Next-Generation Sequencing of Cell-Free DNA Extracted From Pleural Effusion Supernatant: Applications and Challenges.

Ami Patel,Priya D Velu,Jonas J Heymann,Susan A Alperstein,Lucelina Rosado,Hanna Rennert,Hyeon Jin Park,Wei Song,Robert Burkhardt,Lin Cong,Erika Hissong,Momin T Siddiqui

doi:10.3389/fmed.2021.662312

Abstract

Cell-free DNA (cfDNA) extracted from diverse specimen types has emerged as a high quality substrate for molecular tumor profiling. Analytical and pre-analytical challenges in the utilization of cfDNA extracted from pleural effusion supernatant (PES) are herein characterized in patients with metastatic non-small cell lung carcinoma (NSCLC). Pleural effusion specimens containing metastatic NSCLC were collected prospectively. After ThinPrep® (TP) and cell block (CB) preparation, DNA was extracted from residual PES and analyzed by gel electrophoresis for quality and quantity. Libraries were prepared and sequenced with a targeted next-generation sequencing (NGS) platform and panel clinically validated for plasma specimens. Results were compared with DNA extracted from corresponding FFPE samples that were sequenced using institutional targeted NGS assays clinically validated for solid tumor FFPE samples. Tumor (TC) and overall cellularity (OC) were evaluated. Fourteen specimens were collected from 13 patients. Median specimen volume was 180 mL (range, 35–1,400 mL). Median TC and OC on TP slides and CB sections were comparable. Median extracted DNA concentration was 7.4 ng/μL (range, 0.1–58.0 ng/μL), with >5 ng/μL DNA extracted from 10/14 specimens (71%). Mutations were identified in 10/14 specimens, including 1/3 specimens with median molecular coverage <1,000 reads. The minimal detected allelic fraction was 0.6%. NGS was falsely negative for the presence of one driver mutation. No correlation was identified between sample volume or OC, quality or quantity of extracted DNA, or mutation detection. Despite analytical and pre-analytical challenges, PES represents a robust source of DNA for NGS.

Highlights

Cell-free deoxyribonucleic acid (DNA) has emerged as an alternative to DNA extracted from traditional formalin-fixed, paraffin-embedded (FFPE) tissue for molecular analysis of tumors [1]
A rare study has demonstrated that an next-generation sequencing (NGS) platform and methodology clinically validated on plasma specimens can be utilized for malignant pleural effusion supernatant (PES) from patients with metastatic non-small cell lung carcinoma (NSCLC) [25], with promising results
Our study finds PES to be a robust substrate for NGS, it highlights the challenges involved in the pre-analytic and analytic phases of NGS performed on body fluid Cell-free DNA (cfDNA)

Summary

Introduction

Cell-free deoxyribonucleic acid (DNA) has emerged as an alternative to DNA extracted from traditional formalin-fixed, paraffin-embedded (FFPE) tissue for molecular analysis of tumors [1]. A major advantage of using such specimens is that they are largely derived from patients with unresectable, advanced stage malignancy. Their collection is minimally invasive, and their utilization can spare patients the discomfort and morbidity associated with more invasive procedures. In order to better define these variables, DNA extracted directly from pleural effusion supernatants (PES/ES) from patients with metastatic non-small cell lung carcinoma (NSCLC) have been analyzed using a targeted next-generation sequencing (NGS) platform clinically validated for plasma specimens

Methods

Results

Conclusion