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.
APOBEC3B; mutation; lung cancer; epidermal growth factor receptor; LightCycler
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.
EGFR; recurrent; prognosis; tyrosine kinase inhibitor
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.
thymic carcinoma; thymidylate synthase; orotate phosphoribosyltransferase
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.
Keap1; NRF2; lung cancer; mutations; adenocarcinoma
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.
nuclear factor (erythroid derived 2)-like 2; polymorphism; NRF2; forced expiratory volume in 1 second; smoker
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.
Idiopathic pulmonary fibrosis; Acute exacerbation; Lung cancer; KL-6
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.
NRF2; lung cancer; squamous cell carcinoma; RagD; mutation
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.
ANGTL2; angiopoietin; lung cancer; metastasis; LightCycler
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.
programmed cell death 1 ligand 1; immunotherapy; lung cancer; tumor invasion; LightCycler
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.
epidermal growth factor receptor; polymorphism; lung cancer; gefitinib therapy; 8227G/A
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.
FISH; KIF5B/RET; lung cancer; RET expression
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.
thymoma; small-sized; Ki67; podoplanin
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.
discoidin domain receptor 2; mRNA expression; lung cancer; squamous cell carcinoma; polymorphism
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.
Braf; epidermal growth factor receptor; lung cancer; mutations; erbB2; adenocarcinoma
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.
thymoma; myocarditis; myositis; carboplatin; paclitaxel
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.
Sox2; lung cancer; squamous cell carcinoma; copy number; differentiation
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.
hypermethylation; large tumor suppressor gene; lung cancer
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.
methylation; DLEC1 gene; lung cancer