Ultra-deep massively parallel sequencing with unique molecular identifier tagging achieves comparable performance to droplet digital PCR for detection and quantification of circulating tumor DNA from lung cancer patients

Abstract

The identification and quantification of actionable mutations are of critical importance for effective genotype-directed therapies, prognosis and drug response monitoring in patients with non-small-cell lung cancer (NSCLC). Although tumor tissue biopsy remains the gold standard for diagnosis of NSCLC, the analysis of circulating tumor DNA (ctDNA) in plasma, known as liquid biopsy, has recently emerged as an alternative and noninvasive approach for exploring tumor genetic constitution. In this study, we developed a protocol for liquid biopsy using ultra-deep massively parallel sequencing (MPS) with unique molecular identifier tagging and evaluated its performance for the identification and quantification of tumor-derived mutations from plasma of patients with advanced NSCLC. Paired plasma and tumor tissue samples were used to evaluate mutation profiles detected by ultra-deep MPS, which showed 87.5% concordance. Cross-platform comparison with droplet digital PCR demonstrated comparable detection performance (91.4% concordance, Cohen’s kappa coefficient of 0.85 with 95% CI = 0.72–0.97) and great reliability in quantification of mutation allele frequency (Intraclass correlation coefficient of 0.96 with 95% CI = 0.90–0.98). Our results highlight the potential application of liquid biopsy using ultra-deep MPS as a routine assay in clinical practice for both detection and quantification of actionable mutation landscape in NSCLC patients.

Ultra-Deep Massive Parallel Sequencing of Plasma Cell-Free DNA Enables Large-Scale Profiling of Driver Mutations in Vietnamese Patients With Advanced Non-Small Cell Lung Cancer

“…We have previously conducted a study comparing the performance of liquid biopsy and tissue biopsy using matched tissues and plasma samples obtained from the same cohort of patients (15). We observed a high concordance rate of 85% between actionable mutation profiles detected from paired plasma and tissue samples in a cohort of 40 NSCLC patients, consistent with other similar studies (15,17,18).…”

“…We and others have reported high concordance rates between liquid and tissue biopsy testing by comparing mutation profiles of matched plasma and paired tissue samples. However, the major limitation of such studies is that the comparison was mostly carried out in a small cohort of <50 patients due to the difficulties in obtaining sufficient number of matched plasma and tissue biopsies (15,17,18). Hence, the analysis was mostly limited to the most commonly mutated driver gene (EGFR or KRAS).…”

“…There were at least two possible explanations for the lower detection rate by cfDNA testing compared to that of tumor tissue testing. Indeed, previous cross-platform comparison between cfDNA testing and digital PCR reported a sensitivity of cfDNA sequencing of 79% for mutation detection in plasma, suggesting that mutations in cfDNA could not be detected in 20% of the cases ( 15 ). This was consistent with findings from previous studies that cfDNA based sequencing exhibited lower sensitivity in mutation detection compared to matched tissue sequencing ( 14 , 15 , 20 – 22 ).…”

“…It is therefore more suitable for populationscale study involving a large number of samples. Additionally, it has been reported that ultra-deep sequencing of plasma cfDNA of NSCLC patients achieved high concordance rates of driver mutation detection compared to sequencing of matched tumor tissues (13)(14)(15)(16).…”

“…We have previously conducted a study comparing the performance of liquid biopsy and tissue biopsy using matched tissues and plasma samples obtained from the same cohort of patients (15). We observed a high concordance rate of 85% between actionable mutation profiles detected from paired plasma and tissue samples in a cohort of 40 NSCLC patients, consistent with other similar studies (15,17,18). However, the major limitation of such studies is that the conclusion was drawn from a relatively small sample size due to the difficulties in obtaining tissue biopsies from NSCLC patients with metastatic cancer.…”