Yoshiaki Nagai

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.

RATIONALE: Pulmonary alveolar microlithiasis is an autosomal recessive disorder in which microliths are formed in the alveolar space. OBJECTIVES: To identify the responsible gene that causes pulmonary alveolar microlithiasis. METHODS: By means of a genomewide single-nucleotide polymorphism analysis using DNA from three patients, we have narrowed the region in which the candidate gene is located. From this region, we have identified a gene that has mutations in all patients with pulmonary alveolar microlithiasis. MEASUREMENTS AND MAIN RESULTS: We identified a candidate gene, SLC34A2, that encodes a type IIb sodium phosphate cotransporter and that is mutated in six of six patients investigated. SLC34A2 is specifically expressed in type II alveolar cells, and the mutations abolished the normal gene function. CONCLUSION: Mutations in the SLC34A2 gene that abolish normal gene function cause pulmonary alveolar microlithiasis.

Mutation in the epidermal growth factor receptor (EGFR) is frequently seen in non-small cell lung cancers (NSCLCs), especially in Asian females with adenocarcinoma. The frequency of mutation and the factors associated requires to be elucidated by analyzing a large number of consecutive clinical samples. We summarized the result of the EGFR mutation analysis for 1,176 patients performed at the time of diagnosis or relapse. The PNA-LNA PCR clamp, a highly sensitive detection method for the EGFR mutation, was employed. For fresh cases a portion of samples isolated to establish the diagnosis of lung cancer was used. For cases with a relapsed disease archival tissue were tested. The variables associated with the EGFR mutation after removing the confound factors were investigated by the logistic analysis using the samples collected in our university (n = 308) where detailed information on patients were available. The frequency of the EGFR mutation and its subtypes were investigated using all samples (n = 1,176). The EGFR mutation was significantly associated with adenocarcinoma (p = 0.006) and light-smoking (p < 0.0001), but not gender. The deletions in exon 19 were more frequently associated with male gender while exon 21 deletions were with female gender (p = 0.0011). The overall frequency of the EGFR mutation was 31%. Our result suggests that the female predominance in the EGFR mutation rate is a reflection of a higher frequency of adenocarcinoma in females. The gender difference in the mutation subtypes may provide a clue for the mechanism of the occurrence of the EGFR mutation.

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 mutation-positive 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.

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.