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Purpose: The resistance to the EGFR tyrosine kinase inhibitors (TKI) is a major concern in non–small cell lung cancer (NSCLC) treatment. T790M mutation in EGFR accounts for nearly 50% of the acquired resistance to EGFR-TKIs. Earlier studies suggested that T790M mutation was also detected in TKI-naïve NSCLCs in a small cohort. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment T790M in a larger cohort.Experimental Design: ddPCR was established as follows: wild-type or T790M mutation-containing DNA fragments were cloned into plasmids. Candidate threshold was identified using wild-type plasmid, normal human genomic DNA, and human A549 cell line DNA, which expresses wild type. Surgically resected tumor tissues from 373 NSCLC patients with EGFR-activating mutations were then examined for the presence of T790M using ddPCR.Results: Our data revealed a linear performance for this ddPCR method (R2 = 0.998) with an analytical sensitivity of approximately 0.001%. The overall incidence of the pretreatment T790M mutation was 79.9% (298/373), and the frequency ranged from 0.009% to 26.9%. The T790M mutation was detected more frequently in patients with a larger tumor size (P = 0.019) and those with common EGFR-activating mutations (P = 0.022), as compared with the others.Conclusions: The ultra-sensitive ddPCR assay revealed that pretreatment T790M was found in the majority of NSCLC patients with EGFR-activating mutations. ddPCR should be utilized for detailed assessment of the impact of the low frequency pretreatment T790M mutation on treatment with EGFR-TKIs. Clin Cancer Res; 21(15); 3552–60. ©2015 AACR.

Supplementary Figures S1-8. Figure S1: Flowchart for mutation call. Figure S2: Quantification of performance of ddPCR analysis for EGFR T790M mutation. Figure S3: Analytical sensitivity of 0.001% for the ddPCR assay. Figure S4: Determination of false-positive events from wild-type control DNA and normal human genomic DNA. Figure S5: Determination of false-positive events in genomic DNA from formalin-fixed paraffin-embedded (FFPE) A549 cells. Figure S6: Detection of EGFR T790M mutant alleles in patients with EGFR T790M-containing primary tumors. Figure S7: Comparison of T790M mutation detection in 6 samples by different operators. Figure S8: Reproducibility of ddPCR analysis from 16 T790M-positive samples.

Supplementary Materials and Methods, Reference and Supplementary Figure Legends

Supplementary Tables S1-5. Table S1: Primers and probes used for droplet digital PCR (ddPCR). Table S2: Determination of the limit of blank (LOB) from wild-type control DNA, normal human genomic DNA, and EGFR wild-type A549 genomic DNA. Table S3: Determination of the limit of blank (LOB) using genomic DNA from adjacent normal tissue samples of EGFR-activating mutation positive adenocarcinoma and EGFR wild-type squamous cell carcinoma. Table S4: Concordance between ddPCR and deep sequencing of EGFR T790M mutation. Table S5: Distribution of amplifiable DNA concentrations in the 377 analyzed samples.

This interim report presents the results of pleurodesis with 50 ml of 50% glucose solution for patients with inoperable pneumothorax. Twenty patients were enrolled, and treatment was performed for 22 sites in total. The degree of lung collapse was mild in 2 cases, moderate in 12 cases, and severe in 8 cases. The mean number of treatments was 1.4 times (range 1~3). Mild chest pain after injection occurred in one case, and additional chest tube insertion was required for pleural effusion in one case. Other side effects, such as fever or dehydration, were not observed. On day one the mean blood glucose level was 145.0 mg/dl (range 103~259), and the mean pleural effusion volume was 284.6 ml (range 5~910). The air leakage was successfully controlled in 20 of the 22 sites( 91%). Pleurodesis in this manner was thought to be useful intervention for inoperable patients with pneumothorax.

Background The aim of this study was to compare outcomes of primary treatment with stereotactic body radiation therapy (SBRT) versus sublobar resection (SLR) for clinical stage I non-small cell lung cancer (NSCLC) in patients with medical comorbidities. Methods Consecutive patients who underwent SBRT (n = 106) or SLR (100 wedge resection, 41 segmentectomy) because of medical comorbidities associated with stage I NSCLC were enrolled. Lesions located in the outer third of the lung field on computed tomography were defined as external, and others were defined as internal. A propensity score–matched analysis was also performed that compared SBRT and SLR results. Charts were reviewed to determine local tumor recurrence, disease-specific survival (DSS), and overall survival (OS). Results A propensity score–matched analysis, recurrence-free survival (RFS) became significant in favor of surgery (p = 0.036). For large nodules of greater than 2.0 cm in diameter, RFS was significantly better in the surgery group (p = 0.042). No significant differences in OS, DSS, or RFS were observed with small nodules of less than 2.0 cm in diameter. In the external group, a higher recurrence rate was seen for SBRT group. For internal group, there was no statistical difference between each treatment. Local recurrence rate was higher in the SBRT group (p = 0.0082) in the external group. Conclusions In a matched comparison of stage I NSCLC in patients with medical comorbidities, RFS was in favor of surgery comparing SBRT, but there were no significant differences in OS or DSS. The tumor size and tumor location should be considered before deciding whether to perform SBRT or surgery.

An 84-year-old man was referred to our out-patient clinic with an elongated mass localized to the retrosternal area that was incidentally identified by computed tomography. On 18F-fluorodeoxyglucose-positron emission tomography, this lesion showed intense tracer uptake. Thus, a surgical biopsy under thoracoscopy was performed. Histological examination revealed dense fibrous tissue associated with inflammatory cell infiltration. The immunoglobulin (Ig) G4/IgG plasma cell ratio was over 90%. Serum IgG4 levels were normal. According to the Umehara criteria for IgG4-related disease, a final diagnosis of a "possible" IgG4-related fibrosing mediastinitis was made. Oral glucocorticoid treatment with 30 mg/day prednisolone reduced the mass.

