EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics

Abstract

In contrast to other primary epidermal growth factor receptor (EGFR) mutations in lung adenocarcinomas, insertions in exon 20 of EGFR have been generally associated with resistance to EGFR-tyrosine kinase inhibitors. Their molecular spectrum, clinicopathologic characteristics, and prevalence are not well established. Tumors harboring EGFR exon 20 insertions were identified through an algorithmic screen of 1,500 lung adenocarcinomas. Cases were first tested for common mutations in EGFR (exons 19 and 21) and KRAS (exon 2) and, if negative, further analyzed for EGFR exon 20 insertions. All samples underwent extended genotyping for other driver mutations in EGFR, KRAS, BRAF, ERBB2/HER2, NRAS, PIK3CA, MEK1, and AKT by mass spectrometry; a subset was evaluated for ALK rearrangements. We identified 33 EGFR exon 20 insertion cases [2.2%, 95% confidence interval (CI), 1.6-3.1], all mutually exclusive with mutations in the other genes tested (except PIK3CA). They were more common among never-smokers (P < 0.0001). There was no association with age, sex, race, or stage. Morphologically, tumors were similar to those with common EGFR mutations but with frequent solid histology. Insertions were highly variable in position and size, ranging from 3 to 12 bp, resulting in 13 different insertions, which, by molecular modeling, are predicted to have potentially different effects on erlotinib binding. EGFR exon 20 insertion testing identifies a distinct subset of lung adenocarcinomas, accounting for at least 9% of all EGFR-mutated cases, representing the third most common type of EGFR mutation after exon 19 deletions and L858R. Insertions are structurally heterogeneous with potential implications for response to EGFR inhibitors.

Figure 1

Distribution of all primary EGFR mutations identified in the current study. Although, based on our analysis, insertions in exon 20 corresponded to 9% of all EGFR mutated samples, we estimate that the true incidence may be even higher, closer to 11%, factoring in the expected positive cases which would have been detected if the entire negative group had been tested. Figure includes 5 cases with double mutations as follows *2 cases with double mutations G719A/S768I and G719S/E709A, **2 cases with concurrent T790M at baseline, *** 1 case with concurrent T790M at baseline.

Figure 2

Modeling of EGFR exon 20 insertions using the 3-dimensional structure of the EGFR kinase domain predicts different interactions with the erlotinib binding region. The X-ray structure at 2.6 Å resolution (PDB code 1M17) is used to show the drug and the positions of mutations. Yellow – C helix; green – erlotinib; labeled residues identify the region of the insertion.

Figure 3

Positions of the EGFR exon 20 insertions identified over a 3 year period and comparison with the spectrum of EGFR exon 19 and HER2 insertion mutations detected within the same time frame. Insertions in exon 20 of EGFR show higher heterogeneity compared to both HER2 and EGFR exon 19. Most insertions in HER2 are represented by the A775_G776insYVMA while insertions in exon 19 of EGFR all share the inserted sequence PVAI and are located in the same region I744-E746.