PEAC: An Ultrasensitive and Cost-Effective MRD Detection System in Non-small Cell Lung Cancer Using Plasma Specimen

Abstract

Circulating tumor DNA (ctDNA), a tumor-derived fraction of cell-free DNA (cfDNA), has emerged as a promising marker in targeted therapy, immunotherapy, and minimal residual disease (MRD) monitoring in postsurgical patients. However, ctDNA level in early-stage cancers and postsurgical patients is very low, which posed many technical challenges to improve the detection rate and sensitivity, especially in the clinical practice of MRD detection. These challenges usually include insufficient DNA input amount, limit of detection (LOD), and high experimental costs. To resolve these challenges, we developed an ultrasensitive ctDNA MRD detection system in this study, namely PErsonalized Analysis of Cancer (PEAC), to simultaneously detect up to 37 mutations, which account for 70–80% non-small cell lung cancer (NSCLC) driver mutations from low plasma sample volume and enables LOD of 0.01% at a single-site level. We demonstrated the high performance achieved by PEAC on both cfDNA reference standards and clinical plasma samples from three NSCLC patient cohorts. For cfDNA reference standards, PEAC achieved a specificity of 99% and a sensitivity of 87% for the mutations at 0.01% allele fraction. In the second cohort, PEAC showed 100% concordance rate between ddPCR and Next-generation sequencing (NGS) among 29 samples. In the third cohort, 22 of 59 patients received EGFR TKI treatment. Among them, three in four patients identified low level actionable gene mutations only by PEAC had partial responses after targeted therapy, demonstrating high ctDNA detection ability of PEAC. Overall, the developed PEAC system can detect the majority of NSCLC driver mutations using 8–10 ml plasma samples, and has the advantages of high detection sensitivity and lower costs compared with the existing technologies such as ddPCR and NGS. These advantages make the PEAC system quite appropriate for ctDNA and MRD detection in early-stage NSCLC and postsurgical recurrence monitoring.

The use of minimal residual disease in thoracic oncology: Gaps between promises and the on‐the‐ground reality of daily practice

“…Currently, one of the biggest challenges in this field is choosing the most suitable assay with which to detect the molecular MRD of lung cancer patients with a high sensitivity and a high specificity. 16 Several assays can be used, and they are briefly described here.A fixed panel assay that tracks driver mutations and targetable gene alterations can be used with a single plasma sample. Alternatively, a personalised panel, requiring previous tumour DNA sequencing, can be used.…”

Prognostic Value of Circulating Tumor DNA (ctDNA) in Oncogene-Driven NSCLC: Current Knowledge and Future Perspectives

“…However, there have been several technical challenges in the detection rate and sensitivity of MRD detection since ctDNA levels in early-stage cancers and postsurgical patients are low. Today, an array of sensitive ctDNA detection systems in patients with actionable NSCLC driver gene mutations, such as the Amplification Refractory Mutation System (ARMS), the digital PCR (dPCR)/digital droplet PCR (ddPCR), or the Next-generation sequencing (NGS), have been used to overcome challenges such as insufficient DNA input amount and high costs [37]. These advantages have facilitated the conduction of several studies that are looking at using ctDNA to demonstrate the clinical utility of detecting MRD in driver-mutant NSCLC and treating recurrent disease earlier, and in the following section, we present the most impactful ones.…”

“…It is also well documented that L858R mutation has been correlated with a shorter median OS (13.7 months) for mutation carriers versus wild-type patients (27.7 months) [53]. Other researchers used a blocker displacement amplification-derived method as a tool for MRD monitoring, to examine sequential blood samples from an EGFR-mutated NSCLC patient who exhibited no evidence of radiologic recurrence [37]. This patient was then treated with icotinib.…”

Tissue and Plasma-Based Highly Sensitive Blocker Displacement Amplicon Nanopore Sequencing for EGFR Mutations in Lung Cancer

“…LNA is bridged nucleic acid that locks ribose moiety in the 3′-endo conformation, which prevents the cleavage mismatch nucleotide and enhances mismatch discrimination [ 12 ]. BDA uses a DNA oligonucleotide probe with internal three-carbon spacer, which can bind to wild-type DNA and prevent the binding of PCR primer [ 13 , 14 ]. Hence, BDA can limit the amplification of the wild-type alleles and effectively enrich low variant allele frequency (VAF) [ 9 , 13 ].…”