A 65-year-old woman presented with mycosis fungoides and an anterior mediastinal tumor. Stage Ⅱa mycosis fungoides was treated with bath psoralen plus ultraviolet A, topical corticosteroids, and oral bexarotene. One month later, a surgical resection was performed for the anterior mediastinal tumor, which was a stage Ⅱ thymoma with membrane invasion. Furthermore, adjuvant radiotherapy was performed for anterior mediastinum. The mycosis fungoides lesion exacerbated after 3 months;thus, chemotherapies were performed. The patient died of respiratory insufficiency due to multiple pulmonary metastases of mycosis fungoides 1 year after the operation.

The purpose of this study was to validate the ability of the mean computed tomography (m-CT) value to predict tumor invasiveness and recurrence, and further, to compare with other measurements such as consolidation/tumor ratio and solid tumor size.A retrospective study was conducted of 494 patients with clinical stage IA lung cancer who had peripherally located lung adenocarcinoma. Receiver operating characteristic curve analysis was used to compare the ability to predict tumor invasiveness and recurrence between m-CT value, consolidation/tumor ratio, and tumor size. Multiple logistic regression analyses were performed to determine the independent variables for the prediction of pathologic, less invasive lung cancer. Disease-free survival was measured from the date of the operation until any recurrence.The m-CT values were 643.6 ± 9.4 Hounsfield units in the noninvasive cancer group and 365.9 ± 11.4 Hounsfield units in the invasive cancer group (p0.0001). The invasive cancer group was strongly associated with a high CT attenuation value, high consolidation/tumor ratio, large solid tumor size, large tumor size, and high standardized uptake value. Multiple logistic analyses, including the preoperatively determined variables, revealed that standardized uptake value and m-CT are independent predictive factors of less invasive lung cancer. In addition, the hazard ratio of the m-CT value was higher than that of the standardized uptake value value.The evaluation of m-CT value is useful in predicting less invasive lung cancer. The m-CT value can potentially determine operative procedure, particularly limited resection for peripheral lung adenocarcinoma.

The MDM2 protein plays an important role in the regulation of cell proliferation and apoptosis via ubiquitination and proteasome‐mediated degradation of p53. The genetic polymorphism rs2279744 (c.309T>G) of the MDM2 gene is reportedly associated with susceptibility and/or prognosis in various cancers. In this study, we investigated the risk factors for worse survival in patients with lung adenocarcinoma (AC). We examined the association between c.309T>G and the prognosis of lung cancer by retrospectively reviewing 453 lung cancer patients. We studied both, clinicopathological and genetic characteristics, including the c.309T>G, p53 Arg72Pro, EGFR,KRAS, and p53 mutations. Associations between these factors and survival outcome were analyzed using Cox proportional hazards models. The frequencies of MDM2 polymorphisms were T/T, 20.8%; T/G, 48.6%, and G/G, 30.7%. The overall survival (OS) of AC patients with pathological stage I disease and the MDM2 T/T genotype was significantly shorter than that of those with the T/G or G/G genotypes (P = 0.02). Multivariate analysis revealed that the MDM2 T/T genotype was an independent, significant prognostic factor (hazard ratio [HR] = 2.23; 95% confidence interval [CI]: 1.07–4.65; P = 0.03). The MDM2 T/T genotype was predictive of poorer survival in a Japanese population. Genotyping for this polymorphism might predict the clinical outcomes of stage I AC patients.

Purpose A deletion polymorphism of the Bim gene has been reported to be a prognostic factor for patients with non–small-cell lung cancer (NSCLC) treated with epidermal growth factor receptor-tyrosine kinase inhibitors in the Asian population. We investigated the impact of the Bim deletion polymorphism on survival among patients with completely resected NSCLC. Patients and Methods The Bim polymorphism was detected by polymerase chain reaction analysis. We measured overall survival (OS) and recurrence-free survival rates in 411 patients and postrecurrence survival (PRS) in 94 patients who experienced recurrence and received additional anticancer therapy. Results The Bim deletion polymorphism was detected in 61 patients (14.8%). OS rates were significantly lower for patients with the Bim deletion polymorphism than for those with the wild-type sequence. On multivariable analysis, the Bim deletion polymorphism was identified as an independent prognostic factor for OS (hazard ratio, 1.98; 95% CI, 1.17 to 3.36; P = .011). Among the 94 patients who experienced recurrence and were treated with anticancer therapy, patients with the Bim deletion polymorphism showed significantly poorer PRS than those with the wild-type sequence (median, 9.8 months v 26.9 months, respectively; P < .001). Multivariable analysis revealed that the Bim deletion polymorphism was an independent predictor of PRS (hazard ratio, 3.36; 95% CI, 1.75 to 6.47; P < .001). This trend remained apparent in subgroup analyses stratified by EGFR status, histology, and therapeutic modality. Conclusion The Bim deletion polymorphism is a novel indicator of shortened PRS among patients with recurrent NSCLC treated with anticancer therapy in the Asian population.

The aim of this study was to identify risk factors for recurrence in non-small cell lung cancer (NSCLC) patients with lymph node metastases after surgical resection.We reviewed 66 consecutive patients with surgically resected NSCLC who had pathologically proven positive lymph nodes (pN1 or pN2). All patients underwent a preoperative 2-[(18)F]-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) evaluation. We analyzed the recurrence-free survival (RFS) and recurrence-free proportion (RFP) according to the clinicopathological factors.A total of 27 patients were pathologically N1 and 39 were N2. The 5-year overall survival rate and the RFS rate were 47.2 and 27.7 %, respectively. The cut-off values for the SUVmax of the tumor and the lymph node ratio (LNR) were determined to be 6.5 and 0.12, respectively, using a receiver operating characteristics curve analysis. Both univariate and multivariate analyses revealed three significant independent factors for RFS: namely, the SUVmax of the tumor, the LNR, and the use of adjuvant chemotherapy. Only the SUVmax was an independent significant predictor of the RFP.Both the SUVmax and the LNR can serve as prognostic factors for patients with pN + NSCLC. Our study suggests that the LNR could be a stronger prognostic factor than the N classification of the TNM system and the SUVmax may predict recurrence in node-positive NSCLC patients.

