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1.  Intra-tumor heterogeneity of BRAF V600E mutation in lung adenocarcinomas 
BRAF mutations exist in numerous types of cancer, including melanomas, colorectal cancers and lung cancers. The V600E-specific inhibitor vemurafenib has marked clinical activity in patients with BRAF V600E-mutated melanoma. However, there are many cases of resistance to vemurafenib. This may be due to the reported intra-tumor heterogeneity of the BRAF V600E mutation in primary melanomas. BRAF mutations are found in 1–5% of non-small cell carcinomas (NSCLCs), almost exclusively in adenocarcinoma. A few cases have been reported in which vemurafenib was effective against BRAF V600E-mutated lung cancers. In a previous study, five lung adenocarcinomas with BRAF V600E mutation were detected by direct sequencing. The present study analyzed these tumors for the percentage of mutation (%mutation) by competitive allele-specific polymerase chain reaction (CAST-PCR) assay. In addition, sections of all components of the adenocarcinomas were obtained by laser microdissection and analyzed. The %mutations of BRAF V600E within the macrodissected tumors (cases 1–5) were: Case 1, 10.0%; case 2, 8.0%; case 3, 8.9%; case 4, 21.5%; and case 5, 14.9%. In four cases (cases 2–5), the %mutations of each adenocarcinoma component were as follows: Case 2, lepidic growth 6.5–24.5%, papillary 1.3–11.2% and acinar 9.8%; case 3, solid 2.5–69.9%, acinar 12.4–27.1% and papillary 3.7–17.4%; case 4, acinar 10.0–45.0% and papillary 44.0%; and case 5, papillary 3.7–93.4%. Sensitive BRAF mutation detection methods were used and evidence for heterogeneity of the BRAF V600E mutation in these lung adenocarcinoma cases was observed. Targeted therapy with a BRAF V600E inhibitor such as vemurafenib may have potential in the treatment of lung cancer with this mutation; however, it is necessary to consider how the treatment effect of and drug resistance to BRAF V600E inhibitors are affected by the presence of heterogeneity in future studies.
PMCID: PMC4471699  PMID: 26136882
BRAF mutation; lung cancer; heterogeneity; V600E; competitive allele-specific polymerase chain reaction
2.  Immunohistochemical determination of the miR-1290 target arylamine N-acetyltransferase 1 (NAT1) as a prognostic biomarker in breast cancer 
BMC Cancer  2014;14:990.
There are many molecular differences between estrogen receptor α (ERα)-positive and ER-negative breast cancers. Recent analyses have shown that the former can be divided into two subtypes, luminal A and luminal B. These differ in response to endocrine therapy and chemotherapy, and in prognosis. In a previous study, we found that microRNA (miR)-1290 that was significantly down-regulated in luminal A tumors and its potential target arylamine N-acetyltransferase 1 (NAT1). The aim of the present study was to determine whether NAT1 is a bona fide target of miR-1290, and to investigate the impact of NAT1 on breast cancer prognosis.
Luciferase reporter assays were employed to validate NAT1 as a putative miR-1290 target gene. Expression of NAT1, ERα, progesterone receptor (PgR) and HER2 was analyzed in 394 breast cancer samples by immunohistochemistry.
NAT1 was confirmed to be a direct target of miR-1290. Levels of expression of NAT1 were positively correlated with those of ERα (P < 0.0001) and PgR (P < 0.0001), but negatively correlated with both tumor grade and size (P < 0.0001). Kaplan-Meier analysis showed that the presence of NAT1 was significantly associated with increased overall survival (OS) (P = 0.0416) in these patients. Similarly, significant associations of NAT1 with disease-free survival (DFS) (P = 0.0048) and OS (P = 0.0055) in those patients who received adjuvant endocrine therapy with tamoxifen (n = 176) were found. Moreover, NAT1 was also significantly associated with increased DFS (P = 0.0025) and OS (P = 0.0007) in the subset of lymph node-positive patients (n = 147). Univariate and multivariate analyses showed significant associations between levels of NAT1 and DFS (P = 0.0005 and 0.019, respectively).
We report that miR-1290 directly targets the NAT1 3′-UTR and that NAT1 protein expression is correlated with improved OS of breast cancer patients. NAT1 is a possible prognostic biomarker for lymph node-positive breast cancer. Thus, miR-1290 and its target NAT1 are associated with important characteristics of breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-990) contains supplementary material, which is available to authorized users.
PMCID: PMC4364092  PMID: 25528056
Breast cancer; miR-1290; Arylamine N-acetyltransferase 1 (NAT1)
3.  Increased BRAF copy number in lung adenocarcinoma 
Oncology Letters  2014;9(2):709-712.
Point mutation of the BRAF gene is a genetic event that occurs in a subset of lung adenocarcinoma cases. For example, BRAF V600E is a driver mutation that can be effectively targeted using selective BRAF and/or MEK inhibitors. The present study hypothesized that an increase in BRAF copy number may be correlated with certain clinicopathological features of lung adenocarcinoma in Japanese patients. The BRAF gene copy number was analyzed using quantitative polymerase chain reaction amplifications in 29 surgically treated lung adenocarcinoma cases without EGFR or Kras mutations from Nagoya City University Hospital (Nagoya, Japan). Seven BRAF-mutant cases were included. Increased BRAF gene copy number was identified in three lung adenocarcinoma patients (10.3%), all of which exhibited the V600E mutation. Using fluorescence in situ hybridization with BRAF-specific and chromosome 7 centromeric probes, increased copy number status was associated with gene amplification or gain of chromosome 7. Although increased BRAF copy number was correlated with BRAF V600E mutations, numerical changes in BRAF copy number were rare and mild in lung adenocarcinoma, resulting in no significant difference in pathological tumor status or tumor stage.
