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Objectives Circulating tumor cells (CTCs) are associated with tumor spread, whereas cancer stem cells may be related to drug resistance. However, few studies have analyzed the levels of circulating cancer stem cells (CCSCs) and CTCs in patients with advanced non-small cell lung cancer (NSCLC). Materials and Methods Treatment-naive patients with EGFR-mutated NSCLC who received epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy were recruited prospectively. The cell surface vimentin antibody was used for CTC detection and CD133 antibody for CCSC detection. CCSC and CTC levels were measured as cell count per 4 mL of blood, before treatment, after 2 and 12 weeks of treatment, and at disease progression. Data on clinical characteristics and outcomes were also collected. Results At diagnosis (n = 29), the median CCSC and CTC levels were 0 (interquartile range, 0–2) and 3 (2–9), respectively. After 12 weeks, the CCSC and CTC levels were lower than those at diagnosis (CCSC: 0 (0−0), p = 0.14; CTC: 1 (0–4), p = 0.048). At disease progression, the median CCSC and CTC levels were 0 (0–1) and 1 (0–2), respectively. Patients with higher CCSC and CTC levels at diagnosis had a numerically shorter progression-free survival. Conclusion In patients with EGFR-mutated NSCLC, CCSC and CTC levels became lower after 12 weeks of EGFR-TKI therapy and remained low at disease progression. High pre-treatment CCSC and CTC levels may be associated with a trend towards poor treatment outcomes.

Besides being a predictive biomarker of response to immunotherapy in lung cancer in general, programmed death-ligand 1 (PD-L1) is not so well correlated with treatment outcomes of lung adenocarcinoma (ADC) harbouring epidermal growth factor receptor (EGFR) mutations, as reported studies are inconclusive and seldom addressed the issues of response to treatment and resistance. The primary objective is to evaluate the association of PD-L1 and EGFR tyrosine kinase inhibitor (TKI) efficacy, resistance, and relevant clinical outcomes. The secondary objective is to further explore the tumour microenvironments of EGFR mutant tumours with different PD-L1 expression.Using immunohistochemical (IHC) staining, we retrospectively tested PD-L1 expression (Dako 22C3) in the pre-treatment tumours from advanced EGFR mutant lung ADC patients, of whom all were treated with TKIs. Multiplex IHC assay was applied for exploring immune cells in tumour microenvironments.A total of 153 Taiwanese patients were enrolled in our study, of whom a majority of cases were female (58.9%) and non-smokers (75.8%). The objective response rate (ORR) to EGFR TKI and progression-free survival (PFS) were better in patients with PD-L1 expression50% (ORR/PFS in PD-L1 0% versus 1-49% versus ≥50%: 65.6%/12.5 months versus 56.4%/12.8 months versus 38.9%/5.9 months, P 0.05). The multivariate analysis showed that PD-L150% was an independent prognostic factor for longer PFS (hazard ratio (HR) 0.433, 95% confidence interval (CI) 0.250-0.751, P = 0.003). Furthermore, tumours with higher PD-L1 expression were less likely to develop a secondary T790M mutation (T790M+ in PD-L1 0% versus 1-49% versus ≥50%: 53.7% versus 35.7% versus 10%, P = 0.024). Multiplex IHC tests were applied in 15 cases and revealed a potential correlation between PD-L1, immune cells, and EGFR TKI responses.Lower pre-treatment PD-L1 is associated with better ORR, PFS, and higher frequency of T790M resistance in EGFR TKI-treated lung ADC patients.

Constitutive activation of the epidermal growth factor receptor (EGFR) signaling pathway is implicated in the initiation and progression of lung cancer. EGFR tyrosine kinase inhibitor (TKI)-targeted therapy has become the standard treatment for nonsmall cell lung cancer (NSCLC) patients. However, acquired resistance to these agents remains a major obstacle for managing NSCLC. Here, we investigated a novel strategy to overcome EGFR TKI resistance by targeting the stanniocalcin 2 (STC2)-JUN-AXL pathway. We revealed that STC2 was expressed at significantly higher levels in EGFR TKI-resistant cells. Further, clinical analysis showed that STC2 expression was increased after the development of EGFR TKI resistance and that higher levels were correlated with shorter progression-free survival in EGFR TKI-treated lung cancer patients. Moreover, STC2 overexpression in EGFR TKI-sensitive cells resulted in EGFR TKI resistance. Conversely, genetic silencing of STC2 rendered EGFR TKI-resistant cells more sensitive to EGFR TKIs. Mechanically, STC2 enhanced AXL promoter activity by increasing the phosphorylation of c-Jun, which is an indispensable transcription factor that transactivates AXL. STC2 promoted activation of the JUN-AXL-extracellular signal-regulated kinase (ERK) signaling axis in lung cancer cells. Pharmacological or genetic inhibition of AXL-ERK activity inhibited STC2-mediated EGFR TKI resistance. We also demonstrated that PE2988 cells, a C797S-independent osimertinib-resistant primary cancer cell line from a lung cancer patient, responded to combined AXL inhibitor and osimertinib treatment. In conclusion, our research indicates that STC2 overexpression is important for acquired resistance to EGFR TKIs and that STC2-JUN-AXL-ERK signaling might be a potential therapeutic target to overcome resistance to EGFR TKIs.

