Hideki Katayama

Detection of EGFR Gene Mutation in Lung Cancer by Mutant-Enriched Polymerase Chain Reaction Assay

Purpose: Mutations in the epidermal growth factor receptor (EGFR) gene have been reported to be present in non–small cell lung cancer (NSCLC) and related to the responsiveness of tumors to EGFR tyrosine kinase inhibitors, suggesting its usefulness as a biomarker. Because clinical samples contain tumor and normal cells or genes, a highly sensitive assay for detecting mutation is critical for clinical applications. Experimental Design: The mutant-enriched PCR is a rapid and sensitive assay with selective restriction enzyme digestion. We developed the mutant-enriched PCR assay targeting exons 19 and 21 of EGFR and applied the developed assay to detect mutations in 108 cases of surgically resected specimens of NSCLCs, 18 samples of computed tomography (CT)–guided needle lung biopsies, and 20 samples of pleural fluid. In addition, results were then compared with those from direct sequencing and a nonenriched PCR assay. Results: The mutant-enriched PCR that was proved to enrich one mutant of 2 × 103 normal genes detected mutations in 37 cases of 108 resected tumors, seven samples of CT-guided lung biopsies, and seven samples of pleural fluid. Among mutant cases, four resected tumors, two CT-guided lung biopsies, and two pleural fluid were identified as additional mutant cases by the mutant-enriched PCR, which were considered normal based on nonenriched assays. Conclusions: Our results indicate that EGFR mutations are readily detectable by mutant-enriched PCR in various clinical samples. Thus, mutant-enriched PCR may provide a valuable method of potentially detecting a small fraction of mutant genes in heterogeneous specimens, indicating its possible use in clinical application for NSCLC.

Usefulness of EGFR mutation screening in pleural fluid to predict the clinical outcome of gefitinib treated patients with lung cancer

The importance of epidermal growth factor receptor (EGFR) gene mutation has been recognized in nonsmall cell lung cancer (NSCLC), requiring the standardization of mutation screening system including the kind of samples. Here, we examined the EGFR mutation status in 61 pleural fluid samples from NSCLC cases using direct sequencing, nonenriched PCR, mutant‐enriched PCR and peptide nucleic acid‐locked nucleic acid (PNA‐LNA) PCR clamp assay. The mutant‐enriched PCR assay detected 16 mutant cases. Among them, the nonenriched PCR assay failed to detect 3 mutant cases. Regarding the discrepancy between mutant‐enriched PCR and PNA‐LNA PCR clamp assays, 3 cases of exon19‐deletions were detected only by mutant‐enriched PCR assay and no difference at the L858R mutation. There was no difference in results between direct sequencing and nonenriched PCR assay. We also correlated the EGFR mutation with clinical outcome of gefitinib‐treated 29 cases. EGFR mutations were present in 10 cases, revealing 7 partial response and 3 no change (NC). In EGFR wild‐type cases, 10 revealed NC and 9 progressive disease. The responders were significantly more frequent among the EGFR mutant cases than among the wild‐type (p < 0.0001). Overall survival (p = 0.0092) and progression‐free survival (p = 0.018) were significantly longer among the EGFR mutant cases than among the wild‐type. In summary, we evaluated the utility of EGFR mutation screening in pleural fluid using 4 assays that showed some discrepancies arising from the designs of the assays. As clinical importance, the EGFR mutation status in pleural fluid can be a biomarker for the favorable outcome of gefitinib‐treated NSCLC cases.

Aberrant promoter methylation of insulin‐like growth factor binding protein‐3 gene in human cancers

