Unveiling mutational dynamics in non‐small cell lung cancer patients by quantitative EGFR profiling in vesicular RNA

Abstract

The mutational status of the epidermal growth factor receptor (EGFR) guides the stratification of nonsmall cell lung cancer (NSCLC) patients for treatment with tyrosine kinase inhibitors (TKIs). A liquid biopsy test on cell-free DNA is recommended as a clinical decision supporting tool, although it has limited sensitivity. Here, we comparatively investigated the extracellular vesicle (EV)-RNA as an independent source for multidimensional and longitudinal EGFR profiling in a cohort of 27 NSCLC patients. We introduced and validated a new rapid, highly specific EV-RNA test with wild-type-and mutant-sensitive probes (E746-A750del, L858R and T790M). We included a cohort of 20 NSCLC patients with EGFR wild-type tumor tissues and systematically performed molecular EV-RNA and ctDNA analyses with clinical data statistics and biophysical profiles of EVs.At the single-patient level, we detected variegated tumor heterogeneity dynamics supported by combinations of driver EGFR mutations. EV-RNA-based mutation analysis showed an unprecedented sensitivity of over 90%. The resistance-associated mutation T790M frequently pre-existed at baseline with a gained EV-transcript copy-number at progression, while the general mutational burden was mostly decreasing during the intermediate follow-up. The biophysical profile of EVs and the quantitative assessment of T790M revealed an association with tumor size determined by the sum of the longest diameters in target lesions. Vesicular-RNA provides a validated tool suitable for use in clinical practice to investigate the dynamics of common driver EGFR mutations in NSCLC patients receiving TKIs.

Transcriptomic Features in a Single Extracellular Vesicle via Single‐Cell RNA Sequencing

“…The molecular cargo of an individual EV and the heterogeneity within an EV population is lost in bulk‐level analyses. Recently, several new analytical methods, such as total internal reflection fluorescence microscopy (TIRFM), [ 6 ] digital PCR, [ 7 ] microarray‐based chip, [ 8 ] and high‐sensitivity flow cytometry combined with qPCR, [ 9 ] have been used to quantify a specific miRNA or mRNA in an single‐EV or small identical subpopulations. However, a high‐throughput analysis for the RNA profile of a single‐EV is still lacking.…”

Advances in the analysis of single extracellular vesicles: A critical review

“…One group has shown that EVs can be sequestered in the droplets prior to lysing, enabling the detection of nucleic acid biomarkers from a single EV [ 75 ]. Using the ddPCR system from BioRad to digitize EVs into droplets, Pasini et al detected EGFR mutations (specifically L858R and T790M) in vesicular RNA.…”

Epidermal Growth Factor Receptor Mutations Carried in Extracellular Vesicle-Derived Cargo Mirror Disease Status in Metastatic Non-small Cell Lung Cancer

“…Notably, a robust method was developed for detecting mutations in EV-RNA using droplet digital PCR (ddPCR) and was validated with Sanger sequencing with mutations found with over 90% sensitivity. The study profiled EVs before treatment and at the time of progression events in a cohort of metastatic NSCLC patients, further demonstrating the promise of using EV-RNA to monitor mutations through treatment ( Pasini et al, 2021 ). There remains, however, a need to verify whether changes in mutation burden, at either the RNA or protein level, found within EVs, is a predictor of progression events as opposed to a result of disease progression.…”

“…Several recent works have demonstrated the feasibility of using EV-RNA to detect EGFR mutations from blood ( Dong et al, 2019 ; Pasini et al, 2021 ) and bronchoalveolar lavage fluid ( Hur et al, 2019 ; Liam et al, 2020 ). Notably, a robust method was developed for detecting mutations in EV-RNA using droplet digital PCR (ddPCR) and was validated with Sanger sequencing with mutations found with over 90% sensitivity.…”