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Clinical Cancer Research Vol. 12, 710-717, February 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Molecular Context of the EGFR Mutations: Evidence for the Activation of mTOR/S6K Signaling

Esther Conde1, Barbara Angulo1, Moying Tang1, Manuel Morente2, Juan Torres-Lanzas5, Angel Lopez-Encuentra3, Fernando Lopez-Rios4 and Montserrat Sanchez-Cespedes1

Authors' Affiliations: 1 Lung Cancer Group and 2 Tumour Bank, Spanish National Cancer Centre, 3 Pulmonary and 4 Pathology Departments, Hospital Universitario 12 de Octubre, Madrid, and 5 Thoracic Surgery Department, Hospital Virgen de la Arrixaca, Murcia, Spain

Requests for reprints: Montserrat Sanchez-Cespedes, Molecular Pathology Programme, Spanish National Cancer Centre, Melchor Fernandez Almagro 3, 28029 Madrid, Spain. Phone: 34-9-1224-6954; Fax: 34-9-1224-6923; E-mail: msanchez{at}cnio.es.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Activating somatic mutations in the epidermal growth factor receptor (EGFR) gene are present in a small subset of lung adenocarcinomas. These mutations cluster in specific regions and confer sensitivity to inhibitors of the tyrosine kinase activity of EGFR. To further determine the genetic and molecular characteristics of tumors carrying EGFR gene mutations, we investigated the EGFR gene status in lung adenocarcinomas and evaluated its association with specific characteristics of the patients and tumors, such as mutations at KRAS and p53, EGFR and ErbB2 gene amplification, levels of EGFR and HER2 proteins, and levels of downstream effectors of EGFR, such as phospho–extracellular signal-regulated kinase and phospho-S6 proteins.

Experimental Design: The mutational status of EGFR was determined by direct sequencing in 86 primary lung adenocarcinomas and 12 lung cancer cell lines, and was correlated with a number of variables relating to the tumor and patient. A tissue microarray containing 37 lung tumors was constructed to determine, by fluorescence in situ hybridization analysis, the number of copies of EGFR and ErbB2 genes and, by immunohistochemistry, the levels of EGFR, HER2, phospho-ERK, and phospho-S6 proteins.

Results: EGFR gene mutations were identified in 13% of the primary tumors. The type and clustering of the mutations were identical to those previously reported. Amplification of the EGFR occurred in 14% of the tumors and could arise in tumors with EGFR mutations. Interestingly, mTOR activation, as measured indirectly by augmented levels of phospho-S6 protein, was more frequent in tumors with gene alterations in either EGFR or KRAS (P = 0.00005; Fisher's exact test) than in their wild-type counterparts.

Conclusions: Our data agree with the accumulation of EGFR mutations in a subset of patients with lung cancer. Moreover, we report EGFR gene amplification in EGFR-mutant tumors and a positive correlation between EGFR or KRAS alterations and activation of mTOR signaling.

Non–small cell lung tumors carry many molecular abnormalities, including aberrant protein expression, gene promoter hypermethylation and gene mutations (1). Although the levels of many proteins are aberrant in lung tumors, only a few genes exhibit genetic alterations. Recently, somatic mutations in EGFR have been identified in a subset of lung adenocarcinomas, including a new member into the small group of genes that are known to be mutated in lung cancer (2–4).

The epidermal growth factor receptor (EGFR, Erb-B1) belongs to the erbB family of transmembrane tyrosine kinase receptor proteins, which also includes HER2/neu (encoded by erb-B2), HER3 (encoded by erb-B3), and HER4 (encoded by erb-B4; ref. 5). These receptors, by binding to different ligands, trigger a cascade of intracellular signaling reactions that lead to the regulation of several aspects of cell biology, such as cell division, apoptosis, and cytoskeletal rearrangement (5). Mutations in the EGFR gene in lung cancer lead to amino acid changes or in-frame deletions in exons 18 to 21 and arise more frequently in cases of adenocarcinomas, females, nonsmokers, and people of Asian origin (2–4, 6, 7). These mutations seem to enhance the ability of the ligand to induce EGFR autophosphorylation (3, 8, 9). In addition to its role in lung carcinogenesis, the presence or absence of EGFR mutations has important clinical implications because it seems to determine the responsiveness to EGFR tyrosine kinase inhibitors such as gefitinib (Iressa, ZD1839) or erlotinib (Tarceva, OSI-774; refs. 2–4, 10). Furthermore, EGFR and ErbB2 gene amplification and protein overexpression have been observed in a subset of lung tumors [see ref. (11) for review]. Tumors featuring gene amplification also have concomitant protein overexpression, although the opposite is not necessarily true. Interestingly, an association between EGFR mutations and increased EGFR gene copy number has recently been described in lung adenocarcinoma cell lines, suggesting a mechanism for amplifying the effect of these oncogenic mutations (8).