PMCID: PMC4301492  PMID: 25621040
BRAF; lung cancer; adnocarcinoma; copy number; V600E
4.  PD-1 gene promoter polymorphisms correlate with a poor prognosis in non-small cell lung cancer 
Molecular and Clinical Oncology  2014;2(6):1035-1042.
An imbalance to the regulation of the immune system changes the tumor-specific T-cell immunity in the cancer microenvironment and adjusts the tumor progression and metastasis. Inhibiting the interactions of the immune function mediates the antitumor activity in preclinical models. The programmed death 1 (PD-1) gene −606 G/A polymorphism, which may modify promoter activity and is Asian-specific, was investigated by TaqMan quantitative polymerase chain reaction assay in surgically treated non-small cell lung cancer (NSCLC) cases. In the present study, 583 surgically removed NSCLC cases were included for single-nucleotide polymorphism (SNP) analyses. The PD-1 SNP statuses at the promoter region (rs36084323) were 146 AA (25.0%), 293 GA (50.3%) and 144 GG (24.7%). The ratio was extremely similar to the healthy control in a previous study: 24.9% AA, 47.8% GA and 27.3% GG. The ratio of the GG phenotype was not significantly different for gender (25.1% males and 23.9% female), age (25.2% ≤65 years and 24.4% >65 years), smoking status (26.1% smoker and 21.8% non-smoker) and pathological subtypes [25.4% adenocarcinoma (adeno) and 24.2% squamous cell carcinoma (SCC)]. The GG ratio of PD-1 was not significantly different between pathological stage II–IV (25.5%) and stage I cases (24.1%; P=0.6245). The survival time of the patients with the −606 GG phenotype of PD-1 was significantly lower (n=147, 50 succumbed) compared to the patients with −606 GA or −606 AA (n=435, 109 succumbed) (P=0.0183). The GG phenotype patients had a significantly worse prognosis in the SCC population (P=0.009), however, this was not different to the adeno population (P=0.2594). Thus, PD-1 may promote tumor prognosis and provide a candidate for the blockade of its function as a strategy to antagonize the progression process in NSCLC, particularly lung SCC.
PMCID: PMC4179826  PMID: 25279194
programmed death 1; immunotherapy; lung cancer; prognosis; squamous cell carcinomas
5.  Laparoscopic Adrenalectomy for Solitary Adrenal Metastasis From Lung Cancer 
Background and Objectives:
Several studies have been reported on the problem of determining when laparoscopic adrenalectomy is indicated for solitary adrenal metastasis of malignant tumors. Our efforts at answering this question constitute the basis of this report.
From June 2010 to June 2011, laparoscopic adrenalectomy was performed in 10 lung cancer patients with solitary adrenal metastases (5 adenocarcinomas, 1 squamous cell carcinoma, 1 large cell carcinoma, 1 small cell carcinoma, and 2 pleomorphic carcinomas). The surgical results of all 10 patients were examined.
Adrenal swelling was detected by computed tomography in all patients except 1 case of pleomorphic carcinoma. The findings of positron emission tomography–computed tomography were positive in 8 patients, including the 2 cases with pleomorphic carcinomas. Laparoscopic surgery was successfully performed in 9 cases. In the eighth patient (a case of pleomorphic carcinoma with adrenal swelling), laparoscopic adrenalectomy was attempted but conversion to open surgery was required because of clear evidence of pancreatic invasion.
The results obtained in this study, along with other published reports, support 4 criteria as operative indications for laparoscopic adrenalectomy in solitary adrenal metastasis from the lung: (1) the primary lung cancer is resected or can be cured by radical chemotherapy, (2) metastasis is limited to the adrenal gland only, (3) adrenal metastasis does not invade the surrounding organs, and (4) the size of the adrenal tumor does not exceed 10 cm. In cases of pleomorphic carcinoma, laparoscopic adrenalectomy should be performed when positron emission tomography–computed tomography results are positive.
PMCID: PMC4208896  PMID: 25392660
Laparoscopic adrenalectomy; Adrenal metastasis; Lung cancer; Pleomorphic carcinoma
6.  Investigation of neurotrophic tyrosine kinase receptor 1 fusions and neurotrophic tyrosine kinase receptor family expression in non-small-cell lung cancer and sensitivity to AZD7451 in vitro 
Molecular and Clinical Oncology  2014;2(5):725-730.
