College of Medicine, the Catholic Universitiy of Korea. Statistical consultation was supported by the Catholic Research Coordinating Center of the Korea Health 21 R&D Project (A070001), the Ministry of Health & Welfare, Republic of Korea.

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Detection and comparison of EGFR mutations in matched tumor tissues, cell blocks, pleural effusions, and sera from patients with NSCLC with malignant pleural effusion, by PNA clamping and direct sequencing

Abstract

Introduction

Section snippets

Patient Characteristics

Patient characteristics

Discussion

Conflict of interest

Acknowledgments

Lung Cancer

J Thorac Oncol

Lung Cancer

J Thorac Oncol

Tuberc Respir Dis

Lung Cancer

Lung Cancer

Lung Cancer

J Mol Diagn

Epidermal growth factor receptor mutation testing in lung cancer: searching for the ideal method

Clin Cancer Res

Comparative analysis of peptide nucleic acid (PNA)-mediated real-time PCR clamping and DNA direct sequencing for EGFR mutation detection

Tuberc Respir Dis

Management of malignant pleural effusions

Eur Respir J