Ichiro Nakachi

Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

BackgroundAlterations in the processing of the genetic information in carcinogenesis result from stable genetic mutations or epigenetic modifications. It is becoming clear that nucleosomal histones are central to proper gene expression and that aberrant DNA methylation of genes and histone methylation plays important roles in tumor progression. To date, several histone lysine methyltransferases (HKMTs) have been identified and histone lysine methylation is now considered to be a critical regulator of transcription. However, still relatively little is known about the role of HKMTs in tumorigenesis.ResultsWe observed differential HKMT expression in a lung cancer model in which normal human bronchial epithelial (NHBE) cells expressing telomerase, SV40 large T antigen, and Ras were immortal, formed colonies in soft agar, and expressed specific HKMTs for H3 lysine 9 and 27 residues but not for H3 lysine 4 residue. Modifications in the H3 tails affect the binding of proteins to the histone tails and regulate protein function and the position of lysine methylation marks a gene to be either activated or repressed. In the present study, suppression by siRNA of HKMTs (EZH2, G9A, SETDB1 and SUV39H1) that are over-expressed in immortalized and transformed cells lead to reduced cell proliferation and much less anchorage-independent colony growth. We also found that the suppression of H3-K9, G9A and SUV39H1 induced apoptosis and the suppression of H3-K27, EZH2 caused G1 arrest.ConclusionOur results indicate the potential of these HKMTs in addition to the other targets for epigenetics such as DNMTs and HDACs to be interesting therapeutic targets.

Molecular sub-group-specific immunophenotypic changes are associated with outcome in recurrent posterior fossa ependymoma.

Better understanding of ependymoma (EPN) biology at relapse is needed to improve therapy at this critical event. Convincing data exist defining transcriptionally distinct posterior fossa (PF) sub-groups A and B at diagnosis. The clinical and biological consequence of these sub-groups at recurrence has not yet been defined. Genome and transcriptome microarray profiles and clinical variables of matched primary and first recurrent PF EPN pairs were used to identify biologically distinct patterns of progression between EPN sub-groups at recurrence. Key findings were validated by histology and immune function assays. Transcriptomic profiles were partially conserved at recurrence. However, 4 of 14 paired samples changed sub-groups at recurrence, and significant sub-group-specific transcriptomic changes between primary and recurrent tumors were identified, which were predominantly immune-related. Further examination revealed that Group A primary tumors harbor an immune gene signature and cellular functionality consistent with an immunosuppressive phenotype associated with tissue remodeling and wound healing. Conversely, Group B tumors develop an adaptive, antigen-specific immune response signature and increased T-cell infiltration at recurrence. Clinical distinctions between sub-groups become more apparent after first recurrence. Group A tumors were more often sub-totally resected and had a significantly shorter time to subsequent progression and worse overall survival. Minimal tumor-specific genomic changes were observed for either PF Groups A or B at recurrence. Molecular sub-groups of PF EPN convey distinct immunobiologic signatures at diagnosis and recurrence, providing potential biologic rationale to their disparate clinical outcomes. Immunotherapeutic approaches may be warranted, particularly in Group A PF EPN.

An alternative method for screening EGFR mutation using RFLP in non-small cell lung cancer patients

Introduction: Epidermal growth factor receptor (EGFR) mutations are strong determinants of tumor response to EGFR tyrosine kinase inhibitors in non-small cell lung cancers (NSCLCs). Currently available methods of EGFR mutation detection rely on direct sequencing. Here, we describe the use of an alternative way to screen EGFR mutations. Methods: A total of 109 frozen tumor specimens from NSCLC patients were obtained. For mutational analysis of EGFR exons 18, 19, and 21, reverse transcription-polymerase chain reaction was performed on the cDNA using original primers designed for restriction fragment length polymorphism (RFLP). Results: EGFR mutations were detected in 37 patients (34%) by both RFLP and direct sequencing except one case in which it was detected only by RFLP. EGFR mutations were more frequently observed to be significant by multivariate analysis in patients with adenocarcinoma (OR = 5.56), no-smoking history (OR = 4.34), and 65-year-old or younger (OR = 2.64), but not in women (OR = 1.14). Among 37 patients, 18 were treated with gefitinib and 9 responded to the treatment. One patient without any mutation responded. Conclusion: RFLP is a useful method for screening EGFR mutations and can also be applied to predicting the sensitivity of NSCLC patients to EGFR-tyrosine kinase inhibitors.

