Rare mutation-dominant compound EGFR-positive NSCLC is associated with enriched kinase domain-resided variants of uncertain significance and poor clinical outcomes

Abstract

Background: Compound epidermal growth factor receptor (EGFR) mutations are less responsive to tyrosine kinase inhibitors (TKIs) than single EGFR mutations in non-small cell lung cancer (NSCLC). However, the detailed clinical characteristics and prognosis of various compound EGFR mutations remain to be elucidated.

Methods: We retrospectively studied the next-generation sequencing (NGS) data of treatment-naïve tumors from 1025 NSCLC patients with compound EGFR mutations, which were sub-categorized into different combinations of common mutations (19-Del and EGFR exon 21 p.L858R), rare mutations, and variants of uncertain significance (VUSs). Prognosis and drug resistance to first-line TKIs were analyzed in 174 and 95 patients, respectively.

Results: Compound EGFR mutations were enriched with EGFR exon 21 p.L858R and rare mutations, but not 19-Del (P < 0.001). The common + rare and rare + rare subtypes had fewer concurrent mutations in the PI3K pathway (P = 0.032), while the rare + rare and common + VUSs subtypes showed increased association with smoking- and temozolomide-related mutational signatures, respectively (P < 0.001). The rare mutation-dominant subtypes (rare + VUSs and rare + rare) had the worst clinical outcomes to first-line TKIs (P < 0.001), which was further confirmed using an external cohort (P = 0.0066). VUSs in the rare + VUSs subtype selectively reside in the EGFR kinase domain (P < 0.001), implying these tumors might select additional mutations to disrupt the regulation/function of the kinase domain.

Conclusions: Different subtypes of compound EGFR mutations displayed distinct clinical features and genetic architectures, and rare mutation-dominant compound EGFR mutations were associated with enriched kinase domain-resided VUSs and poor clinical outcomes. Our findings help better understand the oncogenesis of compound EGFR mutations and forecast prognostic outcomes of personalized treatments.

Keywords: Compound EGFR mutations; Non-small cell lung cancer; Precision medicine; Resistant mechanism; Tyrosine kinase inhibitors.

Fig. 1

Compound EGFR mutation-positive patients had fewer EGFR 19-Del mutations and more L858R and rare EGFR mutations. A The flowchart of the study. B Comparing the percentage of patients with single EGFR mutation (n = 7460) and compound EGFR mutations (n = 1025) according to their EGFR mutation type. Based on the dominant EGFR mutations, patients with compound EGFR mutations were divided into common (i.e., common + common, common + rare, or common + VUSs), rare (i.e., rare + rare or rare + VUSs), and VUSs (i.e., VUSs + VUSs) groups. C Comparing the percentage of patients with single EGFR mutation (n = 7460) and compound EGFR mutations (N = 998) according to their EGFR mutation type. Patients with concurrent L858R and 19-Del (n = 27) were not included in the analysis. D The difference of the accompanied EGFR mutations between 19-Del and L858R-containing compound EGFR mutations. Patients with concurrent L858R and 19-Del (n = 27) were not included in the analysis. NGS, next-generation sequencing; VUS, variants of uncertain significance; TM, transmembrane domain

Fig. 2

The molecular and genetic characteristics of different types of compound EGFR mutations. A The lollipop plots of EGFR VUSs from various VUS-containing compound EGFR mutations, including L858R + VUSs (n = 416), 19-Del + VUSs (n = 86), and rare + VUSs (n = 185). B The percentage of patients with various mutated genes stratified by different compound EGFR mutation types. Patients’ samples that were characterized by targeted NGS of 139 key lung cancer-related genes were included in the analysis (n = 720). C The percentage of patients with various altered signaling pathways stratified by different compound EGFR mutation types. Patients’ samples that were characterized by targeted NGS of 139 key lung cancer-related genes were included in the analysis (n = 720). The Kruskal–Wallis test was conducted to compare multiple groups. P-value of < 0.05 was considered to be statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). SP, signal peptide; TM, transmembrane domain

Fig. 3

Mutational signature and chromosomal instability of different types of compound EGFR mutations. A The mutational signature analysis for patients with different types of compound EGFR mutations. Patients whose baseline tumor tissue samples were characterized by large panel targeted sequencing of 425 cancer-relevant genes were included in the analysis (n = 408). The contribution of each signature was the proportion of the selected signature over all the detected signatures in that specific patient. The Kruskal–Wallis test was conducted to compare multiple groups. The chromosomal instability score in patients with double vs multiple EGFR mutations (B) or in patients with different types of compound EGFR mutations (C). Patients whose baseline tumor tissue samples were characterized by large panel targeted sequencing of 425 cancer-relevant genes were included in the analysis (n = 408). P-value of < 0.05 was considered to be statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). CIS, chromosomal instability score

Fig. 4

The correlation between the type of compound EGFR mutations and patients’ prognosis to first-line EGFR TKIs. A Kaplan–Meier curve of progression-free survival in NSCLC patients in strata of the number of EGFR mutations. B Kaplan–Meier curve of progression-free survival in dual EGFR mutation-positive patients in the strata of the various combination of EGFR mutations. One patient with the common + common subtype was not included in the analysis. C Kaplan–Meier curve of progression-free survival in compound EGFR mutation-positive patients in the strata of various generations of EGFR TKIs. D Kaplan–Meier curve of progression-free survival in compound EGFR mutation-positive patients who harbored EGFR VUSs, and these patients were in the strata of different types of compound EGFR mutations, as well as the location of the VUSs, which can be inside the EGFR kinase domain (KD +) or outside the EGFR kinase domain (KD −). One patient with the rare + VUSs (KD −) subtype was not included in the analysis. Log‐rank test with P-value < 0.05 was considered to be statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

Drug-resistant mechanism analysis using patients with paired baseline and PD samples (n = 95). A The comparison of EGFR mutation status between paired baseline and PD samples. Each column represented a sample derived from a patient, and the two oncoprint plots (i.e., baseline vs PD to first-line TKIs) used the same order to arrange the paired patient samples. The frequency of mutated genes (B) or altered signaling pathways (C) between the baseline samples and PD samples. D The status of aberrant signaling pathways between the baseline and the paired PD samples, stratified by different compound EGFR mutation subtypes. BL, baseline; PD, progressive disease