[A Patient with Lung Adenocarcinoma, Lymphangitis Carcinomatosa, and Multiple Bone Metastases Who Achieved Long-Term Survival after Successful Treatment with Carboplatin, Paclitaxel, and Bevacizumab].

BACKGROUND: Lymphangitis carcinomatosa of the lung is intractable and associated with a poor prognosis. CASE: A 53-year-old woman was admitted to our hospital due to an uncomfortable feeling on deep inspiration. She was diagnosed with left lung adenocarcinoma with lymphangitis carcinomatosa and bone metastases to the frontal bone of the skull and thoracic vertebrae. The lung carcinoma was positive for an EGFR mutation. Because the patient's performance status (PS) was 0, carboplatin plus paclitaxel plus bevacizumab therapy was initiated and she received zoledronic acid and concurrent radiation therapy of 40 Gy for the metastasis to the thoracic vertebrae. After 2 courses of treatment, the respiratory symptoms had improved. After 6 courses of treatment, a chest CT indicated that the lymphangitis carcinomatosa had disappeared. The serum CEA level, which was 126.2 ng/mL (normal<5.0) before treatment, reduced to 5.0 ng/mL. She was administered 10 courses of bevacizumab as a maintenance therapy; however, the CEA level rose again to 11.7 ng/mL, the lung tumor volume increased, and the metastasis of the frontal bone deteriorated. As second-line chemotherapy, EGFR-TKI was started. However, after 11 months, because of grade 4 liver dysfunction, EGFR-TKI was stopped. She then received fourth-line chemotherapy in our outpatient hospital. This patient has survived 52 months since the initial diagnosis. CONCLUSION: Chemotherapy including bevacizumab facilitated long-term survival (52 months) of a patient with lung adenocarcinoma accompanied by lymphangitis carcinomatosa and multiple bone metastases.

A safe method for marking small pulmonary nodules with crystal violet.

INTRODUCTION: The number of cases of wedge resection of small-sized pulmonary nodules performed under video-assisted thoracoscopic surgery (VATS) is increasing. Computed tomography (CT)-guided marking with hook wires has been used to locate the nodules that are not identifiable under VATS. However, this method is invasive and is associated with a risk of complications. METHODS: We evaluated the usefulness of marking the pleural surface above the nodule using crystal violet for 22 small-sized pulmonary nodules. Following the collapse of the lung, a long stick with a cotton tip dipped in crystal violet was inserted from the thoracic port or a small thoracotomy, and was placed against the inside of the chest wall right above the nodule with reference to the preoperative CT image. The lung was then expanded, and the crystal violet-infiltrated tip stained the visceral pleura. Regardless of the marking point, wedge resection of the lung was performed. To evaluate the accuracy of the marking, we measured the distance from the center of the marking to the point on the visceral pleural nearest to the nodule (DMN) in the resected lung specimen. RESULTS: This marking method caused no morbidity during or after the operation. The DMN ranged between 0 and 50 mm (mean ± SD 18.2 ± 12.6 mm). In 18 of 22 cases (81.8%), the DMN was 20 mm or less. CONCLUSIONS: The intraoperative marking method using crystal violet was performed with reasonable accuracy. It also caused no morbidity. It was easy and non-invasive. This method can be used in the cases in which CT-guided percutaneous marking is not feasible due to the nodule's location.

Clinicopathological analysis of small-sized anterior mediastinal tumors.

PURPOSE: The purpose of this study was to determine the clinicopathological findings and prognosis of small-sized anterior mediastinal tumors (SSAMTs). METHODS: A retrospective study was conducted on 43 patients who underwent surgery between January 1989 and December 2011 for SSAMTs. RESULTS: From the preoperative radiological findings, the tumors were classified into solid (n = 28) and cystic lesions (n = 15). The pathological diagnoses of the solid lesions included thymoma (n = 24), thymic carcinoma (n = 1), mucosa-associated lymphoid tissue lymphoma (n = 1), teratoma (n = 1) and neurofibroma (n = 1), and those of the cystic lesions included thymic cysts (n = 8), thymoma (n = 3), bronchogenic cysts (n = 2), teratoma, (n = 1) and a pericardial cyst (n = 1). The 27 thymomas were composed of stages I (n = 22), II (n = 3), III (n = 1) and IVb (n = 1). The overall survival in the 43 patients was 97.1 % at 5 years. In the 28 patients with solid lesions, the overall survival was 95.8 % at 5 years. All patients with cystic lesions were still alive at the last follow-up. CONCLUSION: Cystic lesions of SSAMTs were benign lesions or stage I thymoma, and most of the solid lesions of SSAMTs were stage I or II thymomas. SSAMTs are good candidates for video-assisted thoracic surgery procedures, as conversion to sternotomy can be selected based on the intraoperative findings of pericardial invasion and a rapid pathological diagnosis of thymic carcinoma.

