Not all epidermal growth factor receptor mutations in lung cancer are created equal: Perspectives for individualized treatment strategy

Somatic mutations in the kinase domain of the epidermal growth factor receptor (EGFR) gene are detected in approximately 40% and 17% of lung adenocarcinoma in Asians1 and in Caucasians,2 respectively. When these biomarkers were first developed, early studies simplified the complexity of tumor genotype by dichotomizing them as mutant or wild type. Fortunately, common mutations (i.e. exon 19 deletions [Del19] and L858R mutation in exon 21) are associated with sensitivity to EGFR tyrosine kinase inhibitors (TKI).3, 4 Targeted therapies for these lung cancers were established based on 7 phase III randomized trials.5-11

EGFR mutations other than Del19 and L858R are variably termed either minor (less common) or uncommon mutations. The need for appropriate management of patients with these uncommon mutations is increasing because the incidence of uncommon EGFR mutations is comparable to rare targetable driver genes such as ROS1 and RET.12-15 We recently reported that second generation EGFR-TKI, afatinib or neratinib, are especially effective for EGFR exon 18 mutations compared with other EGFR-TKI, indicating the significance of mutation-specific EGFR-TKI selection.16

In this review, we comprehensively collected data on the frequency, in vitro sensitivity and treatment response of lung cancers harboring common and uncommon EGFR mutations to provide insight for the future direction of rational therapeutic strategy .

EGFR Pathway and Mutations in the EGFR

EGFR is one of the ERBB family receptor tyrosine kinases that consists of four members: EGFR (also known as ERBB1/HER1), ERBB2/HER2/NEU, ERBB3/HER3 and ERBB4/HER4. Specific ligands bind to the extracellular domain of EGFR, which leads to the formation of homodimers and heterodimers. Dimerization stimulates intrinsic tyrosine kinase activity of the receptors and triggers the autophosphorylation of specific tyrosine residues. Signal transducers initiate multiple downstream pathways such as MAPK, PI3K-AKT and STAT 3 and 5, which regulate proliferation and apoptosis.17

The EGFR gene, located on chromosome 7p12, consists of 28 exons and 27 introns. In 2004, somatic mutations in the kinase domain were discovered in patients with lung cancer whose tumor responded to gefitinib.3, 4 EGFR mutations shift the equilibrium of protein structures from an inactive state into an active state, resulting in the increased and sustained phosphorylation of EGFR and other HER family proteins without ligand stimulation.18

Types of EGFR Mutations According to the COSMIC Database

The catalogue of somatic mutations in cancer (COSMIC) is the largest open access database.19 As of May 2016, approximately 16 000 EGFR mutations are registered. According to this database, as many as 594 types of EGFR mutations are reported. Among them, 93% are present in the first four exons (18–21) of the gene encoding tyrosine kinase domain. Although COSMIC is extremely useful for comprehensive overview of EGFR mutations, including rare mutations, the results should be interpreted cautiously because the database consists of various data. For example, there was a discrepancy in the frequency of Del19 and L858R in conventional published data.20 Del19 accounts for approximately half of L858R (Table 1).

Frequency of EGFR Mutations by Compilation of Recent Large Studies

Three factors appear to complicate estimations of the true frequencies of each EGFR mutation in clinics: methods for detecting mutations, the presence of complex mutations and publication biases.

Sanger sequencing has been performed to detect mutations throughout the exons sequenced (usually exons 18–21), although the sensitivity is relatively low (requiring approximately 10% of the mutation allele).21 Next-generation sequencing can also achieve broad mutation detection. Importantly, rare mutations may include artifactual mutations that are generated during the pre-analytic period.22 In contrast, mutation-specific diagnostic kits have been developed for rapid and easy testing in clinical settings. Therascreen (Qiagen, Manchester, UK) and cobas (Roche, Basel, Switzerland) are approved by health authorities as in vitro diagnostic kits. These assays can detect the following specific mutations with high sensitivities (requiring approximately 1% of the mutation allele): G719A/S/C, Del19, S768I, exon 20 insertions (Ins20: V769_D770insASV, D770_N771insG/SVD and H773_V774insH), T790M, L858R and L861Q (Fig. 1). In other words, there is no way for other mutations to be detected. Although using these diagnostic kits is the standard method for detecting EGFR mutations in clinical practice, it is necessary to improve them to be able to detect rare but targetable mutations.

Multiple EGFR mutations are sometimes detected in the same tumor and these mutations have been referred to as co-mutations, complex mutations or compound mutations.23-26 Numeration for these mutations is not defined: some studies include them as a part of the representative mutation, such as Del19 or L858R, and others count these mutations independently (i.e. double-counting).

