EGFR, ERBB2, and KRAS mutations in Korean non-small cell lung cancer patients

Cancer Genetics and Cytogenetics 173 (2007) 107e113

EGFR, ERBB2, and KRAS mutations in Korean non-small cell lung cancer patients Nack Cheon Baea, Myung Hwa Chaeb, Myung Hoon Leec, Kyung Mee Kimb, Eung Bae Leed, Chang Ho Kima, Tae-In Parkc, Sung Beom Hane, Sanghoon Jheonf, Tae Hoon Junga, Jae Yong Parka,b,* a Department of Internal Medicine, Kyungpook National University Hospital, Samduk 2Ga 50, Daegu, 700-412, Republic of Korea Cancer Research Institute, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu, 700-422, Republic of Korea c Advanced Medical Technology Cluster Center, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu, 700-422, Republic of Korea d Department of Thoracic Surgery, Kyungpook National University Hospital, Samduk 2Ga 50, Daegu, 700-412, Republic of Korea e Department of Internal Medicine, School of Medicine, Keimyung University, Dongsan Dong 194, Daegu, 700-712, Republic of Korea f Department of Thoracic and Cardiovascular Surgery, Seoul National University School of Medicine, Seoul, 110-799, Republic of Korea b

Received 20 July 2006; received in revised form 9 October 2006; accepted 13 October 2006

Abstract

The epidermal growth factor receptor (EGFR), and its family members play an important role in the development and progression of lung cancers. It has been reported that somatic mutations in the tyrosine kinase domain of the EGFR or ERBB2 genes occur in a subset of patients with lung cancer. We searched for mutations of the EGFR, ERBB2, and KRAS genes in surgically resected non-small cell lung cancers (NSCLCs) to determine the prevalence of these mutations in Korean lung cancer patients. In addition, we examined the relationship between the mutations and clinicopathologic features of lung cancers. Mutations of the EGFR, ERBB2, and KRAS genes were determined by polymerase chain reactionebased direct sequencing in 115 surgically resected non-small cell lung cancers. EGFR mutations were present in 20 patients (17.4%). The EGFR mutations were found only in adenocarcinomas (20 of 55 adenocarcinomas, 36.4%). The ERBB2 mutation was found in 1 adenocarcinoma of the 115 NSCLCs (0.9% overall; 1.8% of the 55 adenocarcinomas). KRAS mutations were found in 6 (5.2%) of the 115 NSCLCs (2 of 60 squamous cell carcinomas, or 3.3%, and 4 of 55 adenocarcinomas, or 7.3%). EGFR mutations in adenocarcinomas were more frequent in women (P 5 0.02) and in never-smokers (P 5 0.004). EGFR mutations in adenocarcinomas were not associated with pathologic stage in never-smokers, but were more frequent in pathologic stage IIeIV than in stage I in ever-smokers (P 5 0.01). Of the 55 adenocarcinomas, 25 (45.5%) had mutations of one or another of the three genes; EGFR mutations were never found in adenocarcinomas together with ERBB2 or KRAS mutations. These findings suggest that the EGFR mutation is frequent in Korean lung cancer patients, and that the ERBB2 mutation is rare. Further studies are needed to investigate the role of EGFR mutations in the carcinogenesis of adenocarcinoma among smokers. Ó 2007 Elsevier Inc. All rights reserved.

1. Introduction The epidermal growth factor receptor (EGFR) signal transduction cascade plays an important role in many tumorigenic processes, including cell proliferation, angiogenesis, and metastasis, as well as protection of the cell from apoptosis [1]. These effects are mediated by the activation of downstream signal transduction cascades, including Ras-

* Corresponding author. Tel.: þ82-53-420-5536; fax: 82-53-426-2046. E-mail address: [email protected] (J.Y. Park). 0165-4608/07/$ e see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.10.007

