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Lung Adenocarcinoma Harboring Mutations in the ERBB2 Kinase Domain

2006; Elsevier BV; Volume: 8; Issue: 3 Linguagem: Inglês

10.2353/jmoldx.2006.050132

1943-7811

Makoto Sonobe, Toshiaki Manabe, Hiromi Wada, Fumihiro Tanaka,

Cancer-related Molecular Pathways

Mutations in the ERBB2kinase domain have been reported in non-small cell lung cancer (NSCLC). Here, we describe a detailed search for ERBB2 gene mutations in tumors derived from NSCLC patients. Tumor specimens from 223 patients who underwent resection for NSCLC were examined for the presence of mutations in exons 19 and 20 of the ERBB2gene. Correlations were then made between the expression of these mutations and the clinical characteristics of the patients from which they were derived as well as the tumor's pathological features. ERBB2mutations were observed in four of the above tumors (1.8%), all of which were adenocarcinomas. All ERBB2mutations were in-frame insertions that occurred in exon 20. The patients from whom these tumors were derived were nonsmokers. Three of the tumors were of the papillary subtype, and one was a mixed subtype that consisted of acinar, papillary, and solid components. None of the tumors had a bronchio-alveolar component nor did they have epidermal growth factor receptoror K-rascodon 12 mutations. In conclusion, patients with these tumors tended to be nonsmokers who had clinical features similar to those of lung cancer patients whose tumors expressed epidermal growth factor receptormutations, although their tumors showed slightly different pathological features. Mutations in the ERBB2kinase domain have been reported in non-small cell lung cancer (NSCLC). Here, we describe a detailed search for ERBB2 gene mutations in tumors derived from NSCLC patients. Tumor specimens from 223 patients who underwent resection for NSCLC were examined for the presence of mutations in exons 19 and 20 of the ERBB2gene. Correlations were then made between the expression of these mutations and the clinical characteristics of the patients from which they were derived as well as the tumor's pathological features. ERBB2mutations were observed in four of the above tumors (1.8%), all of which were adenocarcinomas. All ERBB2mutations were in-frame insertions that occurred in exon 20. The patients from whom these tumors were derived were nonsmokers. Three of the tumors were of the papillary subtype, and one was a mixed subtype that consisted of acinar, papillary, and solid components. None of the tumors had a bronchio-alveolar component nor did they have epidermal growth factor receptoror K-rascodon 12 mutations. In conclusion, patients with these tumors tended to be nonsmokers who had clinical features similar to those of lung cancer patients whose tumors expressed epidermal growth factor receptormutations, although their tumors showed slightly different pathological features. Members of c-erb B family of oncogenes, which include epidermal growth factor receptor(EGFR) and ERBB2, play a critical role in the development and progression of non-small cell lung cancer (NSCLC) by promoting cell growth and by preventing apoptosis by regulating downstream effectors such as mitogen-activated protein kinase, protein kinase B, and signal transducer and activator of transcription 3.1Klapper LN Kirschbaum MH Sela M Yarden Y Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors.Adv Cancer Res. 2000; 77: 25-79Crossref PubMed Scopus (518) Google Scholar,2Bunn Jr, PA Franklin W Epidermal growth factor receptor expression, signal pathway, and inhibitors in non-small cell lung cancer.Semin Oncol. 2002; 29: S38-S44Abstract Full Text Full Text PDF PubMed Google Scholar In 2004, Lynch and colleagues,3Lynch 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: 2129-2139Crossref PubMed Scopus (10139) Google Scholar Paez and colleagues,4Paez 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 ML Sellers WR Johnson BE Meyerson M EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8586) Google Scholar and Pao and colleagues5Pao W Miller V Zakowski M Doherty J Politi K Sarkaria I Singh B Heelan R Rusch V Fulton L Mardis E Kupfer D Wilson R Kris M Varmus H 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 USA. 2004; 101: 13306-13311Crossref