Kouya Shiraishi

Identification of Genes Upregulated in ALK-Positive and EGFR/KRAS/ALK-Negative Lung Adenocarcinomas

Activation of the EGFR, KRAS, and ALK oncogenes defines 3 different pathways of molecular pathogenesis in lung adenocarcinoma. However, many tumors lack activation of any pathway (triple-negative lung adenocarcinomas) posing a challenge for prognosis and treatment. Here, we report an extensive genome-wide expression profiling of 226 primary human stage I-II lung adenocarcinomas that elucidates molecular characteristics of tumors that harbor ALK mutations or that lack EGFR, KRAS, and ALK mutations, that is, triple-negative adenocarcinomas. One hundred and seventy-four genes were selected as being upregulated specifically in 79 lung adenocarcinomas without EGFR and KRAS mutations. Unsupervised clustering using a 174-gene signature, including ALK itself, classified these 2 groups of tumors into ALK-positive cases and 2 distinct groups of triple-negative cases (groups A and B). Notably, group A triple-negative cases had a worse prognosis for relapse and death, compared with cases with EGFR, KRAS, or ALK mutations or group B triple-negative cases. In ALK-positive tumors, 30 genes, including ALK and GRIN2A, were commonly overexpressed, whereas in group A triple-negative cases, 9 genes were commonly overexpressed, including a candidate diagnostic/therapeutic target DEPDC1, that were determined to be critical for predicting a worse prognosis. Our findings are important because they provide a molecular basis of ALK-positive lung adenocarcinomas and triple-negative lung adenocarcinomas and further stratify more or less aggressive subgroups of triple-negative lung ADC, possibly helping identify patients who may gain the most benefit from adjuvant chemotherapy after surgical resection.

A genome-wide association study identifies two new susceptibility loci for lung adenocarcinoma in the Japanese population

Lung adenocarcinoma is the most common histological type of lung cancer, and its incidence is increasing worldwide. To identify genetic factors influencing risk of lung adenocarcinoma, we conducted a genome-wide association study and two validation studies in the Japanese population comprising a total of 6,029 individuals with lung adenocarcinoma (cases) and 13,535 controls. We confirmed two previously reported risk loci, 5p15.33 (rs2853677, Pcombined = 2.8 × 10−40, odds ratio (OR) = 1.41) and 3q28 (rs10937405, Pcombined = 6.9 × 10−17, OR = 1.25), and identified two new susceptibility loci, 17q24.3 (rs7216064, Pcombined = 7.4 × 10−11, OR = 1.20) and 6p21.3 (rs3817963, Pcombined = 2.7 × 10−10, OR = 1.18). These data provide further evidence supporting a role for genetic susceptibility in the development of lung adenocarcinoma.

Contribution of nicotine acetylcholine receptor polymorphisms to lung cancer risk in a smoking-independent manner in the Japanese

Recent genome wide association (GWA) studies on European and American populations revealed association with lung cancer risk of single-nucleotide polymorphisms (SNPs) in the locus containing two nicotine acetylcholine receptor (CHRNA) genes, whose involvement in tobacco addiction had been indicated. Association with lung cancer risk in smokers was consistently, but that in non-smokers as well as that with smoking behavior was inconsistently, observed in these studies. To obtain further information on the significance of CHRNA SNPs in lung cancer risk, association of seven SNPs in this locus with lung cancer risk as well as smoking status was examined in a Japanese population by a case-control study of 1250 cases (562 adenocarcinoma, 391 squamous cell carcinoma and 297 small cell carcinoma) and 936 controls. The frequency of the haplotype consisting of minor alleles for three SNPs, rs8034190, rs16969968 and rs1051730, which had been defined as a susceptible haplotype in the GWA studies, was much lower in the Japanese population (0.013) than in European and American populations (0.3-0.4). However, this haplotype was significantly associated with lung cancer risk also in Japanese (odds ratio = 2.3, 95% confidence interval = 1.5-3.7, P = 0.00028, respectively). The association was observed both in smokers and non-smokers and in all histological types of lung cancers. Individuals with this haplotype showed higher smoking doses than those without; however, the difference was not statistically significant. These results strongly indicate that CHRNA SNPs confer lung cancer susceptibility in a small subset of Japanese in a smoking-independent manner.