Insulin‐like growth factor binding protein‐3 (IGFBP‐3) is postulated to be a mediator of growth suppression signals. Here, we examined the methylation status of IGFBP‐3 to correlate to clinicopathological factors in human cancers. The methylation status of IGFBP‐3 was determined by bisulfite DNA sequencing and was correlated with expression semi‐quantified by real‐time RT‐PCR to develop a methylation‐specific PCR (MSP) assay for IGFBP‐3. Using the MSP assay, we examined the methylation status of IGFBP‐3 in gastric cancer (GC), colorectal cancer (CRC), breast cancer (BC) and malignant mesothelioma (MM). IGFBP‐3 methylation was detected in 6 of 13 (46%) and 16 of 24 (67%) GC cell lines and tumors, respectively; 4 of 8 (50%) and 15 of 26 (58%) CRC cell lines and tumors, respectively; 3 of 11 (27%) and 7 of 39 (18%) BC cell lines and tumors, respectively and 1 of 5 (20%) and 18 of 56 (32%) MM cell lines and tumors, respectively. Interestingly, the methylation status of MM specimens from Japanese patients (75%, 12 out of 16 patients) was significantly higher than those from the USA (15%, 6 out of 40 patients) (p < 0.0001), suggesting the presence of ethnic differences in the IGFBP‐3 methylation status. We also found that IGFBP‐3 methylation was preferentially present in GCs arising in the lower‐third of the stomach (p = 0.079). In summary, our results showed that IGFBP‐3 methylation played an important role in the silencing of its expression, suggesting that IGFBP‐3 may act as a tumor suppressor gene in several human cancers examined.

Association between cytokine removal by polymyxin B hemoperfusion and improved pulmonary oxygenation in patients with acute exacerbation of idiopathic pulmonary fibrosis

Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is characterized by severe worsening dyspnea of unknown etiology and high mortality without effective treatment. Recently, direct hemoperfusion with polymyxin B (PMX)-immobilized fiber cartridge (PMX-DHP) has been reported to improve pulmonary oxygenation and survival in patients with AE-IPF although its mechanism of action remains unknown. To gain insights into the pathobiology of AE-IPF through the beneficial effects of PMX-DHP, we analyzed the profile of cytokines adsorbed onto PMX-fibers used in 9 AE-IPF patients. In addition, the sera of these AE-IPF patients collected immediately before and after PMX-DHP, 9 stable IPF patients and 8 healthy individuals were also analyzed. The serum levels of cytokines including IL-9, IL-12, IL-17, PDGF and VEGF were significantly decreased immediately after PMX-DHP (P<0.02), and VEGF and IL-12 were most prominently reduced. In addition to PDGF and VEGF, IL-1β, IL-1ra, IL-8, IL-23, FGF basic, GM-CSF, IP-10, RANTES and TGF-β were eluted from used PMX-fibers. Interestingly, improved pulmonary oxygenation after PMX-DHP was correlated well with the quantities of eluted VEGF. These results suggest that adsorption of proinflammatory, profibrotic and proangiogenic cytokines onto PMX-fibers is one of the mechanisms of action of PMX-DHP in AE-IPF. Notably, removal of VEGF by PMX-DHP may contribute to the rapid improvement in oxygenation by suppressing vascular permeability in the lung.

Aberrant promoter methylation in pleural fluid DNA for diagnosis of malignant pleural effusion.

Accumulating evidence implicates epigenetic changes such as hypermethylation in carcinogenesis. We investigated whether DNA methylation of 5 tumor suppressor genes in pleural fluid samples could aid in diagnosis of malignant effusion. In samples from 47 patients with malignant pleural effusions and 34 with nonmalignant effusions, we used a methylation‐specific polymerase chain reaction to detect aberrant hypermethylation of the promoters of the DNA repair gene O6‐methylguanine‐DNA methyltransferase (MGMT), p16INK4a, ras association domain family 1A (RASSF1A), apoptosis‐related genes, death‐associated protein kinase (DAPK), and retinoic acid receptor β (RARβ). Promoter hypermethylation was associated with malignant effusion for MGMT (Odds ratio (OR) = ∞), p16INK4a (OR = ∞), RASSF1A (OR = 13.8; CI, 1.71–112), and RARβ (OR = 3.17; CI, 1.10–9.11), but not for DAPK. Instead, DAPK methylation was associated with the length of smoking (p < 0.05). Patients with hypermethylation of MGMT, p16INK4a, RASSF1A or RARβ were 5.68 times more likely to have malignant effusions than patients without methylation (p = 0.008). Methylations per patient were more numerous for lung cancer than nonmalignant pulmonary disease (0.915 vs. 0.206, p < 0.001). Sensitivity, specificity, and positive predictive value of methylation in one or more genes for diagnosis of malignant effusion were 59.6%, 79.4%, and 80.0% respectively. In conclusion, aberrant promoter methylation of tumor suppressor genes in pleural fluid DNA could be a valuable diagnostic marker for malignant pleural effusion.