Advances in the molecular segmentation of lung cancer has raised the possibility that neurotrophic tyrosine kinase receptor (NTRK) 1 fusions and NTRK1-3 expression may be promising molecular targets for future therapeutic interventions. We investigated the antitumor effects of a selective pan-NTRK inhibitor, AZD7451, by evaluating its antiproliferative effects on the KM12 cell line (a colorectal cancer cell line harboring a tropomyosin-NTRK1 fusion) and the H460 and H810 cell lines [large-cell neuroendocrine carcinoma (LCNEC) cell lines expressing NTRK2]. Relative quantitative polymerase chain reaction (qPCR) was performed to measure the mRNA levels of the NTRK1-3 tyrosine kinase domain using cDNA extracted from KM12, H460 and H810 cells. The cultures were grown in 6-well plates at a density of 1.0×106 cell/well and treated with AZD7451 at different doses (1, 2.5, 4, 5, 7.5 and 10 nM) using dimethyl sulfoxide as a control. Following a 24-h incubation, the number of surviving cells was measured using a hemocytometer. Furthermore, we performed western blotting of the high-affinity nerve growth factor receptor (TRKA) and NTRK2 (TRKB) proteins and monitored the effects on the downstream signaling pathways Akt and ERK in these cell lines following treatment with AZD7451 (KM12 and H460: 0, 1 and 5 nM; H810: 0 and 5 nM). Immunohistochemical analyses of the surgically resected samples were also performed, using anti-NTRK1,2 antibodies. We performed reverse-transcription PCR and direct sequencing to investigate NTRK fusions in 268 patients; however, were unable to confirm the presence of NTRK fusions in this cohort. Further immunohistochemical analyses of the primary patient samples demonstrated that none of 61 tumors had NTRK1 overexpression and 7 of 39 samples exhibited NTRK2 expression, including 1 LCNEC sample. The qPCR results from the KM12 cell line revealed an apparent increase and overexpression of NTRK1 mRNA levels, while H460 cells exhibited a modest increase and the H810 cell line showed no apparent increase in the expression of any NTRK1-3 isoforms. There were no increases in the NTRK2 mRNA levels in any of the three cell lines, although KM12 and H460 cells exhibited low levels of NTRK2 expression. In vitro growth and proliferation of the KM12 cell line harboring the NTRK1-fusion was found to be potently inhibited by AZD7451 at a concentration of 2 nM. The proliferation of H460 cells was also found to be inhibited at a concentration of 5 nM, while there was no apparent inhibitory effect of AZD7451 on the growth or proliferation of H810 cells. Western blotting of KM12 cells treated with AZD7451 also revealed a potent inhibition of TRKA phosphorylation following AZD7451 treatment. Analysis of H460 cells confirmed the expression and inhibition of phosphorylation of NTRK2, whereas there was little to no expression of TRKA/B in H810 cells. Subsequent in vitro analysis of cell lines treated with the pan-TRK inhibitor AZD7451 suggested that the proliferation of KM12 and H460 cells was significantly inhibited by AZD7451, while H810 cells expressing low levels of wild-type NTRK1-3 were not inhibited. Based on these results, there is potential for a NTRK-dependent proliferation driver in a subpopulation of lung cancer patients with NTRK expression. In addition, pharmacological inhibition with a NTRK inhibitor, such as AZD7451, in cells harboring NTRK1 fusions, may be associated with beneficial antitumor effects.
PMCID: PMC4106662  PMID: 25054037
neurotrophic tyrosine kinase receptor; AZD7451; fusion; KM12
7.  APOBEC3B gene overexpression in non-small-cell lung cancer 
Biomedical Reports  2014;2(3):392-395.
Recent study results have demonstrated that a subclass of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminase may induce mutation clusters in various types of cancer. From the Cancer Genome Altas, an APOBEC mutation pattern was identified in bladder, cervical, breast, head and neck and lung cancers. In the present study, APOBEC3B mRNA expression was investigated using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay using LightCycler in surgically treated non-small-cell lung cancer (NSCLC) cases. Additionally, 88 surgically removed Japanese NSCLC cases were analyzed for mRNA level. The results showed that APOBEC3B/β-actin mRNA levels were significantly higher in lung cancer (1,598.481±6,465.781) when compared to adjacent normal lung tissues (2,116.639±8,337.331, P=0.5453). The tumor/normal (T/N) ratio of APOBEC3B/β-actin mRNA levels was not different within the gender, age, smoking status and pathological stages. The T/N ratio of APOBEC3B/β-actin mRNA levels was not significantly different in epidermal growth factor receptor (EGFR) or Kras mutation-positive cases as compared to the wild-type cases.
PMCID: PMC3990218  PMID: 24748981
APOBEC3B; mutation; lung cancer; epidermal growth factor receptor; LightCycler
8.  Prognosis of recurrent non-small cell lung cancer following complete resection 
Oncology Letters  2014;7(4):1300-1304.
Prognosis following recurrence subsequent to complete resection of non-small-cell lung cancer (NSCLC) is considered a multifactorial process dependent on clinicopathological, biological and treatment characteristics. Gefitinib was approved for lung cancer treatment in Japan in 2002. The aim of the current study was to quantify the prognostic effects of these characteristics on post-recurrence prognosis. In total, 127 NSCLC patients were analyzed who underwent complete resection and subsequently had recurrent cancer. The correlation between characteristics of the initial and recurrent disease with post-recurrence prognosis was investigated. The factors clearly associated with post-recurrence prognosis using Cox proportional hazards models were age at recurrence (those <65 years of age typically had better prognoses) and interval between initial resection and recurrence (intervals of <1 year accompanied a worse prognosis). Epidermal growth factor receptor (EGFR) mutant patients treated with EGFR tyrosine kinase inhibitors (TKIs), exhibited the longest median survival following recurrence (37.4 months) in the sample. Treatment, particularly EGFR TKIs for recurrent NSCLC, was observed to significantly prolong survival. The results of the study highlight that various treatment modalities according to the clinical background of the patient should be considered in patients with postoperative recurrent NSCLC.
