Mutational analysis of EGFR and related signaling pathway genes in lung adenocarcinomas identifies a novel somatic kinase domain mutation in FGFR4

Abstract

Background: Fifty percent of lung adenocarcinomas harbor somatic mutations in six genes that encode proteins in the EGFR signaling pathway, i.e., EGFR, HER2/ERBB2, HER4/ERBB4, PIK3CA, BRAF, and KRAS. We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this signaling pathway that could contribute to lung tumorigenesis.

Methodology/principal findings: We analyzed genomic DNA from a total of 261 resected, clinically annotated non-small cell lung cancer (NSCLC) specimens. The coding sequences of 39 genes were screened for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Mutations were considered to be somatic only if they were found in an independent tumor-derived PCR product but not in matched normal tissue. Sequencing of 9MB of tumor sequence identified 239 putative genetic variants. We further examined 22 variants found in RAS family genes and 135 variants localized to exons encoding the kinase domain of respective proteins. We identified a total of 37 non-synonymous somatic mutations; 36 were found collectively in EGFR, KRAS, BRAF, and PIK3CA. One somatic mutation was a previously unreported mutation in the kinase domain (exon 16) of FGFR4 (Glu681Lys), identified in 1 of 158 tumors. The FGFR4 mutation is analogous to a reported tumor-specific somatic mutation in ERBB2 and is located in the same exon as a previously reported kinase domain mutation in FGFR4 (Pro712Thr) in a lung adenocarcinoma cell line.

Conclusions/significance: This study is one of the first comprehensive mutational analyses of major genes in a specific signaling pathway in a sizeable cohort of lung adenocarcinomas. Our results suggest the majority of gain-of-function mutations within kinase genes in the EGFR signaling pathway have already been identified. Our findings also implicate FGFR4 in the pathogenesis of a subset of lung adenocarcinomas.

Figure 1. Genes sequenced in this study.

The schematic diagram depicts the EGFR signaling pathway. Genes listed in red were sequenced only in genomic DNAs from 217 tumors (“Group 1”). Genes listed in blue were sequenced only in WGA-treated DNA tumor samples (“Group 2”). Genes in black were sequenced in both groups. Gene nomenclature is as reported in GenBank as of December 2006. See Supplemental Table S3 for clinical characteristics of all tumors sequenced.

Figure 2. Schematic of overall results.

A putative variation was defined as a sequence variation compared to a reference sequence in GenBank. After visual inspection and exclusion of known SNPs and silent changes, there were 239 tumor sequences with a variation representing 174 distinct types of variations. The sequence variations were further divided into three groups: 135 variations (99 distinct types) within exons encoding the kinase domains of respective genes, 82 variations (69 types) in exons encoding areas outside the kinase domains of respective kinase genes, and 22 variations (6 types) in RAS family genes. Non-synonymous variations confirmed by sequence analysis of a 2^nd PCR were either somatic mutations or variants found in matched normal tissue (listed in Supplemental Table S4). The significance of two novel variants, ERBB2 (exon 20, Arg784Cys) and MAPK6 (exon 4, Val262Ile), is unclear, because we could not determine if the variants were also found in DNA from corresponding normal tissue.

Figure 3. Analysis of FGFR4.

Forward/reverse sequencing chromatograms for the mutation identified in exon 16 of FGFR4 in tumor and matched normal samples. The nucleotide change is c.2041G>A, that would lead to substitution of lysine for glutamic acid at position 681.

Figure 4. Amino acid alignment of the FGFR4 kinase domain with other tyrosine kinase domains found to be altered in human cancers.

The DFG motif found in all kinases is underlined. The glutamic acid residue at position 681 in FGFR4 (boxed) is highly conserved amongst the various kinases. Amino acids affected by mutations and reported in the COSMIC (Catalogue of Somatic Mutations in Cancer) database appear in yellow. The analogous Glu914 residue in ERBB2 (boxed) has been found to be mutated in a glioblastoma. Figure adapted from a screenshot of the “Mutagrator” bioinformatics tool developed for this study. The previously reported Pro712Thr mutation in FGFR4 was also identified by the Mutagrator tool but is not shown. See methods for more details.

Figure 5. Structural modeling of the FGFR4 Glu681Lys amino acid substitution.

A. The FGFR4 WT and E681K mutant structures are predicted using the PROTINFO software (38) provided by the (PS)2 server (National Chiao Tung University, Taiwan). These predictions are based on crystallographic structure for FGFR1 tyrosine kinase domain (PDB accession 1FGK) (33), as no FGFR4 structure is available, and visualized using VMD (39). FGFR4 Glu681 (yellow), ATP binding site (pink), activation loop (green) and catalytic loop (white). Glu681 (yellow) is nestled between the TK activation and catalytic loops. B. 3D close-up of the surfaces of Glu681 (yellow), Arg650 (green) in the activation loop, and Ala615 (white) in the catalytic loop. Since Glu681 is strongly negatively charged and Arg650 is strongly positively charged, ionic bonding between these two closely juxtaposed residues may be assumed. C. 3D close-up of the surfaces of mutated Lys681 (orange), Arg650 and Ala615. The glutamic acid to lysine substitution at position 681 could structurally and functionally alter the kinase domain by flipping the charge of residue 681 and disrupting ionic bonds with neighboring residues, particularly the closely juxtaposed Arg650.