Activation of EGFR is mediated by autophosphorylation at several key tyrosine residues leading to the binding of different adaptors involved in downstream signaling (5). The EGFR downstream pathways include AKT and RAS/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase, which are involved in cell survival and cell proliferation, respectively. Whether any of these signal transduction pathways is a preferred target of EGFR gene mutations is currently under investigation. Several publications have reported that mutations in EGFR and KRAS, but not in p53, are mutually exclusive (6, 7, 12). These observations may indicate that mutations at both genes are functionally equivalent, hence alterations at only one of them is enough to trigger constant activation of the signaling through RAS/ERK/mitogen-activated protein kinase. However, other studies have reported that EGFR mutants selectively activate AKT-mediated signal transduction but have no effect on ERK signaling (13).

In this study, we have screened a set of primary lung adenocarcinomas from Spanish patients and lung cancer cell lines for EGFR mutations, and evaluated the genetic (KRAS, p53 gene mutations and EGFR, ErbB2 gene amplification) and molecular (EGFR, HER2, phospho-ERK and ribosomal phospho-S6 protein expression) context in which these mutations arise.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and tumor characteristics. Tumor and matched normal tissues from 86 patients diagnosed with lung adenocarcinoma were provided by the Spanish National Cancer Centre Tumor Bank Network, Spanish National Cancer Centre (Madrid, Spain), with the collaboration of the following hospitals: Hospital Universitario 12 de Octubre and Hospital Universitario Puerta de Hierro in Madrid, Hospital Virgen de la Arrixaca in Murcia and Hospital Virgen de la Salud, in Toledo. There were 30 and 56 females and males, respectively, and the median age was 60 years; 71 and 15 were fresh-frozen and paraffin-embedded tumors, respectively. Available slides were reviewed by two pathologists (E. Conde and F. Lopez-Rios) and was classified according to WHO criteria (14). Informed consent was obtained from each patient.

DNA extraction and mutation analysis. Representative sections from tissue used for DNA extraction were stained with H&E. Freshly frozen and paraffin-embedded tissue from tumors was meticulously dissected to ensure that specimens contained at least 70% tumor cells. Approximately 10 to 20 µm sections were collected from normal and tumor samples and placed in 1% SDS/proteinase K (10 mg/mL) at 58°C overnight. Digested tissue was then subjected to phenol-chloroform extraction and ethanol precipitation. The cell lines A427, A549, H23, H661, H1155, H1170, H1299, H1395, H2087, H2126, Calu-3, and U1552 were obtained from the American Type Culture Collection (Rockville, MD), and cultured under the recommended conditions. DNA from cell lines was extracted using standard proteinase K/isoamyl alcohol protocols. For the screening of EGFR gene mutations, exons 18 to 21 of the gene were PCR-amplified and automatically sequenced. For frozen tumors, PCR reactions were done in a total volume of 25 µL. The mix contained 1.25 units of DNA polymerase (Biotools, Madrid, Spain), 2.5 µL of Tth buffer 10x (Biotools), 2 mmol/L of MgCl2, 200 µmol/L of each deoxynucleotide, 2 µL of DMSO, 6 ng/µL of each primer, and 50 ng of genomic DNA. The PCR cycling conditions consisted of an initial denaturation at 94°C for 5 minutes, followed by two cycles of denaturation (30 seconds at 94°C), annealing (1 minute at 60°C), elongation (1 minute at 72°C), followed by 35 cycles of denaturation (30 seconds at 94°C), annealing (1 minute at 58°C), and elongation (1 minute at 72°C). After the last cycle, a final extension (1 minute at 72°C) was added. The primers used for PCR were as follows: exon 18, forward 5'-TCCAAATGAGCTGGCAAGTG-3' and reverse 5'-CAAACACTCAGTGAAACAAAGAG-3'; exon 19, forward 5'-TGCATCGCTGGTAACATCC-3' and reverse 5'-TGGAGATGAGCAGGGTCTAG-3'; exon 20, forward 5'-GATCGCATTCATGCGTCTTC-3' and reverse 5'-TCCCCATGGCAAACTCTTGC-3'; exon 21, forward 5'-GCTCAGAGCCTGGCATGAA-3' and reverse 5'-GCATCCTCCCCTGCATGTG-3'. The following were used for the sequencing reaction: exon 18, 5'-AAATGAGCTGGCAAGTGCCG-3'; exon 19, 5'-CATCGCTGGTAACATCCACC-3'; exon 20, 5'-CGCATTCATGCGTCTTCACC-3'; and exon 21, 5'-CAGAGCCTGGCATGAACAT-3'.