Introduction Recent advances in the detection of genomic DNA from plasma samples allow us to follow tumor DNA shedding in plasma during systemic treatment. Osimertinib is the standard of care for patients with NSCLC with acquired EGFR T790M mutations. We assessed changes in serial plasma cell-free circulating tumor DNA (ctDNA) genomic alterations to predict osimertinib efficacy. Methods We prospectively collected plasma from patients having EGFR-mutated advanced NSCLC previously treated with EGFR tyrosine kinase inhibitor therapy and with acquired EGFR T790M mutation detected by standard methods. Plasma samples were collected before starting osimertinib treatment, 4 weeks after osimertinib treatment, and on progression. ctDNA was analyzed using the Guardant360 assay. Results A total of 15 eligible patients received osimertinib. Before starting treatment, EGFR-activating mutations were detected in the ctDNA of all patients, and EGFR T790M was detected in 93% of the cases. Osimertinib treatment was associated with an objective response rate of 53% and a median progression-free survival of 7.3 months. A total of 12 of the 15 patients had undetectable plasma T790M and decreased activating mutation allelic frequency (AF) at week 4. None of the 12 patients had disease progression within 16 weeks. For the remaining three patients, with detectable plasma T790M (n = 2) or increased activating mutation AF (n = 1) at week 4, two had progressive disease within 16 weeks (p = 0.03). Conclusions In patients with EGFR-mutated advanced NSCLC, persistent EGFR T790M or increasing activating mutation AF as detected in ctDNA 4 weeks after the start of osimertinib treatment may predict disease progression within 16 weeks.

Introduction Recent advances in the detection of genomic DNA from plasma samples allow us to follow tumor DNA shedding in plasma during systemic treatment. Osimertinib is the standard of care for patients with NSCLC with acquired EGFR T790M mutations. We assessed changes in serial plasma cell-free circulating tumor DNA (ctDNA) genomic alterations to predict osimertinib efficacy. Methods We prospectively collected plasma from patients having EGFR-mutated advanced NSCLC previously treated with EGFR tyrosine kinase inhibitor therapy and with acquired EGFR T790M mutation detected by standard methods. Plasma samples were collected before starting osimertinib treatment, 4 weeks after osimertinib treatment, and on progression. ctDNA was analyzed using the Guardant360 assay. Results A total of 15 eligible patients received osimertinib. Before starting treatment, EGFR-activating mutations were detected in the ctDNA of all patients, and EGFR T790M was detected in 93% of the cases. Osimertinib treatment was associated with an objective response rate of 53% and a median progression-free survival of 7.3 months. A total of 12 of the 15 patients had undetectable plasma T790M and decreased activating mutation allelic frequency (AF) at week 4. None of the 12 patients had disease progression within 16 weeks. For the remaining three patients, with detectable plasma T790M (n = 2) or increased activating mutation AF (n = 1) at week 4, two had progressive disease within 16 weeks (p = 0.03). Conclusions In patients with EGFR-mutated advanced NSCLC, persistent EGFR T790M or increasing activating mutation AF as detected in ctDNA 4 weeks after the start of osimertinib treatment may predict disease progression within 16 weeks.