Talc pleurodesis for secondary pneumothorax in elderly patients with persistent air leak.

BACKGROUND: We herein report the usefulness of two types of talc pleurodesis for secondary pneumothorax of elderly patients with persistent air leak who have severe pulmonary emphysema. METHODS: We assessed 17 elderly patients with persistent air leak who received talc pleurodesis for secondary pneumothorax from April 2013 to March 2017. Thoracoscopic talc poudrage (TTP) (n=11) was performed in patients whose general condition was thought to sufficiently stable to tolerate for general anesthesia. Talc slurry pleurodesis (TSP) (n=6) via a chest tube was performed in patients whose general condition was thought to be insufficiently stable to tolerate general anesthesia. RESULTS: The median drainage period after pleurodesis was 6 days in patients who received TTP and 12 days in patients who received TSP. Complications associated with talc pleurodesis included atrial fibrillation (n=1) in the thoracoscopic poudrage group, while the slurry pleurodesis group showed chest pain (n=2), asthmatic attack (n=1), and pneumonia (n=1). All patients who received thoracoscopic poudrage were able to leave the hospital after removal of the chest tube. Five of the six patients who received slurry pleurodesis were able to leave the hospital, but one of them died of acute exacerbation of interstitial pneumonia (IP) on the 45th day after pleurodesis. The success rate was 94% (16/17). There were no cases of recurrence during the observation period. CONCLUSIONS: TTP was deemed likely to be safe and effective for patients able to tolerate general anesthesia. In patients with IP, especially those treated with steroids, the indication of talc pleurodesis should be cautiously considered.

A new approach to left sleeve pneumonectomy: complete VATS left pneumonectomy followed by right thoracotomy for carinal resection and reconstruction.

BACKGROUND: Left sleeve pneumonectomy is a challenging operation that requires individualized approaches. Here, we present a new minimally invasive combined thoracoscopic approach. CASE PRESENTATION: A 61-year-old woman was diagnosed with tracheobronchial adenoid cystic carcinoma. The tumor originated from the left main stem bronchus, and tumor with carinal involvement was observed. We judged that complete resection would be possible via left sleeve pneumonectomy. However, because tumor involvement with the esophagus and descending aorta was suspected, evaluation of resectability in advance was necessary. After confirmation via examination thoracoscopy of no involvement with the surrounding organs, complete VATS left pneumonectomy was performed and followed by right thoracotomy for carinal resection and reconstruction. CONCLUSIONS: When thoracoscopic surgery becomes mainstream, this minimally invasive combined thoracoscopic approach might be an optimal option for patients who require left sleeve pneumonectomy.

Post-irradiation constrictive pericarditis following thymoma treatment: a report of two cases.

Whole pericardial irradiation may be performed for mediastinal malignancies with pericardial dissemination or malignant pericardial effusion. Case 1 is a 57-year-old woman with a B1 thymoma, Masaoka stage IVa. She underwent whole pericardial irradiation and suffered post-irradiation constrictive pericarditis 3 years later. Diuresis, catecholamine infusions, drainage, and pericardiectomy were performed. However, she died of heart failure after 4 years and 1 month due to constrictive pericarditis. Case 2 is a 56-year-old woman with myasthenia gravis and a B2 thymoma, Masaoka stage III. She underwent an extended thymectomy with partial resection of the lung and pericardium and received adjuvant radiation therapy of the whole pericardium. She was affected by post-irradiation constrictive pericarditis 7 months later, for which she underwent pericardiectomy. However, her constrictive pericarditis remained. In conclusion, we report two advanced thymoma cases with post-irradiation constrictive pericarditis. Indicators for whole pericardial irradiation should be determined strictly and carefully.

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.

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.

Genetic profiling of thymic carcinoma using targeted next-generation sequencing.

