PL-1 Detection of deletions, point mutations and single nucleotide polymorphisms in the human EGF receptor tyrosine kinase domain in tumor samples from non-small cell lung cancer patients

Abstract

Introduction: Mutations in the tyrosine kinase (TK) domain of the human EGF receptor (EGFR) have recently been identified in NSCLC specimens. They occur predominantly in the histological subgroup of adeno- and bronchioloalveolar carcinomas and correlate with responsiveness to the EGFR TK inhibitor Gefitinib (Iressa, ZD1839). To determine the frequency of such mutations we have screened a panel of NSCLC samples with that histology. Method: Genomic DNAwas extracted from paraffin-embedded or snap-frozen NSCLC tissue samples. Exons 18 to 21 of the human EGFR were amplified by PCR, purified over spin columns and subsequently subjected to sequencing. Results: The TK domain of the human EGFR of 34 samples with histologically confirmed NSCLC (4 bronchioloalveolar carcinomas, 30 adenocarcinomas, 18 males, 16 females) was investigated for the presence of genetic alterations. Exon 20 harbors a single nucleotide polymorphism (SNP) at codon Q787 (CAG->CAA). 13 samples showed wild-type CAG, 10 specimens were heterozygous at this locus and 11 cases displayed a homozygous CAA sequence. The SNP was similarly detected in four investigated corresponding normal lung tissues. Seven samples (20.6%) showed mutations in the TK domain, five of which were located in the “hotspot“ regions of exons 19 and 21. In four samples in-frame deletions in exon 19 were detected, one of which occurred homozygously. The patient with the homozygous deletion in exon 19 has been treated with Gefitinib for more than one year and showed an objective response. In one sample a heterozygous mutation in exon 21 (L858R) was identified. Sequencing of two samples yielded a previously unreported in-frame deletion of 3 nt in exon 18. Six of the patients with mutations were female. In all but one cases corresponding normal lung tissue was available. The mutations were not detectable in any of them confirming their somatic origin during lung cancer pathogenesis. Conclusion: Our results show an almost evenly d.