Background Programmed death-ligand 1 (PD-L1) expression is associated with clinical outcomes of epidermal growth factor receptor (EGFR) mutant lung adenocarcinoma (ADC) treated with tyrosine kinase inhibitors (TKIs). However, whether PD-L1 expression plays a role in anaplastic lymphoma kinase (ALK)-positive lung ADC is unknown. We aimed to evaluate the impact of PD-L1 in patients with ALK-positive lung ADC receiving crizotinib. Materials and Methods PD-L1 expression was identified by immunohistochemistry (IHC). Reverse transcriptase-polymerase chain reaction was used for ALK variant detection, and immunofluorescence-based multiplex staining was applied for exploring immune cells in tumor microenvironments. Results A total of 78 patients with ALK-positive advanced ADC were enrolled in our study, of whom 52 received crizotinib. Compared with EGFR/ALK wild-type tumors, PD-L1 expression was lower in ALK-positive ADC. ALK fusion variants were identified in 32 patients, and those with variant 3 and 5 (short variants) had higher PD-L1 expression than those with other variants. The crizotinib objective response rate (ORR) and progression-free survival (PFS) was better in tumors with negative PD-L1 expression (ORR/PFS in PD-L1 0% vs. 1%–49% vs. 50%–100%: 60.7%/11.8 months vs. 38.5%/6.5 months vs. 36.4%/4.0 months, p = .007/.022). The multivariate Cox proportional hazards model revealed that PD-L1 0% (vs. ≥1%) was an independent factor for longer PFS (adjusted hazard ratio 0.322, 95% confidence interval 0.160–0.650, p = .002). Multiplex IHC in three cases showed a varied extent of immune cell infiltrations in tumors with different PD-L1 expression. Conclusion Positive PD-L1 expression was associated with unfavorable clinical outcomes in patients with ALK-positive lung ADC receiving crizotinib. Implications for Practice Not all lung adenocarcinoma with sensitizing driver mutations experienced durable responses to small-molecule tyrosine kinase inhibitors (TKIs). Similar to the negative impact of programmed death-ligand 1 (PD-L1) in epidermal growth factor receptor mutant tumors treated with TKIs, this study demonstrated that positive PD-L1 expression was also associated with worse response rate and shorter progression-free survival of anaplastic lymphoma kinase (ALK)-positive adenocarcinoma treated with crizotinib. Among different ALK fusion partners, tumors with short variants (V3 and V5) had higher PD-L1 compared with long variants (V1, V2, and V6). Testing PD-L1 before initiating crizotinib for ALK-positive lung cancer could be a simple method to provide important prognostic information.

A kinesin family member 5b (KIF5B)-MET proto-oncogene, receptor tyrosine kinase (MET) rearrangement was reported in patients with lung adenocarcinoma but its oncogenic function was not fully evaluated. We used one-step reverse transcription-polymerase chain reaction for RNA samples to screen for the KIF5B-MET fusion in 206 lung adenocarcinoma and 28 pulmonary sarcomatoid carcinoma patients. Genomic breakpoints of KIF5B-MET were determined by targeted next-generation sequencing. Soft agar colony formation assays, proliferation assays, and a xenograft mouse model were used to investigate its oncogenic activity. In addition, specific MET inhibitors were administered to evaluate their anti-tumor activities. A KIF5B-MET fusion variant in a patient with a mixed-type adenocarcinoma and sarcomatoid tumor was identified, and another case was found in a pulmonary sarcomatoid carcinoma patient. Both cases carried the same chimeric gene, a fusion between exons 1–24 of KIF5B and exons 15–21 of MET. KIF5B-MET-overexpressing cells exhibited significantly increased proliferation and colony-forming ability. Xenograft tumors harboring the fusion gene demonstrated significantly elevated tumor growth. Ectopic expression of the fusion gene stimulated the phosphorylation of KIF5B-MET as well as downstream STAT3, AKT, and ERK1/2 signaling pathways. The MET inhibitors significantly repressed cell proliferation; phosphorylation of downstream STAT3, AKT, and ERK1/2; and xenograft tumorigenicity. In conclusion, the KIF5B-MET variant was demonstrated to have an oncogenic function in cancer cells. These findings have immediate clinical implications for the targeted therapy of subgroups of non-small cell lung cancer patients.

Summary Background Osimertinib is approved for the treatment of non-small-cell lung cancer in patients who develop the EGFR Thr790Met mutation after treatment with epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitors (TKIs). We assessed outcomes in patients with non-small-cell lung cancer and the EGFR Thr790Met mutation who were treated with osimertinib, a third-generation EGFR TKI, after previous treatment failure with one or more other EGFR TKIs. Methods Eligible patients had been enrolled at one centre in the AURA study, had shown resistance to a previous EGFR TKI, and had EGFR -activating mutations and acquired Thr790Met mutation detectable in tumour tissue or plasma. Patients took 20–240 mg osimertinib per day until disease progression or development of intolerable side-effects. Plasma samples were collected every 6 weeks and tumour tissue biopsy was done at study entry and was optional after disease progression. We tested samples for resistance mechanisms, including EGFR -activating, Thr790Met, and Cys797Ser mutations, and assessed associations with overall survival, progression-free survival, and survival after disease progression. Findings Of 71 patients enrolled in AURA, 53 were eligible for this analysis. Median progression-free survival was 11·1 months (95% CI 8·4–13·9) and overall survival was 16·9 months (11·7–29·1). 47 patients had disease progression. Median overall survival after osimertinib progression was 5·4 months (95% CI 4·1–10·0). Plasma samples were available for 40 patients after disease progression. 12 (30%) of these had the Thr790Met mutation (four of whom also had Cys797Ser mutations). Patients without detectable EGFR -activating mutations in plasma before treatment had the best overall and post-progression survival (22·4 months, 95% CI 15·6–not reached, and 10·8 months, 7·2–not reached, respectively). Loss of the Thr790Met mutation but presence of EGFR -activating mutations in plasma were associated with the shortest progression-free survival (median 2·6 months, 95% CI 1·3–not reached). In 22 post-progression tumour samples, we found one squamous cell and two small-cell transformations. We detected Thr790Met in nine (50%) of 18 samples, Cys797Ser in two (17%) of 12, cMET amplification in five (50%) of ten, BRAF mutation in one (8%) of 13, and KRAS mutation in one (8%) of 13. Interpretation Heterogeneous resistance mechanisms developed in patients receiving osimertinib. Differences in resistance mechanisms might dictate future development strategies for osimertinib in clinical trials. Funding AstraZeneca, Taiwan Ministry of Science and Technology.

