Germ-line mutations in epidermal growth factor receptor (EGFR) are rare but may contribute to oncogenesis: a novel germ-line mutation in EGFR detected in a patient with lung adenocarcinoma

Abstract

Background: A subset of lung cancer patients harbour EGFR somatic mutations in their tumours and are candidates for treatment with EGFR tyrosine kinase inhibitors. In a few cases EGFR mutations have also been found in the germ line, suggesting a role in lung carcinogenesis. Objetives of this study were: 1) To analyze the EGFR gene mutations in a population diagnosed with lung adenocarcinoma from Northern Spain. 2) To determine the frequency of a new germ-line mutation found in our laboratory as well as the frequency in our population of three other EGFR germ-line mutations detected by other authors. 3) To determine whether the novel mutation detected may have a functional effect on the EGFR protein.

Methods: Tumour DNA samples were obtained from frozen or paraffin embedded tumour tissues. Samples of DNA from peripheral blood cells were obtained from 912 individuals with lung cancer recruited from the CAPUA study 12, 477 unrelated healthy donor individuals and 32 individuals with other types of cancer. EGFR gene exons 18 to 21 were studied by direct standard dideoxy sequencing. Specific mutations were determined either by direct sequencing or by specific RFLP analysis. Cell lines were transfected with EGFR-mutant plasmids and analysed by western blot with antibodies specific for total or phosphorylated-EGFR.

Results: We found EGFR mutation in 12 of the 71 tumour samples (17%). One tumour contained two mutations. One mutation (p.R776G) was present as a germ line. Using an RFLP analysis, this mutation was not found in 954 alleles from healthy individuals studied, concluding that it is not a polymorphism. The mutation was not found either in genomic DNA from 912 lung cancer patients. Three additional EGFR germ-line mutations that were already described were not found in any of the studied samples. These observations show that EGFR mutated alleles are rare in the population. In vitro studies revealed that tyrosine autophosphorylation is enhanced in p.R776G-mutant EGFR when compared with wild-type EGFR. This enhanced autophosphorylation in the absence of ligand may be associated with a proliferative advantage.

Conclusions: Germ-line mutations in EGFR are rare but may contribute to oncogenesis.

Figure 1

Strategy developed to resolve if p.R776G and p.L858R were located in cis. Taking into account that c.2326C > G mutation causing p.R776G amino acid change generates a new HaeIII restriction site, when an exon 21 PCR fragment containing these mutations was digested with HaeIII, two different bands could be distinguished from the heterozygous tumour sample: one 276 bp fragment (wt for the position c.2326) and one 248 bp fragment (mutated at this position). Sequence analysis of 248 bp fragment revealed that the p.L858R mutation was present in the same band as the p.R776G mutation.

Figure 2

Anti-Phospho-EGFR immunoblot analysis. COS-7 and 293 EBNA cells were transiently transfected as described. All EGFR constructs are expressed at similar levels. EGFR activity (phospho-EGFR level) is increased in p.R776G mutant, both in presence or in absence of ligand.