The Combination of Multiple Receptor Tyrosine Kinase Inhibitor and Mammalian Target of Rapamycin Inhibitor Overcomes Erlotinib Resistance in Lung Cancer Cell Lines through c-Met Inhibition

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) show antitumor activity in a subset of non–small cell lung cancer (NSCLC) patients. However, the initial tumor response is followed by recurrence. Several studies have suggested the importance of other receptor tyrosine kinases (RTK) and downstream kinases as potential targets in the treatment of NSCLC. We used the multiple-RTK inhibitor AEE788, which inhibits EGFR, vascular endothelial growth factor receptor, and human epidermal growth factor receptor 2, with and without the downstream kinase inhibitor RAD001 (an inhibitor of mammalian target of rapamycin). AEE788 inhibited cell growth more effectively than did erlotinib in three NSCLC cell lines examined (A549, H1650, and H1975). However, in the EGFR-TKI–resistant cell line H1975 harboring T790M resistance mutation, cell growth inhibition by AEE788 was only mild, and the phosphorylation of its leading targets such as EGFR and vascular endothelial growth factor receptor 2 was not inhibited. In H1975, AEE788 induced significantly greater cell growth inhibition when combined with RAD001 than when used alone. This cooperative effect was not seen with the combination of erlotinib and RAD001. We found that c-Met was highly phosphorylated in this cell line, and the phosphorylated c-Met was inhibited effectively by AEE788. Using a phospho-RTK array, the phosphorylation of c-Met and insulin-like growth factor-I receptor was inhibited by AEE788. These results suggest that upstream RTK inhibitor overcomes the acquired resistance to EGFR-TKI when combined with downstream kinase inhibitor. Thus, the combined inhibition of upstream and downstream RTKs is a promising strategy for the treatment of NSCLC. Mol Cancer Res; 8(8); 1142–51. ©2010 AACR.

Bronchoscopic Microsampling is a Useful Complementary Diagnostic Tool for Detecting Lung Cancer

PURPOSE Bronchoscopic microsampling (BMS) is a novel and direct method with which to obtain epithelial lining fluid (ELF) from the lungs. Analysis of DNA hypermethylation of tumor suppressor genes (TSGs) is expected to be a sensitive tool for the early detection of lung cancer. It has been reported that the existence of EGFR mutations and EML4-ALK gene rearrangements are related to the sensitivity of corresponding kinase inhibitors. We aimed to evaluate the suitability of ELF as a sample for analyzing molecular changes specific for lung cancer. PATIENTS AND METHODS We collected ELF from 61 lung cancer patients by BMS from the airway close to the peripheral lung nodule and purified the nucleic acids. We performed methylation specific PCR in each ELF as well as matched serum and tumor tissue for TSGs for DNA methylation analysis. We also examined EGFR mutations and EML4-ALK rearrangement. RESULTS The sensitivity for detecting DNA hypermethylation in ELF vs serum was 74.1% vs 18.5%. We found 60.1% of patients had at least one hypermethylation in ELF, while only 27.9% had it in serum. Of note, DNA hypermethylation was detected even in stage I patients (60.0%) and the detection rate was almost the same level in each stage. We also found the sensitivity for detecting EGFR mutation in ELF vs serum was 58.3% vs 8.3%. We detected an EML4-ALK fusion gene using ELF in one patient. CONCLUSIONS BMS is an alternative method to detect cancer specific genetic and epigenetic alterations and will be a useful complementary diagnostic tool for lung cancer. SUMMARY Investigation of genetic and epigenetic changes associated with lung cancer has clinical importance for its diagnosis and management. The clinical usefulness of bronchoscopic microsampling (BMS) in lung cancer has not yet been evaluated. This study demonstrates that BMS could be useful for detecting lung cancer specific molecular changes and valuable for early diagnosis and determination of treatment options for lung cancer.