Purpose: The resistance to the EGFR tyrosine kinase inhibitors (TKI) is a major concern in non–small cell lung cancer (NSCLC) treatment. T790M mutation in EGFR accounts for nearly 50% of the acquired resistance to EGFR-TKIs. Earlier studies suggested that T790M mutation was also detected in TKI-naïve NSCLCs in a small cohort. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment T790M in a larger cohort. Experimental Design: ddPCR was established as follows: wild-type or T790M mutation-containing DNA fragments were cloned into plasmids. Candidate threshold was identified using wild-type plasmid, normal human genomic DNA, and human A549 cell line DNA, which expresses wild type. Surgically resected tumor tissues from 373 NSCLC patients with EGFR-activating mutations were then examined for the presence of T790M using ddPCR. Results: Our data revealed a linear performance for this ddPCR method (R2 = 0.998) with an analytical sensitivity of approximately 0.001%. The overall incidence of the pretreatment T790M mutation was 79.9% (298/373), and the frequency ranged from 0.009% to 26.9%. The T790M mutation was detected more frequently in patients with a larger tumor size (P = 0.019) and those with common EGFR-activating mutations (P = 0.022), as compared with the others. Conclusions: The ultra-sensitive ddPCR assay revealed that pretreatment T790M was found in the majority of NSCLC patients with EGFR-activating mutations. ddPCR should be utilized for detailed assessment of the impact of the low frequency pretreatment T790M mutation on treatment with EGFR-TKIs. Clin Cancer Res; 21(15); 3552–60. ©2015 AACR.

Objectives General thoracic surgeons must be familiar with anatomical variations in the pulmonary bronchi and vessels. We analyzed variations in the bronchovascular pattern of the right upper lung lobe using three-dimensional CT angiography and bronchography and then compared our results with those of previous reports. Methods We reviewed anatomical variations in the right upper pulmonary bronchus and vessels of 263 patients using 3DCT angiography and bronchography images obtained using a 64-channel multidetector CT and workstation running volume-rendering reconstruction software. Results Variations in the pulmonary vein were classified into four types: the “anterior-plus-central vein type” was the most common, noted in 219 cases (83.2 %). The “anterior vein type” was evident in 23 cases (8.8 %), a significantly lower incidence than in previous reports (p

It is often difficult to control perioperative bleeding in patients with liver cirrhosis and concurrent thrombocytopenia and coagulation factor deficiency. Partial splenic embolization (PSE), an auxiliary treatment strategy in management of liver cirrhosis and hepatocellular carcinoma, can not only increase platelets but also improve liver function. With advances in interventional radiology, PSE is a safer and more reliable procedure compared to a splenectomy. We present the case of a 69-year-old man diagnosed with left lung cancer, with thrombocytopenia, and hepatitis C virus-related cirrhosis. Although he was administered prophylactic platelet transfusion prior to operation, he was noted to be refractory to platelet transfusion. PSE was performed to improve his thrombocytopenia, following which we could safely perform left upper lobectomy of the lung and ND2a-1 lymph node dissection without any major bleeding. PSE is useful induction therapy to provide a wider choice of treatment options for patients with thrombocytopenia.

Purpose A deletion polymorphism of the Bim gene has been reported to be a prognostic factor for patients with non–small-cell lung cancer (NSCLC) treated with epidermal growth factor receptor-tyrosine kinase inhibitors in the Asian population. We investigated the impact of the Bim deletion polymorphism on survival among patients with completely resected NSCLC. Patients and Methods The Bim polymorphism was detected by polymerase chain reaction analysis. We measured overall survival (OS) and recurrence-free survival rates in 411 patients and postrecurrence survival (PRS) in 94 patients who experienced recurrence and received additional anticancer therapy. Results The Bim deletion polymorphism was detected in 61 patients (14.8%). OS rates were significantly lower for patients with the Bim deletion polymorphism than for those with the wild-type sequence. On multivariable analysis, the Bim deletion polymorphism was identified as an independent prognostic factor for OS (hazard ratio, 1.98; 95% CI, 1.17 to 3.36; P = .011). Among the 94 patients who experienced recurrence and were treated with anticancer therapy, patients with the Bim deletion polymorphism showed significantly poorer PRS than those with the wild-type sequence (median, 9.8 months v 26.9 months, respectively; P < .001). Multivariable analysis revealed that the Bim deletion polymorphism was an independent predictor of PRS (hazard ratio, 3.36; 95% CI, 1.75 to 6.47; P < .001). This trend remained apparent in subgroup analyses stratified by EGFR status, histology, and therapeutic modality. Conclusion The Bim deletion polymorphism is a novel indicator of shortened PRS among patients with recurrent NSCLC treated with anticancer therapy in the Asian population.

A 61-year-old woman with an abnormal radiograph shadow in her anterior mediastinum was admitted to our hospital and underwent an extended thymectomy. The pathological diagnosis of the tumor was a non-papillary adenocarcinoma of the thymus in pathological stage IV b using the Masaoka classification owing to mediastinal lymph node metastasis. We found parasternal lymph node metastases 5 months after her first operation, and subsequently, she underwent surgery and adjuvant radiotherapy. We found systemic lymph node metastases and metastatic lesions in distant organs, including her lungs, brain, and kidney 27 months after her first operation. Systemic chemotherapy, such as carboplatin plus paclitaxel and an ADOC regimen were not very effective, so we performed immunohistochemical staining of the primary thymic adenocarcinoma. The levels of both thymidylate synthase and dihydropyrimidine dehydrogenase were low; therefore, we started S-1 100mg/body (2 weeks of administration, 1 week of withdrawal)31 months after her first operation. She entered complete remission 6 months after the initiation of S-1. We surgically resected her solitary lung metastasis 13 months after initiation of S-1, and then continued the S-1 treatment. There was no recurrence for more than 2 years after the lung surgery. We believe that when the expression levels of thymidylate synthase or dihydropyrimidine dehydrogenase are low in cases of recurrent thymic adenocarcinoma, S-1 may be able to induce an effective response.