PubMed Scopus (3918) Google Scholar reported that many NSCLC tumors, obtained from patients who responded to gefitinib or erlotinib treatment, harbored somatic mutations in the tyrosine kinase domain of their EGFRgene. Several descriptive studies showed that EGFRgene mutations in NSCLC tumors were common in the lung adenocarcinomas of patients who were nonsmokers or mild smokers, women, and/or of Asian descent.6Kosaka 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: 8919-8923Crossref PubMed Scopus (1141) Google Scholar7Huang SF Liu HP Li LH Ku YC Fu YN Tsai HY Chen YT Lin YF Chang WC Kuo HP Wu YC Chen YR Tsai SF High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan.Clin Cancer Res. 2004; 10: 8195-8203Crossref PubMed Scopus (516) Google Scholar8Marchetti A Martella C Felicioni L Barassi F Salvatire S Chella A Camolese 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: 857-865Crossref PubMed Scopus (780) Google Scholar9Han SW Kim TY Hwang PG Jeong S Kim J Choi IS Oh DY Kim HJ Kim DW Chung DH Im SA Kim YT Lee JS Heo DS Bang YJ Kim NK 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: 2493-2501Crossref PubMed Scopus (725) Google Scholar10Shigematsu 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: 339-346Crossref PubMed Scopus (2119) Google Scholar11Qin BM Chen X Zhu JD Pei DQ Identification of EGFR kinase domain mutations among lung cancer patients in China: implication for targeted cancer therapy.Cell Res. 2005; 15: 212-217Crossref PubMed Scopus (63) Google Scholar12Yang SH Mechanic LE Yang P Landi MT Bowman ED Wampfler J Merzaman D Hong KM Mann F Dracheva T Fukuoka J Travis W Caporaso NE Harris CC Jen J Mutations in the tyrosine kinase domain of the epidermal growth factor receptor in non-small cell lung cancer.Clin Cancer Res. 2005; 11: 2106-2110Crossref PubMed Scopus (135) Google Scholar13Sonobe M Manabe T Wada H Tanaka F Mutations in the epidermal growth factor receptor gene are linked to smoking-independent, lung adenocarcinoma.Br J Cancer. 2005; 93: 355-363Crossref PubMed Scopus (144) Google Scholar These data suggest that EGFRmutations may play a causal role in the development of lung adenocarcinoma that does not correlate with smoking. Stephens and colleagues14Stephens 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: 525-526Crossref PubMed Scopus (33) Google Scholar first reported the presence of mutations in the kinase domain of the ERBB2gene, which shares a strong homology with the EGFRgene, in lung adenocarcinomas; Shigematsu and colleagues15Shigematsu 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: 1642-1646Crossref PubMed Scopus (606) Google Scholar and Sasaki and colleagues16Sasaki 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: 180-184Crossref PubMed Scopus (151) Google Scholar reported similar mutations in their lung adenocarcinoma patients. Recently, Lee and colleagues17Lee JW Soung YH Kim SY Nam SW Park WS Wang YP Jo KH Moon SW Song SY Lee JY Yoo NJ Lee SH ERBB2 kinase domain mutation in the lung squamous cell carcinoma.Cancer Lett. 2005; (Jul 16; (Epub ahead of print))Google Scholar also reported a similar mutation in a lung squamous cell carcinoma. The regions within which the ERBB2gene mutations occurred, ie, exons 19 and 20, correspond to the kinase domain of EGFRgene. Thus, these mutations could potentially result in the activation of the tyrosine kinase activity of the ERBB2 protein and, as such, may play an important role in oncogenesis in a manner similar to EGFRmutations. In this study, we conducted a detailed search for ERBB2gene mutations in tumors derived from NSCLC patients who underwent tumor resection at a particular Japanese hospital. A total of 223 patients with NSCLC who underwent tumor resection at the Department of Thoracic Surgery, Kyoto University Hospital from November 2002 to May 2005 were included in this study (Table 1). Included in this population were 154 patients whose tumors we previously examined for the presence of EGFRgene mutations.13Sonobe M Manabe T Wada H Tanaka F Mutations in the epidermal growth factor receptor gene are linked to smoking-independent, lung adenocarcinoma.Br J Cancer. 