Development, validation, and comparison of gene analysis methods for detecting EGFR mutation from non-small cell lung cancer patients-derived circulating free DNA

Abstract

The feasibility and required sensitivity of circulating free DNA (cfDNA)-based detection methods in second-line epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment are not well elucidated. We examined T790M and other activating mutations of EGFR by cfDNA to assess the clinical usability. In 45 non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations, cfDNAs were prepared from the plasma samples. EGFR mutations in cfDNA were detected using highly sensitive methods and originally developed assays and these results were compared to tissue-based definitive diagnoses. The specificity of each cfDNA-based method ranged 96–100% whereas the sensitivity ranged 56–67%, indicating its low pseudo-positive rate. In EGFR-TKI failure cohort, 41–46% samples were positive for T790M by each cfDNA-based method, which was comparable to re-biopsy tissue-based T790M positive rates in literature. The concordance of the results for each EGFR mutation ranged from 83–95%. In eight patients, the results of the cfDNA-based assays and re-biopsy-derived tissue-based test were compared. The observed overall agreement ranged in 50–63% in T790M, and in 63–100% in activating EGFR mutations. In this study, we have newly developed three types of assay which have enough sensitivity to detect cfDNA. We also detected T790M in 44% of patients who failed prior EGFR-TKI treatment, indicating that cfDNA-based assay has clinical relevance for detecting acquired mutations of EGFR .

Molecular characterization of advanced non-small cell lung cancer patients by cfDNA analysis: experience from routine laboratory practice

“…The concentration of cfDNA has also been suggested as a plausible biomarker for tumor burden and prognosis in lung cancer patients (45)(46)(47). As observed in our cohort, higher levels of cfDNA are found in stage IV patients compared to stage III patients (48).…”

Validation of a liquid biopsy assay with molecular and clinical profiling of circulating tumor DNA

“…For example, in early-stage disease, when treatments have much higher success rates, many patients have low ctDNA fractions that may be below the LOD for liquid biopsies [29][30][31][32] , limiting clinical utility because of the risk of false negatives. Furthermore, while validation studies of existing liquid biopsy assays have shown high sensitivity and specificity [33][34][35][36][37][38] , few studies have corroborated results with orthogonal methods , or between NGS testing platforms. Kuderer et al compared commercially available liquid and tissue NGS platforms and found only 22% concordance in genetic alterations 39 .…”

The Circulating Biomarkers League: Combining miRNAs with Cell-Free DNAs and Proteins

“…The primary advantage of NGS lies in its capacity to conduct extensive analyses of genes associated with diseases, yielding a wealth of DNA sequencing data. Recently, mutation tests based on cfDNA for the epidermal growth factor receptor (EGFR) gene have received approval for use as in vitro diagnostic tools in clinical environments [ 77 ]. However, a drawback of employing cfDNA for mutation testing, as opposed to tumor tissues, is its lower sensitivity in identifying mutations derived from tumors, with the factors affecting the detection of such genetic changes in cfDNA remaining unclear.…”

“…However, a drawback of employing cfDNA for mutation testing, as opposed to tumor tissues, is its lower sensitivity in identifying mutations derived from tumors, with the factors affecting the detection of such genetic changes in cfDNA remaining unclear. Conversely, mutation testing with ctDNA offers an advantage over tumor tissue samples by potentially capturing a broad spectrum of genetic variations in a patient, irrespective of tumor heterogeneity [ 77 ].…”