Raf-mitogen activated protein kinase, phosphatidylinositol3 kinaseeAkt, and Janus tyrosine kinaseeSTAT (signal transducer and activator of transcription) pathways [2e5]. Several studies have shown that mutations in the tyrosine kinase (TK) domain of the EGFR gene (previously ERBB) are present in a subset of non-small cell lung cancers (NSCLCs), and those tumors with the EGFR mutations have been reported to be highly sensitive to gefitinib, an EGFR TK inhibitor [6e8]. With ERBB (alias ERBB1) reclassified as EFGR, the ERBB gene family consists of ERBB2 (previously NGL; alias EGFR2, HER2, NEU ), ERBB2IP, ERBB3 (alias

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EGFR3, HER3), and ERBB4 (alias EGFR4, HER4) (see http://www.gene.ucl.ac.uk/nomenclature/). Several studies have shown that ERBB2 is overexpressed in ~20% of NSCLCs, and ERBB2 overexpression has been associated with an unfavorable prognosis [9,10]. Stephens et al. [11] reported that 5 of 120 NSCLCs (4.2%) and 5 of 51 adenocarcinomas (9.8%) harbored mutations in the ERBB2 TK domain. Shigematsu et al. [12] have recently reported that, similar to the EGFR mutations in lung cancers, mutations in the ERBB2 TK domain preferentially targeted adenocarcinoma histology, women, never-smokers, and Asian ethnicity. Several studies have observed that simultaneous alterations of genes in the same signaling pathways are rarely found in a single tumor. For example, CDKN2A (alias p16INK4a) methylation and retinoblastoma (RB1) gene mutation, which negatively regulate the phosphorylation of Rb, are rarely found together in individual tumors [13,14]. As in the case of the p16INK4aeRb pathway, it has been reported that EGFR mutations were never found in lung cancers with KRAS mutation [8,15], suggesting that EGFR and KRAS mutations are mutually exclusive. Because the prevalence of genetic alterations often varies depending on the patient’s ethnicity, we searched for mutations of the EGFR, ERBB2, and KRAS genes in surgically resected NSCLC samples to determine the prevalence of these mutations in Korean lung cancer patients. In addition, we examined the relationship between these mutations and clinicopathologic features in lung cancers. We also searched for these mutations in the normal lung tissues of patients with NSCLC.

2. Materials and methods 2.1. Patients and tissue samples Tumor and corresponding normal lung tissue specimens were obtained from 115 NSCLC patients who underwent curative resection at Kyungpook National University Hospital (Daegu, Korea) from January 2003 to December 2005. None of the patients had received chemotherapy or radiotherapy before surgery. Written informed consent was obtained from each patient before the surgery. This study was approved by the institutional review board of the Kyungpook National University Hospital. Histologic type was determined according to WHO criteria [16]: 60 tumors were squamous cell carcinomas (52.2 %) and 55 were adenocarcinomas (47.8%), including 5 bronchioloalveolar cell carcinomas (BACs). Histocytologic subtyping of BACs, according to Barkley and Green [17], revealed that all 5 cases were nonmucinous BAC. There were 87 men (75.7%) and 28 women (24.3%), with age at diagnosis ranging from 41 to 77 in the men (median age, 63) and from 41 to 78 in the women (median age, 65). There were 23 never-smokers (20.0%) and 92 smokers

