Somatic mutations of EGFR, ERBB2, ERBB3 and ERBB4 in juxtamembrane activating domains are rare in non‐small cell lung cancers

Receptor tyrosine kinases (RTK) regulate diverse cellular functions, including cell survival and growth. Epidermal growth factor receptor (EGFR) is a prototypical RTK, and the EGFR family consists of EGFR (ERBB1 ⁄ HER1), ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4) (1). The ERBB genes are mammalian equivalents of viral oncogenes, and are deregulated in many cancers (1). Lung adenocarcinomas harbor EGFR mutations, which predict clinical responses to the EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) (2). ERBB2 amplification is present in about 25% of breast cancers, and trastuzumab (Herceptin) against the ERBB2 is effective in treating breast cancers with ERBB2 amplification (3). Kinase domain mutations of ERBB2 and ERBB4 mutations have also been detected in several types of human cancers (4, 5). Intracellular kinase domain is crucial in activation of ERBB proteins (1). However, in addition to the kinase domain, alterations in other domains such as deletions of extracellular domain of EGFR are known to activate ERBB signaling (1). Recently, Red Brewer et al. (6) discovered that a region in the juxtamembrane domain (JMD) of EGFR functioned as an activating domain of EGFR. The JMD of EGFR is located between the transmembrane domain and the kinase domain. The C-terminal region of the EGFR JMD (amino acid residues 688– 706) contributes to EGFR activation, whereas the N-terminal region of the JMD (amino acid residues 669–687) does not (6). The active JMD region contributes to EGFR dimerization and stabilizes the dimers (6). They also found that point mutations in the JMD (V689A and L703A) in non-small cell lung cancers (NSCLC) were activating mutations. The V689A-mutated EGFR promotes cellular transformation and tumorigenesis in mice (6). These data indicate that mutation of the EGFR JMD may play an important role in NSCLC development. Like EGFR, ERBB2, ERBB3 and ERBB4 possess a JMD and most of the amino acids in each JMD region are conserved (6). It could be hypothesized that the active regions in JMD of ERBB2, ERBB3 and ERBB4 could also be activated by somatic mutations in NSCLC. For this, we have analyzed 200 surgically resected NSCLC tissues, including 120 adenocarcinomas and 80 squamous cell carcinomas from Korean patients. Male to female ratios of the adenocarcinoma patients and the squamous cell carcinoma patients were 58:62 and 62:18, respectively. Malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a hypodermic needle affixed to a micromanipulator. DNA extraction was performed by a modified single-step DNA extraction method. Genomic DNA each from tumor cells and corresponding normal cells was amplified by polymerase chain reaction (PCR) with four primer pairs covering the C-terminal active JMD region of EGFR, ERBB2, ERBB3 and ERBB4 in their exon 18 (Table 1). Radioisotope was incorporated into the PCR products for detection by autoradiogram. After singlestrand conformation polymorphism (SSCP), migration of the PCR products on the SSCP was analyzed by visual inspection. Direct DNA sequencing reactions were performed in the cancers with the mobility shifts in the SSCP. On the SSCP autoradiograms, all of the PCR products from the cancers were clearly seen. However, the SSCPs from them did not reveal any aberrantly migrating band compared with wild-type bands from the normal tissues, indicating that there was no evidence of the somatic mutation of EGFR or ERBB2 or ERBB3 or ERBB4 in the NSCLCs. To confirm the SSCP results, we repeated the experiments twice, including tissue microdissection, PCR and SSCP to ensure the specificity of the results, and found that the data were consistent. Most of the EGFR mutations detected in NSCLC were located in either exon 19 or exon 21 in the kinase domain. However, several