We reported a case of bronchial atresia requiring differentiation from the intralobar sequestration. A 42-year-old man was referred to our institution with suspicion of intralobar sequestration, based on a 3-dimensional computed tomography (CT) angiography that showed abnormal blood vessels from the right inferior phrenic artery flowing into the right lower lobe. CT revealed a lesion between S9 and S10 wherein there were refluxed blood vessels from A9 without an accompanying bronchus, with polycysts and emphysematous changes. Ventilation-perfusion scintigraphy revealed a reduction in uptake in the same sites. He was diagnosed as congenital bronchial atresia preoperatively, and we performed a right basal segmentectomy. Pathological examination confirmed the bronchiectasis and emphysematous changes in the lung parenchyma, but malignant findings were not confirmed.

Murine double minute 2 (MDM2) is a negative regulator of p53. A single-nucleotide polymorphism (SNP) (rs2279744: c.309T>G) in the promoter region of the MDM2 gene has been shown to result in higher levels of MDM2 RNA and protein. Regarding the contribution of c.309T>G in the MDM2 gene to the lung cancer risk, previous studies are conflicting. In order to evaluate the association between c.309T>G and the lung cancer risk, a case-control study was performed. The MDM2 genotypes were determined in 762 lung cancer patients and in 700 cancer-free control subjects using the Smart Amplification Process. Statistical adjustment was performed for gender, age and pack-years of smoking. The distributions of c.309T>G (T/T, T/G, G/G) were 20.1, 49.7, 30.2% in the case group and 21.7, 47.9, 30.4% in the healthy-control group. There were no overall associations between the MDM2 genotypes and the risk of lung cancer [T/G genotype: Adjusted odds ratio (AOR), 1.30; 95% confidence interval (CI), 0.88–1.93; and G/G genotype: AOR, 1.18; 95% CI, 0.78–1.80]. The subgroup analysis of gender, histology, smoking status and epidermal growth factor receptor mutation status also indicated that there was no association with lung cancer. Additionally, the genotypes did not have an effect on the age at the time of diagnosis of lung cancer (P=0.25). In conclusion, the G allele frequency in the lung cancer cases was 0.551, which was similar to other studies. The results of the present study suggest that the c.309T>G is not significantly associated with lung cancer.

Background: On the basis of our recent findings of oncogenic KRAS-induced interleukin-8 (IL-8) overexpression in non-small cell lung cancer, we assessed the clinicopathological and prognostic significances of IL-8 expression and its relationship to KRAS mutations in lung adenocarcinomas. Methods: IL-8 expression was examined by quantitative RT–PCR using 136 of surgical specimens from lung adenocarcinoma patients. The association between IL-8 expression, clinicopathological features, KRAS or EGFR mutation status and survival was analysed. Results: IL-8 was highly expressed in tumours from elderly patients or smokers and in tumours with pleural involvement or vascular invasion. In a non-smokers' subgroup, IL-8 level positively correlated with age. IL-8 was highly expressed in tumours with KRAS mutations compared with those with EGFR mutations or wild-type EGFR/KRAS. Lung adenocarcinoma patients with high IL-8 showed significantly shorter disease-free survival (DFS) and overall survival (OS) than those with low IL8. DFS and OS were significantly shorter in the patients with mutant KRAS/high IL-8 than in those with wild-type KRAS/low IL-8. Cox regression analyses demonstrated that elevated IL-8 expression correlated with unfavourable prognosis. Conclusions: Our findings suggest that IL-8 expression is associated with certain clinicopathological features including age and is a potent prognostic marker in lung adenocarcinoma, especially in oncogenic KRAS-driven adenocarcinoma.

The aim of this study was to investigate the prognosis of pulmonary adenocarcinoma patients following postoperative recurrence, according to epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) gene mutation status and recurrence site. In total 58 adenocarcinoma patients with recurrence following surgical resection were retrospectively evaluated between 2002 and 2011. The patients were divided into groups according to the presence or absence of EGFR and KRAS mutations and the clinicopathological characteristics, recurrence sites and postrecurrence survival were compared. EGFR and KRAS mutations were detected in 26 (45%) and 11 patients (19%), respectively. Initial recurrence was distant in 25 (43%), local in 17 (29%) and both distant and local in 16 cases (28%). In EGFR-mutant (EGFR+) cases, bilateral/contralateral lung recurrence was a frequent finding. EGFR+ cases exhibited significantly better outcomes compared to KRAS+ and EGFR-KRAS- (wild-type) cases. The 2-year post-recurrence survival rates were 81, 18 and 47% in EGFR+, KRAS+ and wild-type cases, respectively. The patients with distant organ recurrence exhibited significantly worse survival compared with those without distant recurrence in wild-type, but not in the EGFR+ cases or the entire cohort. Multivariate analysis revealed that EGFR mutations and a number of recurrent lesions were the only statistically significant independent predictors of postrecurrence prognosis. Our results indicated distinct survival differences in recurrent adenocarcinoma patients according to driver mutations. Patients with EGFR-mutated tumors exhibited increased survival, regardless of recurrence at distant sites, whereas patients with KRAS-mutated adenocarcinoma exhibited poor outcome following postoperative recurrence. Therefore, the assessment of driver mutations is essential for predicting postrecurrence survival following surgical resection.

The case was 83-year-old man who had complete situs inversus, and was pointed out to have peripheral adenocarcinoma with the size of 1.8 cm at the left upper lobe( S3). Because of severe emphysema and other multiple comorbidities, left S3 segmentectomy with hilar lymph node sampling was performed using video-assisted thoracic surgery (VATS). Preoperatively, the simulation of operation was performed using the 3 dimension computed tomography images of pulmonary arteriovenous and bronchus (3DCTAB). Postoperative course was uneventful. 3DCTAB was thought to be useful in understanding the anatomical location of pulmonary arteriovenous and bronchus directly, and in performing segmentectomy in the case of situs inversus like this.