Druggable Oncogene Fusions in Invasive Mucinous Lung Adenocarcinoma

Purpose: To identify druggable oncogenic fusions in invasive mucinous adenocarcinoma (IMA) of the lung, a malignant type of lung adenocarcinoma in which KRAS mutations frequently occur. Experimental Design: From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations and 34 cases without (38%), we conducted whole-transcriptome sequencing of 32 IMAs, including 27 cases without KRAS mutations. We used the sequencing data to identify gene fusions, and then performed functional analyses of the fusion gene products. Results: We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1, EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. NRG1 fusions were present in 17.6% (6/34) of KRAS-negative IMAs. The CD74-NRG1 fusion activated HER2:HER3 signaling, whereas the EZR-ERBB4 and TRIM24-BRAF fusions constitutively activated the ERBB4 and BRAF kinases, respectively. Signaling pathway activation and fusion-induced anchorage-independent growth/tumorigenicity of NIH3T3 cells expressing these fusions were suppressed by tyrosine kinase inhibitors approved for clinical use. Conclusions: Oncogenic fusions act as driver mutations in IMAs without KRAS mutations, and thus represent promising therapeutic targets for the treatment of such IMAs. Clin Cancer Res; 20(12); 3087–93. ©2014 AACR.

Association of DNA Repair Gene Polymorphisms With Response to Platinum-Based Doublet Chemotherapy in Patients With Non–Small-Cell Lung Cancer

PURPOSE To identify polymorphisms in DNA repair genes that affect responses to platinum-based doublet chemotherapy in patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS In total, 640 patients with NSCLC who received platinum-based doublet chemotherapy in the National Cancer Center Hospital in Japan from 2000 to 2008 and whose responses were evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) participated in a study of the association between response and genotypes for 30 single nucleotide polymorphisms (SNPs) in 27 DNA repair genes. Candidate SNPs were selected in a discovery set of 201 patients, and their associations were validated in an independent set of 439 patients by prespecified P value criteria. RESULTS Homozygotes for the minor allele TP53-72Pro of the Arg72Pro SNP in the TP53 gene showed a better response rate (54.3%) than those for the major allele TP53-72Arg (29.1%; P = 4.4 × 10(-5)) irrespective of therapeutic regimens, and minor allele homozygotes had significantly longer progression-free and overall survivals than major allele homozygotes (hazard ratio [HR], 0.85; 95% CI, 0.74 to 0.98; P = .020; and HR, 0.86; 95% CI, 0.74 to 0.99; P = .039). Minor allele carriers for SNP Lys940Arg in the poly (ADP-ribose) polymerase 1 (PARP1) gene showed a better response rate to the paclitaxel regimen (45.8%) than to the gemcitabine regimen (10.5%; P for interaction = .019). CONCLUSION Polymorphisms in the TP53 and PARP1 genes are involved in inter-individual differences in the response to platinum-based doublet chemotherapy in patients with NSCLC.

Regulatory Nexus of Synthesis and Degradation Deciphers Cellular Nrf2 Expression Levels

ABSTRACT Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine.

MYC Amplification as a Prognostic Marker of Early Stage Lung Adenocarcinoma Identified by Whole Genome Copy Number Analysis

Purpose: Even in small-sized (≤2 cm in greatest dimension) and/or pathologic stage I lung adenocarcinoma (ADC), a considerable proportion of the patients will relapse within 5 years and show poor prognosis. The purpose of this study was to identify genetic alterations that define prognosis of patients with early-stage lung ADC. Experimental Design: Regions of copy number alterations in 65 small-sized lung ADCs and 40 ADC cell lines were determined by using GeneChip Human Mapping 10-K and 250-K single-nucleotide polymorphism (SNP) arrays, respectively. A copy number assay based on real-time genomic PCR (RT-G-PCR) was done for 60 small-sized lung ADCs and 162 stage I lung ADCs. Results: Several regions on chromosomes 5p, 7p, 8q, and 14q were frequently (>10%) amplified in both small-sized ADCs and lung ADC cell lines. In particular, the MYC gene was mapped in the minimum common region at chromosome 8q24.21, and therefore was indicated to be a target of gene amplification in lung ADCs. MYC amplification correlated with poor prognosis (P = 0.031) of patients with small-sized ADCs. MYC amplification detected by SNP array analysis was well reproduced by RT-G-PCR analysis. Therefore, to investigate the utility of MYC amplification as a prognostic marker for early-stage lung ADCs, 162 stage I lung ADCs were subjected to the analysis. MYC amplification was associated with relapse-free survival in these patients (P = 0.013 by multivariate Cox proportional hazard model analysis). Conclusions: These results strongly indicate that MYC amplification is a prognostic marker of patients with early-stage lung ADCs. Clin Cancer Res; 17(6); 1481–9. ©2010 AACR.

