EGFR-V834L combined with L858R mutation reduced afatinib sensitivity and associated to early recurrence in lung cancer

Abstract

Background: The third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib is widely used as a first-line treatment for EGFR-mutated non-small cell lung cancer (NSCLC). However, there is no established treatment for osimertinib resistance, so second-generation afatinib is an alternative treatment option. The purpose of this study was to elucidate gene alterations associated with afatinib efficacy and resistance by analyzing cell-free DNA (cfDNA) obtained from patients with EGFR-mutated NSCLC.

Methods: This study was conducted as a prospective clinical trial in patients with EGFR-mutated NSCLC at multiple institutions. We analyzed plasma cfDNA from the patients treated with afatinib as the first-line therapy.

Results: Paired specimens were obtained before and at the time of resistance to afatinib treatment, and specimens only at afatinib resistance were obtained from 22 and 18 patients, respectively. In plasma cfDNA from the 22 cases, driver EGFR mutations were detected in 13 cases (59.1%), and the compound V834L mutation was detected in two cases in cis with EGFR-L858R mutation. The median progression-free survival (mPFS) was remarkably shorter in patients with V834L than in all 22 cases (4.2 vs. 9.2 months). Moreover, we detected V834L and T790M combined with EGFR-L858R in the cfDNA from one patient resistant to afatinib. Preclinical experiments using EGFR-L858R, with or without V834L, in Ba/F3 cells revealed that V834L with L858R conferred resistance to low concentrations of EGFR-TKIs, including afatinib and osimertinib. In three cases of EGFR-L858R+V834L, other co-mutations, including TP53, CTNNB1, and RB1, were detected either before or after afatinib resistance.

Conclusions: These results suggested that V834L cooperates with other coexisting mutations to influence the therapeutic efficacy of EGFR-TKIs.

Keywords: Co-mutation; cell-free DNA (cfDNA); epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI); liquid biopsy; resistance.

Figure 1

Consort diagram. A total of 55 patients were enrolled in this study. Afatinib treatment was discontinued in 49 cases, and plasma samples were collected at the time of resistance in 40 cases. In 22 cases, we were able to collect paired samples before afatinib treatment and at the acquisition of resistance to afatinib treatment. In 18 cases, we collected samples only at the acquisition of resistance to afatinib treatment.

Figure 2

Genomic alterations detected in cfDNA of patients obtained before treatment by afatinib. EGFR driver mutations were detected in 13 patients (59.1%). The most common alteration was KMT2D (8/22; 38.1%), LRP1B (8/22; 38.1%), and NF1 (8/22; 38.1%). EGFR, epidermal growth factor receptor; cfDNA, cell-free DNA.

Figure 3

Preclinical analyses showing the role of EGFR-V834L mutation on sensitivity to EGFR-TKIs. (A) Ba/F3 cells were transfected with EGFR-L858R or EGFR-L858R+V834L in cis. The cells were incubated with afatinib or osimertinib. The cell viability was determined by CCK-8 assay. Means ± standard deviations of triplicate culture are shown. (B) Ba/F3 cells transfected with EGFR-L858R or EGFR-L858R+V834 in cis were treated with afatinib (0.3 nM) for 24 h. The expression of indicated proteins was determined by western blotting. (C) Ba/F3 cells transfected with EGFR-L858R or EGFR-L858R+V834 in cis were inoculated subcutaneously into SHO mice. When the tumor volume reached 50–100 mm³, the mice were orally treated with vehicle (control: n=10 tumors/5 mice), afatinib (2.5 mg/kg: n=10 tumors/5 mice) or osimertinib (5 mg/kg: n=10 tumors/5 mice) daily. Means ± SEs of tumor volumes are shown. *, P<0.05; N.S., not significant. EGFR, epidermal growth factor receptor; p-, phosphorylated-; ERK, extracellular signal-related kinase; cas3, caspase-3; TKIs, tyrosine kinase inhibitors; CCK-8, Cell Counting Kit-8; SHO, SCID Hairless Outbred; SE, standard error.

Figure 4

Genomic alterations detected in cfDNA of patients obtained at the acquisition of afatinib resistance. EGFR, epidermal growth factor receptor; cfDNA, cell-free DNA.

Figure 5

The PFS and case presentation of the three patients with EGFR-L858R positive NSCLC in which EGFR-V834L was detected in cfDNA obtained before treatment or at afatinib resistance. (A) PFS stratified by detection of EGFR-V834L in cfDNA either before afatinib treatment or at the acquisition of afatinib resistance in 40 cases in this study. (B) Case presentation of Case 1. (C) Case presentation of Case 2. (D) Case presentation of Case 3. EGFR, epidermal growth factor receptor; mPFS, median progression-free survival; CI, confidence interval; HR, hazard ratio; NA, not available; PFS, progression-free survival; M, months; PUL, pulmonary; OSS, osseous; PD, progressive disease; PLE, pleural; PR, partial response; NSCLC, non-small cell lung cancer; cfDNA, cell-free DNA.