Yu Hikosaka

RET expression and detection of KIF5B/RET gene rearrangements in Japanese lung cancer

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.

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. non‑adenocarcinoma 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.

Evaluation of Kras Gene Mutation and Copy Number Gain in Non-small Cell Lung Cancer

Introduction: Recent studies for the characterization of the lung cancer genome have suggested that Kras gene was frequently amplified and correlated with activating mutations of Kras, which occur in approximately 5 to 10% of Japanese lung cancers. Methods: We analyzed Kras mutation and Kras copy number in 172 Japanese non-small cell lung cancer (NSCLC) cases and their relation to the survival of patients. We also studied using fluorescence in situ hybridization to provide direct evidence of Kras amplification in 40 clinical specimens. Results: In 172 NSCLC cases, increased Kras copy number existed in 19 (11.0%) cases. Increased Kras gene copy number was correlated with Kras mutation. Nevertheless, Kras gene copy number gain was not correlated with gender, pathological subtypes, stages, and smoking status. Increased Kras copy number was not associated with overall survival in these 172 cases; however, patients with increased Kras copy number and Kras mutant had significantly worse prognosis, when compared with patients with Kras wild type and Kras not increased. From the fluorescence in situ hybridization analysis, Kras polysomy or amplified patients showed significantly worse prognosis, when compared with Kras disomy patients. Conclusion: Kras mutation plus increased copy number was a predictor of poor clinical outcome in patients with NSCLC.

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.

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.

Excision Repair Cross Complementation Group 1 Polymorphisms Predict Overall Survival after Platinum-Based Chemotherapy for Completely Resected Non-Small-Cell Lung Cancer

BACKGROUND It has been reported that the expression of excision repair cross-complementation group 1 (ERCC1) protein predicts the effect of platinum-based chemotherapy and overall survival in the several cancers. And there are some reports suggesting that the polymorphism of the ERCC1 may predict the effect of platinum-based chemotherapy and survival of the patients. We have already reported that the expression of ERCC1 protein predicts survival after platinum-based chemotherapy for 90 completely resected non-small-cell lung cancers (NSCLC). MATERIALS AND METHODS We investigated the ERCC1 polymorphisms (C8092A and C118T) whether these factors influence for the prognosis of these 90 NSCLC patients treated with platinum-based chemotherapy. RESULTS Two of the ERCC1 polymorphisms, C8092A and C118T, affected the prognosis of the NSCLC patients who received adjuvant and/or neoadjuvant platinum-based chemotherapy. The wild type, C/C of the codon 8092, was associated with better prognosis than C/A or A/A types (P=0.0154) and the wild type C/C of the codon 118 was associated with better prognosis than C/T or T/T types (P=0.0307). This effect was not seen in an independent group of 55 completely resected NSCLC patients who were treated with surgery alone. The combination of low expression of ERCC1 protein together with the C/C type codon 8092 and C/C type codon 118 polymorphism of the ERCC1 gene was associated with the best prognosis. CONCLUSIONS Our data seem to suggest that the ERCC1 protein expression and polymorphism of ERCC1 may predict the survival of patients who are treated with platinum-based chemotherapy.

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.

Number of recurrent lesions is a prognostic factor in recurrent thymoma

In advanced stage thymomas, recurrence is not uncommon but prognostic factors in recurrent thymoma have not been determined and standardized treatment for recurrence has not yet been established. A retrospective analysis was conducted on 24 thymoma patients who underwent treatment for recurrence in our institution to determine the prognostic factors for overall survival. Recurrence of thymoma appeared 11.6-109.6 months after the primary operation (34.6±25.7 months). Pleural disseminated recurrence was common (n=21) as the primary recurrent lesions. Single or combined modality therapy was performed in 19 patients; surgical resection in 12, radiotherapy in 10, and chemotherapy in six patients. A third surgical resection was performed in two patients. There was no difference in overall survival between the groups with or without treatment nor in those with or without resection. Old age and chemotherapy were factors for poorer prognosis. Patients with one or two recurrent lesions detected on CT examinations showed better prognosis. Prognosis in thymoma patients with recurrence was reviewed in the present study. Patients with a small number of recurrent lesions showed better prognosis irrespective of the treatment.

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.

NFE2L2 Gene Mutation in Male Japanese Squamous Cell Carcinoma of the Lung

Introduction: Recently, the nuclear factor (erythroid derived 2)-like 2 (NFE2L2) gene mutations were identified in lung cancer. The constitutive activation of NFE2L2 in lung cancer cells promotes tumorigenicity. However, the correlation between NFE2L2 mutation status and clinicopathologic features of lung cancer has not been well characterized. Methods: We have investigated NFE2L2 gene mutation status in 263 surgically treated lung cancer cases at Nagoya City University Hospital. The NFE2L2 mutation was analyzed by direct sequencing of cDNA. Results: We detected 13 cases (5.1%) of NFE2L2 mutation in our cohort; all were male and all had a squamous histology. EGFR mutations were present in 78 patients (30.8%). The NFE2L2 mutation was exclusive with EGFR mutations. The NFE2L2 mutation tended to be more frequently found in patients with advanced stages. The patients with NFE2L2 mutation (n = 13, 8 were dead) had significantly worse prognosis than the patient with wild type NFE2L2 (n = 250, 72 were dead) (Log-rank test, p = 0.0032, Breslow-Gehan-Wilcoxon test, p = 0.0028). Conclusion: NFE2L2 mutations might play a role in tumor prognosis of squamous cell carcinoma of the lung.