Purpose Oncogenic driver mutations are critical for lung cancer development and serve as therapeutic targets. However, their associations with environmental factors are not fully understood. We aimed to elucidate the relationship between tumor developmental biology and exposure to environmental factors. Patients and Methods This was a prospective, multicenter, molecular epidemiology study. Eligible patients were those with newly diagnosed stages I to IIIB non–small-cell lung cancer (NSCLC) who underwent surgery. The tumors were examined for somatic mutations in 72 cancer-associated genes by targeted deep sequencing, estrogen receptor β (ERβ) expression using immunohistochemical staining, and infection with any of 37 types of human papillomavirus (HPV) using a polymerase chain reaction–based microarray system. Detailed information on patient demographics and environmental factors was obtained from a comprehensive questionnaire. Results From July 2012 to December 2013, 957 patients were enrolled, and molecular analyses were performed on 876 samples (from 441 ever- and 435 never-smokers). Oncogenic driver mutations in P53 and KRAS increased proportionally with smoking status, whereas mutations in EGFR and SMAD4 decreased. KRAS mutations in smokers and SMAD4 mutations were observed more frequently in proportion to body mass index. TP53 and NFE2L2 mutations were observed more frequently in advanced NSCLC stages. As for never-smokers, no environmental factors were significantly associated with mutational changes. EGFR mutations and TP53 mutations were observed more frequently in women and in men, respectively. Mutations in these two genes were also potentially associated with ERβ expression. Only three patients (0.3%) were HPV positive. Conclusion The mutational spectrum is associated with smoking, body mass index, and other environmental factors, as well as with ERβ expression. Little association was observed between HPV and NSCLC.

Pulmonary angiosarcomas are usually secondary tumors, and only a few primary cases have been reported. Effective strategies for treating this tumor have not been established, and the prognosis of affected individuals is generally very poor. We report a case of primary angiosarcoma presenting as a hemorrhagic solitary nodule at the bifurcation of the left main bronchus, followed for two years before surgery. Bronchial arteriography revealed a tumor stain sign, and racemose hemangioma of the bronchial artery was excluded. The hemoptysis was not controlled by repeated bronchial artery embolization, and the patient underwent left pneumonectomy with routine mediastinal lymph node dissection. Histopathologically, the excised tissue revealed a highly-cellular growth of atypical spindle cells with a storiform pattern. These atypical cells showed relatively low mitotic activity; the MIB-1 index was 10%. The tumor was diagnosed as a primary angiosarcoma of the lung by the following immunohistological findings: positivity for factor VIII-related antigen and CD31. One year after resection, the patient remains well without signs of recurrence.

BACKGROUND: KRAS and epidermal growth factor receptor (EGFR) mutations are thought to play an important role in the carcinogenesis of lung adenocarcinoma. However, clinicopathological findings of KRAS mutated adenocarcinoma cases have not yet been fully clarified. The authors analyzed the relationship between the KRAS mutation and corresponding clinicopathological findings, focusing on nonmucinous and mucinous bronchioloalveolar elements. METHODS: EGFR and KRAS mutations were detected in DNA samples extracted from 182 surgically resected tissues of lung adenocarcinomas by the Smart Amplification Process. The relations between gene mutation status and clinicopathological features were analyzed. All adenocarcinoma cases were divided into bronchioloalveolar carcinoma (BAC), adenocarcinoma with bronchioloalveolar features, and adenocarcinoma without BAC components (non-BAC). BAC/adenocarcinoma with bronchioloalveolar features tumors were further assessed for the presence of mucinous features. RESULTS: EGFR and KRAS mutations were found in 76 and 30 cases, respectively. In the KRAS mutant group, BAC/adenocarcinoma with bronchioloalveolar features was found in 22 cases, which included 10 nonmucinous and 12 mucinous tumors. Of 19 cases with mucinous BAC/adenocarcinoma with bronchioloalveolar features, KRAS mutations were detected in 12, but no EGFR mutation was detected. In the KRAS mutant group, BAC/adenocarcinoma with bronchioloalveolar features had significantly earlier pathological stages and more favorable prognoses than did non-BAC. Mucinous BAC/adenocarcinoma with bronchioloalveolar features showed less smoking history than did nonmucinous BAC/adenocarcinoma with bronchioloalveolar features and non-BAC. Furthermore, transversion type KRAS mutations were more common in non-BAC. CONCLUSIONS: KRAS mutated adenocarcinomas can be divided into BAC/adenocarcinoma with bronchioloalveolar features and non-BAC types. Non-BAC adenocarcinoma is related to smoking history and has a poor prognosis. BAC/adenocarcinoma with bronchioloalveolar features adenocarcinoma, however, has a more favorable prognosis, and mucinous BAC/adenocarcinoma with bronchioloalveolar features has little relationship to smoking history. Cancer 2011;. © 2011 American Cancer Society.

Background: Thymidylate synthase (TS) expression has been reported in various tumors, including non-small-cell lung carcinoma (NSCLC), but not in high-grade neuroendocrine (HGNE) carcinoma of the lung. Methods: We measured TS expression in surgically resected pulmonary tumors, comparing HGNE carcinomas of the lung (13 large-cell neuroendocrine carcinomas, 8 small-cell lung carcinomas) with squamous cell carcinoma and adenocarcinoma of the lung using laser-capture microdissection for tissue isolation, real-time polymerase chain reaction (PCR), and immunohistochemistry. We also measured TS mRNA expression in small-cell lung carcinoma (SCLC) and NSCLC cell lines using real-time PCR. Results: At both mRNA and protein levels, TS expression was significantly higher in squamous cell carcinoma compared to adenocarcinoma. Moreover, TS expression was significantly higher in HGNE carcinomas of the lung compared to squamous cell carcinoma. A significant correlation was found between mRNA and protein expression. TS mRNA expression in SCLC cell lines was significantly higher than in NSCLC cell lines. Conclusions: TS expression was higher in HGNE carcinomas of the lung than in squamous cell carcinoma, which was higher than in adenocarcinoma. This information may be useful in predicting the effects of TS-inhibiting agents in patients with NSCLC and HGNE carcinomas of the lung. J. Surg. Oncol. 2010;102:11–17. 2010 Wiley-Liss, Inc.

