• Open Access

EGFR point mutation in non-small cell lung cancer is occasionally accompanied by a second mutation or amplification

Authors

  • Toshihiko Yokoyama,

    1. Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1–1 Kanokoden, Chikusa-ku, Nagoya 464-0021,
    2. Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, and
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  • Masashi Kondo,

    1. Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, and
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  • Yasuhiro Goto,

    1. Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1–1 Kanokoden, Chikusa-ku, Nagoya 464-0021,
    2. Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, and
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  • Takayuki Fukui,

    1. Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1–1 Kanokoden, Chikusa-ku, Nagoya 464-0021,
    2. Division of General Thoracic Surgery, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
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  • Hiromu Yoshioka,

    1. Division of General Thoracic Surgery, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
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  • Kohei Yokoi,

    1. Division of General Thoracic Surgery, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
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  • Hirotaka Osada,

    1. Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1–1 Kanokoden, Chikusa-ku, Nagoya 464-0021,
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  • Kazuyoshi Imaizumi,

    1. Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, and
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  • Yoshinori Hasegawa,

    1. Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, and
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  • Kaoru Shimokata,

    1. Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, and
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  • Yoshitaka Sekido

    Corresponding author
    1. Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1–1 Kanokoden, Chikusa-ku, Nagoya 464-0021,
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To whom correspondence should be addressed. E-mail: ysekido@aichi-cc.jp

Abstract

Activating mutations of EGFR are found frequently in a subgroup of patients with non-small cell lung cancer (NSCLC) and are highly correlated with the response to gefitinib and erlotinib. In the present study, we searched for mutations of EGFR, HER2 and KRAS in 264 resected primary NSCLC from Japanese patients and determined whether there is a correlation between genetic alterations of these genes and clinicopathological factors, together with 85 tumors that we reported previously. EGFR mutations were found in 102 of the total 349 tumors, and seven tumors had two missense mutations. Reverse transcription–polymerase chain reaction of EGFR and subsequent subcloning analyses identified that the double mutations occurred in the same allele. Furthermore, in 202 NSCLC analyzed by Southern blotting, we identified 11 tumors with gene amplification of EGFR, with eight tumors containing a mutation in EGFR. Sequence analysis detected only weak or no signals of the wild-type allele in the eight tumors, strongly suggesting that the mutated allele was amplified selectively. These findings indicate that a dual genetic change of EGFR can occur in the same allele either with a possible second-hit mutation or with amplification, which may imply a more selective growth advantage in a cancer cell. Meanwhile, HER2 mutations and amplifications were found in six of 349 tumors and three of 202 tumors, respectively, and KRAS mutations in 21 of 349 tumors. Mutations of the EGFR and HER2 genes were more frequently found in female never or light-smoking patients with adenocarcinoma, and there were no tumors that had two or more mutations simultaneously among EGFR, HER2 and KRAS. The current study further demonstrates that a double genetic event in EGFR can occasionally occur in lung cancer, thus providing new clues for understanding the involvement of epidermal growth factor receptor signaling cascades in the pathogenesis of NSCLC. (Cancer Sci 2006; 97: 753–759)

Abbreviations:
EGFR

epidermal growth factor receptor

FISH

fluorescence in situ hybridization

NSCLC

non-small cell lung cancer

PCR

polymerase chain reaction

RT-PCR

reverse transcription–polymerase chain reaction

SSCP

single-strand conformation polymorphism

TK

tyrosine kinase

TKI

tyrosine kinase inhibitor.

Non-small cell lung cancer is one of the leading causes of death from cancer in Japan and Western countries.(1,2) Although the prognosis for patients with advanced NSCLC has not improved in the last 20 years,(3) the molecular and biological characteristics of lung cancer have been studied intensively(4,5) and many molecules associated with the pathogenesis of lung cancer have been identified as promising new targets for lung cancer therapy.(6) Among these molecules, EGFR, one of the ERBB family of receptors, is thought to be a promising target because of its frequent overexpression (range 40–80%) in NSCLC(7) and its relationship with poor prognosis.(8) Small molecules of EGFR-TKI, such as gefitinib and erlotinib, have been developed, examined for the treatment of NSCLC, and shown to have antitumor activity.(9) Several clinical studies have revealed that Japanese female never-smoking patients with adenocarcinoma have a higher response rate to gefitinib.(10,11) Recently, activating mutations of the TK domain of EGFR were found in a unique subgroup of NSCLC and were highly correlated with the response to EGFR-TKI.(12–14) Subsequent analyses using a large series of NSCLC confirmed somatic mutations in the similar subgroup of patients as known clinical predictors of EGFR-TKI sensitivity.(15–18) Moreover, recent studies have shown that increased copy numbers of the EGFR gene are associated with a better response to EGFR-TKI.(17,19) HER2, another member of the ERBB family, can heterodimerize with EGFR and has also been shown to have somatic mutations in the TK domain in several cancers including NSCLC.(20,21) However, the involvement of HER2 alterations in the pathogenesis of NSCLC is not clearly understood.