Oxnard and Jänne provide insightful comments on publication biases. Not all data on specific genotypes reaches the published literature: common genotypes are often included in prospective trials; less common or rare genotypes may be described in observational series or case reports; and the completeness of the data in meta-analyses inherently depends upon selection criteria as well as publication biases.27

Ideally, prospective large studies using the same method can clarify the actual frequency. Considering these factors as possible, we collected large studies conducted by single institutions or multi-institutional studies using the same protocol (Fig. 1). Subsequently, we focused on rare but targetable subsets including Ins19,28 Del1829 and E709X.29 Pretreatment T790M was excluded from our survey because the majority of them exist as complex mutations and the frequency vary widely from 3.9% to 64% based on the sensitivities of assays.30 In addition, the frequency of germline T790M remain unclear.31-33

First, Second and Third Generation EGFR -Tyrosine Kinase Inhibitors

Gefitinib and erlotinib, first generation (1G) EGFR-TKI, reversibly bind to the ATP-binding pocket of EGFR. Randomized phase III trials have demonstrated the superiority of these TKI, in terms of progression free survival (PFS), to conventional chemotherapy in patients with lung cancers harboring EGFR mutations.5, 6, 11 However, these TKI inevitably acquire resistance after an initial response. The secondary mutation T790M accounts for approximately 50%–60% of acquired resistance to 1G-TKI.34, 35

Irreversible pan-HER (EGFR, HER2 and HER4) TKI, so-called second generation (2G) TKI, were developed to overcome the T790M mutation. Despite the promising preclinical data, clinically available concentrations of the drug did not reach the treatment range for T790M tumors because of relatively severe adverse events compared with 1G-TKI due to the inhibition of wild type EGFR. However, afatinib has been approved as the first-line treatment for patients with EGFR-mutant lung cancers based on phase III trials.9, 10 Dacomitinib had a high objective response rate (ORR) of 76% in a phase II trial and continues to undergo clinical evaluation.36 Neratinib is also one of the 2G-TKI. However, its development for lung cancer was abandoned because it was not effective for common EGFR-mutant tumors, although it was effective for G719X tumors.37

The pyrimidine-based third generation (3G) TKI have been developed targeting T790M as well as common mutations without inhibiting wild-type EGFR.38, 39 Osimertinib has been approved for T790M tumors based on the high ORR of approximately 60% for tumors with T790M mutations as a resistance mechanism of 1G-TKI.38 C797S secondary mutation was detected in T790M-positive tumors that acquired resistance to osimertinib.40 Furthermore, C797S mutation appeared to be sensitive to 1G-TKI, and even C797S+T790M in trans can be treated with a combination of 1G and 3G-TKI.41 In contrast, the development of rociletinib was abandoned because the initially reported ORR of 59% was reduced to 45%: initial data were not unconfirmed partial responses although partial responses must be maintained on a second scan obtained at least 4 weeks later.42 Recently, olmutinib (BI1482694/HM61713) was approved for T790M-positive tumors in South Korea and received FDA breakthrough therapy designation.43 Other 3G-TKI, nazartinib (EGF816) and ASP8273, are undergoing clinical evaluation.44

Treatment Strategy by Mutation-specific Tyrosine Kinase Inhibitors Selection

When we discuss treatment strategies for heterogeneous EGFR populations, biases should be considered, especially for the data on less common or rare mutations. To compensate for the weak evidence for such mutations, we also collected data on in vitro sensitivities using Ba/F3 cells (Table 2) as well as clinical response to TKI (Table 3). Notably, the murine pro-B cell line Ba/F3 depends on interleukin-3 (IL-3) for its survival and growth. Accordingly, the growth of Ba/F3 cells transfected with specific EGFR mutation in the absence of IL-3 indicates oncogenic ability, which can exclude artifactual mutations. Of course, the methodological differences and clinically available concentrations should be considered in the interpretation of in vitro sensitivities.

Table 2. Summary of the in vitro sensitivities of Ba/F3 cells expressing each EGFR mutation to various TKI

Conclusions

EGFR mutations in lung cancer are extremely complicated and each mutation appears to have unique characteristics. Conventionally, EGFR mutations have been classified as sensitive, less sensitive and resistant mutations based on their responses to 1G-TKI. However, recent reports including 2G and 3G-TKI revealed that mutation-specific TKI selection could maximize the benefit for patients with NSCLC harboring less sensitive mutations (Table 4). For further development of targeted therapies with EGFR-TKI, it is important to precisely detect targetable mutations, to select the most appropriate TKI for each mutation and to continue investigating in vitro studies and collecting clinical data for even rare mutations.

Table 4. Our current view on treatment strategy for patients with lung cancer harboring each epidermal growth factor receptor mutation

Disclosure Statement

T. Mitsudomi has received lecture fees (from Astra-Zeneca, Boehringer-Ingelheim, and Chugai) and research funding (from Boehringer-Ingelheim and Chugai). Y. Kobayashi has no conflict of interest to declare.