Interactions between CXCL12 and its receptors CXCR4 or CXCR7 are involved in tumor growth and metastasis in various types of human cancer. However, CXCL12 expression and its role in lung cancer are not fully elucidated. Here we examined the expression of CXCL12 in 54 lung cancer cell lines consisting of 23 small cell lung cancers (SCLCs) and 31 non-small cell lung cancers (NSCLCs). CXCL12 was overexpressed in lung cancer cell lines compared to non-malignant human bronchial epithelial cell lines (N = 6). CXCL12 expression was positively but weakly correlated with the expression of CXCR4 or CXCR7. We also examined CXCL12 expression in 89 NSCLC specimens and found that CXCL12 expression was significantly higher in tumor specimens from female patients, non-smokers and adenocarcinoma patients. Small interfering RNAs targeting CXCL12 inhibited cellular proliferation, colony formation and migration of CXCL12-overexpressing lung cancer cells; however, this inhibition did not occur in lung cancer cells that lacked CXCL12. Furthermore, the anti-CXCL12 neutralizing antibody mediated inhibitory effects in three lung cancer cell lines that overexpressed CXCL12, but not in two CXCL12 non-expressing lung cancer cell lines nor two non-malignant bronchial epithelial cell lines. The present study demonstrates that: CXCL12 is concomitantly overexpressed with CXCR4 or CXCR7 in lung cancers; CXCL12 is highly expressed in NSCLCs from females, non-smokers and adenocarcinoma patients; and disruption of CXCL12 inhibits the growth and migration of lung cancer cells. Our findings indicate that CXCL12 is required for tumor growth and provide a rationale for the anti-CXCL12 treatment strategy in lung cancer.

Anastomosis of the tracheal ends requires sufficient mobilization of the trachea because excessive tension is the principal cause of anastomotic insufficiency. Several mobilization and release techniques can be used to reduce some of the anastomotic tension of the trachea. Of these, Montgomery's suprahyoid release technique is a surgical method that severs three muscles above the hyoid bone: the mylohyoid, geniohyoid, and genioglossus. Of these, we present the suprahyoid release technique here because we believe that it is superior to the other mobilization techniques, particularly supralaryngeal release. This is the first report to approach this subject from both anatomical and functional perspectives. In summary, we performed a tracheoplasty requiring a suprahyoid release for a 55-year-old female with adenoid cystic carcinoma as an oncologic emergency. We should master the supralaryngeal release, particularly wide resection of the trachea, because this technique provides sufficient mobilization, with no dysphagia or complications.

KRAS is an oncogene that can be activated by mutations. Patients with non-small cell lung cancer who have KRAS mutations do not respond to tyrosine kinase inhibitors; therefore, accurate detection of KRAS mutations is important for deciding therapeutic strategies. Although sequencing-related techniques have been frequently used, they are usually too complex, have low sensitivity, and are time-consuming for routine screening in clinical situations. We evaluated peptide nucleic acid (PNA)-clamp smart amplification process version 2 (SmartAmp2) as a detection method for KRAS codon 12 mutations in patient specimens compared with traditional sequencing and polymerase chain reaction-related methods. Among 172 lung adenocarcinoma samples, direct sequencing, enzyme-enriched sequencing, and PNA-enriched sequencing showed that 16 (9.3%), 26 (15.7%), and 28 (16.3%) tumors, respectively, contained KRAS mutations in codon 12. Using PNA-clamp SmartAmp2, we could identify 31 (18.0%) tumors that had KRAS mutations in codon 12 within 60 minutes, three of which were undetected by polymerase chain reaction-related methods. On the other hand, we examined 30 nonmalignant peripheral lung tissue specimens and found no mutations in any of the samples using PNA-clamp SmartAmp2. In this study, we confirmed that PNA-clamp SmartAmp2 has high sensitivity and accuracy and is suitable for the clinical diagnosis of KRAS codon 12 mutations.

Mucosa-associated lymphoid tissue (MALT) lymphoma arising in the thymus is extremely rare and little is known regarding its clinicopathological features. This study examined the clinicopathological features of nine cases of thymic MALT lymphoma. Most patients had autoimmune disease or hyperglobulinemia, and they also had cysts in the tumors. Both increased serum autoantibody levels and polyclonal serum immunoglobulin levels remained essentially unchanged after total thymectomy in all patients. Thymic MALT lymphoma needs to be included in the differential diagnosis in Asian patients with a cystic thymic mass accompanied by autoimmune disease or hyperglobulinemia.

Objective A pulmonary segmentectomy is requires the identification of the segmental planes, making it technically more difficult than a lobectomy. Therefore, we present a new method that uses a butterfly needle to distinguish the intersegmental plane under video-assisted thoracoscopic surgery (VATS). Methods From May 2005 to August 2008, 15 patients underwent anatomic segmentectomy using VATS. In this approach, a working port 4 to 7 cm in length was made in the fifth intercostal space. Additional 1.2-cm thoracic ports were made in the seventh intercostal space on the anterior axillary line and the ninth intercostal space on the posterior axillary line. Each segment was selectively isolated, and the targeted bronchovascular pedicle was divided. For the segmentectomy, the lung was deflated, and the pulmonary artery and vein to the involved segment were divided. The segmental bronchus was divided using a stapling device. Using a butterfly needle, oxygen/air (1–2 L) was used to inflate the involved segment, and the involved segment was severed and removed using electrocautery or a stapling device. The raw surface was covered with an absorbable sealing material such as polyglycolic acid to prevent air leaks. Results Using this method, apical segment of the right upper lobe (S1), apical posterior segment of the left upper lobe (S1 + 2), upper division, and posterior segment of the right upper lobe (S2), superior segment of the right or left lower lobe (S6), and basal segmentectomies could be performed with VATS. However, the technique did not work in one patient with severe emphysematous changes, because the plane was not readily identifiable. Conclusions Butterfly-needle video-assisted segmentectomy is a useful technique. Selective segmental inflation allows the intersegmental plane to be identified completely under the surgeon's control, eliminating the need for an anesthesiologist to pass a bronchoscope or insufflate the lung in a particular manner.

