Molecular Testing of Non–Small Cell Lung Carcinoma Diagnosed by Endobronchial Ultrasound–Guided Transbronchial Fine-Needle Aspiration: The Cleveland Clinic Experience

Abstract

Context.— Given the increasing demand for molecular testing of non–small cell lung carcinoma specimens to guide therapeutic decision-making and the trend toward minimally invasive techniques for obtaining diagnostic tissue, cytopathology laboratories must devise strategies to maximize DNA yield for necessary molecular testing. Objective.— To describe our experience at Cleveland Clinic with epidermal growth factor receptor (EGFR) mutation testing by next-generation sequencing and anaplastic lymphoma kinase (ALK) gene rearrangement testing by fluorescence in situ hybridization of non–small cell lung carcinomas diagnosed by cytology, with an emphasis on specimens obtained by endobronchial ultrasound–guided transbronchial fine-needle aspiration. Data Sources.— Data sources include a review of the current literature, including published articles from our institution. Conclusions.— At our institution, liquid-based cytology specimens are the primary resource used for molecular testing of non–small cell lung carcinomas; in most instances, adequate DNA can be extracted from the residual cell pellet for next-generation sequencing, and ThinPrep slides can be used reliably for fluorescence in situ hybridization testing for ALK gene rearrangements. In occasional cases where the cell pellet material is not adequate for molecular testing, cell blocks and/or surgical pathology specimens are secondary options. The cytopathologist's role in specimen handling and triage is essential to ensure that molecular testing can be carried out successfully.

Diff-Quik Cytology Smears from Endobronchial Ultrasound Transbronchial Needle Aspiration Lymph Node Specimens as a Source of DNA for Next-Generation Sequencing Instead of Cell Blocks

“…Endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) sampling of lymph nodes has become an important alternative for obtaining material for next-generation sequencing (NGS) for lung cancer [1-3]. Detailed mutation profiling is expected to benefit patient management by enabling therapy against targetable somatic mutations [4-7].…”

Genotyping and cytomorphological subtyping of lung adenocarcinoma based on liquid‐based cytology

“…Cytologic samples provide high‐quality DNA for molecular testing and are comparable, or even superior to, formalin‐fixed, paraffin‐embedded surgical pathology specimens for the evaluation of clinically relevant mutations . Liquid‐based cytology (LBC) makes it possible to perform additional techniques, such as immunocytochemistry and in situ hybridization, and also allows for molecular testing using DNA and RNA extracted from the remaining fixed cells . Some studies have demonstrated that LBC systems provide excellent cell preservation for routine cytopathology and can be used for DNA‐ and RNA‐based molecular testing …”

“…4,5 Liquid-based cytology (LBC) makes it possible to perform additional techniques, such as immunocytochemistry and in situ hybridization, and also allows for molecular testing using DNA and RNA extracted from the remaining fixed cells. [4][5][6] Some studies have demonstrated that LBC systems provide excellent cell preservation for routine cytopathology and can be used for DNA-and RNAbased molecular testing. 4,5 The 2015 World Health Organization classification 7 classifications.…”

Molecular testing of residual cytology samples: Rethink, reclaim, repurpose

“…Wei et al described using residual PreservCyt (Cytyc Corporation, Marlborough, Massachusetts) specimens from FNA samples and body fluids (either fresh unconcentrated specimens or residual PreservCyt specimens) to extract DNA for multigene NGS analysis . A recent study has described using residual CytoLyt (Hologic Inc, Marlborough, Massachusetts) from endobronchial ultrasound–guided transbronchial needle aspirates for evaluation using NGS . Similarly, another study has reported that the postcentrifuged supernatant fluid from FNA samples combined with the residual PreservCyt yields adequate DNA for mutational analysis by NGS …”