DNA associated with large extracellular vesicles outperforms circulating cell-free DNA for the detection of oncogenic mutations in liquid biopsies of non-small cell lung cancer patients
Background
Liquid biopsy has emerged as a promising approach for detecting genetic alterations in cancer patients and guiding treatment decisions. Although conventionally considered as a single analytic compartment, circulating tumor DNA (ctDNA) can be detected both, as cell-free DNA (cfDNA) and as cargo of extracellular vesicles (EVs). However, the clinical value of these compartments for ctDNA detection is still debated. This study aimed to compare the performance of EV-DNA and cfDNA in detecting tumor-derived mutations in plasma of non-small cell lung cancer (NSCLC) patients.
Methods
From the NSCLC cell line H1975 or plasma samples of NSCLC patients, small and large EVs (sEVs/lEVs) were isolated using differential ultracentrifugation. Both EV subpopulations were characterized by electron microscopy, nanoparticle tracking analysis and immunoblotting. DNA isolation kits were used to isolate plasma EV-DNA and paired cfDNA from the EV-depleted supernatant. Quantitative and qualitative DNA analysis were performed by fluorometry (Qubit), electrophoresis (TapeStation) and next generation sequencing (NGS).
Results
An analysis of the EV subpopulations from H1975 cells as well as NSCLC patients demonstrated that lEVs contained higher amounts of DNA as well as larger fragments compared to sEVs. EV isolation from plasma samples did not affect cfDNA yields and thereby allowed the preparation of EV-DNA and cfDNA from a single plasma sample. EV-DNA yields were significantly lower compared to cfDNA. However, NGS revealed that the number of detected tumor-specific mutations was significantly higher for EV-DNA than cfDNA.
Conclusion
This study identified lEVs as novel and promising compartment for the detection of ctDNA in liquid biopsies. Further correlation with the mutation status of the primary tumor and clinical parameters will help to evaluate their potential as novel tool for molecular diagnostics in cancer.
Keywords
cancer, NSCLC, liquid biopsy, cfDNA, EV-DNA
Funding/Acknowledgments
We thank everyone who has contributed to this work especially Annette Westermann and Buket Celik for their excellent experimental support and also the West German Cancer Center for providing us with patient samples.
Authors
Katharina Maria Richter1,2* (Corresponding Author: Katharinamaria.Richter[at]ukmuenster[dot]de), Smiths S. Lueong3,4,7,8*, Marcel Kemper1,2, Georg Evers1,2, Michael Mohr1,2, Georg Lenz1,2, Balazs Hegedüs5,7,8, Martin Metzenmacher6,7,8, Martin Schuler6,7,8, Annalen Bleckmann1,2*, Kerstin Menck1,2*