2005; 93: 355-363Crossref PubMed Scopus (144) Google Scholar The patients' clinical data were obtained from in-patient and out-patient medical records and included chest X-ray films, whole body computed tomography films, bone scanning data, and operation records. Patients were classified as being either nonsmokers (who never smoked in a lifetime), former smokers (who stopped smoking at least 6 months before the time of diagnosis of lung cancer), or current smokers. Two hundred and five patients underwent complete lung, lobe, or segment removal as well as hilar and mediastinal lymph node dissection. Eighteen patients underwent partial lung resection and lymph node sampling. No patients were, at any time, exposed to gefitinib, erlotinib, or trastuzumab before their tumor was resected. Pathological staging was determined using the current tumor-node-metastasis classification system.18Mountain CF Revisions in the International System for Staging Lung Cancer.Chest. 1997; 111: 1710-1717Crossref PubMed Scopus (4549) Google Scholar Pathological diagnosis was determined, using the World Health Organization (WHO) classification system, by two pathologists of Kyoto University Hospital Laboratory of Anatomical Pathology who were unaware of the tumors' status, vis-à-vis the presence of mutations, and was confirmed by a third pathologist (T.M.).19Travis WD Colby TV Corrin B Shimosato Y Brambilla E World Health Organization: histological typing of lung and pleural tumors. International Histological Classification of Tumors. ed 3. Springer, New York1999Crossref Google Scholar Many of adenocarcinomas were classified into mixed subtype according to the WHO classification system. Thus, to clarify the impact of ERBB2 gene mutations on subtypes of adenocarcinoma, classification according to presence or absence of each component [bronchiolo-alveolar (BAC), papillary, acinar, and solid carcinoma with mucin (solid)] in a tumor was also used in this study. Written informed consent to perform genetic analyses was obtained from all patients before their surgery, and the study itself was approved by the Ethics Committee of Kyoto University Graduate School and Faculty of Medicine.Table 1Characteristic Features of the 223 Patients that Participate in This StudyCharacteristicsn(%)Age (years) Median67 Range31 to 88Sex (no.) Men137(61.4) Women86(38.6)Smoking status (no.) Nonsmoker79(35.4) Smoker144(64.6) Former35(15.7) Current109(48.9)Tumor histology (no.) Adenocarcinoma153(68.6) Squamous cell52(23.3) Large cell11(4.9) Other7(3.1)Operation mode (no.) Pneumonectomy (right)1(0.4) Bilobectomy1(0.4) Lobectomy166(74.4) Segmentectomy37(16.6) Partial resection18(8.1)Pathological stage (no.) IA87(39.0) IB57(25.6) IIA7(3.1) IIB18(8.1) IIIA35(15.7) IIIB15(6.7) IV4(1.8) Open table in a new tab The method used to collect tumor samples was as we previously described.13Sonobe M Manabe T Wada H Tanaka F Mutations in the epidermal growth factor receptor gene are linked to smoking-independent, lung adenocarcinoma.Br J Cancer. 2005; 93: 355-363Crossref PubMed Scopus (144) Google Scholar Since ERBB2mutations that were recently reported in lung cancer specimens were located in exons 19 and 20,14Stephens 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: 525-526Crossref PubMed Scopus (33) Google Scholar15Shigematsu 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: 1642-1646Crossref PubMed Scopus (606) Google Scholar16Sasaki 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: 180-184Crossref PubMed Scopus (151) Google Scholar17Lee JW Soung YH Kim SY Nam SW Park WS Wang YP Jo KH Moon SW Song SY Lee JY Yoo NJ Lee SH ERBB2 kinase domain mutation in the lung squamous cell carcinoma.Cancer Lett. 2005; (Jul 16; (Epub ahead of print))Google Scholar polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) was used to screen for mutations in exons 19 and 20 of the ERBB2gene. PCR amplification was performed using HotStarTaq master mix (Qiagen K.K., Tokyo, Japan). The primers used for exon 19 were forward 5′-CACTCATATCCTCCTCTTTCTG-3′ and reverse 5′-TGTCCTCCTAGCAGGAGAG-3′, amplifying the 160-bp fragment; and for exon 20 were forward 5′-CCATACCCTCTCAGCGTAC-3′ and reverse 5′-CGGAGAGACCTGCAAAGAG-3′, amplifying the 249-bp fragment. The PCR was initially run at 95°C for 15 minutes, followed by 35 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, and a final extension was run at 72°C for 10 minutes. SSCP analyses were performed