After earlier reporting 17 mutations of EGFR in 85 NSCLC,(22) we further extended our analysis with another 264 NSCLC. In a total of 349 tumors, we found 102 with EGFR mutation, six with HER2 mutation, and 21 with KRAS mutation. We also identified seven tumors with double mutations of EGFR and eight tumors with both mutation and amplification of EGFR. We therefore investigated the double genetic event of EGFR in detail. Our results provide new insights into the involvement of alterations of the receptor TK family in NSCLC development.

Materials and Methods

Samples

Primary lung tumor tissues were obtained from 349 randomly selected patients with NSCLC who underwent potentially curative pulmonary resection at Nagoya University Hospital, Nagoya First Red Cross Hospital, Nagoya Second Red Cross Hospital, Kasugai Municipal Hospital and Social Insurance Chukyo Hospital in Japan from August 1999 through to November 2003. In each case, tumor and corresponding normal lung tissue samples (taken as far as possible from the neoplastic area) were snap frozen and stored at −80°C. Ethical approval was obtained from each of the five hospitals and fully informed written consent was obtained from all patients prior to tissue collection.

The clinicopathological characteristics of the 349 primary NSCLC patients are summarized in Table 1. The study population consisted of 226 men and 123 women, with a mean age of 65 years (range 29–85 years). Histological type was determined according to the third World Health Organization/International Association for the Study of Lung Cancer classifications, with 242 adenocarcinomas, 89 squamous cell carcinomas, seven large cell carcinomas, three adenosquamous carcinomas, one pleomorphic carcinoma, one mucoepidermoid carcinoma, and six unclassified NSCLC. Smoking history was obtained by anamnesic data at admission or first outpatient visit. There were 107 never smokers, 24 light smokers (pack-year < 20) and 201 heavy smokers (pack-year  20), with 17 patients of undetermined smoking status. P-stage was determined according to the TNM staging system. One hundred and eighty patients were classified at stage I, 46 at stage II, 88 at stage III and nine at stage IV. The P-stage of 26 patients was not determined.

Table 1. Clinicopathological characteristics of 349 primary non-small cell lung carcinoma patients
Parametersn%
  • Pack-year < 20.

Age (years)
 Mean65 (range 29–85) 
Sex
 Male22665
 Female12335
Histology
 Adenocarcinoma24269
 Squamous cell carcinoma 8926
 Large cell carcinoma  7 2
 Adenosquamous carcinoma  3 0.9
 Pleomorphic carcinoma  1 0.2
 Mucoepidermoid carcinoma  1 0.2
 Unclassified non-small cell carcinoma  6 1.7
Smoking history
 Never or light smokers13138
 Heavy smokers20157
 Undetermined 17 5
P-stage
 I or II22665
 III or IV 9728
 Undetermined 26 7

DNA extraction and direct sequencing

Genomic DNA was prepared from portions of frozen tumors or non-cancerous lung tissues by a standard technique.(23) Genomic DNA (20 ng) was amplified by PCR. The first four (exons 18–21) of seven exons (exons 18–24) encoding the TK domain of EGFR were examined. The primer sequences were 5′-GGCACTGCTTTCCAGCATGG-3′ and 5′-CAGCTTGCAAGGACTCTGGG-3′ for exon 18, 5′-CATGTGGCACCATCTCACAA-3′ and 5′-CCTGAGGTTCAGAGCCATGG-3′ for exon 19, 5′-ATGCGAAGCCACACTGACGT-3′ and 5′-GTATCTCCCTTCCCTGATTA-3′ for exon 20, and 5′-GGCATGAACATGACCCTGAA-3′ and 5′-ACAGCTAGTGGGAAGGCAGC-3′ for exon 21. Mutations in exon 20 of HER2 and codons 12 and 13 of KRAS were examined by direct sequencing. The primer sequences were 5′-GCCATGGCTGTGGTTTGTGATGG-3′ and 5′-ATCCTAGCCCCTTGTGGACATAGG-3′ for exon 20 of HER2, and 5′-GGCCTGCTGAAAATGACTGA-3′ and 5′-GTCCTGCACCAGTAATATGC-3′ for codons 12 and 13 of KRAS.