Background: Immunotherapy that targets programmed death protein-1 (PD-1) provides improved treatment efficacy and survival in patients with metastatic non-small cell lung cancer (NSCLC), especially those with high tumor expression of PD-L1. However, data on this treatment are mostly from clinical trials enrolling highly selected patients. The real-world experience of anti-PD-1 treatment and the usefulness of tumor PD-L1 expression in prediction of treatment response are largely unknown. Methods: We retrospectively reviewed patients with stage IIIB/ IV NSCLC who received monotherapy with nivolumab or pembrolizumab, and evaluated response using RECIST 1.1 criteria. Factors associated with treatment response, progression free survival (PFS), and overall survival (OS) were determined. Results: Seventy-four NSCLC patients out of 116 examined patients were included, most of whom had adenocarcinoma (48/74, 64.9%) and received immunotherapy as a third-line or subsequent treatment (51/74, 68.9%). The median PFS and OS were 1.8 and 7.9 months, respectively. The objective response rate was 32%, but only 47 of 74 patients were evaluable. Through multivariate analysis, epidermal growth factor receptor (EGFR) mutation was independently associated with a poor treatment response. Good performance status (ECOG≤1) and smoking were independently associated with better PFS and OS. Data on tumor PD-L1 expression were available in 43 patients (58%); higher PD-L1 expression correlated with better treatment response and longer PFS. Severe treatment-related adverse events were uncommon. Conclusion: The efficacy and safety of anti-PD-1 medications for advanced NSCLC were comparable in real-world and clinical settings, except in those with poor ECOG scores. Prediction of treatment response from tumor PD-L1 expression seemed practical.

Background Immediate whole brain radiation (WBRT) has been the standard for patients with lung cancer with brain metastases. The study aims to evaluate the effect of immediate cranial irradiation in patients with epidermal growth factor receptor (EGFR) mutant lung cancer in the era of a new generation of EGFR inhibitors. Materials and Methods Medical records of 198 patients with EGFR mutant non-small cell lung cancer and brain metastases at initial metastatic diagnosis were reviewed. Patients were categorized into four groups: immediate WBRT, immediate cranial stereotactic radiosurgery (SRS), delayed radiation upon progression of cranial lesions (DRT), and never cranial irradiation (NRT). Overall survival (OS) and progression-free survival related to EGFR inhibitors were analyzed. Results The SRS group had the fewest brain metastases and fewest extracranial lesions, and the DRT and NRT groups had the smallest brain metastases. Median survival were 18.5, 55.7, 21.1, and 18.2 months for the WBRT, SRS, DRT, and NRT groups, respectively. Patients who had received EGFR T790M inhibitors survived longer (41.1 vs. 19.8 months). In multivariate analysis, the OS of patients in the SRS group was longer than that in the NRT group (adjusted hazard ratio [aHR]: 0.315). Patients who had fewer extracranial lesions and who had received EGFR T790M inhibitor treatments also survived longer (aHR: 0.442 and 0.357, respectively). Conclusion Immediate stereotactic radiosurgery but not whole brain radiation was associated with longer survival. Because of patient heterogeneity and the introduction of EGFR T790M inhibitors, the timing and modality of cranial irradiation should be determined individually, and cranial irradiation may be omitted for selected patients. Implications for Practice Immediate whole brain radiation has been the standard for patients with lung cancer with brain metastases. In this study, it was observed that, for patients with epidermal growth factor receptor (EGFR) mutant advanced lung cancer who had brain metastases, there was no difference in survival between patients who never received cranial irradiation and those who received whole brain radiation immediately. Patients who received immediate stereotactic radiosurgery or who had ever received EGFR T790M inhibitors survived longer. Patients who received immediate stereotactic radiosurgery have fewer brain metastases. These findings suggest that the timing and modality of cranial irradiation should be determined individually, and cranial irradiation may be omitted in selected patients.

