Elsevier

Lung Cancer

Volume 73, Issue 1, July 2011, Pages 96-102
Lung Cancer

Detection of EGFR mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumor EGFR status and response to EGFR inhibitors

https://doi.org/10.1016/j.lungcan.2010.10.014Get rights and content

Abstract

Aims

EGFR mutations now guide the clinical use of EGFR-targeted therapy in lung cancer. However, standard EGFR mutation analysis requires a minimum amount of tumor tissue, which may not be available in certain situations. In this study, we combined a mass spectrometry genotyping assay (Sequenom) with a mutant-enriched PCR (ME-PCR) to detect EGFR mutations in free plasma DNA from patients with lung cancer.

Method

DNAs were extracted from 31 plasma samples from 31 patients and analyzed by both methods for EGFR Exon 19 deletion and EGFR L858R mutation. Results in plasma DNA samples were compared with EGFR mutation status obtained in tumor DNA (18/31 EGFR mutant). The relationship of EGFR mutation status in tumor and/or plasma samples to overall survival was assessed.

Results

The EGFR mutation status in plasma DNA was identical to the primary tumor in 61% of patients (19/31). By mass spectrometry genotyping, the plasma samples contained mutant DNA corresponding to 5/14 EGFR Exon 19 deletions and 3/4 EGFR L858R mutations previously diagnosed in the matched tumors. Two samples were positive in plasma DNA but negative in primary tumor tissue. Results were similar for samples studied by ME-PCR. For patients treated with erlotinib, overall survival was correlated with the presence of EGFR mutation in plasma and/or tumor tissue (p = 0.002), with the two patients positive only in plasma DNA showing responses and favorable outcomes.

Conclusion

The detection of EGFR mutations in plasma DNA samples by mass spectrometry genotyping and ME-PCR is feasible. A positive EGFR result in plasma DNA has a high predictive value for tumor EGFR status and for favorable clinical course on EGFR-targeted therapy and could therefore be useful in guiding clinical decisions in patients with insufficient or unavailable tumor specimens.

Introduction

The detection of EGFR mutations in lung adenocarcinomas has become a routine molecular test with important implications for patient prognosis and selection of therapy. The presence of an activating mutation predicts response to the EGFR tyrosine kinase inhibitors (TKI) erlotinib or gefitinib, and is prognostically favorable regardless of therapy [1]. Unfortunately, in some cases, tumor tissue is either inadequate for molecular testing because of its small quantity or very low tumor content or is not readily available. Therefore, there is a need to develop new techniques for detecting clinically significant EGFR mutations in patients with little or no available tumor DNA.

Plasma samples from patients with lung cancer contain much higher levels of DNA than plasma from cancer-free patients. Most of this excess circulating DNA is believed to be released from the dying lung cancer cells at primary or metastatic sites [2]. As such, plasma DNA may therefore provide a noninvasive source of genotypic information which could be used as a substitute for tumor tissue for detecting cancer-specific molecular markers that could be used to predict response and prognosis. Several groups have detected EGFR mutations in DNA isolated from plasma [3], [4], [5], [6], [7] or serum samples [8], [9] and show some correlation between mutation status in plasma and tumor tissue [3], [4], [6], [8], [9], [10]. Furthermore, EGFR mutation detected in plasma or serum may, by itself, be predictive of response to EGFR TKI [3], [5], [6], [7], [9].

In this study, we report the detection of EGFR L858R mutations and EGFR Exon 19 deletions in plasma samples from patients with NSCLC using a novel, mass spectrometry assay. The detection of these mutations in plasma samples is correlated with better survival when patients are treated with TKIs.

Section snippets

Patients characteristics

We studied 31 patients with a biopsy-proven diagnosis of stage III or IV NSCLC and available plasma and tumor tissue. All patients gave informed consent, and the collection and analysis of their health information was approved by the Memorial Sloan-Kettering Cancer Center (MSKCC) Institutional Review Board. The patients were followed for tumor responses and survival outcomes.

EGFR Exon 19 deletion assay

Detection of the small in-frame deletions in exon 19 of EGFR was performed by fragment analysis of fluorescently labeled

Patients and tumor characteristics

Patient characteristics are summarized in Table 2. There were 16 women and 15 men, 30 with lung adenocarcinoma and one patient with poorly differentiated squamous cell carcinoma. Seventeen patients were former or current-smokers and 14 were never smokers. At time of blood collection, all patients were stage IV except one with stage III disease. By the mutation-specific PCR assays used in routine practice at MSKCC to establish EGFR mutation status in tumor tissue [10], 14 tumors had EGFR Exon 19

Discussion

The detection of EGFR mutations in tumor tissue has important prognostic and predictive value, and can be used to select therapy for the treatment of lung adenocarcinoma. Many patients with stage IV adenocarcinoma are diagnosed with small biopsies, or by fine needle aspiration of tumors which often yields insufficient DNA for molecular testing. Detection of EGFR mutations in plasma samples would be of value for patients in whom sufficient tumor tissue is not available.

In the present study, we

Conflict of interest statement

None declared.

Acknowledgments

This work was supported by NIH P01 grant CA129243 (M.L.), the Gretchen and Samuel Feldman Fellowship (M.B.) and La Fondation de France (M.B.).

References (21)

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