The PCR products were sequenced using ABI PRISM 3100 (Applied Biosystems, Foster City, CA, USA) with a PCR primer and a BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) as described previously.(24)

Mutation-specific PCR and SSCP analyses for L858R EGFR mutation

Mutation-specific PCR analysis for the L858R mutation was carried out using a forward primer with T substituted to G at the 3′ end (5′-AAGATCACAGATTTTGGGCG-3′) and a reverse primer (5′-ACAGCTAGTGGGAAGGCAGC-3′). Genomic DNA (20 ng) was amplified by touch-down PCR. The conditions used were 10 cycles at 95°C for 30 s, 67–57°C (decreasing 1°C/cycle) for 30 s, and 72°C for 30 s, followed by 20 cycles at 95°C for 30 s, 57°C for 30 s and 72°C for 30 s, and a 7-min extension at 72°C. The PCR products were then applied to a 3% agarose gel, and the band of expected size detected by ultraviolet light was defined as positive.

Single-strand conformation polymorphism analysis was carried out for exon 21 to cover the L858R mutation on mutation detection enhancement gels (FMC Bioproducts, Rockland, ME, USA) containing 10% glycerol with a primer set of 5′-CAGGGCATGAACTACTTGGA-3′ and 5′-TTGCCTCCTTCTGCATGGTA-3′, as described previously.(25)

Cloning of EGFR cDNA

Total RNA was extracted from tumor samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was generated using SuperScript II (Invitrogen) and random primers (Takara Bio, Tokyo, Japan) according to the manufacturer's instructions. RT-PCR was carried out using the primer set 5′-TAAGATCCCGTCCATCGCCA-3′ and 5′-TTGCCTCCTTCTGCATGGTA-3′, covering exons 18–21 of EGFR. Amplified products were subcloned into pGEM-T Easy Vectors (Invitrogen) using XL-1 and XL-1 mutS strains (Stratagene, La Jolla, CA, USA) as competent cells. More than 25 clones were picked up for each sample, plasmids were purified using QIAprep Spin Miniprep KIT (Qiagen, Valencia, CA, USA), and the inserts were sequenced bidirectionally.

Southern blot analysis

A total of 7 µg of genomic DNA digested with EcoRI endonuclease (New England Biolabs, Beverly, MA, USA) was electrophoresed on a 0.7% agarose gel, transferred to Hybond N+ membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK), hybridized with 32P-labeled probes synthesized with random primers, and subjected to autoradiography after washing, as described previously.(24) The DNA probes of EGFR, HER2 and β-actin were prepared by PCR with primers set of 5′-GGCACTGCTTTCCAGCATGG-3′ and 5′-CCTGAGGTCAGAGCCATGG-3′ for EGFR, 5′-GCCATGGCTGTGGTTTGTGATGG-3′ and 5′-ATCCTAGCCCCTTGTGGACATAGG-3′ for HER2, and 5′-CTGTGGCATCCACGAAACTA-3′ and 5′-AGGAAAGACACCCACCTTGA-3′ for β-actin.

Hybridized membranes were exposed to scientific imaging films (Eastman Kodak, Rochester, NY, USA) or BAS-IP (Fuji Photo Film, Tokyo, Japan), which were analyzed using a BAS 2500 image analyzer (Fuji Photo Film) to quantify the intensity of signals. The samples with EGFR copies more than 3.5-fold the normal lung DNA were defined as gene amplification positive.

Statistical analyses

The variables evaluated in the present study were investigated for association with mutation status of EGFR, HER2 and KRAS using Fisher's exact test or the χ2-test. To identify which independent variables had a significant influence on EGFR mutation, a logistic regression model was used. P < 0.05 was regarded as statistically significant. All analyses were carried out using StatView version 5 software (SAS Institute, Cary, NC, USA).