For paraffin-embedded tumors, PCR reactions were done in a total volume of 25 µL. The mix contained: 21.5 µL of Taq Platinum PCR SuperMix [22 units/mL complexed recombinant Taq DNA polymerase with Platinum Taq antibody, 22 mmol/L Tris-HCl (pH 8.4), 55 mmol/L KCl, 1.65 mmol/L MgCl2, 220 µmol/L deoxynucleotide triphosphates, and stabilizers; Invitrogen Life Technologies, Karlsruhe, Germany], 6 ng/µL of each primer, 100 ng of genomic DNA. Thermal cycling conditions for exons 19 and 21 included 5 minutes at 94°C, followed by two cycles of 94°C for 30 seconds, 58°C for 1 minute, 72°C for 1 minute, and 40 cycles of 94°C for 30 seconds, 56°C for 1 minute, 72°C for 1 minute, and finally one cycle of 72°C for 1 minute; and for exons 18 and 20, 5 minutes at 94°C, followed by two cycles of 94°C for 30 seconds, 60°C for 1 minute, 72°C for 1 minute, and 40 cycles of 94°C for 30 seconds, 58°C for 1 minute, 72°C for 1 minute, and finally one cycle of 72°C for 1 minute. The primers used for PCR were as follows: exon 18, forward 5'-TGACCCTTGTCTCTGTGTTC-3' and reverse 5'-AGAGGCCTGTGCCAGGGAC-3'; exon 19, forward 5'-CAGTTAACGTCTTCCTTCTC-3' and reverse 5'-CACACAGCAAAGCAGAAACTC-3'; exon 20, forward 5'-ACTGACGTGCCTCTCCCTC-3' and reverse 5'-CCCGTATCTCCCTTCCCTG-3'; exon 21, forward 5'-TCACAGCAGGGTCTTCTCTG-3' and reverse 5'-CTGACCTAAAGCCACCTCC-3'. The following were used for the sequencing reaction: exon 18, 5'-CCCTTGTCTCTGTGTTCTTG 3'; exon 19, 5'-GTTAACGTCTTCCTTCTCTC-3'; exon 20, 5'-CTGACGTGCCTCTCCCTCC-3'; and exon 21, 5'-AGCAGGGTCTTCTCTGTTTC-3'.

The PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide. The bands were excised, purified with an UltraClean 15 Kit (MO Bio Laboratories, Solana Beach, CA) and sequenced using Big Dye Terminator chemistry (Applied Biosystems, Foster City, CA) with an ABI PRISM 3700 DNA Analyzer (Perkin-Elmer Life Sciences, Inc., Boston, MA). All variants were confirmed by resequencing independent PCR products. Mutations in p53 and KRAS genes were screened as previously described (15).

Tissue microarray. Formalin-fixed, paraffin-embedded tissue blocks from 37 patients were used for tissue microarray construction. A tissue arrayer (Beecher Instruments, Silver Spring, MD) was used to construct the tissue microarray (16). Slides were reviewed by pathologists (E. Conde and F. Lopez-Rios), who selected areas containing tumor cells, avoiding those areas featuring necrosis, inflammation and keratinization. To assess reproducibility, we selected two 1-mm diameter cylinders from different areas of the tumor. Normal lung tissue and reactive tonsil tissue were included as control specimens to ensure quality, reproducibility, and homogenous staining.

Fluorescence in situ hybridization analysis. Fluorescence in situ hybridization analysis was carried out for the detection of ErbB2 and EGFR amplification using commercially available probes from Vysis, Inc. (Downers Grove, IL). We used 4-µm sections of all the tissue microarrays. In both cases, the specified gene probes, which span the entire gene, were labeled in SpectrumOrange. These probes also contain a centromeric probe for chromosome 7 (for the EGFR probe) and chromosome 17 (for the ErbB2 probe), which are labeled in SpectrumGreen and hybridize to the alpha satellite DNA located at their centromeres.

The slides were deparaffinized, boiled in a pressure cooker with 1 mmol/L EDTA (pH 8.0) for 10 minutes, and incubated with pepsin at 37°C for 30 minutes. The slides were then dehydrated. The probes were denatured at 75°C for 2 minutes after overnight hybridization at 37°C in a humid chamber. Slides were washed with 0.4x SSC and 0.3% NP40.

