Next-generation sequencing of tissue and circulating tumor DNA: Resistance mechanisms to EGFR targeted therapy in a cohort of patients with advanced non-small cell lung cancer

Abstract

Background: Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) has been considered as an effective treatment in epidermal growth factor receptor-mutant (EGFR-mutant) advanced non-small cell lung cancer (NSCLC). However, most patients develop acquired resistance eventually. Here, we compared and analyzed the genetic alterations between tissue assay and circulating tumor DNA (ctDNA) and further explored the resistance mechanisms after EGFR-TKI treatment.

Methods and materials: Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), Cobas® ARMS-PCR and next-generation sequencing (NGS) were performed on tissue samples after pathological diagnosis. Digital droplet PCR (ddPCR) and NGS were performed on plasma samples. The association between genetic alterations and clinical outcomes was analyzed retrospectively.

Results: Thirty-seven patients were included. The success rate of re-biopsy was 91.89% (34/37). The total detection rate of EGFR T790M was 62.16% (23/37) and the consistency between tissue and ctDNA was 78.26% (18/23). Thirty-four patients were analyzed retrospectively. For tissue re-biopsy, 24 patients harbored concomitant mutations. Moreover, tissue re-biopsy at resistance showed 21 patients (21/34, 61.76%) had the concomitant somatic mutation. The three most frequent concomitant mutations were TP53 (18/34, 52.94%), MET (4/34, 11.76%), and PIK3CA (4/34, 11.76%). Meanwhile, 21 patients (21/34, 61.76%) with EGFR T790M mutation. Progression-free survival (PFS) and overall survival (OS) were better in patients with T790M mutation (p = 0.010 and p = 0.017) or third-generation EGFR-TKI treatment (p < 0.0001 and p = 0.073). Interestingly, concomitant genetic alterations were significantly associated with a worse prognosis for patients with EGFR T790M mutation receiving third-generation EGFR-TKIs (p = 0.037).

Conclusions: Multi-platforms are feasible and highly consistent for re-biopsy after EGFR-TKI resistance. Concomitant genetic alterations may be associated with a poor prognosis for patients with EGFR T790M mutation after third-generation EGFR-TKIs.

Trial registration: ClinicalTrials.gov NCT03309462.

Keywords: epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI); genetic alterations; non-small cell lung cancer (NSCLC); re-biopsy.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Figures

FIGURE 1

Flow chart of eligible population. A total of 39 patients with lung cancer diagnosed with EGFR 19del or EGFR 21L858R mutation positive were resistant to the first‐ or second‐generation EGFR‐TKIs after treatment and enrolled. Among them, when NGS re‐examined the genes in the first biopsy sample, 1 case was excluded due to the result indicating that EGFR 19del was false positive, and 1 case was excluded due to the result indicating mutation containing EGFR 20T790M. Thirty‐seven patients had tissue gene testing and blood gene testing. ARMS‐PCR, amplification refractory mutation system‐polymerase chain reaction; ctDNA, circulating tumor DNA; ddPCR, digtal droplet polymerase chain reaction; EGFR, epidermal growth factor receptor; EGFR‐TKI, epidermal growth factor receptor‐tyrosine kinase inhibitor; NGS, next‐generation sequencing; PD, progressive disease.

FIGURE 2

Genetic alterations of enrolled patients by ctDNA and tissue NGS. (A) Oncoprint of ctDNA NGS alterations (n = 37). Synonymous alterations and variants of unknown significance were excluded. All 37 patients were tested for ctDNA, but only 34 of them were also tested for tissue NGS. Each vertical bar represents a patient. (B) Most frequent alterations identified by plasma‐derived ctDNA NGS (n = 37). (C) Oncoprint of tissue NGS (n = 34). (D) Most frequent alterations identified by tissue NGS (n = 34). ctDNA, circulating tumor DNA; EGFR, epidermal growth factor receptor; EGFR‐TKI, epidermal growth factor receptor‐tyrosine kinase inhibitor; NGS, next‐generation sequencing.

FIGURE 3

The detection efficiency of different detection platforms for EGFR T790M mutation. (A) EGFR T790M mutation was detected and compared by both ctDNA and tissue assays and as illustrated by the Venn diagrams. (B) EGFR T790M mutation was detected and compared by NGS and ddPCR in plasma samples. (C) EGFR T790M mutation was detected and compared by ARMS‐PCR, Cobas® ARMS‐PCR and NGS in tissue samples. ARMS‐PCR, amplification refractory mutation system‐polymerase chain reaction; ctDNA, circulating tumor DNA; ddPCR, digtal droplet polymerase chain reaction; EGFR, epidermal growth factor receptor; NGS, next‐generation sequencing.

FIGURE 4

Maximum change in tumor size from baseline in individual patients over the course of treatment. Changes in tumor size (diameter) were assessed in patients with or without EGFR T790M mutation treated with the third‐generation EGFR‐TKIs or other treatments. Tumor shrinkage relative to baseline was observed in 70.59% of patients. EGFR‐TKI, epidermal growth factor receptor‐tyrosine kinase inhibitor.

FIGURE 5

Survivals analysis of patients after resistance to first‐generation TKI therapy. (A) Kaplan‐Meier curves of PFS in 34 patients with first‐generation EGFR‐TKI treatment whose tissue re‐biopsy had concomitant mutations compared with those without concomitant mutations. (B) Kaplan‐Meier curves of OS in 34 patients with first‐generation EGFR‐TKI treatment whose tissue re‐biopsy had concomitant mutations compared with those without concomitant mutations. (C) Kaplan‐Meier curves of PFS in 21 patients with EGFR T790M mutation whose tissue re‐biopsy had concomitant mutations compared with those without concomitant mutations after receiving third‐generation EGFR‐TKIs treatment. (D) Kaplan‐Meier curves of OS in 21 patients with EGFR T790M mutation whose tissue re‐biopsy had concomitant mutations compared with those without concomitant mutations after receiving third‐generation EGFR‐TKIs treatment. EGFR, epidermal growth factor receptor; EGFR‐TKI, epidermal growth factor receptor‐tyrosine kinase inhibitor; OS, overall survival; PFS, progression‐free survival.