using the GenePhor System and GeneGel Excel 12.5/24 (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions; the gel temperature was maintained at 15°C. To measure the sensitivity, we performed SSCP analysis of samples with a ratio of ERBB2-mutated copy [2322ins/dup(GCATACGTGATG)] to ERBB2-normal copy of 0.5, 0.2, 0.1, 0.05, 0.02, or 0.01, and we found that SSCP method was able to detect the mutation from the sample with a ratio of ERBB2-mutated copy to ERBB2-normal copy of 0.02 (data not shown). To determine the gel temperature for adequate optimization of altered band, we performed SSCP analyses under 12°C, 15°C, or 18°C of gel temperature, and we found that 15°C of gel temperature was most appropriate (data not shown). After the gels were stained with silver carbonate, altered bands were cut from the gels, and DNA fragments were eluted for direct sequencing. When an ERBB2mutation was observed, a DNA sample from the corresponding normal lung tissue was also analyzed for the presence of ERBB2mutations. We previously described the methods used for the detection of mutations in the EGFRgene, p53gene, and K-rascodon 12.13Sonobe M Manabe T Wada H Tanaka F Mutations in the epidermal growth factor receptor gene are linked to smoking-independent, lung adenocarcinoma.Br J Cancer. 2005; 93: 355-363Crossref PubMed Scopus (144) Google Scholar ERBB2gene mutations were found in four of our patients (4 of 223, 1.8%); these mutations only occurred within exon 20. PCR-SSCP analysis revealed two types of altered bands in exon 20 (Figure 1), and nucleotide sequencing confirmed the presence of the following corresponding in-frame insertion mutations: 2322 ins/dup(GCATACGTGATG) was observed in three tumors, and 2326ins(TGT) with 2326G>T was observed in one tumor (Table 2). ERBB2mutations were exclusively observed in adenocarcinomas; thus, the incidence of ERBB2mutations in our adenocarcinoma patients was 2.6% (4 of 153). Corresponding normal lung tissues had no ERBB2mutations.Table 2Patients Harboring ERBB2Gene Mutations in Their TumorAge (years) SexSmokingTumor size/TNM classificationDifferentiation grade/adenocarcinoma subtypeNucleotide alterationAmino acid alteration45Non15 mmWell2322 ins/dupins774(AYVM)Womant1n0m0, IAPapillary(GCATACGTGATG)65Non22 mmModerate2326G>TG776L, ins776(C)Mant1n1m0, IIAMixed (acinar/papillary/solid)2326ins(TGT)79Non8 mmModerate2322ins/dupins774(AYVM)Womant1n0m0, IAPapillary(GCATACGTGATG)82Non20 mmModerate2322 ins/dupins774(AYVM)Womant2n0m0, IBPapillary(GCATACGTGATG) Open table in a new tab Mutations in the kinase domain of the EGFRgene (from exon 18 to exon 21) were found in 80 patients (80 of 223, 35.9%); all of these patients had an adenocarcinoma (80 of 153, 52.3%). Mutations within codon 12 of the K-ras gene were observed in the following 18 patients (8.1%): 15 adenocarcinoma patients (15 of 153, 9.8%), two squamous cell carcinoma patients (2 of 52, 3.8%), and one large cell carcinoma patient (1 of 11, 9.1%). Mutation types were as follows: substitution of cysteine (TGT) (n = 7), valine (GTT) (n = 5), aspartic acid (GAT) (n = 4), serine (AGT) (n = 1), and phenylalanine (TTT) (n = 1) in the place of glycine (GGT). Seventy-one mutations within exons 5, 6, 7, and 8 of the p53gene were observed in the following 69 patients (69 of 223, 30.9%): 43 adenocarcinoma patients (43 of 153, 30.7%), 22 squamous cell carcinoma patients (22 of 52, 42.3%), and two large cell carcinoma patients (2 of 11, 18.2%), and in two patients with tumors of another histological type (two of seven, 28.6%). The frequency distribution of these mutations was 14 missense/nonsense point mutations and two insertions within exon 5; seven missense point mutations, three deletions, and three insertions within exon 6; nine missense point mutations, three deletions, and three insertions within exon 7; and 21 missense/nonsense point mutations and six deletions within exon 8. Because ERBB2mutations were observed exclusively in adenocarcinoma patients in our series, we investigated the relationship between the presence of ERBB2mutations and clinicopathological features/other genetic alterations in these adenocarcinoma patients (n = 153). Three of our four patients with ERBB2-mutated tumors were women, and all were nonsmokers (Table 3). Although statistical