Fluorescence in situ hybridization was evaluated without previous knowledge of other genetic, clinical, or immunohistochemical results. Fluorescence signals in each sample were scored by counting the number of single-copy gene and centromeric signals in an average of 130 (60-210) well-defined nuclei. Amplification was defined as the presence (in >5% of tumor cells) of either >10 gene signals or more than three times as many gene signals as centromere signals in the same chromosome. The cutoff values for copy-number changes were obtained from the analysis of normal adjacent cells in each experiment.

Immunohistochemistry. Immunohistochemical staining of EGFR, HER2, phospho-ERK, and phospho-S6 (phospho-Ser235/236) proteins was done on 3-µm-thick sections from the tissue microarray and transferred to silanized glass slides. The protocols used were those previously described (16). After incubation, immunodetection was done with the DAKO EnVision Visualization Method (Dako, Glostrup, Denmark), with diaminobenzidine chromogen as the substrate. Sections were counterstained with hematoxylin. The antibodies used in the analyses, indicating source, dilution, and corresponding internal controls are described in Table 1. Immunostaining was evaluated by two different pathologists (E. Conde and F. Lopez-Rios), using uniform criteria and without prior knowledge of the clinical and pathologic characteristics of the patients.


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  		Table 1. Antibodies used, indicating source, dilution, and corresponding internal controls

 
The following criteria were used for individual evaluation of the immunostaining:
EGFR immunostaining was scored as positive (or 1) and negative (or 0) when ≥10% and <10% of tumor cells, respectively, showed unequivocal membrane staining (17, 18). Basal cells, in the normal respiratory epithelium, showed immunoreactivity and were used as an internal positive control. The alveolar epithelium was used as an internal negative control.
HER2 immunostaining was scored using the HercepTest scoring system. No staining or membrane staining in <10% of tumor cells were scored as 0; weak membrane staining in >10% of tumor cells or cells with incomplete membrane staining were scored as 1+; weak/moderate complete membrane staining in >10% was scored as 2+; strong, complete membrane staining in >10% of tumor cells was scored 3+. Scores of 2+ and 3+ were judged as positive, whereas scores of 0 or 1+ were considered negative. Appropriate positive and negative controls were included (19).
Phospho-ERK immunostaining was scored as positive (or 1) and negative (or 0) when ≥15% and <15% of the cells, respectively, were immunoreactive. Specimens were also scored according to the location of the staining (nuclear and/or cytoplasmic). Endothelial cells were used as internal positive controls. Normal lung tissue was used as an internal negative control (20).
Phospho-S6 staining was scored as negative (or 0); weak cytoplasmic positive staining (or 1); moderate cytoplasmic positive staining (or 2) and strong cytoplasmic positive staining (or 3). Extent of staining was scored as 0 (0%), 1 (≤50%), 2 (51-75%), 3 (76-85%), and 4 (>85%) according to the percentage of tumor cells that were immunoreactive. The sum of the intensity and extent of scoring was used as the final staining score (0-7). Tumors with a final score of ≥5 were considered positive or high-intensity, whereas those with a final score of <5 were negative or low-intensity. Endothelial cells and lymphocytes were used as internal positive and negative controls, respectively. In normal respiratory epithelium, weak to moderate phospho-S6 reactivity was detected in the cytoplasm of all layers. The alveolar epithelium also showed weak/medium immunoreactivity for phospho-S6 (21).

Statistical analysis. Frequencies were compared either by Fisher's exact test or the {chi}2 contingency test, as appropriate. Differences of P < 0.05 were considered statistically significant. Analyses were carried out with the SSPS program, version 10.0.5 (SSPS Inc., Chicago, IL).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
EGFR gene mutations in lung adenocarcinomas: genetic and pathologic context. Tumor DNA from 86 unselected lung adenocarcinoma patients was screened for mutations within exons 18 to 21 of the EGFR gene. Eleven (13%) somatic mutations were identified. All of them were previously described changes, either amino acid substitutions in exon 21 or in-frame deletions in exon 19 (Table 2). Examples of some of these alterations are shown in Fig. 1A-B. In addition to primary tumors, 12 lung adenocarcinoma cell lines (A427, A549, H23, H661, H1155, H1170, H1299, H1395, H2087, H2126, Calu-3, and U1552) were tested, but showed no mutations. Other variants that did not lead to amino acid changes and were confirmed to be germ line polymorphisms are also listed.