(80.0%). Of the 55 adenocarcinoma cases, 22 cases were from never-smokers (2 men and 20 women, or 40.0%). Pathologic staging of lung cancers was determined according to the revised International System for Staging Lung Cancer [18]: 66 (57.4%) of the 115 patients had stage I disease, 14 (12.2%) stage II, 33 (28.7%) stage III, and 2 (1.7%) stage IV (Table 1). All of the tumor and macroscopically normal lung tissue samples were obtained at the time of surgery, and these were rapidly frozen in liquid nitrogen and stored at 80 C. Only tumors with O80% tumor component were sent for DNA extraction and mutational analysis. The normal lung tissue specimens were obtained from either the opposite end of resected surgical samples or as distant as possible from the tumor. All of the macroscopically normal samples were confirmed as normal under hematoxyline eosin stain. 2.2. DNA extraction and sequencing of EGFR, ERBB2, and KRAS genes Genomic DNA was obtained from primary tumors and corresponding normal lung tissues by overnight digestion with sodium dodecyl sulfate and proteinase K (Life Technologies, Rockville, MD) at 37 C followed by standard phenolechloroform (1:1) extraction and ethanol precipitation. Mutations of the first four exons (exon 18e21) of the tyrosine kinase (TK) domain of the EGFR gene, mutations of exons 19 and 20 of the TK domain of the ERBB2 gene, and mutations of codons 12 and 13 of the KRAS gene were detected using polymerase chain reactionebased (PCRbased) direct sequencing. The PCR reactions were performed in a total volume of 20 mL containing 100 ng genomic DNA, 0.2 mmol/L of each primer, and 0.2 mmol/L dNTPs, 1 unit of Taq polymerase (Takara, Shuzo Company, Otus, Shiga, Japan), and 1 reaction buffer (10 mmol/L Tris-HCl, pH 8.3; 50 mmol/L KCl; and 1.5 mmol/L MgCl2). The PCR cycle conditions consisted of an initial denaturation step at 95 C for 5 minutes followed by 35 cycles of 30 seconds at 95 C; 30 seconds at 56 C to 62 C; 30 seconds at 72 C; and a final elongation at 72 C for 10 minutes. The primers and conditions for PCR amplification are shown in Table 2. The PCR products were purified using a GENECLEAN Turbo kit (Q-Biogene, Carlsbad, CA). Sequencing was done using an ABI Prism 3100 Genetic Analyzer (PE Biosystems, Foster City, CA). 2.3. Statistical analysis The relationship between mutations of the three genes studied and the clinicopathologic characteristics was analyzed using Student’s t-test for continuous variables, and either c2 test or Fisher’s exact test for categorical variables. Fisher’s exact test was used if there were <5 observations in a group. Logistic regression models were used to further

N.C. Bae et al. / Cancer Genetics and Cytogenetics 173 (2007) 107e113 Table 1 Patient characteristics Variables Sample size Age, years, mean 6 SD Sex, no. (%) Male Female Smoking history, no. (%) Never-smoker Ever-smoker Pathologic stage, no. (%) I II III þ IV

109

Table 2 Primer sequences and annealing temperatures for direct sequencing SCC

AC

Total

n 5 60 62.4 6 7.97

n 5 55 62.7 6 8.20

n 5 115 62.5 6 8.05

56 (93.3) 4 (6.7)

31 (56.4) 24 (43.6)

87 (75.7) 28 (24.3)

1 (1.7) 59 (98.3)

22 (40.0) 33 (60.0)

23 (20.0) 92 (80.0)

33 (55.0) 7 (11.7) 20 (33.3)

33 (60.0) 7 (12.7) 15 (27.3)

66 (57.4) 14 (12.2) 35 (30.4)

Gene

Exon Primer sequence

EGFR

18 19 20

Abbreviations: AC, adenocarcinoma; SCC, squamous cell carcinoma; SD, standard deviation.

explore observed differences and to identify baseline factors that may independently predict for mutation. All statistical tests were two-sided, and P-values ! 0.05 were considered statistically significant.