Application of SNP microarrays to the Genome-wide Analysis of Chromosomal Instability in Premalignant Airway Lesions

Chromosomal instability is central to the process of carcinogenesis. The genome-wide detection of somatic chromosomal alterations (SCA) in small premalignant lesions remains challenging because sample heterogeneity dilutes the aberrant cell information. To overcome this hurdle, we focused on the B allele frequency data from single-nucleotide polymorphism microarrays (SNP arrays). The difference of allelic fractions between paired tumor and normal samples from the same patient (delta-θ) provides a simple but sensitive detection of SCA in the affected tissue. We applied the delta-θ approach to small, heterogeneous clinical specimens, including endobronchial biopsies and brushings. Regions identified by delta-θ were validated by FISH and quantitative PCR in heterogeneous samples. Distinctive genomic variations were successfully detected across the whole genome in all invasive cancer cases (6 of 6), carcinoma in situ (3 of 3), and high-grade dysplasia (severe or moderate; 3 of 11). Not only well-described SCAs in lung squamous cell carcinoma, but also several novel chromosomal alterations were frequently found across the preinvasive dysplastic cases. Within these novel regions, losses of putative tumor suppressors (RNF20 and SSBP2) and an amplification of RASGRP3 gene with oncogenic activity were observed. Widespread sampling of the airway during bronchoscopy demonstrated that field cancerization reflected by SCAs at multiple sites was detectable. SNP arrays combined with delta-θ analysis can detect SCAs in heterogeneous clinical sample and expand our ability to assess genomic instability in the airway epithelium as a biomarker of lung cancer risk. Cancer Prev Res; 7(2); 255–65. ©2013 AACR.

Exhaustion of Airway Basal Progenitor Cells in Early and Established Chronic Obstructive Pulmonary Disease

Rationale: Up to 40% of smokers develop chronic obstructive pulmonary disease (COPD) over a period that spans decades. Despite the importance of COPD, much remains to be learned about susceptibility and pathogenesis, especially during early, prediagnostic stages of disease. Airway basal progenitor cells are crucial for lung health and resilience because of their ability to repair injured airways. In COPD, the normal airway epithelium is replaced with increased basal and secretory (mucous) cells and decreased ciliated cells, suggesting that progenitors are impaired. Objectives: To examine airway basal progenitor cells and lung function in smokers with and without COPD. Methods: Bronchial biopsies taken from smokers at risk for COPD and lung cancer were used to acquire airway basal progenitor cells. They were evaluated for count, self‐renewal, and multipotentiality (ability to differentiate to basal, mucous, and ciliated cells), and progenitor count was examined for its relationship with lung function. Measurements and Main Results: Basal progenitor count, self‐renewal, and multipotentiality were all reduced in COPD versus non‐COPD. COPD progenitors produced an epithelium with increased basal and mucous cells and decreased ciliated cells, replicating the COPD phenotype. Progenitor depletion correlated with lung function and identified a subset of subjects without COPD with lung function that was midway between non‐COPD with high progenitor counts and those with COPD. Conclusions: Basal progenitor dysfunction relates to the histologic and physiologic manifestations of COPD and identifies a subset that may represent an early, prediagnostic stage of COPD, indicating that progenitor exhaustion is involved in COPD pathogenesis.

Real-world Efficacy and Safety of Nivolumab for Advanced Non-Small-cell Lung Cancer: A Retrospective Multicenter Analysis