analyses could not be applied because of the low number of ERBB2mutations, clearly the incidence of ERBB2mutations was higher in female than male patients (3.8% versus 1.3%) and in nonsmokers than in smokers (5.3% versus 0%).Table 3Correlation Between the Presence of ERBB2Mutations and the Patients' Clinicopathological FeaturesVariableMutated geneAdenocarcinoma patients (n = 153)ERBB2 (n = 4)EGFR (n = 80)K-rascodon 12 (n = 15)Not detected (n = 54)Age (years) Median (range)67 (31 to 83)72 (45 to 82)65 (33 to 83)65.5 (50 to 79)63 (31 to 83)Gender (no.) Men751221042 Women78358512Smoking history (no.) Nonsmoker7546038 Former smoker25012310 Current smoker5308936Tumor differentiation (no.) Well42128310 Moderate65337520 Poor46015724Subtypes of adenocarcinoma (no.) Mixed1151611142 Bronchio-alveolar60312 Papillary2631517 Acinar30102 Solid30021Acinar component (no.) Present961461138 Absent57334416Bronchio-alveolar component (no.) Present59043313 Absent944371241Papillary component (no.) Present1284701044 Absent25010510Solid component (no.) Present48115824 Absent105365730 Open table in a new tab One of the above four tumors was staged as a grade 1 tumor, whereas the other three were staged as grade 2 (Table 2). Using the WHO classification system, three of these tumors were characterized as being of the papillary subtype, and one was characterized as a mixed subtype that consisted of acinar, papillary, and solid components. None of the tumors had a BAC component, which is in contrast to tumors with EGFRgene mutations, approximately half of which had a BAC component. Only one of our tumors had a solid component, and approximately half of those with a K-rasgene mutation had a solid component (Table 3). EGFRmutations and K-rascodon 12 mutations were not detected in any of our ERBB2-mutated tumors. One of four ERBB2-mutated tumors had p53gene mutation [substitution of arginine (AGG) in the place of lysine (AAG) at the codon 164]. This type of point mutation (A:T to G:C) is not characteristic of mutations caused by tobacco smoking.20Pfeifer 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: 7435-7451Crossref PubMed Scopus (857) Google Scholar The presence of a p53mutation did not correlate with the presence of an ERBB2gene mutation (Table 4).Table 4Correlation Between the Presence of ERBB2Mutations and Other Genetic Alterations in 153 Adenocarcinoma PatientsMutation statusNumber of adenocarcinoma patients (%)ERBB2EGFRK-rascodon 12p53(mutated/wild)4 (2.6%)MutatedWildWild1/380 (52.2%)WildMutatedWild17/6315 (9.8%)WildWildMutated4/1154 (35.3%)WildWildWild21/33 Open table in a new tab ERBB2gene mutations were detected in four of 223 patients (1.8%) who underwent resection for NSCLC; our results are comparable to those of previous reports (prevalence, 0.9 to 4.2%; Table 5).14Stephens 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: 525-526Crossref PubMed Scopus (33) Google Scholar15Shigematsu 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: 1642-1646Crossref PubMed Scopus (606) Google Scholar16Sasaki 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: 180-184Crossref PubMed Scopus (151) Google Scholar17Lee JW Soung YH Kim SY Nam SW Park WS Wang YP Jo KH Moon SW Song SY Lee JY Yoo NJ Lee SH ERBB2 kinase domain mutation in the lung squamous cell carcinoma.Cancer Lett. 2005; (Jul 16; (Epub ahead of print))Google Scholar A 12-bp in-frame insertion mutation within exon 20, which was the major type of mutation found by other investigators,14Stephens 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: 525-526Crossref PubMed Scopus (33) Google Scholar15Shigematsu 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: 1642-1646Crossref PubMed Scopus (606) Google Scholar16Sasaki 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: 180-184Crossref PubMed Scopus (151) Google Scholar17Lee JW Soung YH Kim SY Nam SW Park WS Wang YP Jo KH Moon SW Song SY Lee JY Yoo NJ Lee SH ERBB2 kinase domain mutation in the lung squamous cell carcinoma.Cancer Lett. 2005; (Jul 16; (Epub ahead of print))Google Scholar was the most frequent mutation in our patients. Other mutations that we found included a novel 2326G>T, 2326ins(TGT). In this type of mutation, the point in exon 20 at which the insertion mutation occurred corresponded to the point in exon 