Absence of EGFR mutation in the kinase domain in common human cancers besides non‐small cell lung cancer (2004)

The epidermal growth factor receptor (EGFR), a receptor protein tyrosine kinase, contributes to a number of processes in tumor survival and growth activity, thus making it a promising target for the cancer therapies.1 One of the agents that target the EGFR is the orally active EGFR tyrosine kinase inhibitor gefitinib (Iressa).2 The potential clinical benefits of gefitinib have been demonstrated in non-small cell lung cancer (NSCLC) patients.3 However, since the response rates with gefitinib were varied, identification of molecular and genetic markers for sensitivity and resistance to gefitinib has been required to select patient populations who are most likely benefit. Recently, 2 research groups discovered that somatic mutations of EGFR gene in the NSCLC could predict significant clinical responses to gefitinib.4, 5 They found the EGFR somatic mutations in the adenocarcinomas, bronchioloalveolar carcinomas and large cell carcinomas of lung that all correlated with a clinical response to gefitinib. The mutations were detected in the exon 18, 19 and 20 that encode the intracellular kinase domain. The mutations detected in the exon 18 would substitute the amino acid G719 in the P-loop, while those detected in the exon 21 would substitute amino acids in the activation domain. The mutations in the exon 19 were in-frame deletions that may alter the structure of αC helix. All of the EGFR mutations affect amino acids near the ATP-binding pocket that is targeted by gefitinib. Although most of the clinical benefits of gefitinib have been reported in NSCLC patients, there is evidence that gefitinib could be a potential agent for the treatment of other tumors. Gefitinib shows in vitro growth inhibition of several tumor cells, including head/neck, oral, urinary bladder, prostate, breast, ovarian and colon tumor cells.6 Also, gefitinib has shown antitumoral activity against a range of human tumor xenografts.7 To see if any other types of human cancers besides NSCLC carry the EGFR mutations, we analyzed 537 tissue samples from common human cancers, including 98 colon adenocarcinomas, 185 gastric adenocarcinomas, 93 breast ductal carcinomas, 73 hepatocellular carcinomas and 88 acute adulthood leukemias by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. Malignant cells and normal cells 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 Genomic DNA each from tumor cells and corresponding normal cells were amplified with 3 primer pairs. Radioisotope was incorporated into the PCR products for detection by autoradiogram. DNA sequence analysis of aberrantly migrating bands on the SSCP led to the identification of 1 EGFR mutation in a breast invasive ductal carcinoma but no EGFR mutation was detected in the other 536 samples (Fig. 1). However, the EGFR mutation detected was a silent mutation, which showed a G-to-A transition at the nucleotide 2121 (2121G>A; L707L) in exon 18. We repeated the experiments 3 times, including tissue microdissection, PCR and SSCP analysis to ensure the specificity of the results, and found that the data were consistent (data not shown). As potential positive controls, we analyzed the genomic DNA from 10 lung adenocarcinoma tissues to detect the EGFR mutations by the same method. Four mutations (2 mutations in the exon 19 and another 2 in the exon 21) that had been reported in the earlier studies were identified by the PCR-SSCP and subsequent DNA sequencing, demonstrating that the PCR-SSCP methods could detect the EGFR mutations (data not shown). SSCP analysis of EGFR gene in human cancers. (a) The PCR products of the exon 19 from the representative 12 cases of gastric carcinomas and a known positive control mutation (2235_2249delGGAATTAAGAGAAGC, arrows) were visualized on SSCP. (b) The PCR products of the exon 21 from the representative 10 cases of colon carcinomas and a known positive control mutation (2573T>G, arrows) were visualized on SSCP. (c) The PCR products of the exon 18 from the representative 6 cases of breast carcinomas were visualized on SSCP. The DNA from 1 cancer exhibits 2 aberrant bands (arrows) in addition to the wild-type bands. Because gefitinib had an anti-cancer activity in wide types of cancer cell lines and the EGFR kinase domain mutation is known to mediate the gefitinib sensitivity,6, 7 we expected that human cancer tissues would widely carry EGFR mutations. However, our data showed that EGFR kinase domain is very rarely mutated in common human cancers besides NSCLC. Therefore, the anti-cancer activity of gefitinib in a wide range of tumors identified in the preclinical studies may not be mediated by the EGFR mutations. Phase II/III studies are currently investing gefitinib as monotherapy or combination therapy against breast, head/neck, gastric, prostate and colon cancers. If clinical benefits by gefitinib treatment are not identified in these clinical studies, the in vitro anti-cancer activity of gefitinib could be interpreted as a nonspecific effect. It is unknown whether there exist any other predictors of gefitinib sensitivity. However, currently, the EGFR kinase domain mutation is the only known mechanism for mediating the gefitinib sensitivity.4, 5 Therefore, the present study suggests that before the identification of clinical benefit of gefitinib in cancer patients besides NSCLC patients, clinical application of gefitinib should be restricted to the NSCLC patients with the EGFR mutations. 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.