Results

EGFR mutations in Japanese NSCLC

We previously reported that 17 tumors had EGFR mutations identified by direct sequencing of exons 18, 19 and 21 among 85 resected Japanese NSCLC.(22) In the present study, we first carried out direct sequencing of exon 20 for the 85 tumors and detected another four tumors with mutations (Fig. 1). Taken together, we identified 21 tumors with EGFR mutations and further extended our analysis using another 264 tumors. We then identified 72 tumors with EGFR mutations in exons 18–21. In total, we found that 93 (27%) of 349 tumors had EGFR mutations by direct sequencing (Fig. 1).

Figure 1.

A schematic representation of mutation analysis of the epidermal growth factor receptor (EGFR) gene. We previously reported 17 tumors with EGFR mutation detected by direct sequencing of exons 18, 19 and 21 among 85 non-small cell lung carcinomas (NSCLC; as indicated in parentheses), and further carried out direct sequencing of exon 20. Direct sequencing analysis of a further 264 NSCLC revealed 93 tumors with EGFR mutation among a total of 349 NSCLC. As the L858R mutation was then detected by mutation-specific polymerase chain reaction (PCR) in another nine tumors, we identified a total of 102 tumors with EGFR mutations (95 tumors with a single mutation and seven tumors with double mutations of EGFR).

As some recent reports suggest that SSCP or mutation-specific PCR analysis may be more sensitive than direct sequencing,(18) we used mutation-specific PCR to search for the mutation L858R in exon 21, which is one of the most frequent EGFR mutations, in 349 NSCLC. After confirming that all tumors with L858R detected by direct sequencing were positive for the mutation, we found another nine with the L858R mutation in 349 tumors. In total, we detected 102 (29%) EGFR-mutated tumors (95 tumors with a single mutation and seven with double mutations) among 349 NSCLC, with the overall number of mutations being 109 (Fig. 1; Table 2). The mutations consisted of three different types: in-frame deletions, single-nucleotide substitutions, and in-frame duplications or insertions. Forty-four tumors had an in-frame deletion at codons 746–750 in exon 19, with eight of these deletions being accompanied by a point mutation or insertion. Fifty-three tumors had missense mutations in exons 18, 20 or 21, and the L858R mutation was the most common missense mutation. Five tumors had an in-frame duplication or insertion of one or three amino acids at codons 769–776 in exon 20. None of the mutations were present in the corresponding normal lung tissue, indicating that these mutations were somatic. Single-nucleotide polymorphisms were identified at 2361 (G/A) and 2457 (G/A) in exon 20 (data not shown).

Table 2. One hundred and two tumors with epidermal growth factor receptor gene mutations among 349 non-small cell lung carcinomas
Mutation typeExonNucleotide changeAmino acid changen
  • Includes 17 cases reported previously:(22) one of G719S, six of del(2235–2249), three of del(2236–2250), one of del(2240–2254), one of del(2237–2254)2255C > T, and five of L858R. del, deletion; dup, duplication; ins, insertion.

In-frame deletion19del(2236–2250)E746-A750 del18
19del(2235–2249)E746-A750 del15
19del(2240–2254)L747-T751 del3
19del(2240–2257)L747-P753 del S ins3
19del(2237–2251)E746-T751 del A ins2
19del(2237–2254)2255C > TE746-S752 del V ins1
19del(2239–2247)2248G > CL747-A750 del P ins1
19del(2240–2257)2261A > CL747-K754 del ST ins1
Total  44
Single-nucleotide substitution212573 T > GL858R43
202303 G > TS768I2
18 + 212125 G > A + 2573 T > GE709K + L858R1
18 + 212126 A > G + 2573 T > GE709G + L858R1
18 + 212126 A > C + 2573 T > GE709A + L858R1
18 + 202155 G > A + 2303 G > TG719S + S768I1
18 + 202155 G > T + 2303 G > TG719C + S768I1
20 + 202303 G > T + 2305 G > TS768I + V769L1
20 + 202303 G > T + 2320 G > AS768I + V774M1
212582 T > GL861Q1
Total  53
In-frame duplication and/or insertion202304–2312 ins TGTGGCCAGVAS 769–771 ins1
202308–2316 ins GACAACGTGDNV 770–772 ins1
202311–2319 ins GCACCGTGGAPW 771–773 ins1
202314–2316 ins/dup AACN 772 ins1
202320–2328 ins/dup AACCCCCACNPH 774–776 ins1
Total  5
Overall tumors   102
Overall mutations   109