3. Results Because EGFR mutations in lung cancers were limited to the first four exons (exons 18e21) of the TK domain in previous studies [6e8,15,19,20], we searched for mutations in these four exons. Of 115 NSCLC patients who underwent surgical resection of their tumors, EGFR TK domain mutations were found in 20 cases (17.4%). All EGFR mutations were found in adenocarcinomas only (20 of 55 adenocarcinomas, 36.4%). Of these 20 mutations, 9 (45%) were in-frame deletions in exon 19, 10 (50%) were missense point mutations (L858R) in exon 21, and 1 (5%) was an in-frame duplicationeinsertion mutation in exon 20. In-frame deletions in exon 19, involving four to seven codons, centered around the uniformly deleted codons 747e749 (Leu-Arg-Glu sequences). Among the nine in-frame deletion mutations in exon 19, five mutations were simple in-frame deletions, and four in-frame deletions were coupled with missense mutations at the carboxyl-terminal amino acid position flanking the deletion (Table 2). Representative nucleotide sequences of the EGFR mutation are shown in Figure 1. The ERBB2 mutation was found in 1 adenocarcinoma of the 115 NSCLCs (0.9% overall, and 1.8% of the adenocarcinomas). This single adenocarcinoma case with the ERBB2 mutation was from a never-smoker woman. The mutation was a heterozygous in-frame insertion, 2327_2329 ins TCT (G776 del VC ins), in exon 20 (Fig. 1). This mutation differed in the inserted base pair (TCT) from the mutations (TTT and TGT) that have been previously identified [12]. KRAS mutations were found in 6 (5.2%) of the 115 NSCLCs (2 of 60 squamous cell carcinomas, or 3.3%, and 4 of 55 adenocarcinomas, or 7.3%). All 6 cases with

21

ERBB2 19 20 KRAS

1

F, 50 -CTGCTGGGCCATGTCTGGCA-30 R, 50 -GCTTGCAAGGACTCTGGGCTC-30 F, 50 -GTGCATCGCTGGTAACATCCA-30 R, 50 -AGCAGCTGCCAGACATGAGA-30 F, 50 -TCTTCACCTGGAAGGGGTCCA-30 R, 50 -CCATGGCAAACTCTTGCTATC-30 F, 50 -CTCAGAGCCTGGCATGAACAT-30 R, 50 -CAATACAGCTAGTGGGAAGGC30 F, 50 -CCCACGCTCTTCTCACTCAT-30 R, 50 -GGGTCCTTCCTGTCCTCCTA-30 F, 50 -CTCTCAGCGTACCCTIGTCC-30 R, 50 -AGGTGCATACCTTGGCAATC-30 F,50 -GTACTGGTGGAGTATTIGAT-30 R,50 -TGAAAATGGTCAGAGAAACC-30

Tm, Product  C size, bp 58

284

60

270

60

360

62

324

62

183

62

220

55

285

Abbreviations: F, forward primer; R, reverse primer; Tm, annealing temperature.

KRAS mutation were from ever-smoker men (Table 3). We also examined 27 nonmalignant lung tissues in which the corresponding tumor harbored mutation of any of the three genes (EGFR, ERBB2, and KRAS ), and found no mutations. Because all of the EGFR mutations were found in adenocarcinoma cases, we analyzed the relationship between EGFR mutations and clinicopathologic features of adenocarcinomas (Table 4). EGFR mutations in adenocarcinomas were significantly more frequent in women than in men (54.2 versus 22.6%, P 5 0.02), and in never-smokers than in ever-smokers (59.1 versus 21.2%, P 5 0.004). EGFR mutations in adenocarcinomas were more frequent in pathologic stage IIeIV than in stage I (54.5 versus 24.2%, P 5 0.02). For never-smokers, EGFR mutation was not associated with pathologic stage of disease; for ever-smokers, EGFR mutations were more frequent in stage IIeIV than in stage I (P 5 0.01). Of the 55 adenocarcinomas, 25 (45.5.0%) had mutations of one or another of the three genes studied. EGFR mutations were never found in adenocarcinomas with ERBB2 or KRAS mutations (Table 4), suggesting a mutually exclusive relationship.

