Application of liquid biopsy-based targeted capture sequencing analysis to improve the precision treatment of non-small cell lung cancer by tyrosine kinase inhibitors

Abstract

Background: Targeted therapy of patients with non-small cell lung cancer (NSCLC) who harbour sensitising mutations by tyrosine kinase inhibitors (TKIs) has been found more effective than traditional chemotherapies. However, target genes status (eg, epidermal growth factor receptor (EGFR) TKIs sensitising and resistant mutations) need to be tested for choosing appropriate TKIs. This study is to investigate the performance of a liquid biopsy-based targeted capture sequencing assay on the molecular analysis of NSCLC.

Methods: Plasma samples from patients with NSCLC who showed resistance to the first/second-generation EGFR TKIs treatment were collected. The AVENIO ctDNA Expanded Kit is a 77 pan-cancer genes detection assay that was used for detecting EGFR TKIs resistance-associated gene mutations. Through comparison of the EGFR gene testing results from the Cobas EGFR Mutation Test v2, and UltraSEEK Lung Panel, the effectiveness of the targeted capture sequencing assay was verified.

Results: A total of 24 plasma cell-free DNA (cfDNA) samples were tested by the targeted capture sequencing assay. 33.3% (8/24) cfDNA samples were positive for EGFR exon 20 p.T790M which leads to EGFR dependent TKIs resistance. 8.3% (2/24) and 4.2% (1/24) samples were positive for mesenchymal-epithelial transition gene amplification and B-Raf proto-oncogene, serine/threonine kinase exon 15 p.V600E mutations which lead to EGFR independent TKIs resistance. The median value of the p.T790M variant allele fraction and variant copy numbers was 2% and 36.10 copies/mL plasma, respectively. The next-generation sequencing test showed higher than 90% concordance with either MassArray or qPCR-based methods for detecting either EGFR TKIs sensitising or resistance mutations.

Conclusion: The targeted capture sequencing test can support comprehensive molecular analysis needed for TKIs treatment, which is promising to be clinically applied for the improved precision treatment of NSCLC.

Keywords: drug reactions; lung cancer; non-small cell lung cancer.

Figure 1

Detection of EGFR sensitising and TKIs resistance variants in plasma cell-free DNA (cfDNA) of patients with NSCLC by the NGS-based Avenio panel. (A) Variant allele fraction of the EGFR gene in plasma cfDNA; (B) molecular concentration of different EGFR variants in the plasma of patients with NSCLC. EGFR, epidermal growth factor receptor; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; TKI, tyrosine kinase inhibitor.

Figure 2

Percentages of tumour biomarker genes variant alleles located in different ranges of fraction ratio and copy numbers. (A) Distribution of tumour biomarker genes variant alleles in different ranges of fraction ratio; (B) distribution of tumour biomarker genes variant alleles in different ranges of copy numbers.

Figure 3

Testing of TP53 gene in the plasma cell-free DNA (cfDNA) of non-small cell lung cancer (NSCLC) patients by next-generation sequencing-based Avenio panel. (A) TP53 gene’s variant allele fraction in plasma cfDNA of NSCLC; (B) molecular concentration of TP53 gene variants in the plasma of NSCLC.

Figure 4

Testing of gene copy number variants in the plasma cell-free DNA of patients with non-small cell lung cancer by using next-generation sequencing-based Avenio panel. EGFR, epidermal growth factor receptor; MET, mesenchymal-epithelial transition.