Abstract
Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20.Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay. Within the framework of the CIRCAN project set-up at the Lyon University Hospital, plasma samples were collected to establish a pre-analytical and analytical workflow of cfDNA analysis. We evaluated all of the steps from blood sampling to mutation detection output, including shipping conditions (4H versus 24H in EDTA tubes), the reproducibility of cfDNA extraction, the specificity/sensitivity of ddPCR (using external controls), and the comparison of different PCR assays for the detection of the three most important EGFR hotspots, which highlighted the increased sensitivity of our in-house primers/probes. Hence, we have described a new protocol facilitating the molecular detection of somatic mutations in cancer patients from liquid biopsies, improving their diagnosis and introducing a less traumatic monitoring system during tumor progression.
Highlights
In the last two decades, the discovery of somatic oncogenic drivers, such as mutations in the epidermal growth factor receptor (EGFR), has revolutionized the treatment of advanced non-small cell lung cancer (NSCLC) [1]
Molecular diagnosis based on circulating free DNA offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19, the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20
To evaluate the concordance between nextgeneration sequencing (NGS) and droplet digital polymerase chain reaction (ddPCR) results, we assessed the level of homology between the EGFR molecular profile of 10 selected formalin-fixed paraffin-embedded (FFPE) biopsies from patients with known mutational profiles using NGS and ddPCR assays
Summary
In the last two decades, the discovery of somatic oncogenic drivers, such as mutations in the epidermal growth factor receptor (EGFR), has revolutionized the treatment of advanced non-small cell lung cancer (NSCLC) [1]. Some of these sensitive mutations, including L858R and exon 19 deletions (delEX19), are targetable using tyrosine kinase inhibitors (TKIs). Most patients progress after a median progression-free survival time of around 9-12 months [2] This tumoral progression may be due to the acquisition and/or activation of several emerging oncogenic drivers in tumor cells [3], including the acquisition of resistance EGFR mutations, such as the T790M substitution on exon 20. This mutation can be successfully targeted by 3rd generation EGFR TKIs, such as osimertinib (Astra-Zeneca, United Kingdom), the administration of which is currently approved in the context of advanced NSCLC [4]
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