4. Discussion Although EGFR mutations in lung cancers have been extensively studied, most previous studies have investigated mutations in patients with advanced stage of lung cancer. In the present study, we searched for EGFR mutations in Korean NSCLC patients who underwent curative surgical resection and explored the relationship between EGFR mutational status and clinicopathologic features, including the presence of ERBB2 or KRAS mutations. Mutations in the TK domain of either the EGFR or ERBB2

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Fig. 1. Mutations in EGFR, ERBB2, and KRAS genes in non-small cell lung cancers. (A) Seven patterns of in-frame deletions in EGFR exon 19 and representative electropherograms of in-frame deletion mutations. (B) A 9-bp insertion mutation, 2308_2316 ins GCCAGGGTG (ASV770-772 ins), in EGFR exon 20. (C) L858R mutations in EGFR exon 21. (D) In-frame insertion, 2327_2329 ins TCT (G776 del VC ins), in ERBB2 exon 20. (E) Mutations in the KRAS gene.

genes were found exclusively in adenocarcinomas. EGFR mutations in adenocarcinomas were more frequent in women than in men, and in never-smokers than in eversmokers; they were never found in tumors with ERBB2 or KRAS mutations. A meta-analysis [21] of nine published studies showed that EGFR mutations were limited to the first four exons (exons 18e21) of the TK domain, which encode the N-lobe

and the 50 portion of the C-lobe of EGFR. The mutations consisted of three different types (deletions, insertions, and missense point mutations) and they all targeted key structures around the adenosine triphosphate binding cleft, including the glycine-rich GXGXXG motif of the phosphate binding loop (P-loop), the aC-helix, and the aspartic acidephenylalanineeglycine sequence (DFG motif) in the activation loop (A-loop).

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111

Table 3 Genetic alterations in the kinase domain of the EGFR or ERBB2 gene, and in the KRAS gene, in non-small cell lung cancers Genea EGFR

Case Age, Smoking status no. Histology Sex years (pack-years) Exon Nucleotide alteration

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ERBB2 21 KRAS 22 23 24 25 26 27

AC AC AC AC AC AC AC AC AC AC AC AC (BAC) AC AC AC AC AC AC (BAC) AC AC AC SCC SCC AC AC AC AC

M F M M F F F M F M F F F F F F M F F M F M M M M M M

67 57 66 68 60 49 52 65 51 47 41 65 69 72 66 69 62 68 54 66 59 59 73 52 63 58 64

Never Never Current (20) Current (30) Never Never Current (5) Current (40) Never Current (25) Never Never Never Never Never Never Former (9) Never Never Former (30) Never Current (15) Current (40) Current (52) Former (30) Current (40) Current (45)

19 19 19 19 19 19 19 19 19 20 21 21 21 21 21 21 21 21 21 21 20 1 1 1 1 1 1

2235 2249 del GGAATTAAGAGAAGC 2240 2257 del TAAGAGAAGCAACATCTC 2235 2249 del GGAATTAAGAGAAGC 2239_2251 del TTAAGAGAAGCA, 225lAOC 2237_2255 del AATTAAGAGAAGCAACAT, 2255COT 2240 2254 del TAAGAGAAGCAACAT 2236 2250 del GAATTAAGAGAAGCA 2235_2252 del GGAATTAAGAGAAGCAAC, 2254TOA, 2255COT 2235 2249 del GGAATTAAGAGAAGC 2308 2316 ins GCCAGGGTG 2573TOG 2573TOG 2573TOG 2573TOG 2573TOG 2573TOG 2573TOG 2573TOG 2573TOG 2573TOG 2327 2329 ins TCT 34GOT 34GOC 34GOC 35GOA 35GOT 35GOC

Amino acid alteration E746 A750 del L747 P753 del S ins E746 A750 del L747 TI5l del P ins E746 S752 del V ins L747 TI5l del E746 A750 del E746 S752 del I ins E746 A750 del ASV770 772 ins L858R L858R L858R L858R L858R L858R L858R L858R L858R L858R G776 del VC ins G12C Gl2R Gl2R G12D G12V G12A

Abbreviations: AC, adenocarcinoma; BAC, bronchioloalveolar cell carcinoma; SCC, squamous cell carcinoma; M, male; F, female. a Accession numbers are NM_005228 (EGFR), NM_004448 (ERBB2) and AF493917 (KRAS ).