Contribution of the TP53, OGG1, CHRNA3, and HLA-DQA1 Genes to the Risk for Lung Squamous Cell Carcinoma

Introduction: Recent genome-wide association studies (GWASs) have identified polymorphisms in several genes associated with lung cancer risk. Nevertheless, functional polymorphisms in DNA repair and metabolic genes that had been reported as being associated with risk for lung cancer, particularly for lung squamous cell carcinoma (SQC), were not examined in those studies. Therefore, significance of these functional polymorphisms was evaluated in a population, in which polymorphisms in the GWAS genes showed associations with lung SQC risk. Methods: Polymorphisms in three DNA repair genes, TP53, MDM2, and OGG1, and two metabolic genes, CYP1A1 and GSTM1, were examined for associations with lung SQC risk in a hospital-based case-control study consisting of 377 cases and 325 controls, which had been previously subjected to association studies on GWAS genes, CHRNA3, TERT, and HLA-DQA1. Results: Genotypes for two DNA repair genes, TP53 and OGG1, showed significant associations with SQC risk (p < 0.05), and those for two GWAS genes, CHRNA3 and HLA-DQA1, showed significant associations with SQC risk (P < 0.05) with odds ratios between 1.65 (95% confidence interval = 1.06–2.57 for OGG1) and 2.57 (95% confidence interval = 1.03–6.87 for CHRNA3). Marginally significant associations were also observed for MDM2 and CYP1A1 genes. Interactions among these polymorphisms on SQC risk were not observed. Conclusions: Association of functional polymorphisms in DNA repair and metabolic genes with lung SQC risk was appreciated. This result indicates the necessity of reevaluation for the significance of functional polymorphisms in DNA repair and metabolic genes on lung cancer risk in other populations subjected to GWASs.

Gene aberrations for precision medicine against lung adenocarcinoma

Lung adenocarcinoma (LADC), the most frequent histological type of lung cancer, is often triggered by an aberration in a driver oncogene in tumor cells. Examples of such aberrations are EGFR mutation and ALK fusion. Lung adenocarcinoma harboring such mutations can be treated with anticancer drugs that target the aberrant gene products. Additional oncogene aberrations, including RET, ROS1, and NRG1 fusions, skipping of exon 14 of MET, and mutations in BRAF, HER2, NF1, and MEK1, were recently added to the list of such “druggable” driver oncogene aberrations, and their responses to targeted therapies are currently being evaluated in clinical trials. However, approximately 30% and 50% of LADCs in patients in Japan and Europe/USA, respectively, lack the driver oncogene aberrations listed above. Therefore, novel therapeutic strategies, such as those that exploit the vulnerabilities of cancer cells with non‐oncogene aberrations, are urgently required. This review summarizes the current status of research on precision medicine against LADC and enumerates the research priorities for the near future.

Individuals susceptible to lung adenocarcinoma defined by combined HLA-DQA1 and TERT genotypes.

Adenocarcinoma (ADC) is the commonest histological type of lung cancer, and its weak association with smoking indicates the necessity to identify high-risk individuals for targeted screening and/or prevention. By a genome-wide association study (GWAS), we identified an association of polymorphisms in the 6p21.31 locus containing four human leukocyte antigen (HLA) class II genes with lung ADC risk. DQA1*03 of the HLA-DQA1 gene was defined as a risk allele with odds ratio (OR) of 1.36 [95% confidence interval (CI) = 1.21-1.54, P = 5.3 x 10(-7)] by analysis of 1656 ADC cases and 1173 controls. DQA1*03 and the minor allele for a polymorphism, rs2736100, in TERT, another lung cancer susceptibility locus identified in recent GWASs on Europeans and Americans, were indicated to independently contribute to ADC risk with per allele OR of 1.43 (95% CI = 1.31-1.56, P = 7.8 x 10(-16)). Individuals homozygous both for the DQA1*03 and minor TERT alleles were defined as high-risk individuals with an OR of 4.76 (95% CI = 2.53-9.47, P = 4.2 x 10(-7)). The present results indicated that individuals susceptible to lung ADC can be defined by combined genotypes of HLA-DQA1 and TERT.