Micro‐Abstract We evaluated the real‐world efficacy and safety of nivolumab in 142 patients with advanced non–small‐cell lung cancer in Japan and identified the clinical characteristics that influence the efficacy. Negative EGFR/ALK mutation status and previous radiotherapy were significantly associated statistically with the treatment response. These findings might aid in the efficient immunotherapeutic management of lung cancer. Background Nivolumab, an immune checkpoint inhibitor, is now a standard treatment for previously treated advanced non–small‐cell lung cancer based on the results from phase III clinical trials. We evaluated the real‐world efficacy and safety of nivolumab in a nonselected population and identified the clinical characteristics that influence efficacy. Materials and Methods A total of 142 patients with advanced non–small‐cell lung cancer who were administered nivolumab at Keio University and affiliated hospitals in Japan from January to July 2016 were enrolled. The treatment efficacy and adverse events were retrospectively reviewed, and the clinical characteristics associated with the nivolumab response were evaluated using univariate and stratified analyses and the Cochran‐Mantel‐Haenszel test. Results The objective response rate was 17.0% (95% confidence interval [CI], 12.0%‐24.0%), the median progression‐free survival (PFS) was 58 days (95% CI, 50‐67 days), and the proportion of patients with adverse events of any grade was 45.0%. EGFR/ALK mutation status was inversely associated with the treatment response (P < .05), and the difference in PFS for the mutation‐positive versus mutation‐negative patients was statistically significant (49 vs. 63 days; hazard ratio, 1.9; 95% CI, 1.1‐5.2; P = .029). Previous radiotherapy also had a positive association with the treatment response (P = .012). Conclusion The objective response rate, PFS, and adverse event profiles were comparable to those observed in previous clinical trials. EGFR/ALK mutation‐negative status and previous radiotherapy might be key clinical characteristics associated with a positive treatment response. Our findings could aid in the efficient immunotherapeutic management of lung cancer.

Intraoral ulcer due to non-invasive positive pressure ventilation: an overlooked complication

A male patient aged 81 years reported with dyspnoea and loss of consciousness, at our emergency department. His respiratory rate was 30 breaths/min, and his level of consciousness determined using the Glasgow Coma Scale was eye (1), verbal (1) and motor (4). Despite oxygen administration via a bag valve mask, his percutaneous oxygen saturation level was measured as only 81%. Physical examination revealed weak vesicular sound on auscultation, suggesting he had severe emphysema. Arterial blood gas analysis after …

Abstract 3006: Hyperoxia may be a treatment option for NSCLC

Introduction: Many anti-tumor drugs have been developed, however, prognosis of NSCLC remains poor and new approaches for NSCLC treatment are expected. Lung is a unique organ whose epithelial cells are directly exposed to oxygen. Hyperoxia produces reactive oxygen species (ROS) and induces cell damage. Therefore we attempted to investigate the potential of hyperoxia as an anti-tumor treatment in NSCLC cells in this study. Methods: We used oxygen-concentration-adjustable incubators and validated effect of hyperoxia on various lung cancer cell lines in vitro. The effect of hyperoxia on cell proliferation, apoptosis and gene expression was evaluated using MTS assay, flowcytometry and cDNA microarray, respectively. Results: We found that the growth of lung cancer cell lines (A549, NCI-H1975) was inhibited by 50% of hyperoxia in a dose-dependent manner, while that of normal airway epithelial cells (NHBE, BEAS-2B) was not. Two independent pathway analysis using cDNA microarray revealed the activation of NF-kB and AMPK pathways, and the production of nitric oxide and ROS in hyperoxia (50%) treated lung cancer cell lines compared to untreated ones. The combined treatment with ABT-263, Bcl-2 inhibitor, and hyperoxia (50%) induced synergistic growth inhibition and apoptosis in NCI-H1975 and SK-MES-1 cells. Moreover, the combination of ABT-263 and activator of either ROS, NF-kB or AMPK also showed synergistic growth inhibition in those cells. Conclusions: Hyperoxia (50%) showed anti-tumor effect in NSCLC cell lines through the activation of NF-kB and AMPK pathways, and the production of ROS. The effect was augumented in combination with Bcl-2 inhibitor. Hyperoxia may be a treatment option for NSCLC patients. Citation Format: Junko Hamamoto, Hiroyuki Yasuda, Ichiro Nakachi, Michael G. Edwards, Masayoshi Miyawaki, Makoto Nishino, Aoi Kuroda, Tetsuo Tani, Daisuke Arai, Kota Ishioka, Ichiro Kawada, Katsuhiko Naoki, Tomoko Betsuyaku, Kenzo Soejima. Hyperoxia may be a treatment option for NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3006. doi:10.1158/1538-7445.AM2015-3006