Radial endobronchial ultrasound (R-EBUS) is one important diagnostic approach in non-small cell lung cancers (NSCLC). However, the small samples obtained from R-EBUS-guided transbronchial biopsies are sometimes insufficient for pathological and molecular diagnosis. Herein, we investigated the suitability of R-EBUS-guided bronchial brushing specimens for NSCLC diagnosis and EGFR genotyping. We enrolled 941 consecutive patients with peripheral pulmonary lesions who underwent R-EBUS. Cytology-positive brushing specimens from non-squamous NSCLC patients were tested for EGFR mutations. Non-squamous NSCLC was diagnosed in 624 patients (66.3%). Positive cytology was documented in the brushing samples of 376 patients (60.3%). Higher diagnostic yields were obtained in patients exhibiting bronchus signs on chest tomography, and those with R-EBUS probe located within the lesion. EGFR genotyping was successfully performed in 363 samples (96.5% of cytology-positive brushing samples). EGFR genotyping concordance between brushing specimens and matched tissue samples was 88.7% (kappa = 0.745, P

// Yi-Nan Liu 1 , Tzu-Hua Chang 1 , Meng-Feng Tsai 2 , Shang-Gin Wu 3 , Tzu-Hsiu Tsai 1 , Hsuan-Yu Chen 4 , Sung-Liang Yu 5 , James Chih-Hsin Yang 6, 7 , Jin-Yuan Shih 1, 8 1 Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan 2 Department of Molecular Biotechnology, Dayeh University, Changhua, Taiwan 3 Department of Internal Medicine, National Taiwan University Hospital, Yun-Lin Branch, Yun-Lin, Taiwan 4 Institute of Statistical Science, Academia Sinica, Taipei, Taiwan 5 Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan 6 Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan 7 Graduate Institute of Oncology, Cancer Research Center, National Taiwan University, Taipei, Taiwan 8 Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan Correspondence to: Jin-Yuan Shih, e-mail: jyshih@ntu.edu.tw Keywords: IL-8, EGFR, gefitinib, resistance, stemness Received: October 15, 2014 Accepted: February 15, 2015 Published: March 18, 2015 ABSTRACT Epidermal growth factor receptor (EGFR)-targeted strategy is limited by resistance. We identify the potential genes involved in EGFR TKI (tyrosine kinase inhibitor) resistance and study the therapeutic mechanism in the non-small cell lung cancers. Potential genes involved in resistance were examined by analyzing datasets from a pair of EGFR TKI-sensitive (PC9) and TKI-resistant cells (PC9/gef). Blood specimens from patients taking EGFR TKI as first-line treatment were used to examine the correlation between drug's efficacy and IL-8 level. The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines. We identified IL-8 was up-regulated in gefitinib-resistant cells, and high plasma IL-8 level was correlated with shorter progression-free-survival time. IL-8 overexpression suppressed gefitinib-induced apoptosis in gefitinib-sensitive cells. By contrast, suppression of IL-8 enhanced gefitinib-induced cell death in gefitinib-resistant cells. IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity. Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells. Our study demonstrates an important role for IL-8, and suggests IL-8 is a potential therapeutic target for overcoming EGFR TKI resistance.

Objectives Polymorphisms of DNA repair genes may affect DNA repair capacity and the sensitivity of platinum doublets chemotherapy in non-small-cell lung cancer (NSCLC). We prospectively evaluated whether single nucleotide polymorphisms (SNPs) of ERCC1, ERCC2, XRCC1, and XRCC3 were associated with treatment outcome in advanced non-squamous NSCLC patients receiving pemetrexed/platinum as their first-line chemotherapy. Materials and methods Genotyping of six SNPs in four DNA repair genes in 58 patients treated with first-line pemetrexed/platinum was performed using TaqMan SNP Genotyping Assays. Results The wild-type ERCC1 8092 (C/C) was significantly associated with a better objective response compared to the variant genotypes (C/A + A/A) (48% vs 10%, P = .005). In the multivariate Cox proportional hazards model, we found that individuals with a wild-type genotype of ERCC1 Asn118Asn, ERCC1 C8092A and ERCC2 Asp312Asn had significantly better overall survival (OS) than those with a heterozygous or homozygous variant genotype. On the other hand, the heterozygous variant genotype of ERCC2 Lys751Gln was associated with better OS than that of the wild-type genotype. We further explored the combined effect of SNPs on OS, and found a significant allele/dose-dependent trend toward decreasing OS in patients with an increasing number of unfavorable alleles among four SNPs in ERCC1 and ERCC2. The median OS of patients with two or three unfavorable alleles (30.1 and 30.5 months, respectively) was significantly longer than that of patients with 4 unfavorable alleles (11.8 months, log-rank test for trend, P = .001). Conclusion A combination of ERCC1 and ERCC2 polymorphisms may predict OS among pemetrexed/platinum treated advanced non-squamous NSCLC patients.

