Case Report: Afatinib Treatment in a Patient With NSCLC Harboring a Rare EGFR Exon 20 Mutation

Abstract

Unlike most other primary epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC), exon 20 insertions, comprising approximately 4% to 10% of all EGFR mutations, are generally considered to be resistant to EGFR tyrosine kinase inhibitors (TKIs). However, EGFR exon 20 insertions are structurally and pharmacologically heterogeneous, with variability in their position and size having implications for response to different EGFR TKIs. The second-generation ErbB family blocker, afatinib, is approved for the first-line treatment of EGFR mutation-positive NSCLC and has been shown to have a broad inhibitory profile against common and uncommon EGFR mutations. Here, we describe a patient with bilateral multifocal lung adenocarcinoma harboring a very rare EGFR exon 20 insertion (c.2317_2319dup3; p.H773dup), who has been receiving treatment with afatinib for 4.5 years. To our knowledge, this is the first report describing long-term benefit for a patient treated with afatinib with this rare exon 20 insertion. We are aware of two further cases with this rare EGFR mutation. One patient, also reported here, has early-stage lung adenocarcinoma and has not yet received systemic therapy for NSCLC. The other patient received afatinib in the context of a global compassionate use program and had progressive disease. Our findings may be of clinical relevance for patients carrying tumors with this rare mutation as epidemiological evidence suggests that p.H773dup may function as a driver mutation in NSCLC. Together with previous preclinical and clinical evidence for the activity of afatinib against certain EGFR exon 20 insertions, these findings warrant further investigation.

Keywords: EGFR mutation; H773dup; NSCLC; afatinib; exon 20 insertion; long-term response; uncommon mutation.

Figure 1

Inhibition of EGFR mutant protein autophosphorylation by afatinib and erlotinib in cellular assays. NIH-3T3 cells (ATCC; #CRL-1658) were cultured in supplemented Dulbecco’s Modified Eagle Medium and cultivated at 37°C/5% CO₂ in a humidified atmosphere to maintain <80% confluence. Cells were then transfected with one of 12 EGFR mutant plasmid constructs, using 4 µg of DNA for EGFR variants G598V, D770_N771insNPG, P772_H773insV, WHins774, and T790M, and 2.5 µg of constructs R108K, A289D, A289T, A289V, G719S, WASVins770, and L861Q, diluted in 250 µl serum-free culture medium. A diluted Lipofectamine-DNA mix was then added drop-wise to the cells. 48 h post-transfection, cells were treated for 2 h with afatinib or erlotinib (1–10,000 nM) or were left untreated. At 2 h post-treatment, protein lysates were prepared using lysis buffer and the effect of TKI treatment on EGFR tyrosine-1068 phosphorylation was analyzed by Western blot (primary antibody: phospho-specific anti-EGFR [Y1068] antibody; Abcam, #ab40815, 1:1,000 dilution; secondary antibody: goat anti-rabbit; Dako, #P0448, 1:1,000 dilution). Actin was used as a loading control. EGFR, epidermal growth factor receptor.

Figure 2

Patient 1 case history and time line of key events. PD, progressive disease; SD, stable disease.

Figure 3

Patient 1 clinical course, including treatment history and CT scans. CT, computed tomography.

Figure 4

Insertion site of EGFR exon 20 insertion mutation. Sequencing electropherogram of EGFR exon 20 showing: (A) wild-type sequence of the adenocarcinoma from patient 1 resected in 2014; (B) duplication of a CAC base triplet in the adenocarcinoma from patient 1 biopsied in 2015. EGFR, epidermal growth factor receptor; CAC, cytosine adenine cytosine; (C) wild-type sequence of the adenocarcinoma from patient 2 resected in 2019; (D) duplication of a CAC base triplet in the adenocarcinoma from patient 2 biopsied in 2019.