OBJECTIVES: Thymic carcinoma is a rare mediastinal neoplasm and little is known about its tumorigenesis. There is no effective treatment except for complete resection, and the prognosis of advanced cases is poor. To identify the mutations associated with tumorigenesis, we analyzed genetic profile of thymic carcinoma using targeted next-generation sequencing. MATERIALS AND METHODS: We sequenced about 409 cancer-related genes in 12 thymic squamous cell carcinoma tissues including 10 tumor/normal tissue pairs using Ion AmpliSeq Cancer Panel and Ion PGM Sequencer. We filtered the mutations with Ingenuity Variant Analysis, SIFT, PolyPhen-2, and PROVEAN. RESULTS AND CONCLUSION: Twenty-five candidate mutations in 24 genes were identified, including five tyrosine kinase genes (KIT, DDR2, PDGFRA, ROS1, IGF1R). There was no recurrent mutation among the samples studied. The KIT exon 11 deletion mutation in 1 patient was an activating mutation and may be an oncogenic driver mutation. Genetic profiling of thymic carcinoma using targeted next-generation sequencing was performed. The mutation status of thymic squamous cell carcinoma is highly heterogeneous.

Prognosis of recurrent non-small cell lung cancer following complete resection.

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.

APOBEC3B gene overexpression in non-small-cell lung cancer.

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/ß-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/ß-actin mRNA levels was not different within the gender, age, smoking status and pathological stages. The T/N ratio of APOBEC3B/ß-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.

Genotype analysis of the NRF2 gene mutation in lung cancer.

Nuclear factor (erythroid derived 2)-like 2 (NRF2, gene name NFE2L2) gene mutations have been previously identified in lung cancers. The constitutive activation of NRF2 resulting from gene mutations has been correlated with the poor prognosis of patients with squamous cell lung cancer. However, DNA sequencing using PCR methods described to date is time-consuming and requires significant quantities of DNA. Thus, this existing approach is not suitable for a routine pre-therapeutic screening program. We genotyped the NRF2 gene mutation status in 262 surgically treated lung cancer cases using LightCycler analysis. The presence of the NRF2 gene mutation was confirmed by direct sequencing. We detected 6 cases (2.3%) with NRF2 gene mutations in our cohort, particularly smokers (P=0.04) with squamous histology (P=0.0001). NRF2 gene mutations were present in 10% (6/60) of the lung squamous cell carcinoma (SqCC) cases. The NRF2 gene mutation was exclusive of epidermal growth factor receptor mutations. The NRF2 gene mutation occurred with a tendency towards a higher frequency in male patients. Patients with the NRF2 gene mutation (n=22, 11 succumbed to disease) had a significantly worse prognosis when compared with the patients with the wild-type NRF2 gene (n=521, 98 succumbed to disease) from a larger cohort study (log-rank test, P<0.0001) even upon multivariate analysis. In our study, NRF2 gene mutations played a role in the prognosis of patients with SqCC of the lung.

Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in Japanese lung adenocarcinoma.

PURPOSE: Mutations in components of the mitogen-activated protein kinase (MAPK) cascade may be a new candidate for target for lung cancer. The usefulness of immunohistochemistry (IHC) as a new approach for the detection of BRAF V600E in cancer patients has been recently reported. METHODS: To increase the sensitivity, we modified BRAF V600E expression detection assay by IHC using mutation specific antibody. From the screening step, we found a novel 599 insertion T BRAF mutation in lung adenocarcinoma. In this study included 26 surgically removed cases with EGFR, Kras, erbB2, EML4-ALK and KIF5B-RET wild-type (wt) lung adenocarcinomas, including 7 BRAF mutants (5 V600E, 1 N581I, and 1 novel 599 insertion T mutation) analyzed by DNA sequencing. Detection of the BRAF V600E mutation was carried out by the Dako EnVision™ FLEX detection system using the VE1 clone antibody and compared with the results of direct sequencing. RESULTS: The autostainer IHC VE1 assay was positive in 5 of 5 (100%) BRAF V600E-mutated tumors and negative in 20 of 21 (95.2%) BRAF non-V600E tumors, except for a novel 599 insertion T case. CONCLUSION: IHC using the VE1 clone and FLEX linker is a specific method for the detection BRAF V600E and may be an alternative to molecular biology for the detection of mutations in lung adenocarcinomas. This method might be useful for screening to use molecular target therapy for lung adenocarcinomas.

PD-L1 gene expression in Japanese lung cancer patients.

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/ß-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/ß-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/ß actin mRNA levels among factors inlcuding gender, age, smoking status and pathological subtypes. The T/N ratio of PD-L1/ß 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.

Polymorphisms of NRF2 gene correlated with decreased FEV1 in lung cancers of smokers.

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.

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.

Keap1 mutations in lung cancer patients.

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.

RagD gene expression and NRF2 mutations in lung squamous cell carcinomas.