PMCID: PMC3961426  PMID: 24944713
EGFR; recurrent; prognosis; tyrosine kinase inhibitor
9.  Proposal for a new mediastinal compartment classification of transverse plane images according to the Japanese Association for Research on the Thymus (JART) General Rules for the Study of Mediastinal Tumors 
Oncology Reports  2013;31(2):565-572.
There is no existing worldwide published method for mediastinum compartment classification based on transverse section images for the differential diagnosis of mediastinal tumors. Herein, we describe a new method for anatomic mediastinal compartment classification using transverse section computed tomography (CT) images and the use of this method to classify mediastinal lesions, and thus evaluate whether the method is sufficiently user-friendly and useful. In a publication of the Japanese Association for Research on the Thymus (JART), we proposed the following four mediastinal compartments based on transverse CT images: superior portion of mediastinum, anterior mediastinum (prevascular zone), middle mediastinum (peri-tracheoesophageal zone), and posterior mediastinum (paravertebral zone). In the present study, we retrospectively analyzed 445 pathologically proven mediastinal mass lesions, and categorized them into the proposed four compartments by consensus reading. Mass lesions were classified into compartments based on the location of the lesion centroid, and each lesion was satisfactorily categorized into a compartment. Almost all thymic epithelial tumors (99%, 244/246), all 24 thymic malignant lymphomas and a majority of germ cell neoplasms (93%, 54/58) were classified as being in the anterior mediastinum compartment. The majority of intrathoracic goiters (82%, 14/17) were categorized as being in the superior portion of the mediastinum compartment. Approximately two-thirds of mass lesions in the middle mediastinum were cysts, including foregut and pericardial cysts. Approximately 80% of 37 mass lesions in the posterior mediastinum were neurogenic tumors. Correspondingly, 29 of the 49 neurogenic tumors (60%) were categorized as being in the posterior mediastinum, while 10 (20%) were in the superior portion of the mediastinum, 4 (8%) in the anterior mediastinum, and 6 (12%) in the middle mediastinum. Our findings showed that the newly proposed mediastinal compartment classification using transverse images appears to be user-friendly enough for practical clinical application and may be helpful in differential diagnoses.
PMCID: PMC3896522  PMID: 24317723
mediastinum; compartment; mediastinal mass; mediastinal neoplasm; computed tomography; differential diagnosis
10.  Expression of thymidylate synthase and orotate phosphoribosyltransferase in thymic carcinoma 
Thymic carcinoma is a rare thymic epithelial tumor in which chemotherapy for advanced disease has not yet been established. Thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) protein expression levels in thymic carcinoma were evaluated as possible indicators of the anticancer activity of 5-fluorouracil (5-FU) drugs using immunohistochemistry (IHC). A total of 24 samples of thymic carcinoma were used in the present study. The tumor sections were immunohistochemically stained for TS and OPRT. As a comparison with thymic carcinoma, we also assessed the TS and OPRT protein expression levels in 55 lung cancer samples. The TS expression was positive in 12 of 24 thymic carcinoma samples (50%) and OPRT expression was positive in 10 (42%). The association between TS and OPRT expression and Masaoka stages of thymic carcinoma was analyzed. The TS and OPRT expressions in stage IV were significantly higher compared to that in stages I, II or III. We also compared the TS and OPRT expression levels between thymic carcinoma and lung cancer (33 adenocarcinomas and 22 squamous cell carcinomas). TS expression in thymic carcinoma was significantly lower compared with lung squamous cell carcinoma. OPRT expression in thymic carcinoma was significantly higher compared to lung adenocarcinoma. The combination of a relatively low expression of TS and high expression of OPRT suggests an improved antitumor effect of 5-FU drugs in thymic carcinoma compared to in lung carcinoma.
PMCID: PMC3501379  PMID: 23170110
thymic carcinoma; thymidylate synthase; orotate phosphoribosyltransferase
11.  Keap1 mutations in lung cancer patients 
Oncology Letters  2013;6(3):719-721.
Kelch-like ECH-associated protein 1 (Keap1) inhibits nuclear factor erythroid 2-related 2 (NEF2L2; also named NRF2)-induced cytoprotection and has been hypothesized to represent a candidate tumor suppressor. We have previously reported the somatic mutations of the NRF2 gene (NFE2L2), however, the correlation between the Keap1 mutation and the clinicopathological features of lung cancer has not been well investigated. Therefore, in the present study, the Keap1 mutational status in non-small cell lung cancer (NSCLC) patients was investigated by reverse transcription PCR and direct sequencing. The study included 76 surgically-removed lung cancer cases from patients of the Nagoya City University Hospital in which the EGFR and NFE2L2 mutation status was already established. Keap1 mutations were identified in 2 (2.6%) adenocarcinoma patients with a history of heavy smoking. These mutations were identified to exist exclusively. The Keap1 mutation was only detected in patients with advanced adenocarcinoma (4.3%) and the completely exclusive status of this mutation and others, including EGFR, Kas, erbB2 and NRF2L2, is likely to improve the selection of personalized therapy for lung cancer.
