Absence of the ERBB2 kinase domain mutation in lung adenocarcinomas in Korean patients

Abstract

Protein tyrosine kinases regulate cell-signaling pathways that mediate a number of processes in tumor survival and growth activity. Epidermal growth factor receptor (EGFR) is a prototypical receptor tyrosine kinase, and the EGFR gene is the mammalian equivalent of the avian viral oncogene v-erb.1 Two research groups have discovered that lung adenocarcinomas (including bronchioloalveolar carcinomas) harbor EGFR gene mutations.2, 3 In addition, the EGFR gene mutations in lung cancers could predict significant clinical responses to gefitinib (Iressa),2, 3 an orally active EGFR tyrosine kinase inhibitor that has given significant clinical benefit to a subset of lung cancer patients.4 EGFR mutations have been detected in the exons that encode the intracellular kinase domain.2, 3 The EGFR family consists of 4 receptor tyrosine kinases, EGFR (ERBB1, HER1), ERBB2 (HER2), ERBB3 (HER3) and ERBB4 (HER4).1 Like EGFR, the other 3 members possess an extracellular ligand-binding domain, a transmembrane domain, a tyrosine kinase domain and a regulatory domain. They share great sequence homologies in the tyrosine kinase domains.1 Binding of the EGFR family members with their ligands enables the formation of homodimers or heterodimers with other family members, which subsequently results in kinase activation.1 The high sequence homologies and the presence of heterodimerization among the EGFR family members indicate that the EGFR signaling pathway is not linear and that other EGFR members besides EGFR (ERBB1) may possess activating mutations in human cancers. ERBB2 is amplified in approximately 25% of invasive breast cancers, and the humanized antibody (trastuzumab, Herceptin) against overexpressed ERBB2 has proven to be effective at treating breast cancers with ERBB2 amplification.5, 6 Stephens et al.7 reported that 5 of 51 lung adenocarcinomas (10%) harbored ERBB2 somatic mutations in the DNA sequences encoding the kinase domain. Also, they identified 3 additional ERBB2 mutations in gastric, ovarian and brain tumors at lower frequencies.7 The majority of the mutations in the lung adenocarcinomas (4/5) were in-frame insertions in exon 20, while the remaining one was detected in exon 19. Interestingly, the positions of the ERBB2 mutations were similar to those of EGFR mutations detected in lung cancers, suggesting that the ERBB2 mutations activate ERBB2. Incidence of genetic alterations in some genes varies depending on ethnicity. For example, EGFR mutations in lung cancers were more frequent in patients from Japan than in those from the United States.2 Our aim was to confirm the presence of ERBB2 mutations in lung adenocarcinomas and to determine whether there is any significant difference in the frequency of ERBB2 mutations between lung adenocarcinomas in Caucasians (the UK sample reported in previously7) and Asians (Koreans in the present study). We analyzed 103 lung adenocarcinoma tissue samples, including 53 adenocarcinomas without bronchioloalveolar features, 46 adenocarcinomas with bronchioloalveolar carcinoma features and 4 pure bronchioloalveolar carcinomas. The male to female ratio of the patients was 54:49. Ages of the patients ranged 37–78 years, with an average of 59.0 years. Patients consisted of 33 current smokers, 10 former smokers and 60 nonsmokers. Malignant cells and normal cells from the same patients were selectively procured from hematoxylin and eosin–stained slides using a 30G1/2 hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a micromanipulator, as described previously.8 DNA extraction was performed by a modified single-step method, as described previously.8 Because all of the ERBB2 mutations in lung cancers were detected within the DNA sequences encoding the kinase domain in the previous study,7 genomic DNA samples from tumor cells and corresponding normal cells were amplified with 8 primer pairs covering the DNA sequences in exons 18–24, encoding the kinase domain. For the detection of ERBB2 mutations, we analyzed these 7 exons by PCR-SSCP. Radioisotope ([32P]dCTP) was incorporated into PCR products for detection by SSCP autoradiogram. However, SSCP from the tumors did not reveal any aberrantly migrating band compared to the wild-type bands from the normal tissues (Fig. 1a,b). To confirm the SSCP results, we also analyzed the PCR products of exon 20, where most of the ERBB2 mutations were detected in the previous study,7 by direct DNA sequencing; but we could not detect any evidence of DNA sequence alteration in normal tissues or in tumor samples (Fig. 1c). We repeated the experiments twice, including tissue microdissection, PCR, SSCP and direct DNA sequencing analysis to ensure the specificity of the results and found that the data were consistent (data not shown). By comparison, we found EGFR mutations in 42% (44/103) of adenocarcinomas (unpublished data) in agreement with a previous study.2 SSCP analysis of the ERBB2 gene in human cancers. (a) PCR products of exon 19 from the 10 representative cases of lung adenocarcinoma were visualized on SSCP. (b) PCR products of exon 20 from the 10 representative cases of lung adenocarcinoma were visualized on SSCP. (c) PCR products of exon 20 from a lung adenocarcinoma (T) and the normal tissue of the same patient (N) were directly sequenced. Arrows indicate the DNA sequence region where the most common ERBB2 mutations [2322ins/dup (GCATACGTGATG)] were detected in the previous study.7 Because the previous study showed a modest frequency (10%) of ERBB2 mutation in lung adenocarcinomas,7 we expected some ERBB2 mutations in the 103 samples. However, we detected no ERBB2 kinase domain mutations in the samples. There is a difference in the ERBB2 mutation frequency between the previous study, which detected 5 mutations in 51 lung adenocarcinomas, and our study (0 mutations in 103 lung adenocarcinomas, Fisher's exact test p = 0.003). One explanation for this discrepancy may be a racial difference in the occurrence of ERBB2 mutation, as identified in the case of EGFR mutations.2 Although the clinical trial data on the ERBB2 kinase inhibitor trastuzumab did not reveal any benefit for lung cancer patients,9 the discovery of ERBB2 mutation in lung cancer again raises hope of treating lung cancer using ERBB2 kinase inhibitors. However, our data could not confirm that ERBB2 was frequently mutated in lung adenocarcinomas.7 Therefore, the present study suggests that the incidence of ERBB2 mutation should further be analyzed in a wide range of countries and that the clinical application of ERBB2 kinase inhibitor should be restricted to lung cancer patients after confirmation of ERBB2 mutation. Yours sincerely, Jong Woo Lee, Young Hwa Soung, Su Young Kim, Won Sang Park, Suk Woo Nam, Jung Young Lee, Nam Jin Yoo, Sug Hyung Lee.