In-frame deletions in the region spanning codons 746 to 750 in exon 19 accounted for 44% of all mutations, and inframe duplicationseinsertions in exon 20 accounted for 5% of all mutations [21]. These two types of mutation (in-frame deletions and in-frame duplicationseinsertions) occur on either side of the aC-helix. They are hypothesized to result in similar configurational changes, causing a shift of the helical axis and narrowing the adenosine triphosphate binding cleft, which leads to increases in both gene activation and TK-inhibitor sensitivity [22]. The third type of mutation, missense point mutation occurs in all four exons, particularly L858R located near the DFG motif in exon 21 (accounting for 41% of all mutations) and G719X (where the X indicates A, C, or S) in the GXGXXG motif in exon 18 (accounting for 4% of all mutations). Consistent with previous studies [20], we found that O90% of the mutations were either in-frame deletions in exon 19 or L858R in exon 21, and all in-frame deletions in exon 19 targeted the four-codon region (codons 747e750). All 20 EGFR mutations have been reported previously [8,15,19]. Stephens et al. [11] reported that 5 (9.8%) of 51 lung adenocarcinomas harbored mutations in the ERBB2 TK domain, and Shigematsu et al. [12] reported that ERBB2 TK mutations were found in 11 (2.8%) of 394 lung adenocarcinomas, and 10 out of the 11 ERBB2 mutations were detected in Asian populations. Sasaki et al. [23], however, found only

1 ERBB2 mutation in 122 Japanese lung adenocarcinoma patients, and Lee et al. [24] found no ERBB2 mutation in 103 Korean patients with lung adenocarcinoma. In the present study, we also found only 1 ERBB2 mutation in 55 adenocarcinomas. This finding suggests that ERBB2 kinase domain mutations infrequently occur in Korean lung cancer patients, and also suggests that the incidence of ERBB2 mutations should be further analyzed in diverse ethnic populations. Several studies have shown that, similar to KRAS mutations, EGFR mutations strikingly target adenocarcinoma; however, EGFR mutations are more frequent in East-Asian populations, never-smokers, and women, whereas KRAS mutations are more frequent in Western populations, smokers, and men [6e8,15,19e21]. A meta-analysis [21] of nine published studies showed that, in adenocarcinoma cases, EGFR mutations were present in 48% of East Asian patients but in only 12% for other ethnicities. Consistent with previous studies, we found EGFR mutations exclusively in adenocarcinoma cases, and more frequently in women and never-smokers. The frequency of EGFR mutations in our series of adenocarcinomas was 36.4%, which is lower than that previously reported for East Asian patients [21]. This difference may be due to ethnic and environmental differences. In addition, different distributions of sex and smoking status for the study subjects should also be considered.

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112

Table 4 EGFR mutations in 55 cases of adenocarcinoma of the lung EGFR mutation Variables Age, years <62 O62 Sex Male Female Smoking status Never-smokers Ever-smokers Histology Non-BAC BAC Pathologic stage Stage I Stage IIeIV Never-smokers Stage I Stage IIeIV Ever-smokers Stage I Stage IIeIV KRAS status Mutated Wild-type Overall subjects a b

n

Present

Absent

P-value 0.57a

22 33

9 11

13 22

31 24

7 13

24 11

0.02a

0.004a 22 33

13 7

9 26

50 5

18 2

32 3

1.00b

0.02a 33 22

8 12

25 10

13 9

7 6

6 3

20 13

1 6

19 7

4 51 55

0 20 20

4 31 35

0.67b

0.01b

0.29b

c2 test. Fisher’s exact test.