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  		Table 2. Mutations in the EGFR gene detected in primary lung adenocarcinomas and germ line polymorphisms in primary tumors and lung cancer cell lines

 

Figure 1
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  		Fig. 1. Identification of EGFR gene and protein alterations in lung adenocarcinomas. A, example of a point mutation in a lung primary tumor compared with matched normal DNA. B, example of a lung adenocarcinoma showing an in-frame deletion in exon 19 with only traces of corresponding normal DNA. A normal sequence is also shown for comparison. C, fluorescence in situ hybridization analysis showing the increase in EGFR gene copy number. D, positive immunostaining for the EGFR protein in the same tumor.

 
As indicated in Table 3, EGFR mutations were more common in females and in nonsmokers (P < 0.05). Among the 47 cases available for reevaluation of histology according the most recent WHO classification (14), there were 7 mutated and 40 nonmutated cases. Of those seven adenocarcinomas mixed subtypes harboring EGFR mutations, five exhibited prominent bronchioloalveolar features (AD-BAC), one had a conspicuous papillary (AD-PP) pattern and the remaining tumor was a poorly differentiated adenocarcinoma. Among those 40 reviewed adenocarcinomas without EGFR mutations, 20 (50%) were well-differentiated adenocarcinomas (including five with AD-BAC and one with a predominant papillary pattern). No pure BACs were observed in either group. None of the tumors carrying EGFR mutations had concomitant KRAS mutations, implying an inverse relationship between the activation of these two oncogenes. In contrast, the frequency of p53 mutations was similar in EGFR mutant or wild-type tumors. None of the EGFR mutant tumors had G:C-to-T:A transversions, which have been associated with smoking status.


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  		Table 3. Relationships with pathologic characteristics and other gene alterations in primary lung adenocarcinomas from patients with available data

 
EGFR gene mutations coexist with EGFR but not with ErbB2 gene amplification in lung adenocarcinomas. To date, there has been a paucity of information regarding the correlation of EGFR and ErbB2 gene copy number, levels of protein expression and presence of EGFR mutations. Thus, to further our knowledge about the associations between EGFR and ErbB2 gene alterations and their relationships with protein levels, we constructed a tissue microarray containing all available EGFR mutant tumors (n = 9), 28 EGFR wild-type tumors and appropriate controls. The 28 EGFR wild-type tumors were chosen arbitrarily from our set of tumor specimens. First, we used fluorescence in situ hybridization analysis to assess the EGFR and ErbB2 gene copy number. Only 2 of the 35 analyzable (6%) tumors featured ErbB2 gene amplification (>10 copies). This alteration occurred in a background of wild-type EGFR and KRAS, but in mutant p53. Conversely, high levels of EGFR gene amplification (copy numbers from 5 to 15) were observed in 5 (14%) of the 35 tumors. None of these featured simultaneous KRAS mutations or ErbB2 gene amplification, but three tumors had concomitant mutant EGFR. Based solely on the appearance of the electropherograms, EGFR gene amplification seemed to target the mutant allele specifically in one case, as depicted in Fig. 1C. This tumor had a mutation in exon 19 and showed only traces of the wild-type allele, implying that EGFR gene amplification targeted the mutant allele. In contrast, the other two cases featured a mutation in exon 21 but also a strong contamination of the normal allele, probably due to gene amplification of the wild-type allele. To determine whether gene amplification and EGFR gene mutations lead to increased protein levels, we performed immunohistochemistry for EGFR and HER2 proteins. Following previously described criteria, EGFR and HER2 immunostaining was categorized as 0 (or negative) and 1 (or positive) as indicated in Materials and Methods. According to these criteria, 22 (63%) and 13 (37%) of the tumors were classified as negative and positive for EGFR protein, respectively. Two tumors were not available for analysis. Positive EGFR immunostaining was more common in EGFR mutant than in wild-type tumors (4 of 9, 44% versus 9 of 28, 32%), respectively, although the numbers were not sufficient to allow definite conclusions to be drawn. All tumors with increased EGFR gene copy numbers were also positive for EGFR immunostaining (Fig. 1D). Table 4 summarizes the data on EGFR gene copy number and protein levels of EGFR mutant tumors. On the other hand, 32 (86%) of the tumors were negative and 5 (13%) were positive for HER2 immunostaining. This latter category includes tumors scored as 2+ (four tumors) and 3+ (one tumor). ErbB2 gene amplification was detected in two primary lung adenocarcinomas, one of them with the highest level (3+) and the other with level 2+ of HER2 protein staining. Both featured wild-type EGFR and KRAS genes.


