Epidermal Growth Factor Receptor Double Activating Mutations Involving Both Exons 19 and 21 Exist in Chinese Non-small Cell Lung Cancer Patients

Abstract

Aims

It has been shown that the introduction of a second mutation into the already mutated epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) will alter the sensitivity to tyrosine kinase inhibitors (TKIs). EGFR double activating mutations involving both exons 19 and 21 were previously detected in Asian patients, but the sensitivity to TKIs had not yet been characterised. Our objective was to profile the status of EGFR double mutations in Chinese NSCLC patients and to further ascertain the biological properties.

Materials and methods

In total, 145 NSCLC tumour samples from unselected Chinese NSCLC patients were sequenced to screen mutations in exons 18, 19 and 21 of EGFR. Five patients were detected to harbour the delE746–A750 + L858R double activating mutations. Subcloning experiments were carried out, expression vectors inserted with corresponding full-length EGFR were constructed, and in vitro transient transfections were performed in 293T cells. Whole cell lysates were collected to assess the sensitivity to TKIs using immunoblotting.

Results

All five patients had adenocarcinoma. The frequency of double mutations was 3.4% (5/145). Three patients received and responded to gefitinib treatment. Subcloning experiments showed that all the subclones were either wild type or double mutated. At a concentration of TKIs of 0.1 μM, the autophosphorylation of the double mutant was inhibited greater than that of either single mutated EGFR. However, the difference disappeared when the concentration increased to 1 μM.

Conclusions

delE746–A750 + L858R double activating EGFR mutations exist in Chinese NSCLC patients and both locate on the same allele. These patients tend to respond well to TKIs and the sensitivity to TKIs of this double mutated EGFR is enhanced compared with either single mutant. Nonetheless, the alteration in downstream signal transduction of the double mutant remains to be determined.

Introduction

Non-small cell lung cancer (NSCLC) comprises about 75–85% of all lung cancers and many patients are with advanced disease on diagnosis, without any surgery opportunity [1]. Systemic chemotherapy for these patients has improved slightly during the past two decades [2]. In recent years, epidermal growth factor receptor (EGFR) targeted tyrosine kinase inhibitors (TKIs), either gefitinib or erlotinib, have shown promise in the treatment of relapsed refractory NSCLC, with response rates of 10–20% when used as second or third line 3, 4, 5.

Recent studies have revealed that activating mutations in the EGFR gene correlates to sensitivity to TKIs and at least 28 mutations have been reported in exons 18–21 of the EGFR tyrosine kinase domain. More than 90% of these mutations are either deletions around codons 746–750 in exon 19 or the L858R point mutation in exon 21, both of which have been shown to be activating. Other rare mutations include some missense mutations in exons 18 and 21, several overlapping deletions in exon 19 and small in-frame insertions in exon 20 6, 7, 8.

It is now clear that the introduction of another mutation will change the amino acid sequences of the already mutated EGFR and possibly the molecular conformation. As a result, biological properties of EGFR would differ from the original single mutant and therefore alter its sensitivity to TKIs. For example, the T790M + L858R double mutant is resistant to TKIs [9] and the double mutations L858R + E884K sensitise the receptor to gefitinib and confers resistance to erlotinib [10].

With studies in this field accruing continuously, more multiple mutations have been profiled, especially in Asia, where the mutation frequency of EGFR is much higher than in other parts of the world 11, 12, 13. EGFR point mutations in NSCLC were occasionally accompanied by a second mutation in Japanese NSCLC patients [14] and the delE746–A750 + L858R double mutations were detected in Thai NSCLC patients [13]. Nonetheless, the biological properties of these double mutants have not yet been characterised.

In the present study, we sequenced exons 18, 19 and 21 of EGFR in 145 Chinese NSCLC in an attempt to profile the status of EGFR multiple mutations in Chinese NSCLC. Five patients with the delE746–A750 + L858R double activating mutations were detected. To further ascertain the biological properties of this type of double mutation, expression vectors inserted with the full length of this double mutated EGFR were constructed and in vitro transfections were carried out to assess the sensitivity to TKIs.