Similar to the present study, Yokoyama et al. [25] investigated EGFR, ERBB2, and KRAS mutations in 349 Japanese NSCLC patients. They found ERBB2 and KRAS mutations at frequencies (1.7 and 6.0%, respectively) comparable to those of the present study; however, they found EGFR mutations at a frequency of 29.2%, which is significantly higher than what we found. Notable from their study is that an additional 9 EGFR mutations were detected when they performed a mutation-specific PCR or singlestrand conformation polymorphism (SSCP) after direct sequencing for detection of the L858R mutation; this result suggests that a mutation-specific PCR analysis or SSCP is more sensitive than direct sequencing for the detection of missense mutations of EGFR [19,25]. In addition, Yokoyama et al. [25] showed that 7 among 349 tumors had double missense mutations of EGFR, indicating that two genetic events targeting EGFR occasionally occur in NSCLC [8,15,25]. In the present study, however, we identified no tumors with double mutations of EGFR. A few studies have investigated EGFR mutations in NSCLC patients undergoing curative surgical resection [8,15,19,23]. Sasaki et al. [23] found that EGFR mutations were more frequent in pathologic stage I than in stage IIeIV (72.4 versus 28.6%, P 5 0.03). In other studies, however, the frequency of EGFR mutations was not significantly associated with tumor stage, which suggests that EGFR mutations occur relatively early in the clinical

course and are associated with pathogenesis of adenocarcinoma rather than progression [8,15,19]. Consistent with such findings [8,15,19], in our study EGFR mutations were not associated with pathologic stage of adenocarcinoma in never-smokersdbut in the group of ever-smokers, EGFR mutations in adenocarcinomas were more frequent in pathologic stage IIeIV than in stage I. This latter finding suggests that EGFR mutations might occur as a late event in the carcinogenesis of adenocarcinoma in smokers, and suggests that the role of EGFR mutations in adenocarcinoma may be different in never-smokers and ever-smokers. It is possible, however, that the finding was attributable to chance, due to the small numbers of cases in the subgroups. Thus, additional studies with a greater number of subjects are needed to confirm this finding. In conclusion, we found that EGFR mutations occurred frequently in Korean patients with lung adenocarcinoma, but ERBB2 mutation was rare. EGFR mutations in adenocarcinomas were more frequent in women and in neversmokers. EGFR mutations correlated with pathologic stage of adenocarcinoma in smokers. Further studies are needed to investigate the role of EGFR mutations in the carcinogenesis of adenocarcinoma among smokers.

Acknowledgments This study was supported by Grant no. RTI04-01-01, The Regional Technology Innovation Program of The Ministry of Commerce, Industry and Energy (MOCIE), Republic of Korea, and in part by the Brain Korea 21 Project in 2006.

References [1] Yarden Y, Sliwkowski MX. Untangling the ErbB signaling network. Nat Rev Mol Cell Biol 2001;2:127e37. [2] Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 2003;3:11e22. [3] Weinstein-Oppenheimer CR, Blalock WL, Steelman LS, Chang F, McCubrey JA. The Raf signal transduction cascade as a target for chemotherapeutic intervention in growth factor-responsive tumors. Pharmacol Ther 2000;88:229e79. [4] Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 2004;9:667e76. [5] Calo V, Migliavacca M, Bazan V, Macaluso M, Buscemi M, Gebbia N, Russo A. STAT proteins: from normal control of cellular events to tumorigenesis. J Cell Physiol 2003;197:157e68. [6] Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE, Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497e500. [7] Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129e39.