PMCID: PMC3789102  PMID: 24137397
Keap1; NRF2; lung cancer; mutations; adenocarcinoma
12.  Polymorphisms of NRF2 gene correlated with decreased FEV1 in lung cancers of smokers 
Biomedical Reports  2013;1(3):484-488.
The metabolism of xenobiotics plays a fundamental role in smoking-related lung function loss and the development of pulmonary disease. An NRF2-dependent response is a key protective mechanism against oxidative stress. In the present study, we evaluated the effect of single nucleotide polymorphisms in NRF2 genes on the level of forced expiratory volume in one second (FEV1) in lung cancers of smokers. We genotyped the status of NRF2 gene polymorphisms in 209 surgically treated lung cancer cases of smokers using TaqMan polymerase chain reaction (PCR). The results demonstrated the mean FEV1 in patients with rs2364723 C/C, C/G and G/G to be 2143.9, 2294.2 and 2335.4 ml, respectively, and there was a tendency towards lower FEV1 in C/C phenotype (P=0.0944). The mean FEV1 was significantly lower in the C/C phenotype (2143.9±566.0 ml) compared to C/G or G/G (2308.9±642.9 ml, P=0.05). The mean FEV1 in patients with rs6726395 A/A, G/A and G/G was 66.7, 71.2 and 72.3%, respectively, and there was a significant difference between A/A and G/G phenotype (P=0.043). A tendency towards a lower mean FEV1 in A/A phenotype (66.7±11.7%) was observed when compared to A/G or G/G (71.9±10.7%, P=0.07). This study demonstrated that an NRF2-dependent response to cigarette smoking has the potential to affect FEV1 decrease in a lung cancer population. In conclusion, the results have shown that NRF2 genetic changes may play a role in FEV1 loss in smokers with lung cancer.
PMCID: PMC3917728  PMID: 24648973
nuclear factor (erythroid derived 2)-like 2; polymorphism; NRF2; forced expiratory volume in 1 second; smoker
13.  Post-operative acute exacerbation of pulmonary fibrosis in lung cancer patients undergoing lung resection 
Acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) in lung cancer patients is a critical factor in post-operative mortality. The cause of AE development is unknown and AE may occur in patients without the diagnosis of IPF. We have conducted a retrospective study of consecutive patients who underwent lung cancer surgery since January 2004. Sixty-two patients with fibrous findings in preoperative high-resolution computed tomography were enrolled in the present study and clinicopathological factors were analysed. AE was observed in 6 of 62 patients. The frequency of AE according to the type of fibrous changes classification was 1/7 in the usual interstitial pneumonia (UIP) pattern, 1/16 in the cellular non-specific interstitial pneumonia (NSIP) pattern, 4/25 in the fibrotic NSIP pattern and 0/14 in the unclassified or focal fibrous changes pattern. Preoperative Krebs von den Lungen-6 (KL-6) was higher in patients with AE than in those without AE. In patients who underwent partial resection, AE did not develop even with high KL-6 levels. In conclusion, in patients with both the UIP and the NSIP patterns, AE development is possible. In patients with a high risk of AE, such as those with high KL-6 values, limited surgery may be an option to prevent AE development.
PMCID: PMC3279985  PMID: 22159236
Idiopathic pulmonary fibrosis; Acute exacerbation; Lung cancer; KL-6
14.  RagD gene expression and NRF2 mutations in lung squamous cell carcinomas 
Oncology Letters  2012;4(6):1167-1170.
RagD is a member of the small G protein family, which encodes a recently discovered activator of the mTOR pathway. In vitro, RagD plays an important role in the proliferation of NRF2 gene (NFE2L2) mutated cancer cells. We hypothesized that tumor RagD expression may be correlated with the mutation status of NRF2 in lung cancers. RagD mRNA levels were analyzed by quantitative real-time polymerase chain reaction (qPCR) in 90 surgically-treated lung squamous cell cancer cases, including 14 NRF2 mutation cases, and normalized by β-actin mRNA levels. Mean RagD/β-actin mRNA levels of lung squamous cell carcinoma patients did not differ with age (≤65 vs. >65), Brinkman index (<400 vs. ≥400) or gender. RagD/β-actin mRNA levels were significantly higher in stage III samples (3.204±3.623) compared to stage I samples (1.357±1.560) (P= 0.0039). In addition, higher RagD/β-actin mRNA levels were identified in NRF2 mutant samples (3.107±3.633) compared to wild-type samples (1.774±2.301) (P=0.074). These results suggest that RagD induction by NRF2 activation plays a role in the proliferation of lung squamous cell cancers.
PMCID: PMC3506764  PMID: 23226795
NRF2; lung cancer; squamous cell carcinoma; RagD; mutation
15.  Angiopoietin-like protein ANGPTL2 gene expression is correlated with lymph node metastasis in lung cancer 
Oncology Letters  2012;4(6):1325-1328.