Double mutations of EGFR on the same allele

To determine whether the double point mutations of EGFR, which were identified in the same sample, resulted from mutational events on the same allele or different alleles, we carried out cDNA cloning analysis. After extraction of total RNA from five samples available among the seven tumors that had double mutations, we carried out RT-PCR and TA-cloning using transformation into XL-1 bacteria, picked up at least five to 10 subclones for each sample, and sequenced the inserts bidirectionally. To avoid unsolicited mismatch repairs of PCR heteroduplex products during transformation, we also used the XL-1 mutS strain of bacteria. Collectively, over 30% clones were double-mutation positive, whereas most of the rest were wild type, possibly from RNA of contaminated normal cells or from the remaining wild-type allele (Table 3). Thus, the subcloning analyses revealed that all five tumors had mRNA containing both mutations, indicating that the mutations were localized in the same allele.

Table 3. Analysis of five tumors with double mutations of the epidermal growth factor receptor gene
Sample5′ mutation3′ mutationNo. coloniesSame allele
Total5′ Mt/3′ Mt
  1. Mt, mutation.

KD641G719SS768I5216 (31%)Yes
KD826G719CS768I4038 (95%)Yes
KD530S768IV769L2614 (54%)Yes
KD514E709AL858R2814 (50%)Yes
KD921E709GL858R3616 (44%)Yes

Gene amplification of EGFR

We carried out Southern blotting analysis of 202 tumors and found that 11 (5.4%) tumors had amplification of the EGFR gene. Among them, amplification was found in eight (8%) of 96 patients with EGFR mutations and three (3%) of 106 patients without EGFR mutations. The characteristics of tumors with amplification of EGFR are summarized in Table 4. Because sequence analysis demonstrated that the mutation signals of these tumors were dominant, with five being completely lost for wild-type signal, the amplification was suggested to have originated from the mutant allele (data not shown).

Table 4. Clinicopathological features and mutation status of 11 non-small cell lung carcinoma with epidermal growth factor receptor (EGFR) gene amplification
SampleHistologySexAge (years)Smoking (pack-years)P-stageEGFR amplificationEGFR mutationHER2 mutationKRAS mutation
  1. Ad, adenocarcinoma; del, deletion; F, female; ins, insertion; M, male; ND, not determined; Sq, squamous cell carcinoma.

KD190AdM6345IIIA+
KD215AdM71 8IA+NPH 774–776 ins
KD280SqM7350IIIB+
KD284AdM51 0IIIA+E746-A750 del
KD377SqF7757IIIA+
KD534AdF70 0ND+L858R
KD564AdF75 0IA+APW 771–773 ins
KD566AdM72 0IB+E746-A750 del
KD587AdF75 0IIIA+L747-P753 del S ins
KD591AdM50 0IIIA+L858R
KD938AdM7820IB+L858R

Alteration of HER2 and KRAS mutations

To determine whether alterations of other molecules involved in the EGFR signaling cascades may have accounted for the pathogenesis of NSCLC, we analyzed mutation and amplification of HER2 and mutation of KRAS. We found six (1.7%) mutations in exon 20 in the TK domain of HER2 among 349 NSCLC (Table 5). Four tumors had YVMA 776–779 insertion mutations and two had G776V C (cystein) insertions. The corresponding normal lung tissues did not have the same mutations as the tumors, indicating that these alterations were somatic. We carried out Southern blotting analysis of 202 tumors including five tumors with HER2 mutation and found amplification of HER2 in three tumors without HER2 mutation (data not shown). Meanwhile, KRAS mutations at codons 12 and 13 were present in 21 (6%) of the 349 NSCLC. There were no tumors that had two or more mutations simultaneously among the EGFR, HER2 and KRAS mutations.

Table 5. Clinicopathological features of six tumors with HER2 mutations among 349 non-small cell lung carcinomas
SampleHistologySexAge (years)Smoking (pack-years)P-stageNucleotide changeAmino acid change
  1. Ad, adenocarcinoma; F, female; ins, insertion; M, male.