Information about the impacts of disasters on health is useful for establishing hazard prediction maps and action plans of disaster management. This study aims at learning effective asthma management from the volcano disaster of Mount Asama eruption in Japan on September 1, 2004. We conducted a cross-sectional study to assess the acute impact of volcanic ash on asthma symptoms and their treatment changes by using a questionnaire completed by 236 adult asthmatic patients and their physicians. In the ashfall over 100g/m2 area, 42.9% of asthma patients suffered exacerbations, PEF decreased, asthma treatments increased, and inhalation of β2 stimulants was used most for exacerbated asthma. Compared to severe asthma patients, mild and moderate asthma patients were most at risk. Severe asthma patients were not affected since most of them knew their asthma status was severe, and did not go outside and kept windows closed. Deteriorated asthma symptoms of wheezing, chest tightness and cough appeared in the ashfall over 100g/m2 area. Ash contained inhalable 10μm diameter particles, and included high concentrations of airway toxic substrates of silica. These data suggest that ashfall over 100 g/m2 is harmful, access to these areas by asthma patients needs to be restricted, and these areas need to improve asthma treatment. In addition, the increase in the proportion of asthma patients with wheeze and cough are diagnostic clues for ash-induced asthma in affected areas, and can be used by doctors to tell whether patients are receiving sufficient asthma treatment.

症例は70歳，男性で，左肺扁平上皮癌に対し左上葉切除ND2a郭清を施行した．術後第4病日に39℃に発熱し，第6病日の胸腔ドレーンからの培養で黄色ブドウ球菌が証明された．第7病日の胸部CTで左胸腔内背側に膿瘍腔を認めたため，CTガイド下で膿瘍腔内に12Frアスピレーションカテーテルを挿入し，間欠的胸腔内洗浄を開始した．その後肺炎および敗血症も併発し，全身状態も徐々に悪化したため，第11病日から250ml/時の大量生理食塩水での胸腔内点大量点滴持続洗浄を行ったところ，全身状態が改善し，第27病日には治癒した．点滴持続洗浄法で問題となる大量の洗浄液の回収のために，チェストドレーンバッグと大型吸引装置を利用し，頻回に回路を外すことなく，簡便に廃液の回収が可能な回路を考案した．本装置を用いた点滴大量持続洗浄は，胸腔内を効率良く洗浄でき，急性膿胸に対する効果的な治療手段のひとつと考えられた．

Objectives: Based on recent findings of aromatase and estrogen receptor beta (ERβ) expression in non-small-cell lung cancer, we assessed the clinicopathological and prognostic significance of aromatase and ERβ expression and their relationship to epidermal growth factor receptor (EGFR) mutation in lung adenocarcinoma. Materials and methods: We evaluated 150 resected primary lung adenocarcinoma specimens. Expression of aromatase, ERα, ERβ, progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) was evaluated by immunostaining, and EGFR and KRAS mutations were analyzed. Overall survival (OS) and recurrence-free survival (RFS) were calculated using the Kaplan-Meier method. Results: Expression of aromatase, ERα, ERβ, PR, and HER2 was detected in 88.0%, 1.3%, 79.3%, 2.7%, and 39.3% of specimens, respectively. In patients with EGFR wild-type lung adenocarcinoma, high aromatase expression was an independent predictor of poor OS (hazard ratio [HR]=2.638; 95% confidence interval [CI], 1.173-5.936; P=.019) and RFS (HR=2.505; 95% CI, 1.154-5.434; P=.020). Positive ERβ expression was also an independent predictor of poor RFS (HR=4.013; 95% CI, 1.219-13.207; P=.022). Furthermore, high aromatase expression was a significant predictor of poor survival only in females (OS, P=.010; RFS, P=.007), whereas positive ERβ expression was an important predictor of poor survival only in males (OS, P=.073; RFS, P=.051). No prognostic significance was observed in patients with EGFR mutations. Conclusions: Our findings suggest that EGFR wild-type lung adenocarcinoma is an estrogen-dependent carcinoma, and aromatase expression and ERβ expression are potent prognostic markers for EGFR wild-type lung adenocarcinoma.

To the Editor: We report a patient with a pulmonary squamous dysplastic lesion that appears as pure ground-glass opacity (GGO) on computed tomography (CT). An ill-defined, 15-mm nodular lesion in the left lower lung was noted on a radiograph during an annual health check-up of a 61-year-old female who had never smoked. A physical examination and laboratory study results were unremarkable. Chest CT demonstrated a well-demarcated pure GGO nodule in the inferior lobe of the left lung. The nodule, measuring 15×13 mm, was located in the anteromedial basal segment (S8) (fig. 1a). A positron emission tomography scan showed no accumulation of radioactivity in the lesion. The patient underwent S8 segmentectomy of the left lung with lymph node sampling. Figure 1– a) Chest computed tomography (CT) shows a well-demarcated pure ground-glass opacity nodule (arrowhead), measuring 15×13 mm, in the anteromedial basal segment of the left lung. b) Polygonal cells are present along the alveolar walls, but the alveolar structure is preserved (haematoxylin and eosin; scale bar=100 μm). c) Immunohistochemical staining for p63. The atypical cells were positive for p63. d) Immunohistochemical staining for TTF-1. The atypical cells were …