20 of the EGFRgene at which insertion mutations were found. This finding suggests that one or just a few compounds with DNA editing capabilities may be responsible for the development of mutations within the tyrosine kinase domain of the ERBB2and EGFRgenes. Clarification of the mechanism by which these mutations occur would undoubtedly lead to advances in our understanding of oncogenesis and its prevention.Table 5Summary of Previous Reports on ERBB2Mutations in Lung CancerReferenceFrequency inPatient ethnicityAge/genderSmoking (pack-year)Nucleotide alterationOverallAdenocarcinomaStageHistologyStephens et al14Stephens 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: 525-526Crossref PubMed Scopus (33) Google Scholar5/1205/51UKNDUnknown*Unknown: in the study reported by Stephens et al,14 four of five patients were smokers, but further information was not described.NDAd2322ins/dup(GCATACGTGATG)(4.2%)(9.8%)UKNDUnknown*Unknown: in the study reported by Stephens et al,14 four of five patients were smokers, but further information was not described.NDAd2322ins/dup(GCATACGTGATG)UKNDUnknown*Unknown: in the study reported by Stephens et al,14 four of five patients were smokers, but further information was not described.NDAd2322ins/dup(GCATACGTGATG)UKNDUnknown*Unknown: in the study reported by Stephens et al,14 four of five patients were smokers, but further information was not described.NDAd2335ins(CTGTGGGCT)UKNDUnknown*Unknown: in the study reported by Stephens et al,14 four of five patients were smokers, but further information was not described.NDAd2263-2264TT>CCShigematsu et al15Shigematsu 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: 1642-1646Crossref PubMed Scopus (606) Google Scholar11/67111/394Japan74 years/FNonIAd2324ins/dup(ATACGTGATGGC)(1.6%)(2.8%)Japan58 years/FNonIAd2324ins/dup(ATACGTGATGGC)Japan58 years/MNonIAd2324ins/dup(ATACGTGATGGC)Japan63 years/MSmoker (40)IAd2324ins/dup(ATACGTGATGGC)Japan58 years/FSmoker (0.5)IAd2326ins(TTT)Japan66 years/FNonIIIAd2326ins(TTT), 2326G>CJapan52 years/FSmoker (5)IIIAd2239ins(GGGCTCCCC)Japan68 years/FNonIAd2240ins(GGCTCCCCA)Taiwan76 years/FNonIAd2324ins/dup(ATACGTGATGGC)Taiwan72 years/MNonIIAd2326ins(TTT)Australia83 years/MNonIAd2325ins/dup(TACGTGATGGCT)Sasaki et al16Sasaki 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: 180-184Crossref PubMed Scopus (151) Google Scholar1/951/71JapanND/FNonNDAd, well-2324ins/dup(ATACGTGATGGC)(1.1%)(1.4%)differentiatedLee et al17Lee JW Soung YH Kim SY Nam SW Park WS Wang YP Jo KH Moon SW Song SY Lee JY Yoo NJ Lee SH ERBB2 kinase domain mutation in the lung squamous cell carcinoma.Cancer Lett. 2005; (Jul 16; (Epub ahead of print))Google Scholar1/114Not analyzedKorea65 years/MSmoker (60)IBSq2305G>C(0.9%)Ad, adenocarcinoma; Sq, squamous cell carcinoma; ND, not described.* Unknown: in the study reported by Stephens et al,14Stephens 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: 525-526Crossref PubMed Scopus (33) Google Scholar four of five patients were smokers, but further information was not described. Open table in a new tab Ad, adenocarcinoma; Sq, squamous cell carcinoma; ND, not described. As with EGFRmutations, ERBB2mutations were more prevalent in female and nonsmoking patients although, again, our numbers were small. These findings are similar to those of Shigematsu and colleagues15Shigematsu 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: 1642-1646Crossref PubMed Scopus (606) Google Scholar and Sasaki and colleagues16Sasaki 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: 180-184Crossref PubMed Scopus (151) Google Scholar but different from those of Stephens and colleagues14Stephens 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: 525-526Crossref PubMed Scopus (33) Google Scholar and Lee and colleagues.17Lee JW Soung YH Kim SY Nam SW Park WS Wang YP Jo KH Moon SW Song SY Lee JY Yoo NJ Lee SH ERBB2 kinase domain mutation in the lung squamous cell carcinoma.Cancer Lett. 2005; (Jul 16; (Epub ahead of print))Google Scholar Further investigation will be needed to clarify the relationship between these variables although, at least in Japanese patients, it appears that the absence of a smoking history and being of the female sex are factors that are linked to the expression of ERBB2mutations. All adenocarcinomas that harbored ERBB2mutations had a papillary component while none had a BAC component. This histological predilection is slightly different from that seen for EGFRmutations in adenocarcinomas, in which approximately half of the tumors displayed BAC features.13Sonobe M Manabe T Wada H Tanaka F Mutations in the epidermal growth factor receptor gene are linked to smoking-independent, lung adenocarcinoma.Br J Cancer. 