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  		Table 4. Context of EGFR gene copy number and EGFR protein levels of tumors carrying EGFR gene mutations

 
Levels of phosphorylation of ERK (phospho-ERK) and ribosomal protein S6 (phospho-S6) in lung primary adenocarcinomas according to EGFR gene status. To determine whether EGFR mutations lead to constitutive activation of downstream targets, we measured the levels of phosphorylation of ERK and S6 proteins in tumors included in the tissue microarray. Phospho-ERK mediates the RAF/MAP/ERK kinase signaling pathway, whereas phosphorylation of S6 protein was used as a surrogate marker for measuring S6K and mTOR activity. Examples of phospho-ERK and phospho-S6 immunostaining are depicted in Fig. 2A. Eight (22%) and 28 (78%) of the 36 lung primary tumors analyzed were positive and negative for phospho-ERK immunostaining, respectively. The frequency of positive immunostaining for phospho-ERK was similar among tumors carrying mutations in EGFR or KRAS as in the wild-type genes. On the other hand, most tumors were positive for phospho-S6 immunostaining. However, according to the criteria described in Materials and Methods, 20 (54%) and 17 (46%) of the 37 tumors had high and low levels of phospho-S6 immunostaining, respectively. Interestingly, most EGFR-mutant tumors had high levels of phospho-S6 protein relative to EGFR wild-type (8 of 9, 89%, compared with 12 of 28, 43%; P = 0.02; Fisher's exact test). We then compared the levels of these proteins in tumors carrying genetic alterations at either EGFR and KRAS and wild-type tumors. Remarkably, we observed that, in the former group, increased levels of phospho-S6 were present in 16 of 18 tumors (89%), significantly more than in the latter group (4 of 19 tumors, 21%; P = 0.00005; Fisher's exact test; Fig. 2B).


Figure 2
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  		Fig. 2. A-B, representative examples of positive and negative immunostaining for the phospho-ERK (original magnification, x400) and phospho-S6 (original magnification, x200) proteins. C, distribution of the immunostaining of phospho-ERK and phospho-S6 proteins among the tumors carrying EGFR and KRAS gene alterations, including gene mutations at EGFR or KRAS and gene amplification at EGFR (EGFR/KRAS-positive) compared with the remaining (EGFR/KRAS-negative) tumors. Gray columns, positive immunostaining; red columns, negative immunostaining. High levels of phospho-S6 immunostaining were more common in (EGFR/KRAS-positive) tumors (P = 0.00005; Fisher's exact test).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The recent identification of activating somatic mutations in the EGFR gene in lung adenocarcinomas and their proposed relevance in predicting the response to tyrosine kinase inhibitors of EGFR have had a significant effect in the field of lung cancer treatment (2–4). It is now critical to focus on unraveling the mechanisms and characteristics underlying the presence of EGFR mutations. In the present study, we have found a frequency of EGFR gene mutations in lung adenocarcinomas of Spanish patients similar to that reported for patients of Caucasian origin, and slightly lower than that of patients of Asian origin (6, 7, 22). Our observations support previous results on the clustering of these mutations in individuals who have never used tobacco and whose tumors are classified as adenocarcinomas with bronchioloalveolar features (BAC; refs. 4, 6, 23). Regarding the latter, it was previously known that the presence of BAC or papillary features were significant predictors of the response to tyrosine kinase inhibitors in lung adenocarcinomas, (24, 25). More recent reports proposed that the pathologic features of tyrosine kinase inhibitor responders overlaps with those of terminal respiratory unit adenocarcinoma (23), which according to the WHO classification includes most nonmucinous BAC, most of the adenocarcinomas mixed subtype with BAC features and a subset of papillary carcinomas (14). However, given the lack of consensus in the use of the term "BAC" versus "adenocarcinoma" it is extremely difficult to compare published reports.

The activation of the tyrosine kinase activity of EGFR or HER2 switches on intracellular signaling cascades that include the ras/MAP signal transducers (5). We observed a lack of simultaneous mutations at EGFR and KRAS genes, which is consistent with the widely accepted hypothesis that only one component of a cancer pathway needs to be genetically inactivated/activated for neoplastic clonal expansion to occur and with previously published data (6, 12). On the other hand, although p53 and EGFR mutations frequently coexisted in the same tumor specimen, here we confirmed previous observations on the low frequency of G:C-to-T:A transversions in the p53 gene in EGFR-mutant tumors (6). Because these types of p53 mutations are typically associated with tobacco carcinogens (26), our observations are further evidence of the biological relevance of EGFR mutations in non–tobacco-related lung adenocarcinomas.