Inflammation plays key roles at various stages of tumor development, including invasion and metastasis. In mice, the angiopoietin-like protein (ANGPTL2) gene has been implicated in inflammatory carcinogenesis. ANGPTL2 mRNA expression was investigated by real-time polymerase chain reaction (RT-PCR) assay using LightCycler in surgically treated non-small cell lung cancer (NSCLC) cases. In total, 110 surgically resected NSCLC cases were used for mRNA level analyses. The ANGPTL2/β-actin mRNA levels were not significantly different between lung cancer (1598.481±6465.781) and adjacent normal lung tissues (2116.639±8337.331, P=0.5453). The tumor/normal (T/N) ratio of ANGPTL2/β-actin mRNA levels was not different between gender, age, smoking status and pathological stages. The T/N ratio of ANGPTL2/β-actin mRNA levels was significantly higher in lymph node metastasis-positive cases (2.173±3.151) compared with lymph node metastasis-negative cases (1.212±1.778, P=0.0464). However, ANGPTL2 mRNA status was not correlated with tumor invasion status. Thus, ANGPTL2 may drive metastasis and provide a candidate for blockade of its function as a strategy to antagonize the metastatic process in NSCLC.
PMCID: PMC3506759  PMID: 23205131
ANGTL2; angiopoietin; lung cancer; metastasis; LightCycler
16.  PD-L1 gene expression in Japanese lung cancer patients 
Biomedical Reports  2012;1(1):93-96.
An imbalance in immune regulation affects tumor-specific T-cell immunity in the cancer microenvironment and reshapes tumor progression and metastasis. Blockade of interactions of immune function mediates anti-tumor activity in preclinical models. In the present study, we investigated programmed cell death 1 ligand 1 (PD-L1) mRNA expression by real-time polymerase chain reaction (RT-PCR) using a LightCycler in surgically treated non-small cell lung cancer (NSCLC) cases. This study included 123 surgically removed NSCLC cases for mRNA level analyses. The PD-L1/β-actin mRNA levels showed no marked difference in lung cancer (131.398±421.596) and adjacent normal lung tissues (78.182±254092, P=0.1482). The tumor/normal (T/N) ratio of PD-L1/β-actin mRNA levels was more than 2 in 49 cases and more than 1 in 63 cases. No difference was found in the T/N ratio of PD-L1/β actin mRNA levels among factors inlcuding gender, age, smoking status and pathological subtypes. The T/N ratio of PD-L1/β actin mRNA levels was markedly higher in pathological T4 cases (15.811±36.883) compared to T1 cases (3.492±8.494, P=0.0235). However, the PD-L1 mRNA status did not correlate with lymph node metastasis status. Thus, PD-L1 may drive tumor invasion, while providing a candidate for blockade of its function as a strategy to antagonize the progression process in NSCLC.
PMCID: PMC3956825  PMID: 24648901
programmed cell death 1 ligand 1; immunotherapy; lung cancer; tumor invasion; LightCycler
17.  Polymorphisms in intron 1 of the EGFR gene in non-small cell lung cancer patients 
The epidermal growth factor receptor (EGFR) gene is highly polymorphic and its expression and activity may be affected by various polymorphisms. There have been several studies examining associations between EGFR polymorphisms and clinical outcome of lung cancer therapy; however, the underlying mechanism is largely unknown. The present study investigated EGFR polymorphism status and its correlation with clinicopathological features in Japanese non-small cell lung cancer (NSCLC) patients. We investigated 5 polymorphisms in the EGFR gene (−216G/T, −191C/A, 8227G/A, D994D and R497K) in 274 surgically-treated NSCLC patients. TaqMan single nucleotide polymorphism (SNP) genotyping assays and a PCR-based assay were used to analyze these polymorphisms. In our cohort of patients we did not find any evidence of the −191C/A polymorphism. Our results showed that the patients with the 8227GA or AA type in intron 1 had a significantly better prognosis with the anti-EGFR therapy than the patients with the GG type (p=0.0448) in terms of recurrence of lung cancer. No significant association was observed between 3 other SNPs (−216G/T, D994D and R497K) and clinicopathological features. The EGFR 8227G/A polymorphism in intron 1 may be associated with clinical outcome in NSCLC patients treated with EGFR tyrosine kinase inhibitors.
PMCID: PMC3493703  PMID: 23226726
epidermal growth factor receptor; polymorphism; lung cancer; gefitinib therapy; 8227G/A
18.  RET expression and detection of KIF5B/RET gene rearrangements in Japanese lung cancer 
Cancer Medicine  2012;1(1):68-75.
RET encodes the tyrosine kinase receptor of growth factors belonging to the glial-derived neurotrophic factor family. Recently, RET gene rearrangements with N-terminal of KIF5B gene were identified in lung adenocarcinomas from large-scale sequencing. We investigated RET mRNA expression by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay using LightCycler, and KIF5B/RET gene rearrangements using newly established fluorescence in situ hybridization (FISH) analysis in surgically treated nonsmall cell lung cancer (NSCLC) cases. RET protein expression was also investigated by immunohistochemistry (IHC). This study included 157 surgically removed NSCLC cases for mRNA level analyses. The RET/β actin mRNA levels were not significantly different between lung cancer (6.359 ± 15.268) and adjacent normal lung tissues (8.205 ± 28.931, P = 0.6332). Tumor/normal (T/N) ratio of RET/β actin mRNA levels was not different within gender, stage, smoking status, and pathological subtypes. T/N ratio of RET/β actin mRNA levels was significantly higher in KIF5B/RET rearrangement samples (161.763 ± 123.488) than in wild-type samples (5.9013 ± 17.148, P = 0.044). Although RET IHC positivity was not perfectly correlated with KIF5B/RET arrangement, we have detected the KIF5B/RET rearrangements using FISH analysis. Thus, we have successfully introduced FISH for diagnosing KIF5B/RET positive lung adenocarcinoma. This method facilitates the molecular evaluation for RET fusions and could be applicable in clinical practice to detect lung cancer that may be responsive to RET inhibitors.