The prevalence, demographic features, and clinical outcomes of lung adenocarcinoma patients with novel ROS1 oncogenic rearrangement in East Asian populations are not clear. This study aimed to investigate the clinical and prognostic characteristics of lung adenocarcinoma in patients with ROS1 fusion compared with other driver mutations.Multiplex reverse transcription-polymerase chain reaction was used to detect the ROS1 fusion gene in lung adenocarcinoma cases. Immunohistochemistry was used to confirm the expression of ROS1. The demographic data and clinical outcomes of patients with the ROS1 fusion gene were compared with those of patients without the ROS1 fusion gene, including those with the EGFR mutation, EML4-ALK fusion, KRAS mutation, and quadruple-negative patients.Of 492 patients with lung adenocarcinoma, 12 (2.4%) had the ROS1 fusion gene. Their median age was 45.0 years, significantly younger than that of the ROS1 fusion-negative cohorts (p0.001). Acinar (including cribriform) and solid patterns were the two most common histologic subtypes in the ROS1 fusion tumors (7 of 12, 58.3%) and were predominantly seen in CD74-ROS1 fusion tumors (66.7%). There was no significant survival difference between the ROS1 fusion-positive and ROS1 fusion-negative cohorts in surgical group, but ROS1 fusion-positive patients might have worse outcomes than EGFR-mutant patients in the stage IV group.The ROS1 fusion gene can be successfully detected in East Asian patients with lung adenocarcinoma using multiplex reverse transcription-polymerase chain reaction. These patients tend to be younger and have characteristic histologic subtypes. Due to the small number of ROS1 fusion patients, the prognostic value of ROS1 fusion need further studies to confirm.

The diagnosis of malignant pleural effusions is an important issue in the management of malignancy patients. Generally, cytologic examination is a routine diagnostic technique. However, morphological interpretation of cytology is sometimes inconclusive. Here an ancillary method named BMVC test is developed for rapid detection of malignant pleural effusion to improve the diagnostic accuracy at low cost. A simple assay kit is designed to collect living cells from clinical pleural effusion and a fluorescence probe, 3,6-Bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), is used to illuminate malignant cells. The fluorescence intensity is quantitatively analyzed by ImageJ program. This method yields digital numbers for the test results without any grey zone or ambiguities in the current cytology tests due to intra-observer and inter-observer variability. Comparing with results from double-blind cytologic examination, this simple test gives a good discrimination between malignant and benign specimens with sensitivity of 89.4% (42/47) and specificity of 93.3% (56/60) for diagnosis of malignant pleural effusion. BMVC test provides accurate results in a short time period, and the digital output could assist cytologic examination to become more objective and clear-cut. This is a convenient ancillary tool for detection of malignant pleural effusions.

Objectives Although flexible bronchoscopy with the assistance of miniature radial-probe endobronchial ultrasound (EBUS) is increasingly employed to diagnose peripheral lung cancer, transbronchial biopsies typically offer an insufficient amount of tissue to conduct additional molecular analysis. We evaluated the feasibility of multi-gene analyses from waste brushing samples obtained by EBUS-assisted bronchoscopy. Materials and methods For lung cancer patients with positive brushing cytology, analysis of EGFR, K-ras and EML4-ALK fusions were carried out, utilizing reverse transcription-polymerase chain reaction and Sanger sequencing on the cell-derived RNA retrieved from waste brushing samples. Results EBUS-guided brushings were judged positive for tumor cells in 84 (68.9%) of the 122 patients with peripheral lung cancer receiving flexible bronchoscopy. Genotyping of EGFR and K-ras was successfully implemented in 80 (95.2%) of the 84 cytology-proven brushing samples, along with satisfactory yields to detect EGFR (55.0%) and K-ras (2.5%) mutations. The results of EGFR genotyping from the brushing specimens were highly concordant with those provided from other corresponding samples (concordance rate: 94%, kappa: 0.92). Of the 19 patients with adenocarcinoma or non-small cell lung cancer not otherwise specified harboring wild-type EGFR and K-ras, two cases (10.5%) were identified to harbor EML4-ALK fusions. Conclusion Our results suggest that multi-gene analyses from waste brushing specimens using RNA-based Sanger sequencing is highly feasible. This approach offers an opportunity to overcome the dilemma of flexible bronchoscopy in molecular diagnostics for lung cancer, and could potentially recruit more patients for targeted therapy according to the molecular characteristics of the tumor cells.