KD318AdF680IA2325–2336 ins ATACGTGATGGCYVMA 776–779 ins
KD583AdF362IIIA2337–2339 ins TGTG776V C ins
KD617AdM604IB2325–2336 ins ATACGTGATGGCYVMA 776–779 ins
KD1007AdF540IA2325–2336 ins ATACGTGATGGCYVMA 776–779 ins
KD1024AdF710IB2337–2339 ins TGGG776V C ins
KD1027AdM6745IIA2325–2336 ins ATACGTGATGGCYVMA 776–779 ins

Relationship between clinicopathological features and genetic alterations

In univariate analysis of the correlation with clinicopathological features, EGFR mutations were significantly more frequent in women than in men (54 vs 15%; P < 0.0001), in patients with adenocarcinoma than in other histological subtypes (42 vs 1%; P < 0.0001), in never or light smokers (pack-year < 20) than in heavy smokers (pack-year  20) (54 vs 14%; P < 0.0001), and in patients with early stage (I or II) than in advanced stage (III or IV) (33 vs 21%; P = 0.024) (Table 6). HER2 mutations were more frequent in female never or light smokers, and in early stage patients with adenocarcinoma, but did not reach statistical significance except in those with a lower smoking history. KRAS mutations were more frequent in heavy smoking and early stage patients with adenocarcinoma. In multivariate analysis, EGFR mutations were found to be independently associated with women, a lower smoking history and adenocarcinoma histology (Table 7).

Table 6. Univariate analysis of the relationship between clinicopathological features and epidermal growth factor receptor (EGFR), HER2 and KRAS gene mutations
VariableEGFR mutationHER2 mutationKRAS mutation
n%P-value*n%P-value*n%P-value*
  • Patients whose smoking history (n = 17) and P-stage (n = 26) could not be determined were excluded from the analyses.

  • Pack-years < 20.

  • *

    *Fisher's exact test. NS, not significant.

Total (n = 349)10229 61.7 216 
Sex   <0.0001  NS (0.19)  NS (0.85)
 Male (n = 226)3515 20.9 146 
 Female (n = 123)6754 43.2 76 
Histology   <0.0001  NS (0.18)  0.0061
 Adenocarcinoma (n = 242)10142 62.5 208 
 Non-adenocarcinoma (n = 107)11 00.0 11 
Smoking history   <0.0001  0.037  NS (0.14)
 Never or light smoker (n = 131)7154 53.8 43 
 Heavy smoker (n = 201)2914 10.5 157 
 Undetermined (n = 17)212 00.0 212 
P-stage   0.024  NS (0.67)  NS (0.07)
 I or II (n = 226)7533 52.2 178 
 III or IV (n = 97)2021 11.0 22 
 Undetermined (n = 26)727 00.0 28 
Table 7. Logistic regression analysis of the association between epidermal growth factor receptor (EGFR) gene mutation and clinicopathological features
VariableOdds ratio95% CIP-value
  • Subjects without information on smoking and P-stage were assigned dummy variables, and lack of information was adjusted in a model. CI, confidence interval.

Female 2.681.34–5.39 0.0056
Adenocarcinoma 18.44.32–78.4 <0.0001
Smoking history
 <20 pack-years 2.521.25–5.08 0.0098
 Undetermined 3.50.58–21.3 0.17
P-stage
 I or II 1.810.92–3.56 0.088
 Undetermined 1.070.33–3.46 0.91

Discussion

In the present report, we searched for mutations in the EGFR, HER2 and KRAS genes in primary NSCLC resected from Japanese patients. Among the 349 NSCLC tumors, we found 109 EGFR mutations in 102 tumors, with seven tumors harboring two missense mutations, six HER2 mutations in six tumors and 21 KRAS mutations in 21 tumors. Notably, all the double missense mutations of EGFR were in the same allele, and both mutation and amplification of EGFR were detected in eight tumors, indicating that two genetic events that target EGFR occasionally occur in NSCLC.