PMCID: PMC3544433  PMID: 23342255
FISH; KIF5B/RET; lung cancer; RET expression
19.  Clinicopathological analysis of small-sized thymoma with podoplanin and Ki 67 expression analysis 
Thymoma is the most common tumor of the anterior mediastinum for which surgical resection is currently the primary form of treatment. An increase in the incidence of a small-sized (≤3 cm) thymoma (SST) has recently been noted. Clinicopathological factors and prognosis of SST have not been reported previously. In this study, the clinicopathological data of 21 SST patients were reviewed and podoplanin and Ki67 immunohistochemistry were assessed to determine the biological behavior of SSTs. Pathological diagnosis of SSTs revealed the following types: A (n=1), AB (n=8), B1 (n=5), B2 (n=6) and B3 (n=1). The Masaoka-Koga stages of 21 thymoma patients were I (n=16), II (n=3), III (n=1) and IVb (n=1). In the case of the stage IVb thymoma, phrenic nerve, mediastinal pleura invasion and anterior mediastinal lymph node metastasis were observed. The Ki67 labeling index of this stage IVb was found to be 3.2. This case was also positive for podoplanin and was one of the only 2 cases that were positive for podoplanin. This patient succumbed to thymoma. Advanced stage thymomas are possibly included in SSTs although the majority of SSTs are classified into early stages of disease. Findings of this study suggest that podoplanin analyzed by immunohistochemistry may be useful to determine the malignant behavior of SSTs.
PMCID: PMC3956257  PMID: 24649128
thymoma; small-sized; Ki67; podoplanin
20.  DDR2 polymorphisms and mRNA expression in lung cancers of Japanese patients 
Oncology Letters  2012;4(1):33-37.
Discoidin domain receptor 2, DDR2, is a tyrosine kinase receptor for fibrillar collagen that is involved in postnatal development, tissue repair and primary and metastatic cancer progression. Recently, mutations in the DDR2 kinase gene were identified in squamous cell lung cancer from large-scale Sanger sequencing. The present study investigated the DDR2 gene mutations and mRNA expression in surgically treated non-small cell lung cancer (NSCLC) of squamous histology cases. The presence or absence of DDR2 mutations at the kinase and discoidin domain was analyzed by direct sequencing. In this cohort, DDR2 mutations were not observed in the 166 patients with lung cancer, although DDR2 polymorphisms were observed (H136H, n=14) at the discoidin domain. mRNA levels of DDR2 in lung tumor samples and the adjacent normal lung samples were simultaneously analyzed. DDR2 mRNA levels were significantly decreased in tumor samples compared with normal lung samples. However, the DDR2 mRNA levels were elevated in the DDR2 polymorphism cases.
PMCID: PMC3398371  PMID: 22807955
discoidin domain receptor 2; mRNA expression; lung cancer; squamous cell carcinoma; polymorphism
21.  Braf and erbB2 mutations correlate with smoking status in lung cancer patients 
The erbB pathway involves a family of tyrosine kinases and contributes to resistance or sensitivity to chemotherapy in many tumor types. Somatic mutations of the epidermal growth factor receptor (EGFR) gene at the kinase domain have been found in lung cancer patients. These mutations are correlated with clinical response to targeted molecular therapy. Although Caucasian lung cancer patients have been shown to harbor Braf and erbB2 mutations, only a few reports exist concerning Braf and erbB2 mutations in Japanese lung cancer patients. We investigated the Braf and erbB2 mutation status in non-small cell lung cancer (NSCLC) patients by reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing. The study included 305 surgically removed lung cancer samples from the Nagoya City University Hospital, which were EGFR and Kras wild-type centric. Six Braf mutations were found in the adenocarcinoma cases. Among the adenocarcinoma cases, Braf mutations were more frequently noted in heavy smokers (Brinkman index >400, p=0.0476). We also detected five erbB2 mutations all in the non-smokers. All of these mutations existed exclusively. The erbB2 gene mutations were predominantly found in non-smokers with adenocarcinomas. However, the completely exclusive mutation status could help us design individually tailored targeted molecular therapy for lung cancer.
PMCID: PMC3438531  PMID: 22969966
Braf; epidermal growth factor receptor; lung cancer; mutations; erbB2; adenocarcinoma
22.  Thymoma associated with fatal myocarditis and polymyositis in a 58-year-old man following treatment with carboplatin and paclitaxel: A case report 
Oncology Letters  2011;3(2):300-302.
