Lung cancer is one of the leading causes of the cancer death worldwide. Gefitinib is an inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and has been introduced in the treatment of advanced lung cancers. The responsiveness to gefitinib has been linked to the presence of EGFR mutations. Clinical samples contain many normal cells in addition to cancer cells. A method capable of detecting EGFR mutations in a large background of wild-type EGFR genes could provide a superior clinical test. We developed a rapid and sensitive detection system for EGFR mutations named the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp that can detect EGFR mutations in the presence of 100-to 1,000-fold background of wild-type EGFR. We used this method to screen 30 non-small cell lung cancer cell lines established from Japanese patients. In addition to 11 cell lines that have mutations, we found 12 cell lines in which specific mutations are observed only in the subpopulation(s) of the cells. Genetic heterogeneity of EGFR suggests that the EGFR gene is unstable in established cancers and the heterogeneity may explain variable clinical responses of lung cancers to gefitinib. (Cancer Res 2005; 65(16): 7276-82)

This study was prospectively designed to evaluate a phase II study of gefitinib for non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. Clinical samples were tested for EGFR mutations by peptide nucleic acid-locked nucleic acid PCR clamp, and patients having EGFR mutations were given gefitinib 250 mg daily as the second treatment after chemotherapy. Poor PS patients omitted chemotherapy. Of 107 consecutive patients enrolled, samples from 100 patients were informative, and EGFR mutations were observed in 38 patients. Gefitinib was given to 27 patients with EGFR mutations, and the response rate was 78% (one complete response and 20 partial responses; 95% confidence interval: 58 -93%). Median time to progression and median survival time (MST) from gefitinib treatment were 9.4 and 15.4 months, respectively. Grade 3 hepatic toxicity and skin toxicity were observed in one patient each. There were significant differences between EGFR mutations and wild-type patients in response rates (78 vs 14%, P ¼ 0.0017), and MST (15.4 vs 11.1 months, P ¼ 0.0135). A Cox proportional hazards model indicated that negative EGFR mutation was a secondary prognostic factor (hazards ratio: 2.259, P ¼ 0.036). This research showed the need for screening for EGFR mutations in NSCLC patients.

Gefitinib is an inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR). Accumulating evidence suggests that gefitinib may provide a survival benefit to EGFR mutationpositive non-small lung cancer patients. We have established a clinical test that can detect EGFR mutations from cytological specimens or paraffin-embedded tissue specimens that are contaminated by normal cells. This test is based on the peptide nucleic acid, locked nucleic acid polymerase chain reaction clamp method that can detect G719S, G719C, L858R, L861Q and seven different exon 19 deletions in the presence of 100-1000-fold wild-type alleles. Consequently, using a small aliquot of samples isolated to establish a cancer diagnosis, the EGFR mutation status is determined soon after the diagnosis of cancer is made. We investigated the EGFR mutation status in 86 patients using a variety of cytological specimens (59 bronchoscopy specimens, 16 pleural effusion, 9 sputum, and 2 pericardial effusion) and in 46 patients who had a disease relapse and paraffin-embedded tissues were available. Forty-five patients (34%) were positive for mutation (29 exon 19 deletions, 16 L858R and 1 L861Q). The sensitivity and the specificity of this test was 97% and 100%, respectively. EGFR mutation status thereby obtained was used to determine each patient's therapeutic regimen. This test is easily integrated into the normal clinical practice for lung cancer, while allowing the medical staff to select therapeutic regimen depending on the EGFR mutation status. (Cancer Sci 2007; 98: [246][247][248][249][250][251][252]

Mutations in the epidermal growth factor receptor (EGFR) are observed in a fraction of non-small-cell lung cancers (NSCLS). EGFR mutation-positive NSCLS responds to gefitinib. Secondary T790M mutation confers gefitinib resistance to NSCLS. A detection test for the T790M mutation was designed based on the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method. The specificity and sensitivity of the test were both greater than 0.99. The test revealed that only a small population of the PC-13 cells carried the T790M mutation. The test also revealed that the T790M mutation was found in none of 151 NSCLC specimens obtained before gefitinib treatment, whereas it was found in four of four specimens obtained from NSCLS that had become refractory to gefitinib. In one patient in whom the L858R-positive EGFR allele was amplified to multiple copies, an L858R-T790M double-mutant allele emerged during the gefitinib therapy. This allele was expressed highly. The T790M mutation detection test based on the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method is sensitive and specific, and is applicable to clinical practice. It detects T790M-positive cells in the course of gefitinib treatment, and thus will help to devise therapies effective for T790M-positive NSCLS. (Cancer Sci 2008; 99: 595-600)

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