EGFR gene amplification is also present in a subset of lung tumors (27, 28). Our data imply that EGFR amplification is always accompanied by increased protein expression and that this prevails in tumors carrying EGFR mutations. The biological significance of the coexistence of EGFR mutations and gene amplification in the same tumor specimen is not obvious. Because of the large number of extra copies, amplification of EGFR gene in lung tumors is unlikely to arise stochastically and should confer a growth advantage on the tumor cell, even in the presence of gene mutations. An important question requiring an answer is whether there is selective amplification of the mutant or wild-type allele. Previous investigations reported only EGFR mutant bands in the chromatograms in some lung primary tumors and cell lines, without a detectable background of normal DNA (4, 6). This is especially intriguing in primary tumors in which contamination with normal stromal cells is unavoidable. Several mechanisms, including gene amplification of the mutant allele and loss of the wild-type allele, have been proposed (6, 12, 29). For example, in one of the largest series to date, 40% of the electropherograms suggested amplification of the mutant allele (12). Our present data support the hypothesis that gene amplification of the mutant allele is the most plausible explanation for these observations. However, and although interpreted solely on the basis of DNA sequencing, the mutant allele did not always seem to be the target of EGFR amplification. Regardless of the biological significance, these observations raise concerns about the masking effect that gene amplification of the wild-type allele might have in the detection of EGFR gene mutations and so in the identification of patients who might benefit from tyrosine kinase inhibitor treatments. In this regard, it is interesting to note that high EGFR gene copy number has been recently proposed as a predictor for gefitinib efficacy (28, 30, 31).

Finally, we observed that, unlike in their wild-type counterparts, lung adenocarcinomas carrying activating gene alterations in EGFR or KRAS have higher levels of phospho-S6, a ribosomal protein that is phosphorylated by the mTOR-substrate, S6 kinase. Our present data confirm, in primary tumors, recent observations that the phosphoinositide-3-kinase/AKT/mTOR pathway can mediate signaling of tyrosine kinase receptors, also via RAS, and that RAS plays a critical role in activating mTOR in both normal and tumorigenic settings (32).

On the other hand, increased levels of phopsho-AKT protein have been proposed as a marker of gefitinib sensitivity (28, 33), although no significant correlation has been observed between the presence of EGFR mutations and phospho-AKT overexpression (33). Following this line of reasoning and taking into account that 40% to 90% of adenocarcinomas may contain areas of BAC (25), phospho-S6 immunohistochemistry may prove useful to select tumors for EGFR mutation testing.

In conclusion, we have further determined the genetic and molecular context in which EGFR mutations arise, which helps to elucidate their biological role and provides additional information for the selection of patients who could be candidates for EGFR mutation analysis.


   Acknowledgments
 
We thank our collaborators in the Tumour Bank Network, especially Irene Cuenca, Laura Cereceda, and Alicia Maroto, for their support in providing normal and lung tumor tissues. We also wish to acknowledge Lydia Sanchez-Verde and Ana Díez from the Histology and Immunohistochemistry, and Juan C Cigudosa and M. Carmen Martín Guijarro from the Cytogenetics Units of the Spanish National Cancer Centre.