Aberrant DNA methylation profile in pleural fluid for differential diagnosis of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) usually develops pleural fluid. We investigated the value of DNA methylation in the pleural fluid for differentiating MPM from lung cancer (LC). Pleural fluid was collected from 39 patients with MPM, 46 with LC, 25 with benign asbestos pleurisy (BAP) and 30 with other causes. The methylation of O6‐methylguanine‐DNA methyltransferase (MGMT), p16INK4a, ras association domain family 1A (RASSF1A), death‐associated protein kinase (DAPK), and retinoic acid receptor β (RARβ) was examined using quantitative real‐time PCR. DNA methylation of RASSF1A, p16INK4a, RARβ, MGMT and DAPK was detected in 12 (30.8%), 3 (7.7%), 11 (28.2%), 0 (0.0%) and five patients (12.8%) with MPM, and in 22 (47.8%), 14 (30.4%), 24 (52.2%), 1 (2.2%) and six patients (13.0%) with LC, respectively. The mean methylation ratios of RASSF1A, p16INK4a and RARβ were 0.37 (range 0.0–2.84), 0.11 (0.0–2.67) and 0.44 (0.0–3.32) in MPM, and 0.87 (0.0–3.14), 1.16 (0.0–5.35) and 1.69 (0.0–6.49) in LC, respectively. The methylation ratios for the three genes were significantly higher in LC than in MPM (RASSF1A, P = 0.039; p16INK4a, P = 0.005; and RARβ, P = 0.002). Patients with methylation in at least one gene were 3.51 (95% confidence interval, 1.09–11.34) times more likely to have LC. Hypermethylation seemed no greater with MPM than with BAP. Extended exposure to asbestos (≧30 years) was correlated with an increased methylation frequency (P = 0.020). Hypermethylation of tumor suppressor genes in pleural fluid DNA has the potential to be a valuable marker for differentiating MPM from LC. (Cancer Sci 2012; 103: 510–514)

Effect of gefitinib on N-nitrosamine-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced lung tumorigenesis in A/J mice

Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). N-Nitrosamine-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent carcinogen found in tobacco smoke, induces lung tumors in A/J mice. NNK induces cellular transformation resulting in the over-expression of EGFR. Accordingly, EGFR may be a target for cancer prevention. In this study, we investigated the effect of gefitinib on NNK-induced tumorigenesis and the carcinogenicity of gefitinib in A/J mice. A total of 180 four-week-old female A/J mice were randomly divided into six groups: group 1 (controls), treated with deionized water; group 2, treated with 5 mg/kg p.o. gefitinib; group 3, treated with 50 mg/kg p.o. gefitinib (to test the carcinogenicity of gefitinib); group 4 (controls for NNK treatment), treated with deionized water; group 5, treated with 5 mg/kg p.o. gefitinib; and group 6, treated with 50 mg/kg p.o. gefitinib and injected with NNK once at 8 weeks of age to test the chemopreventive activity of gefitinib. Gefitinib was given once a day, 5 days a week by gavage, beginning at 4 weeks of age and continuing for 26 weeks. All mice were sacrificed at 30 weeks of age. The multiplicities of the NNK-induced lung tumors were significantly suppressed in a dose-dependent manner. Gefitinib had no effect on body weight at a low dose. The administration of gefitinib alone for 26 weeks did not induce tumorigenesis; instead, it significantly suppressed the incidence of spontaneous tumors in the mice, in contrast with other anti-cancer agents. Gefitinib did not induce lung fibrosis when compared with control mice by Azan-Mallory staining. Our results suggest that gefitinib has a weak but significant chemopreventive effect with no carcinogenicity or pulmonary toxicity in A/J mice.