Introduction Tumor tissue is often not obtainable or suitable for molecular-based epidermal growth factor receptor ( EGFR ) mutational analysis in advanced non–small-cell lung cancer (NSCLC). This retrospective and single-institution study was conducted to evaluate the role of effusion immunocytochemistry using two EGFR mutant-specific antibodies for the detection of relevant EGFR mutations in NSCLC, along with the selection of candidates for first-line therapy with EGFR tyrosine kinase inhibitors (TKIs). Methods Immunocytochemistry using two antibodies binding specifically to the major forms of mutant EGFR, L858R, and E746-A750 deletion (delE746-A750), was performed on cell blocks of malignant pleural effusion (MPE) from 78 patients with lung adenocarcinoma, who received first-line EGFR TKIs. The yield of EGFR -mutation detection and prediction of response rate and progression-free survival to TKI treatment by immunocytochemistry were compared with those by clinical characteristics and EGFR sequencing using cell-derived RNA from MPEs. Results Of the 78 MPE samples, direct sequencing using cell-derived RNA identified L858R mutation in 42 cases, deletions in exon 19 in 12 cases (delE746-A750 in eight cases), other types of mutations in three cases, and wild-type EGFR in 21 cases. Effusion immunocytochemistry with these two mutant-specific antibodies exhibited a sensitivity of 71% and 88% and a specificity of 86% and 96% for identifying predefined L858R and delE746-A750 mutations, respectively. Effusion immunocytochemistry provided a superior prediction of tumor response and progression-free survival to first-line EGFR TKIs than did clinical characteristics like sex and smoking status. Patients whose effusion immunocytochemistry showed a reaction to either of the two antibodies had a comparable TKI response rate (67% versus 72%) to those with EGFR mutations assessed by direct sequencing from cell-derived RNA. Conclusions Effusion immunocytochemistry could be introduced into clinical practice to identify more NSCLC patients likely to have benefit from first-line TKI treatment, especially for those without adequate tissue for molecular-based EGFR analysis.

The soft sensors for monitoring respiratory and heart sound were composed of polyurethane and microphones. In this study, silica was blended with polyurethane to change the hardness of the chambers. The hardness would influence the frequency response of the sensors. The material composed of 60 phr silica was chosen to make the chamber of the sensor. It had higher hardness and resulted in the flatten frequency response across the range of 100–1200 Hz. By the filter band designed for heart sound and respiratory sound signal, the heart sound and respiratory sound can be collected. The measured sound was verified by the physician and showed no distortion.

SummaryObjectivesTo evaluate the prevalence and associated presentations of hypoglycemia in bacteremic pneumococcal infections, and serotypes of the isolates.MethodsThis was a retrospective study of 70 episodes of pneumococcal bacteremia that occurred in 2004 and 2005.ResultsWe found hypoglycemia (plasma glucose

Transthoracic ultrasound (US) has become an important diagnostic tool in modern chest medicine. The range of thoracic lesions for which transthoracic US may yield useful diagnostic information has expanded to include not only chest wall and pleural lesions, but also peripheral lung nodules, pulmonary consolidations, necrotizing pneumonias and lung abscesses, tumors with obstructive pneumonitis, mediastinal masses, and peridiaphragmatic lesions. A variety of ultrasound features and signs of chest diseases have been well characterized and widely applied in clinical practice. US guidance increases the diagnostic success rate and decreases the complications associated with interventional procedures such as thoracentesis, closed tube drainage for pleural effusion, and needle biopsy of the pleura. Transthoracic needle aspiration or biopsy, under real-time US guidance, is a relatively safe and easy procedure, and may provide adequate tissue sampling of lesions for cytologic, histologic or microbiologic analysis. This article presents the general techniques and wide applications of transthoracic US and US-guided invasive procedures in the diagnosis and management of various chest diseases.