Previous studies showed that in-frame deletional mutations in exon 19 and L858R mutation in exon 21 account for approximately 90% of EGFR mutations.(12–18) Although the direct sequencing method is accurate and can pick up various kinds of mutation within target regions, it is an expensive and complicated examination tool. Moreover, it may yield a false-negative result when the genomic DNA used is extracted directly from frozen tissues, which sometimes may be contaminated with a significant amount of genomic DNA from non-malignant cells. In this regard, Marchetti et al. reported that SSCP analysis was more sensitive than direct sequencing, especially for missense mutations of EGFR.(18) We used a mutation-specific PCR method for detecting the L858R mutation and still found another nine mutations in 349 tumors. We also used SSCP analysis for the nine tumors with L858R mutation detected only by mutation-specific PCR, as well as 94 tumors that included 20 EGFR-mutated and 74 wild-type tumors, and detected the L858R mutation in all but one of the nine tumors. Thus, there were several tumors with the L858R mutation that were detected only by mutation-specific PCR or SSCP analyses, indicating that these two methods are more sensitive than the direct sequencing method and may be equally useful for the first-step screening of L858R mutation.

We identified seven tumors harboring double missense mutations of EGFR that have been reported by other groups.(15,16,26) However, no report thus far has analyzed how the double mutations occur and whether they are involved in NSCLC development. We analyzed these tumors with the cloning method and found that all of the tumors had mRNA with two mutations. This indicates that EGFR was mutated at two different positions in the same allele, although we could not determine whether the two mutations occur sequentially or at one time. However, it may well be that a second-hit mutation of EGFR occurred in the same allele, which might give a greater growth advantage than a cancer cell with only a first-hit mutation of EGFR. To determine this, an experiment should be conducted to show whether transfectants made using an expression construct with an EGFR double mutation can induce more active biological properties than with a single mutation.

Gene amplification of EGFR has also been shown to be a good predictor of EGFR inhibitor response.(17,19,27) In the present study, the frequency of gene amplification was 5.4%, which seems to be consistent with previous reports using Southern blotting analysis,(28) although other studies using FISH analysis have indicated more frequent amplification.(17,29) Considering the contamination by genomic DNA from normal cells, FISH may be more sensitive than Southern blotting for detecting amplification, although reagents for detection and criteria of definition of amplification levels of EGFR vary among researchers, which seem to have caused confusion in the comparison of EGFR studies using FISH. Together with EGFR mutation status, we identified eight (8%) amplifications among 96 tumors with EGFR mutation and three (3%) among 106 without EGFR mutations. As sequence analysis demonstrated that the mutation signals of all of these eight tumors were dominant, of which five completely lost the wild-type signal, it suggested strongly that the mutated allele was amplified selectively.

Overexpression and amplification of HER2 have been found frequently in several tumors such as breast cancer,(30,31) and mutations of HER2 have also been reported in lung adenocarcinomas.(20,21) In the present study, we found six (1.7%) in-frame duplication or insertion mutations in 349 NSCLC and three (1.5%) amplifications of HER2 in 202 tumors. KRAS is a downstream molecule of EGFR/HER2 in the signaling cascades, and we found 21 (6%) mutations of KRAS among 349 tumors. In agreement with a previous report,(21) there were no tumors with two or more simultaneous mutations among EGFR, HER2 and KRAS, suggesting that the mutations are mutually exclusive.

In the present study, EGFR mutations were significantly more frequent in women and in lighter smokers with adenocarcinoma by multivariate analysis. Although HER2 mutation and amplification were infrequent, at least one genetic alteration of EGFR or HER2 was found in 64 (68%) out of 94 tumors categorized as female, adenocarcinoma, and light smoking history. These results indicated that constitutive activation of the EGFR signaling cascades is a key event in the pathogenesis of this subset of lung cancer. Furthermore, we demonstrated that a double genetic event of EGFR, containing a possible second-hit mutation or amplification, occasionally occurs on the same allele. Hence, we speculate that the second genetic event for EGFR might provide a greater growth advantage to one of the cancer cells with a single mutation. Taken together, our findings may provide new clues to understand the multistep activation mechanisms of EGFR that are involved in the pathogenesis of NSCLC and sensitivity and resistance to EGFR-TKI.

Acknowledgments

This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and a grant from the Aichi Cancer Research Foundation. We would like to thank Dr Koichi Fujita and Dr Norio Mukoyama for the surgical specimens, Dr Keitaro Matsuo, Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, for advice on statistical analyses and Ms Hiroko Kaga for her skillful technical support.

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