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. 2009;4(2):e4576.
doi: 10.1371/journal.pone.0004576. Epub 2009 Feb 24.

Alterations in genes of the EGFR signaling pathway and their relationship to EGFR tyrosine kinase inhibitor sensitivity in lung cancer cell lines

Jeet Gandhi  1 , Jianling Zhang, Yang Xie, Junichi Soh, Hisayuki Shigematsu, Wei Zhang, Hiromasa Yamamoto, Michael Peyton, Luc Girard, William W Lockwood, Wan L Lam, Marileila Varella-Garcia, John D Minna, Adi F Gazdar
Affiliations

Alterations in genes of the EGFR signaling pathway and their relationship to EGFR tyrosine kinase inhibitor sensitivity in lung cancer cell lines

Jeet Gandhi et al. PLoS One. 2009.

Abstract

Background: Deregulation of EGFR signaling is common in non-small cell lung cancers (NSCLC) and this finding led to the development of tyrosine kinase inhibitors (TKIs) that are highly effective in a subset of NSCLC. Mutations of EGFR (mEGFR) and copy number gains (CNGs) of EGFR (gEGFR) and HER2 (gHER2) have been reported to predict for TKI response. Mutations in KRAS (mKRAS) are associated with primary resistance to TKIs.

Methodology/principal findings: We investigated the relationship between mutations, CNGs and response to TKIs in a large panel of NSCLC cell lines. Genes studied were EGFR, HER2, HER3 HER4, KRAS, BRAF and PIK3CA. Mutations were detected by sequencing, while CNGs were determined by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). IC50 values for the TKIs gefitinib (Iressa) and erlotinib (Tarceva) were determined by MTS assay. For any of the seven genes tested, mutations (39/77, 50.6%), copy number gains (50/77, 64.9%) or either (65/77, 84.4%) were frequent in NSCLC lines. Mutations of EGFR (13%) and KRAS (24.7%) were frequent, while they were less frequent for the other genes. The three techniques for determining CNG were well correlated, and qPCR data were used for further analyses. CNGs were relatively frequent for EGFR and KRAS in adenocarcinomas. While mutations were largely mutually exclusive, CNGs were not. EGFR and KRAS mutant lines frequently demonstrated mutant allele specific imbalance i.e. the mutant form was usually in great excess compared to the wild type form. On a molar basis, sensitivity to gefitinib and erlotinib were highly correlated. Multivariate analyses led to the following results: 1. mEGFR and gEGFR and gHER2 were independent factors related to gefitinib sensitivity, in descending order of importance. 2. mKRAS was associated with increased in vitro resistance to gefitinib.

Conclusions/significance: Our in vitro studies confirm and extend clinical observations and demonstrate the relative importance of both EGFR mutations and CNGs and HER2 CNGs in the sensitivity to TKIs.

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Conflict of interest statement

Competing Interests: Dr. Gazdar is a paid consultant/lecturer for AstraZeneca PLC. Dr. Garcia recieves Research Funding >10,000 from AstraZeneca, Genentech and OSI; Honorarium <10,000 from Roche. Dr. Minna receives research support from AstraZeneca PLC.

Figures

Figure 1
Figure 1. Mutations (m) and Copy number gains (g) of EGFR pathway genes in NSCLC.
Fig 1a. shows the frequency of mutations and copy number gains of EGFR pathway genes (EGFR, KRAS, BRAF, PIK3CA, HER2, HER3 and HER4). Forty mutations were identified in 39 cell lines. Mutations and copy number gains were more frequent for EGFR (13%, 40.3%) and KRAS (24.7%, 14.3%) than other gene. CNGs for HER2 (18.2%) were also common. We identified only one HER2 and one HER4 somatic mutation. The numbers above the columns indicate the number of cell lines with mutations (blue columns) or copy number gains (red columns). Fig 1b. The figure depicts the number of genes demonstrating CNGs in mutant and wild type cell lines. Of the 77 cell lines examined, 39 (50.6%) had a mutation in at least one of seven the EGFR pathway genes examined. CNGs were frequent in both mutant and wild type cell lines. Fig 1c. shows frequency of mutations on the basis of NSCLC subtype. Mutations of EGFR and BRAF were exclusively found in adenocarcinoma subtype. The single HER2 mutation was in a adenocarcinoma as compared to the HER4 somatic mutation which was identified in a squamous cell ca. Fig 1d. shows frequency of copy number gains (CNGs) (g>4 by qPCR) on the basis of NSCLC subtype. CNGs for BRAF and PIK3CA were seen predominantly in adenocarcinoma and squamous cell carcinoma respectively. CNGs for the rest of the genes did not favor any subtype.
Figure 2
Figure 2. Copy number gains are not mutually exclusive with either other copy number gains or with mutations.
Fig 2a. shows that copy number gains and mutations are not mutually exclusive. As evident from the figure CNGs for EGFR and KRAS are significantly more frequent in EGFR and KRAS mutant cell lines respectively (p<0.05). There was only one HER2 and HER4 mutant NSCLC cell line and thus they were not included in this figure. Fig 2b. shows that copy number gains are not mutually exclusive and gains of one gene may occur in the presence of gains for other genes.
Figure 3
Figure 3. Mutant allele specific imbalance (MASI) for EGFR and KRAS genes.
EGFR and KRAS genes preferentially have copy number gains (CNG) in cell lines harboring the respective mutations (panels a and b). In mutant lines, the mutant allele almost always is in excess compared to the wild type allele (panels c and d), a phenomenon we have termed MASI. In most MASI cases the mutant allele demonstrates CNGs; however MASI may also be present in cell lines having a diploid copy number of the oncogene, (acquired uniparental disomy) either uniform (NCI-H460) or heterogeneous (NCI-H1975).
Figure 4
Figure 4. Concordance between IC50 values for gefitinib vs erlotinib.
Forty five cell lines were tested for sensitivity to both drugs and the concordance was excellent (p<0.0001).
Figure 5
Figure 5. Rank Order of NSCLC Cell Line depending on the Iressa IC50.
Fig 5. shows a log curve of the gefitinib IC50 values for 45 NSCLC cell lines. They are classified into three categories on the basis of gefitinib IC50: Sensitive (IC50<1 µM), Intermediate (IC50>1 but <10 µM) and Resistant (IC50>10 µM). Of the nine sensitive cell lines, seven of them harbor EGFR mutations, one has CNGs for EGFR and one has CNG for HER2. Of the remaining EGFR mutant cell lines, two had T790M mutation (one intermediate and one resistant) and one had a homozygous deletion of PTEN (resistant). KRAS mutant and wild type cell lines were all resistant to gefitinib.
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