2005; 93: 355-363Crossref PubMed Scopus (144) Google Scholar It remains to be seen whether this discrepancy is real or merely a reflection of the low number of mutated tumors that were examined in our study; detailed histological examinations of this type were not performed in previously published studies. K-rasand EGFRmutations were not observed in any of the ERBB2-mutated tumors in our patients, confirming previous reports.14Stephens 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: 525-526Crossref PubMed Scopus (33) Google Scholar,15Shigematsu 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: 1642-1646Crossref PubMed Scopus (606) Google Scholar,17Lee JW Soung YH Kim SY Nam SW Park WS Wang YP Jo KH Moon SW Song SY Lee JY Yoo NJ Lee SH ERBB2 kinase domain mutation in the lung squamous cell carcinoma.Cancer Lett. 2005; (Jul 16; (Epub ahead of print))Google Scholar ERBB2, EGFR, and K-ras are important molecules that are responsible for the regulation of the mitogen-activated protein kinase pathway.21Rom WN Hay JG Lee TC Jiang Y Tchou-Wong KM Molecular and genetic aspects of lung cancer.Am J Respir Crit Care Med. 2000; 161: 1355-1367Crossref PubMed Scopus (139) Google Scholar22Flanklin WA Veve R Hirsch FR Helfrich BA Bunn Jr, PA Epidermal growth factor receptor family in lung cancer and premalignancy.Semin Oncol. 2002; 29: S3-S14Abstract Full Text Full Text PDF Scopus (282) Google Scholar23Hirsch FR Franklin WA Veve R Varella-Garcia M Bunn Jr, PA HER/neu expression in malignant lung tumors.Semin Oncol. 2002; 29: S51-S58Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar ERBB2 and EGFR also play a role in the regulation of the phosphatidyl inositol triphosphate kinase-Akt pathway.22Flanklin WA Veve R Hirsch FR Helfrich BA Bunn Jr, PA Epidermal growth factor receptor family in lung cancer and premalignancy.Semin Oncol. 2002; 29: S3-S14Abstract Full Text Full Text PDF Scopus (282) Google Scholar,23Hirsch FR Franklin WA Veve R Varella-Garcia M Bunn Jr, PA HER/neu expression in malignant lung tumors.Semin Oncol. 2002; 29: S51-S58Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar It is likely that ERBB2, EGFR, and K-ras codon 12 mutations have similar effects vis-à-vis the oncogenesis of lung adenocarcinomas. Unlike K-rasmutations that appear to be involved in the development of adenocarcinomas in smokers, ERBB2and EGFRgene mutations may play a key role in the development of adenocarcinomas in nonsmokers. The above conclusions notwithstanding, our study had two limitations. Mutations outside of exons 19 and 20 were not examined, and the PCR-SSCP method could have overlooked mutations that did not appear as band pattern alterations. Thus, the incidence of ERBB2mutations that we detected may have been underestimated. However, our results on the incidence of ERBB2mutations seemed to support those of previous investigators, and condition settings in the SSCP analysis were stringently performed in this study. Therefore, we think our results have sufficient validity. In conclusion, mutations in the ERBB2gene were found in 2.6% of our Japanese adenocarcinoma patients and relatively infrequent. ERBB2, EGFR, and K-ras codon 12 mutations were mutually exclusive. On the basis of four adenocarcinomas with ERBB2gene mutations in this series, ERBB2gene mutations tended to be found in nonsmokers and in adenocarcinomas that had a different histological pattern from those with EGFRgene mutations. We thank Drs. Kazumasa Takenaka, Seiji Matsumoto, Shinya Ito, Masatsugu Nakagawa, Shinjiro Nagai, Shoutaro Iwakiri, and Nobuya Mino (Department of Thoracic Surgery, Kyoto University) for their help in collecting clinical samples and for their technical assistance; and Drs. Daisuke Harada, Hirokazu Kotani, and Yoshiaki Ueda (Laboratory of Anatomical Pathology, Kyoto University) for pathological diagnosis of tumors included in this study.