Primary chest wall tumor is relatively rare. According to the annual report by The Japanese Association for Thoracic Surgery in 2012, 447 primary chest wall tumors were resected in 2010. It was only 0.66% of the total number of operations in general thoracic surgery in Japan. From January 1992 to December 2012, 3,022 cases in general thoracic surgery were operated in our department. Of these, 30 patients (1%) with primary chest wall tumor were surgically treated. We retrospectively reviewed the medical records of them and investigated the details of this tumor. The patients group included 11 males and 19 females, with a mean age 57.6 years (range, 16 to 79 years). The majority of these patients were referred to us because of radiographical abnormalities on chest X-ray( 56.7%) or clinical symptoms( 33.3%). The operative procedure was tumor extirpation in 25 cases and chest wall resection in 5 cases. Histologically, 23 cases (76.7%) were benign tumors, 7 cases (23.3%) were malignant tumors. Malignant tumors included aggressive and poor prognostic cases such as malignant fibrous histiocytoma or malignant peripheral nerve sheath tumor, on the other hand, extremely rare tumor with low grade malignancy such as parachordoma arising from the chest wall soft tissue was included. In conclusion, although, the standard therapy for malignant primary chest wall tumors has not been established, aggressive surgical resection remains the treatment of choice and to provide an accurate diagnosis.

We herein report an extremely rare case of a patient chylothorax at an interval of 20 years after thoracic vertebrae fractures, who underwent a successful thoracoscopic thoracic duct ligation and pleurodesis. A 51-year-old man was referred to our hospital with shortness of breath on effort about 1 month after participating in archery. Twenty years previously, he was involved in a traffic accident. At that time, the patient sustained trauma to the spine and suffered a spinal injury, thus resulting in paralysis in the lower part of his body. A chest roentgenogram and computed tomogram revealed a large amount of bilateral pleural effusion. After thoracentesis was performed, a diagnosis of chylothorax was made and the patient was hospitalized. Conservative management by a low-fat diet proved to be unsuccessful. The patient did not request pleurodesis, because pleural adhesions might impair pulmonary function. As a result, we decided to perform surgery. On the right side, we performed video-assisted thoracoscopic surgery by clipping the thoracic duct and applying an absorbable sealing material. Thereafter, pleurodesis was performed and OK-432 was instilled. Thereafter, the pleural fluid flow was almost completely stopped. On the left side, pleurodesis was effective. The patient has since remained symptom free and has been followed up on an outpatient basis for 9 months after the 100th postoperative day. We assumed that the chylothorax in this case was related to the earlier traffic accident.

BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as a reliable predictor of response to EGFR tyrosine kinase inhibitor (TKI) treatment. Two common EGFR mutations (L858R, and exon19 deletion) and EGFR T790M mutation are clinically important for decision-making in clinical practice. Picodroplet digital PCR (ddPCR) has recently emerged as an accurate and ultra-sensitive method for tumor genotyping and have a great advantage over conventional mutation detection methods. We have previously reported far more frequent incidence of pretreatment EGFR T790M mutation than previous reports utilizing ddPCR method. Here, we developed a multiplex ddPCR assay to detect three clinically relevant EGFR mutations in one reaction. METHODS: Positive and negative control plasmids for the EGFR mutation assay were prepared by cloning DNA fragments containing wild-type or mutant EGFR. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. We then evaluated the utility of multiplex ddPCR to detect the three clinically relevant mutations in EGFR from FFPE samples of patients with non-small cell lung cancer. 45 FFPE samples were also genotyped for EGFR mutations by conventional qPCR-based methods for validation. The concordance between duplex and multiplex ddPCR assay was also evaluated. RESULTS: Serial dilutions experiments using genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. All of the 33 EGFR-activating mutations detected in FFPE samples by conventional qPCR-based method were also detected by multiplex ddPCR assay. Owing to its ultra-high sensitivity, additional T790M mutation at ultra-low allele frequency ( CONCLUSIONS: Using ddPCR technology, we established multiplex and ultra-sensitive genotyping platform for three clinically relevant EGFR mutations. Results of a proof-of-principle study using clinical samples indicated that the clinical utility of multiplex ddPCR assay to screen for multiple EGFR-activating mutations concurrently with low-frequency T790M mutation. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1376.

Background Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. Methodology In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named “Duplex SmartAmp,” which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. Results and Conclusions By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

We describe the use of the Starfish 2 heart positioning device as an aid to pericardium reconstruction after en bloc resection of mediastinal tumors of the left pericardium by use of median sternotomy with anterolateral thoracotomy. The Starfish device, which is a tool for off-pump coronary artery procedures, allows excellent cardiac positioning and hemodynamic stability during pericardium reconstruction through a median sternotomy with anterolateral thoracotomy.

Convenient and reliable multiple organ metastasis model systems might contribute to understanding the mechanism(s) of metastasis of lung cancer, which may lead to overcoming metastasis and improvement in the treatment outcome of lung cancer. We isolated a highly metastatic subline, PC14HM, from the human pulmonary adenocarcinoma cell line, PC14, using an in vivo selection method. The expression of 34,580 genes was compared between PC14HM and parental PC14 by cDNA microarray analysis. Among the differentially expressed genes, expression of four genes in human lung cancer tissues and adjacent normal lung tissues were compared using real-time reverse transcription polymerase chain reaction. Although BALB/c nude mice inoculated with parental PC14 cells had few metastases, almost all mice inoculated with PC14HM cells developed metastases in multiple organs, including the lung, bone and adrenal gland, the same progression seen in human lung cancer. cDNA microarray analysis revealed that 981 genes were differentially (more than 3-fold) expressed between the two cell lines. Functional classification revealed that many of those genes were associated with cell growth, cell communication, development and transcription. Expression of three upregulated genes (HRB-2, HS3ST3A1 and RAB7) was higher in human cancer tissue compared to normal lung tissue, while expression of EDG1, which was downregulated, was lower in the cancer tissue compared to the normal lung. These results suggest that the newly established PC14HM cell line may provide a mouse model of widespread metastasis of lung cancer. This model system may provide insights into the key genetic determinants of widespread metastasis of lung cancer.