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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies 
Nature medicine  2012;18(3):382-384.
Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non–small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8–3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.
PMCID: PMC3916180  PMID: 22327622
8.  Characteristics of Lung Cancers Harboring NRAS Mutations 
We sought to determine the frequency and clinical characteristics of patients with lung cancer harboring NRAS mutations. We used preclinical models to identify targeted therapies likely to be of benefit against NRAS mutant lung cancer cells.
Patients and Methods
We reviewed clinical data from patients whose lung cancers were identified at 6 institutions or reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) to harbor NRAS mutations. 6 NRAS mutant cell lines were screened for sensitivity against inhibitors of multiple kinases (i.e. EGFR, ALK, MET, IGF-1R, BRAF, PI3K and MEK).
Among 4562 patients with lung cancers tested, NRAS mutations were present in 30 (0.7%; 95% confidence interval, 0.45% to 0.94%); 28 of these had no other driver mutations. 83% had adenocarcinoma histology with no significant differences in gender. While 95% of patients were former or current smokers, smoking-related G:C>T:A transversions were significantly less frequent in NRAS mutated lung tumors compared to KRAS-mutant NSCLCs (NRAS: 13% (4/30), KRAS: 66% (1772/2733), p<0.00000001). 5 of 6 NRAS mutant cell lines were sensitive to the MEK inhibitors, selumetinib and trametinib, but not to other inhibitors tested.
NRAS mutations define a distinct subset of lung cancers (~1%) with potential sensitivity to MEK inhibitors. While NRAS mutations are more common in current/former smokers, the types of mutations are not those classically associated with smoking.
PMCID: PMC3643999  PMID: 23515407
NRAS mutation; EGFR mutation; KRAS mutation; lung cancer; non-small cell lung cancer; driver mutation; MEK inhibitor; erlotinib; gefitinib; crizotinib
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  The landscape of somatic copy-number alteration across human cancers 
Nature  2010;463(7283):899-905.
A powerful way to discover key genes playing causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here, we report high-resolution analyses of somatic copy-number alterations (SCNAs) from 3131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across multiple cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κB pathway. We show that cancer cells harboring amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend upon expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in multiple cancer types.
PMCID: PMC2826709  PMID: 20164920
23.  Hypermethylation of the large tumor suppressor genes in Japanese lung cancer 
Oncology Letters  2010;1(2):303-307.
Large tumor suppressor (LATS) 1 and 2 are tumor suppressor genes implicated in the regulation of the cell cycle. The methylation statuses of the promoter regions of these genes were studied in Japanese lung cancers. The methylation statuses of the promoter regions of LATS1 and LATS2 were investigated by methylation-specific PCR. The findings were compared to clinicopathological features of lung cancer. Methylation-specific PCR showed that the LATS1 promoter region was hypermethylated in 95 out of 119 (79.8%) lung cancers. The methylation status of LATS1 was significantly associated with squamous histology (p=0.0267) and smoking status (never smoker vs. smoker; p=0.0399). LATS1-ummethylated patients harbored more EGFR mutations (p=0.0143). The LATS2 promoter region was hypermethylated in 160 out of 203 (78.8%) lung cancers. However, the methylation status had no association with the clinicopathological characteristics of the lung cancers cases. Both the LATS1 and LATS2 methylation statuses did not correlate with survival of lung cancer patients. Thus, the EGFR methylation status of the LATS genes has limited value in Japanese lung cancers.
PMCID: PMC3436364  PMID: 22966299
hypermethylation; large tumor suppressor gene; lung cancer
24.  Methylation of the DLEC1 gene correlates with poor prognosis in Japanese lung cancer patients 
Oncology Letters  2010;1(2):283-287.
The incidence of chromosome 3p gene alterations is one of the most frequent and earliest documented events in lung cancer. This study aimed to investigate promoter methylation in the deleted in lung and esophageal cancer 1 (DLEC1) gene, as well as the p16 and CDH1 genes in Japanese lung cancer cases. The methylation status of the promoter regions of DLEC1, p16 and CDH1 was investigated using methylation-specific PCR. The findings were compared to the clinicopathological features of lung cancer. Methylation-specific PCR showed that the DLEC1 promoter region was methylated in 65 out of 116 (56%) lung cancers. Patients with DLEC1-methylated cancer were associated with a significantly worse prognosis than those with unmethylated cancer (p=0.0368; hazard ratio=1.83). The p16 methylation status correlated with squamous histology (p=0.03) and smoking status (never smoker vs. smoker; p=0.0122). Patients with p16 ummethylated cancer harbored more EGFR mutations (p=0.0071). The CDH1 promoter region was hypermethylated in 65 out of 118 (55.1%) lung cancer cases. However, the CDH1 methylation status was not associated with the clinicopathological characteristics of the lung cancer types. p16 and CDH1 methylation status did not correlate with survival in the lung cancer patients. Thus, in our Japanese cohort, the methylation status of the DLEC1 gene was a marker of poor prognosis independent of stage.
PMCID: PMC3436471  PMID: 22966295
methylation; DLEC1 gene; lung cancer
25.  Frequently increased epidermal growth factor receptor (EGFR) copy numbers and decreased BRCA1 mRNA expression in Japanese triple-negative breast cancers 
BMC Cancer  2008;8:309.
Triple-negative breast cancer (estrogen receptor-, progesterone receptor-, and HER2-negative) (TNBC) is a high risk breast cancer that lacks specific therapy targeting these proteins.
We studied 969 consecutive Japanese patients diagnosed with invasive breast cancer from January 1981 to December 2003, and selected TNBCs based on the immunohistochemical data. Analyses of epidermal growth factor receptor (EGFR) gene mutations and amplification, and BRCA1 mRNA expression were performed on these samples using TaqMan PCR assays. The prognostic significance of TNBCs was also explored. Median follow-up was 8.3 years.
A total of 110 (11.3%) patients had TNBCs in our series. Genotyping of the EGFR gene was performed to detect 14 known EGFR mutations, but none was identified. However, EGFR gene copy number was increased in 21% of TNBCs, while only 2% of ER- and PgR-positive, HER2-negative tumors showed slightly increased EGFR gene copy numbers. Thirty-one percent of TNBCs stained positive for EGFR protein by immunohistochemistry. BRCA1 mRNA expression was also decreased in TNBCs compared with controls. Triple negativity was significantly associated with grade 3 tumors, TP53 protein accumulation, and high Ki67 expression. TNBC patients had shorter disease-free survival than non-TNBC in node-negative breast cancers.
TNBCs have an aggressive clinical course, and EGFR and BRCA1 might be candidate therapeutic targets in this disease.
PMCID: PMC2612006  PMID: 18950515

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