Detecting Ultralow Frequency Mutation in Circulating Cell‐Free DNA of Early‐Stage Nonsmall Cell Lung Cancer Patients with Unique Molecular Identifiers

Abstract

The performance of next generation sequencing in profiling somatic mutations from plasma samples, especially in early‐stage cancer, is limited by its sensitivity due to the extremely low amount of cell‐free DNA (cfDNA). Polymerase‐chain reaction amplification of limited amounts of DNA often results in product redundancy and the amplification of contaminant DNA. Numerous methods have been developed to detect and quantify ultralow frequency mutations. In this study, the potential of tagging each DNA molecule with a unique molecular identifier (UMI) is explored to detect ultralow frequency mutations in early‐stage lung cancer patients. Paired tissue and blood samples from 32 patients with early‐stage (stage IA‐IIB) nonsmall cell lung cancer are sequenced using a panel consisting of 168 cancer‐related genes. Collectively, 93.8% of tissue samples and 59.4% of plasma samples have mutations detected. The concordance rates are acceptable for all patients except for stage IA. Notably, mutations at an alleleic frequency as low as 0.1% are detected in UMI‐tagged cfDNA samples. Moreover, patients with nonadenocarcinoma histology have a significantly higher detection rate than patients with adenocarcinoma (92.9% vs 33.3%, P = 0.002). The study demonstrates the potential of UMI in early detection of lung cancer from plasma samples.

Potential utility of longitudinal somatic mutation and methylation profiling for predicting molecular residual disease in postoperative non‐small cell lung cancer patients

“…In our study, 63.1% of patients had somatic mutation detected at baseline from plasma as compared with 100% from paired tumor tissues. Despite the use of UMI‐based sequencing, the lower detection of somatic mutations from blood samples of early stage patients was due to the smaller tumor bulk and the lower concentration of ctDNA present in their circulation, particularly from patients with stage IA disease and LUAD histology as reported by Yang et al 35 Our cohort was also comprised of 75% LUAD, which had been shown as a histology with lower plasma‐based mutation detection rate and lower tissue to plasma mutation concordance as compared with LUSC 39 . Among the eight relapsed patients confirmed by radiologic imaging in the enrolled cohort, one patient had no somatic mutation detected from his plasma sample throughout the follow‐up period, and another patient had ctDNA clearance.…”

“…Detection limit for regular NGS assays is insufficient for analyzing such low‐frequency variants in cfDNA; the use of UMI‐based capture probes has been demonstrated to enable sequencing depths to reach up to 50,000× and improve the accurate detection of mutations with ultralow frequency of up to 0.05% from blood samples of patients with early stage lung cancer 35 . UMI‐tagging of ctDNA fragments from early stage patients improves the accuracy of detecting ultralow frequency mutations by allowing the distinction between the original DNA template and contaminants, which minimizes false detection of somatic mutations 35 . In our study, 63.1% of patients had somatic mutation detected at baseline from plasma as compared with 100% from paired tumor tissues.…”

“…Between 1 and 5 mL of plasma were used for cfDNA extraction. CfDNA and tumor tissue DNA were extracted using appropriate Qiagen DNA extraction kits for tissue and circulating nucleic acid (Qiagen) following the manufacturer's instructions as previously described 35 …”

Investigation on the potential of circulating tumor DNA methylation patterns as prognostic biomarkers for lung squamous cell carcinoma

“…Targeted sequencing was performed as described previously (44). The cfDNA fragments were ligated with adapters containing UMIs.…”

“…The concentration of ctDNA from the plasma-derived cfDNA is highly dependent on the tumor burden. Therefore, the detection of somatic mutations from ctDNA is severely limited in early-stage tumors (44).…”