   Footnotes
 
Grant support: Spanish Ministerio de Ciencia y Tecnología (SAF2002-01595) and the Fondo de Investigaciones Sanitarias (03/0049-03/0046). The Ramon y Cajal Program of the Ministerio de Ciencia y Tecnología, Spain (M. Sanchez-Cespedes) and the Institucion Fundacion Carolina (M. Tang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/22/05; revised 10/ 7/05; accepted 11/22/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sanchez-Cespedes M. Dissecting the genetic alterations involved in lung carcinogenesis. Lung Cancer 2003;40:111–21.[CrossRef][Medline]
  2. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;352:2129–39.
  3. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–500.[Abstract/Free Full Text]
  4. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A 2004;101:13306–11.[Abstract/Free Full Text]
  5. Holbro T, Civenni G, Hynes NE. The ErbB receptors and their role in cancer progression. Exp Cell Res 2003;284:99–110.[CrossRef][Medline]
  6. Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T. Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res 2004;64:8919–23.[Abstract/Free Full Text]
  7. Yang SH, Mechanic LE, Yang P, et al. Mutations in the tyrosine kinase domain of the epidermal growth factor receptor in non-small cell lung cancer. Clin Cancer Res 2005;11:2106–10.[Abstract/Free Full Text]
  8. Amann J, Kalyankrishna S, Massion PP, et al. Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer. Cancer Res 2005;65:226–35.[Abstract/Free Full Text]
  9. Tracy S, Mukohara T, Hansen M, Meyerson M, Johnson BE, Janne PA. Gefitinib induces apoptosis in the EGFRL858R non-small-cell lung cancer cell line H3255. Cancer Res 2004;64:7241–4.[Abstract/Free Full Text]
  10. Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 2005;352:786–92.[Abstract/Free Full Text]
  11. Scagliotti GV, Selvaggi G, Novello S, Hirsch FR. The biology of epidermal growth factor receptor in lung cancer. Clin Cancer Res 2004;10:4227–32s.[CrossRef]
  12. Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97:339–46.[Abstract/Free Full Text]
  13. Sordella R, Bell DW, Haber DA, Settleman J. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 2004;305:1163–67.[Abstract/Free Full Text]
  14. Travis WD, Brambilla E, Müller-Hermelink HK, Harris CC. Editors. World Health Organization classification of tumours. Pathology and genetics of tumours of the lung, pleura, thymus and heart. IARC Press: Lyon 2004.
  15. Fernandez P, Carretero J, Medina PP, et al. Distinctive gene expression of human lung adenocarcinomas carrying LKB1 mutations. Oncogene 2004;23:5084–91.[CrossRef][Medline]
  16. Rodriguez-Pinilla M, Rodriguez-Peralto JL, Hitt R, et al. Cyclin A as a predictive factor for chemotherapy response in advanced head and neck cancer. Clin Cancer Res 2004;10:8486–92.[Abstract/Free Full Text]
  17. Selvaggi G, Novello S, Torri V, et al. Epidermal growth factor receptor overexpression correlates with a poor prognosis in completely resected non-small-cell lung cancer. Ann Oncol 2004;15:28–32.[Abstract/Free Full Text]
  18. Cappuzzo F, Gregorc V, Rossi E, et al. Gefitinib in pretreated non-small-cell lung cancer (NSCLC): epidermal growth factor receptor expression in locally advanced or metastatic NSCLC. J Clin Oncol 2003;21:2658–63.[Abstract/Free Full Text]
  19. Cox G, Vyberg M, Melgaard B, Askaa J, Oster A, O'Byrne KJ. Herceptest: HER2 expression and gene amplification in non-small cell lung cancer. Int J Cancer 2001;92:480–3.[CrossRef][Medline]
  20. Vincent S, Lopez-Picaro JM, Toledo G, et al. ERK1/2 is activated in non-small-cell lung cancer and associated with advanced tumours. Br J Cancer 2004;90:1047–52.[CrossRef][Medline]
  21. Wislez M, Loreto Spencer M, et al. Inhibition of mammalian target of rapamycin reverses alveolar epithelial neoplasia induced by oncogenic K-ras. Cancer Res 2005;65:3226–35.[Abstract/Free Full Text]
  22. Cortes-Funes H, Gomez C, Rosell R, et al. Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients. Ann Oncol 2005;16:1081–6.[Abstract/Free Full Text]
  23. Yatabe Y, Kosaka T, Takahashi T, Mitsudomi T. EGFR mutation is specific for terminal respiratory unit type adenocarcinoma. Am J Surg Pathol 2005;29:633–9.[CrossRef][Medline]
  24. Kim KS, Jeong JY, Kim YC, et al. Predictors of the response to gefitinib in refractory non-small cell lung cancer. Clin Cancer Res 2005;11:2244–51.[Abstract/Free Full Text]
  25. Miller VA, Hirsch FR, Johnson DH. Systemic therapy of advanced bronchioloalveolar cell carcinoma: challenges and opportunities. J Clin Oncol 2005;23:3288–93.[Abstract/Free Full Text]
  26. Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P. Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers. Oncogene 2002;21:7435–51.[CrossRef][Medline]
  27. Hirsch FR, Varella-Garcia M, Bunn PA, Jr, et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 2003;21:3798–807.[Abstract/Free Full Text]
  28. Cappuzzo F, Hirsch FR, Rossi E, et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 2005;97:643–5.[Abstract/Free Full Text]
  29. Minna JD, Gazdar AF, Sprang SR, Herz J. Cancer. A bull's eye for targeted lung cancer therapy. Science 2004;304:458–61.[CrossRef]
  30. Takano T, Ohe Y, Sakamoto H, et al. Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer. J Clin Oncol 2005;23:6829–37.[Abstract/Free Full Text]
  31. Hirsch FR, Varella-Garcia M, McCoy J, et al. Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study. J Clin Oncol 2005;23:6838–45.[Abstract/Free Full Text]
  32. Johannessen CM, Reczek EE, James MF, Brems H, Legius E, Cichowski K. The NF1 tumor suppressor critically regulates TSC2 and mTOR. Proc Natl Acad Sci U S A 2005;102:8573–8.[Abstract/Free Full Text]
  33. Han SW, Kim TY, Hwang PG, et al. Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib. J Clin Oncol 2005;23:2493–501.[Abstract/Free Full Text]



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