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 ß-actin mRNA levels. Mean RagD/ß-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/ß-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/ß-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.

Increased FGFR1 copy number in lung squamous cell carcinomas.

The basic fibroblast growth factor (bFGF), via activation of its receptor, FGFR1, has been postulated to be an important inducer of host stromal response and angiogenesis. More recently, FGFR1 amplifications were investigated using large-scale single nucleotide polymorphism arrays in lung cancer. We hypothesized that FGFR1 overexpression may be correlated with the clinicopathological features of lung cancers. The increased copy number of the FGFR1 gene was analyzed by real-time polymerase chain reaction amplifications in 100 surgically treated non-small cell lung cancer cases from Nagoya City University Hospital. Sixty-five squamous cell carcinoma cases were included. An increased FGFR1 gene copy number was found in 32 (32%) lung cancer patients. The increased FGFR1 copy number status significantly correlated with gender (females 13.8% vs. males 39.4%, p=0.0173), smoking status (never smoker 4.2% vs. smoker 40.8%, p=0.0004) and pathological subtypes (squamous cell carcinoma 41.5% vs. non-squamous cell carcinoma 14.3%, p=0.0066). However, within the squamous cell carcinomas the FGFR1 copy number status did not significantly correlate with gender, smoking status, pathological stages and differentiation status of the lung cancers. Thus, the FGFR1 copy number is common within squamous cell carcinoma.

Overexpression of GLUT1 correlates with Kras mutations in lung carcinomas.

Glucose is the major source of energy for cells, and glucose transporter 1 (GLUT1) is the most common glucose transporter. GLUT1 has been found to be aberrantly expressed in several tumor types. From the results of the microarray and serial analysis of gene expression (SAGE), GLUT1 transcript expression was found to be higher in clones with mutant Kras alleles. We hypothesized that GLUT1 overexpression might be correlated with clinicopathological features of Japanese lung cancers. Immunohistochemistry for GLUT1 was performed in 283 surgically treated non-small cell lung cancer (NSCLC) cases from Nagoya City University Hospital. Thirty-six Kras mutant carcinoma cases were included. GLUT1 overexpression was found in 138 (48.8%) lung cancer patients. The GLUT1 overexpression status was significantly correlated with gender (women 31.9% vs. men 54.5%, P<0.0001), smoking status (never smoker 31.4% vs. smoker 59.4%, P<0.0001) and pathological subtypes (adenocarcinoma 36.4% vs. non­adenocarcinoma 74.5%, P<0.0001). In addition, the GLUT1 overexpression status was significantly correlated with gene mutation status, including EGFR (mutation-positive 23.4% vs. -negative 58.3%, P<0.0001) and Kras (mutation-positive 66.7% vs. -negative 46.6%, P=0.038). The survival of patients with GLUT1 overexpression (n=137, 50 were deceased) was significantly worse when compared to the patients with normal expression of GLUT1 (n=142, 31 were deceased) (Log-rank test, P=0.0009). Thus, GLUT-1 overexpression correlates with an aggressive phenotype of lung carcinoma.

Increased NRF2 gene (NFE2L2) copy number correlates with mutations in lung squamous cell carcinomas.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor belonging to the cap 'n' collar subfamily of the basic-leucine zipper (bZIP) family of transcription factors, which plays a significant role in adaptive responses to oxidative stress. Previously, we reported that NRF2 gene (NFE2L2) mutations correlate with poor prognosis of lung squamous cell carcinomas. We therefore hypothesized that an increased NRF2 gene copy number may correlate with clinicopathological features in lung cancer patients. In this study, the increased copy number of the NRF2 gene was analyzed by real-time polymerase chain reaction (real-time-PCR) amplifications in 90 surgically-treated non-small cell lung cancer (NSCLC) cases. In total, 16 NRF2 mutation cases were included. An increased NRF2 gene copy number was found in 7 (7.8%) lung squamous cell carcinoma patients. Increased NRF2 copy number status significantly correlated with mutation status (mutant, 31.25% vs. wild-type, 2.7%; p=0.0017). The mean NRF2 gene copy number was significantly higher in mutant (2.478 ± 0.668) compared to wild-type NRF2 (1.917 ± 0.737) (p=0.0048). However, the copy number did not correlate with smoking status (p=0.3741), gender (p=0.1545), age (≥65 vs. <65, p=0.1237) and pathological stage. Although an increased NRF2 copy number correlates with mutations in squamous cell carcinoma, the percentage of the increased copy number was low; therefore, another mechanism may exist for the activation of NRF2 mutations in cancer.