Epidermal growth factor receptor gene mutation in non-small cell lung cancer using highly sensitive and fast TaqMan PCR assay
Introduction
Lung cancer is the deadliest cancer in many developed countries. Gefitinib (Iressa®, Astra Zeneca, London, UK), an inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, has been approved in Japan and the United States for the treatment of non-small cell lung cancer (NSCLC). Recently, erlotinib (Tarceva®, Roche, Basel, Switzerland), another inhibitor of EGFR tyrosine kinase, has been approved in the United States and Switzerland for the treatment of NSCLC. Gefitinib caused significant tumor shrinkage in 27.5% of Japanese NSCLC patients but in only 10.4% of Caucasian population [1], [2], [3], [4]. Unfortunately, the addition of gefitinib to the traditional chemotherapy did not add any benefit to the patient survival [3], although overexpression of EGFR protein was seen in relatively high frequencies [5]. We and others have shown that the somatic mutation in tyrosine kinase (TK) domain of EGFR is associated with sensitivity of NSCLC to gefitinib [6], [7], [8]. Gefitinib targets the ATP-binding cleft with the TK domain and the reported mutations are either deletion or single amino acid substitutions in exon 18, 19, or 21 clustered around the ATP-binding pocket of the TK domain.
In vitro, EGFR mutations have been reported to confer enhanced tyrosine kinase activity in response to epidermal growth factor (EGF) and increased sensitivity to inhibition by gefitinib [6], [7], [9], [10]. Thus, it is highly likely that EGFR mutation is a critical determinant of the patient's response to gefitinib. To determine the EGFR gene status may bring important information whether gefitinib is a therapeutic option for the NSCLC patient. If we can avoid unnecessary prescription of gefitinib in patients who are in fact non-responders, we will avoid fatal side effects of the drug and significantly reduce the health care cost. Fluorescent dye-based genotyping technology using the 5′ nuclease assay (TaqMan PCR assay) was developed as a large-scale and highly sensitive method in SNP scoring [11], [12], [13], [14], [15], [16]. For SNP genotyping, one pair of TaqMan probes and one pair of PCR primers are used. Two TaqMan probes differ at the polymorphic site, with one probe complementary to the wild-type allele and the other to the variant allele. Recently, this method is being applied for genotyping of insertion/deletion polymorphism as a simple and cost-effective method [17].
We applied this genotyping technique with TaqMan probe to detect EGFR somatic mutations. Probes were designed according to the 13 different EGFR mutations including 11 that have already been reported. We show in this paper that this method is sensitive enough to detect the mutation in samples contaminated with 9-fold excess of wild-type samples. It is also fast and could be applied in large-scale screening.
Section snippets
Patients and genomic DNA
NSCLC tissues were obtained by surgical excision between 1997 and 1999 from 67 patients at Nagoya City University Hospital in Japan. NSCLC tissues were also obtained from 27 patients at National Hospital Organization, Kinki-Chuo Chest Medical Center who were subsequently treated with gefitinib. Of 27 gefitinib treated samples, six trans-bronchial biopsy samples were obtained. These 94 samples were sequenced and also analyzed using TaqMan PCR assay. We have also analyzed additional 182 recent
Genotyping by genomic DNA sequencing
We have already published the EGFR genomic DNA sequencing data of 67 NSCLC samples [7], [18]. Seventeen cases had a mutated allele and there were six different mutations. Other groups have also reported additional somatic mutations in the same region of the EGFR gene [6], [8]. We first sequenced some additional samples to find previously unknown mutations. Twenty-seven NSCLC tumor samples from National Hospital Organization, Kinki-Chuo Chest Medical Center were subjected to conventional genomic
Discussion
Gefitinib was developed as an inhibitor of EGFR tyrosine kinase that is often over-expressed in many cancers. It showed a promising effect on a few cancers in phase I trial [1]. Subsequently, however, in phase II randomized trials in which the drug was used in combination with other traditional chemotherapy, the effect was marginal in patients with NSCLC [7]. Last year, our group and others have reported identification of genetic mutations in the EGFR kinase domain [6], [7]. The mutation was
Acknowledgment
The authors would like to thank Dr. Xiaojun Zhao for his excellent technical assistance for DNA amplification assay, and Dr. Naoya Hosono for TaqMan PCR assay. This work was supported by a grant for cancer research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Astra Zeneca Research grant 2004.
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