Multi-gene analyses from waste brushing specimens for patients with peripheral lung cancer receiving EBUS-assisted bronchoscopy

Abstract

Objectives

Although flexible bronchoscopy with the assistance of miniature radial-probe endobronchial ultrasound (EBUS) is increasingly employed to diagnose peripheral lung cancer, transbronchial biopsies typically offer an insufficient amount of tissue to conduct additional molecular analysis. We evaluated the feasibility of multi-gene analyses from waste brushing samples obtained by EBUS-assisted bronchoscopy.

Materials and methods

For lung cancer patients with positive brushing cytology, analysis of EGFR, K-ras and EML4-ALK fusions were carried out, utilizing reverse transcription-polymerase chain reaction and Sanger sequencing on the cell-derived RNA retrieved from waste brushing samples.

Results

EBUS-guided brushings were judged positive for tumor cells in 84 (68.9%) of the 122 patients with peripheral lung cancer receiving flexible bronchoscopy. Genotyping of EGFR and K-ras was successfully implemented in 80 (95.2%) of the 84 cytology-proven brushing samples, along with satisfactory yields to detect EGFR (55.0%) and K-ras (2.5%) mutations. The results of EGFR genotyping from the brushing specimens were highly concordant with those provided from other corresponding samples (concordance rate: 94%, kappa: 0.92). Of the 19 patients with adenocarcinoma or non-small cell lung cancer not otherwise specified harboring wild-type EGFR and K-ras, two cases (10.5%) were identified to harbor EML4-ALK fusions.

Conclusion

Our results suggest that multi-gene analyses from waste brushing specimens using RNA-based Sanger sequencing is highly feasible. This approach offers an opportunity to overcome the dilemma of flexible bronchoscopy in molecular diagnostics for lung cancer, and could potentially recruit more patients for targeted therapy according to the molecular characteristics of the tumor cells.

Introduction

The development of targeted therapy represents an important advance in the treatment of lung cancer. Recently, pivotal trials have demonstrated that the presence of specific mutations in tumor cells is an important factor for individualizing treatment with targeted agents in non-small cell lung cancer (NSCLC) [1]. For example, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are indicated in the first-line setting for patients whose tumor harbors a sensitizing EGFR mutation, while those with an anaplastic lymphoma kinase (ALK) fusion oncogene are preferentially treated with crizotinib [2], [3]. Although the selection of targeted therapy based on tumor molecular characteristics offers the possibility of improved responses in lung cancer, the application of personalized treatment with targeted therapy requires an effective system to obtain clinical samples, along with appropriate molecular testing for mutational analysis [4].

In the past decade, the advent of miniature radial-probe endobronchial ultrasound (EBUS) has greatly improved the capacity of flexible bronchoscopy in the evaluation of peripheral lung cancer [5], [6], [7], [8]. One issue accompanied with this advance is whether the specimens offered by this technique can also be used for molecular testing, especially as “druggable” genetic alterations in lung cancer predominantly arise in the adenocarcinoma cell type, which tends to be located peripherally. Unfortunately, analysis of specimens obtained through routine bronchial biopsies has demonstrated that the quantity of the tumor cells in these samples is often low, thereby limiting the amount of additional molecular testing that can be carried out after other sophisticated analysis such as immunohistochemistry used to subclassify NSCLC [9], [10]. In order to obtain sufficient amounts of tissue for genetic analysis, excessive biopsies or other invasive procedures that increase the risk of complications may be required for the patients who receive flexible bronchoscopy as the primary means of diagnosis.

Attendant brushing is an alternative source to collect tumor cells in diagnostic bronchoscopy, although these cytological specimens are generally considered to be inadequate for genomic DNA-based molecular analysis [4]. However, we recently found that Sanger sequencing using cell-derived RNA as the amplification template is much more sensitive than genomic DNA-based sequencing for the detection of EGFR mutations from cytological specimens of malignant pleural effusion [11], [12]. It is worth noting that this method permits detection not only of point mutations and insertions/deletions, but also of gene rearrangements such as ALK and c-ros oncogene 1 (ROS1) fusions that have become emerging biomarkers for NSCLC [3], [13]. In this study, we explored the feasibility of RNA-based Sanger sequencing to conduct multi-gene analyses from waste brushing contents in patients receiving EBUS-assisted bronchoscopy for the diagnosis of peripheral lung cancer.