We report the case of a 58-year-old male presenting with giant cell myocarditis and myositis associated with thymoma following treatment with carboplatin and paclitaxel. The patient was diagnosed as having stage IVa thymoma. Acetylcholine receptor binding antibody titers were positive at 42 nmol/l, although the patient exhibited no symptoms of myasthenia gravis (MG). The patient was treated with a combination of carboplatin and paclitaxel. However, 18 days following administration of this second cycle of chemotherapy, the patient developed a low-grade fever. Twenty-one days after receiving the second cycle of chemotherapy, the patient was admitted to the Nagoya City University Medical School complaining of general fatigue. Serum levels of creatinine phosphokinase (7,271 U/l), alanine aminotransferase (469 U/l) and aspartate aminotransferase (561 U/l) were elevated. Electromyography revealed no evidence of a neuromuscular junction defect or myopathic process. The patient developed progressive muscle weakness and succumbed to the disease in hospital on day 9. An autopsy revealed thymoma invasion of the left parietal and visceral pleura, pericardium and diaphragm. Numerous skeletal muscle groups and myocardium exhibited diffuse lymphocytic infiltration. Although it has been suggested that myocardial disorders may occur in patients with thymoma and/or MG, the mechanism involved remains unknown. This second report may provide new data regarding giant cell myocarditis and myositis associated with thymoma following treatment with carboplatin and paclitaxel.
PMCID: PMC3362376  PMID: 22740899
thymoma; myocarditis; myositis; carboplatin; paclitaxel
23.  Increased Sox2 copy number in lung squamous cell carcinomas 
The transcription factor Sox2 is necessary for foregut morphogenesis. Sox2 is also required for the normal development of the trachea and lung. Recently, Sox2 amplifications were investigated using large-scale single nucleotide polymorphism arrays in esophageal and lung cancer. We hypothesized that Sox2 overexpression might be correlated with clinicopathological features of lung cancers. The increased copy number of the Sox2 gene was analyzed by real-time polymerase chain reaction amplifications in 127 surgically treated non-small cell lung cancer cases from Nagoya City University Hospital, Japan. A total of 87 squamous cell carcinoma (SCC) cases were involved. An increased Sox2 gene copy number was found in 42 (33.1%) lung cancer patients. Increased Sox2 copy number status was significantly correlated with gender (females, 9.5% vs. males, 34.1%; p=0.0026), smoking status (never smoker, 4.8% vs. smoker, 32.9%; p=0.0003) and pathological subtypes (squamous cell carcinoma, 44.8% vs. non-squamous cell carcinoma, 7.5%; p<0.0001). However, among the SCCs, the Sox2 copy number status was not significantly correlated with gender, smoking status, pathological stage or differentiation status. An increased Sox2 copy number is common within SCC.
PMCID: PMC3438854  PMID: 22969842
Sox2; lung cancer; squamous cell carcinoma; copy number; differentiation
24.  Expression of CD44v6 is an independent prognostic factor for poor survival in patients with esophageal squamous cell carcinoma 
Oncology Letters  2011;2(3):429-434.
CD44v6 has been causally associated with the development of metastases and with poor prognosis in various human malignancies. To elucidate the clinicopathological significance of CD44v6 expression in esophageal squamous cell carcinoma (ESCC), the present study aimed to investigate the expression of CD44v6 using immunohistological techniques. Using specific antibodies against CD44v6 and CD44s, expression of the proteins was analyzed immunohistochemically in 63 primary esophageal ESCCs, which were previously resected at the Nagoya City University Hospital without pre-operative induction therapy. Using light microscopy, the positive expression of CD44v6 was divided into a low- or high-expression group. The expression of CD44v6 in ESCC was analyzed with respect to various clinicopathological characteristics. The frequency of CD44v6 expression was 90.5% (57/63). The CD44v6 high-expression group comprised 55.6% of the patients (n=35) and the low expression group included 44.4% of the patients (n=28). In this study, no significant difference was observed between any clinicopathological factor and the immunohistochemical expression of CD44v6. In patients with high levels of CD44v6 expression, survival was markedly worse (p=0.0327). Favorable outcomes were observed for the clinicopathological characteristics of 6 patients whose tissue immunohistochemical expression of CD44v6 was not detected. Moreover, multivariate analysis confirmed that expression of CD44v6 was an independent prognostic indicator (risk ratio =2.793; p=0.0301). Overexpression of CD44v6 is a useful prognostic indicator of ESCC. Therefore, CD44v6 should be investigated as a potential target for therapy.
PMCID: PMC3410448  PMID: 22866099
CD44v6; esophageal squamous cell carcinoma; immunohistochemistry
25.  Decreased expression of FBXW7 is correlated with poor prognosis in patients with esophageal squamous cell carcinoma 
FBXW7 is a tumor suppressor gene that induces the degradation of positive cell-cycle regulators such as c-Myc, cyclin E, c-Jun and Notch. The loss of FBXW7 promotes cell-cycle progression and cell proliferation. In the present study, we investigated the relationship between FBXW7 expression and the clinicopathological characteristics of patients with esophageal squamous cell carcinoma (ESCC). The expression of FBXW7 was quantified by real-time reverse transcription polymerase chain reaction in 43 primary ESCCs and their paired normal esophageal mucosa in patients who had not received preoperative therapy. FBXW7 expression levels were significantly correlated with the progression of the cancer and with local invasiveness. In muscle-invasive tumor cases (T2–4), lymphatic invasive tumor cases and stage II–IV cases, FBXW7 expression levels were significantly decreased (P=0.0315, P=0.0336 and P=0.0289, respectively). Decreased expression of FBXW7 was correlated with poor prognosis (P=0.0255). In conclusion, this study examined the relationship between FBXW7 expression and tumor progression in ESCC. We suggest that FBXW7 is a molecular prognostic marker and can be used to elucidate the mechanism of carcinogenesis.
PMCID: PMC3445893  PMID: 22993608
esophageal cancer; prognosis; FBXW7

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