N.C. Bae et al. / Cancer Genetics and Cytogenetics 173 (2007) 107e113 [8] Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, Fong KM, Lee H, Toyooka S, Shimizu N, Fujisawa T, Feng Z, Roth JA, Herz J, Minna JD, Gazdar AF. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97:339e46. [9] Shi D, He G, Cao S, Pan W, Zhang HZ, Yu D, Hung MC. Overexpression of the c-erbB-2/neu-encoded p185 protein in primary lung cancer. Mol Carcinog 1992;5:213e8. [10] Scheurle D, Jahanzeb M, Aronsohn RS, Watzek L, Narayanan R. HER-2/neu expression in archival non-small cell lung carcinomas using FDA-approved HercepTest. Anticancer Res 2000;20:2091e6. [11] Stephens P, Hunter C, Bignell G, Edkins S, Davies H, Teague J, Stevens C, O’Meara S, Smith R, Parker A, Barthorpe A, Blow M, Brackenbury L, Butler A, Clarke O, Cole J, Dicks E, Dike A, Drozd A, Edwards K, Forbes S, Foster R, Gray K, Greenman C, Halliday K, Hills K, Kosmidou V, Lugg R, Menzies A, Perry J, Petty R, Raine K, Ratford L, Shepherd R, Small A, Stephens Y, Tofts C, Varian J, West S, Widaa S, Yates A, Brasseur F, Cooper CS, Flanagan AM, Knowles M, Leung SY, Louis DN, Looijenga LH, Malkowicz B, Pierotti MA, Teh B, Chenevix-Trench G, Weber BL, Yuen ST, Harris G, Goldstraw P, Nicholson AG, Futreal PA, Wooster R, Stratton MR. Lung cancer: intragenic ERBB2 kinase mutations in tumours. Nature 2004;431:525e6. [12] Shigematsu H, Takahashi T, Nomura M, Majmudar K, Suzuki M, Lee H, Wistuba II, Fong KM, Toyooka S, Shimizu N, Fujisawa T, Minna JD, Gazdar AF. Somatic mutations of the HER2 kinase domain in lung adenocarcinomas. Cancer Res 2005;65:1642e6. [13] Sekido Y, Fong KM, Minna JD. Molecular genetics of lung cancer. Ann Rev Med 2003;54:73e87. [14] Serrano M. The tumor suppressor protein p16INK4a. Exp Cell Res 1997;237:7e13. [15] 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;64:8919e23. [16] Brambilla E, Travis WD, Colby TV, Corrin B, Shimosato Y. The new World Health Organization classification of lung tumours. Eur Respir J 2001;18:1059e68.

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[17] Barkley JE, Green MR. Bronchioloalveolar carcinoma. J Clin Oncol 1996;14:2377e86. [18] Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest 1997;111:1710e7. [19] Marchetti A, Martella C, Felicioni L, Barassi F, Salvatore S, Chella A, Camplese PP, Iarussi T, Mucilli F, Mezzetti A, Cuccurullo F, Sacco R, Buttitta F. EGFR mutations in non-small cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. J Clin Oncol 2005;23: 857e65. [20] Tokumo M, Toyooka S, Kiura K, Shigematsu H, Tomii K, Aoe M, Ichimura K, Tsuda T, Yano M, Tsukuda K, Tabata M, Ueoka H, Tanimoto M, Date H, Gazdar AF, Shimizu N. The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancer. Clin Cancer Res 2005;11:1167e73. [21] Shigematsu H, Gazdar AF. Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. Int J Cancer 2006;118:257e62. [22] Gazdar AF, Shigematsu H, Herz J, Minna JD. Mutations and addiction to EGFR: the Achilles ‘heal’ of lung cancers? Trend Mol Med 2004;10:481e6. [23] Sasaki H, Shimizu S, Endo K, Takada M, Kawahara M, Tanaka H, Matsumura A, Iuchi K, Haneda H, Suzuki E, Kobayashi Y, Yano M, Fujii Y. EGFR and ErbB2 mutation status in Japanese lung cancer patients. Int J Cancer 2006;118:180e4. [24] Lee JW, Soung YH, Kim SY, Park WS, Nam SW, Kim SH, Lee JY, Yoo NJ, Lee SH. Absence of the ERBB2 kinase domain mutation in lung adenocarcinomas in Korean patients. Int J Cancer 2005;116: 652e3. [25] Yokoyama T, Kondo M, Goto Y, Fukui T, Yoshioka H, Yokoi K, Osada H, Imaizumi K, Hasegawa Y, Shimokata K, Sekido Y. EGFR point mutation in non-small cell lung cancer is occasionally accompanied by a second mutation or amplification. Cancer Sci 2006;97: 753e9.