Targeted deep sequencing of cell‐free DNA in serous body cavity fluids with malignant, suspicious, and benign cytology

Abstract

BACKGROUND: Liquid biopsy using cell-free DNA (cfDNA) presents new opportunities for solid tumor genotyping. While studies have demonstrated the utility of cfDNA from plasma, cfDNA from other body fluids remains underexplored. METHODS:We evaluated the molecular features and clinicopathologic correlates of cfDNA from serous body cavity fluids by performing hybrid capture-based next-generation sequencing (NGS) on cfDNA isolated from residual effusion supernatants. Twenty-one serous effusions from pleural (n = 15), peritoneal (n = 5), and pericardial (n = 1) cavity were analyzed. RESULTS:The supernatants provided a median cfDNA concentration of 10.3 ng/µL. Notably, all effusions were sequenced successfully to a median depth >1000×, revealing a broad range of genetic alterations including single nucleotide variants, small insertions and deletions, amplifications, and fusions. Specifically, pathogenic alterations were identified in all malignant fluids (13/13), all fluids suspicious for malignancy (2/2), and 1 benign fluid (1/6) from a patient with metastatic cancer. To validate our findings, we examined matching results from 11 patients who underwent additional testing using formalin-fixed, paraffin-embedded (FFPE) specimens. In 8 patients, the paired results between FFPE and supernatant testing were concordant, whereas in the remaining 3 patients, supernatant analysis identified additional variants likely associated with resistance to targeted therapies. Additional comparison between FFPE and supernatant testing showed no difference in DNA concentration (P = .5), depth of coverage (P = .6), or allele frequency of pathogenic mutations (P = .7).CONCLUSION: cfDNA isolated from serous body cavity fluids represents a promising source of genomic input for targeted

Use of cytology centrifuged supernatants improves cost and turnaround time for targeted next generation sequencing

“…Although the cytology formalin fixed paraffin embedded (FFPE) cell block (CB) is commonly used for molecular testing, post-centrifugation cytology supernatant (CCS) has been shown to serve as a viable alternative. [5][6][7][8][9][10][11][12][13] Previous reports have suggested that CCS for NGS analysis may be associated with improved turnaround time (TAT) and cost savings. [6][7][8][9] In this study, we reviewed our implementation process to evaluate its performance and share our experience with the improved molecular cytopathology workflow using previously discarded CCS.…”

“…Communication among clinical treatment teams, cytopathology, and molecular pathology laboratories is essential to ensure these limited samples are sufficient for both primary diagnosis and subsequent molecular testing. Although the cytology formalin fixed paraffin embedded (FFPE) cell block (CB) is commonly used for molecular testing, post‐centrifugation cytology supernatant (CCS) has been shown to serve as a viable alternative 5‐13 . Previous reports have suggested that CCS for NGS analysis may be associated with improved turnaround time (TAT) and cost savings 6‐9 .…”

Diving into the Pleural Fluid: Liquid Biopsy for Metastatic Malignant Pleural Effusions

“…Despite good results in PF cell pellets, other studies suggest that NGS on body fluid supernatants possesses higher performance for detecting EGFR mutations in cfDNA compared to body fluid sedimentary tumor cells and plasma cfDNA specimens [ 49 ]. In addition, targeted hybrid capture deep sequencing for the analysis of 130 cancer-related genes confirmed the presence of mutations in 15 PF supernatants where median depth of target was >1000× [ 50 ]. High concordance was seen between PF and FFPE samples.…”

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

“…More recently, tumor-derived cfDNA from other specimen types, including cerebrospinal fluid, urine, serous effusion fluid, and fine needle aspiration (FNA) supernatant has also been sequenced with favorable results (8)(9)(10)(11)(12)(13)(14)(15). Effusion fluid, in particular, is enriched in cfDNA species, making it a perfect medium for molecular testing (12,(16)(17)(18)(19)(20). 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. Several studies have demonstrated that effusion specimens represent a robust source of genetic material for tumor profiling (12,16,17,21). However, several important pre-analytical variables in sequencing of effusionderived cfDNA testing remain unclear.…”