Objectives RET rearrangements represent one of the newest molecular targets in non-small cell lung cancer (NSCLC). However, the prevalence, clinical characteristics, and outcome of patients with RET -rearranged lung adenocarcinoma in metastatic disease remain uncertain. Materials and methods Multiplex reverse transcription-polymerase chain reaction (RT-PCR) was used to detect KIF5B – RET and CCDC6 – RET fusions from specimens of malignant pleural effusion (MPE) in patients with metastatic lung adenocarcinoma. The demographic data and outcome of patients with RET -rearranged tumors were compared with those with EGFR -mutant, KRAS -mutant, EML4 – ALK -rearranged, and quadri-negative tumors. Results Of the 722 patients with MPE of lung adenocarcinoma screened, 17 (2.4%) had RET -rearranged tumors. The detected RET rearrangements comprised 11 (65%) KIF5B – RET and 6 (35%) CCDC6 – RET fusions, including 2 novel fusion variants identified. The presence of RET rearrangements was not associated with age at diagnosis, gender or smoking history, but predominantly seen in solid histological subtype. None of patients with RET -rearranged tumors had received kinase inhibitors with activity against RET kinase. The median overall survival was 22.4 months (95% CI, 8.8–36.0) for the 17 patients with RET -rearranged tumors, compared with 21.3 months (95% CI, 18.7–23.9; P =0.57) for the 451 patients with EGFR -mutant tumors, 5.4 months (95% CI, 2.7–8.1; P =0.002) for the 13 patients with KRAS -mutant tumors, 18.9 months (95% CI, 10.7–27.1; P =0.82) for the 51 patients with EML4 – ALK -rearranged tumors, and 12.0 months (95% CI, 9.0–15.0; P =0.07) for the 190 patients with quadri-negative tumors. Conclusion Multiplex RT-PCR from specimens of MPE is feasible for the screening of RET rearrangements in NSCLC. Metastatic RET -rearranged lung adenocarcinoma patients with MPE might have favorable survival comparable to those with metastatic EGFR -mutant tumors.

Objectives: To compare the clinical characteristics of community-acquired thoracic empyema in older and younger patients and to analyze the effect of various factors on outcome. Design: A retrospective, comparative observational study. Setting: A university-affiliated tertiary medical center. Participants: Forty-six patients aged 65 and older (older group) and 86 patients aged 18 to 64 (younger group). Measurements: Demographic, clinical, and microbiological data were reviewed. Results: Older patients were more likely to have dyspnea but less likely to have chest pain or fever (P

The authors summarize the current applications of chest ultrasonography in the diagnosis and management of various pleural diseases. Ultrasound has been proved to be valuable for the evaluation of a wide variety of chest diseases, particularly when the pleural cavity is involved. Chest ultrasound can supplement other imaging modalities of the chest and guides a variety of diagnostic and therapeutic procedures. Pleural effusion, pleural thickening, pleural tumors, tumor extension into the pleura and even the chest wall, pleuritis, and pneumothorax can be detected easily and accurately with chest ultrasound. Many ultrasound features and signs of these diseases have been well characterized and widely applied in clinical practice. Under real-time ultrasound guidance the success rates of invasive procedures on pleural diseases increase significantly whereas the risks are greatly reduced. The advantages of low-cost, bedside availability and no radiation exposure have made ultrasound an indispensable diagnostic tool in modern pulmonary medicine.

Department of Laboratory Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan; Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan

Bacteremic pneumonia caused by Mycobacterium simiae in non-human immunodeficiency virus (HIV)-infected patients has rarely been reported. We describe a non-HIV-infected adult with refractory bacteremia, osteomyelitis, and colonization of M. simiae in the respiratory tract who subsequently developed fatal bacteremic pneumonia. The isolate was confirmed as M. simiae by 16S rRNA gene analysis.

Pulmonary disease, such as asthma or chronic obstructive pulmonary disease (COPD), has been a major health concern throughtout the world. It would thus be necessary to develop an effective monitorning device and a real-time diagnosis algorithm for targeted populations, especially for children. Wheezing sound induced by asthma is a critical index for clinicians to make diagnosis. The traditional way to detect wheezing sound is to utilize the digital image process (DIP) method for tracking the specific wheezing pattern appeared in the short-time Fourier transform (STFT) spectral graph of respiratory sound. However, this method requires intensive computation and thus is difficult to implement for real-time diagnosis and low power consumption for personal health care system. In this study, we developed a new wheezing detection algorithm which is based on the estimation of correlation-coefficient of respiratory sound spectrum, called respiratory spectrum correlation-coefficient method (RSCM) in place of DIP step. Because of low memory quest of RSCM process, it can be installed easily in the microcontroller or PDA. We have implanted RSCM to a personal asthma daily care system based on both laptop and PDA. User can measure the respiratory sound by designed microphone input and real-time diagnose the occurrence of asthma. In the initial test, thirteen cases (six for wheezing and seven for normal) of respiratory sound were collected from the public domain websites. The result shows that the sensitivity and specificity for wheezing detection are 83% and 86%, respectively. This result assures the possibility to meet the demands of personal health care.

To compare the clinical characteristics of community-acquired thoracic empyema in older and younger patients and to analyze the effect of various factors on outcome.A retrospective, comparative observational study.A university-affiliated tertiary medical center.Forty-six patients aged 65 and older (older group) and 86 patients aged 18 to 64 (younger group).Demographic, clinical, and microbiological data were reviewed.Older patients were more likely to have dyspnea but less likely to have chest pain or fever (P.05 for all). The causative organisms were similar between the two groups, with anaerobes and facultative streptococci the most common pathogens. Older patients had increased morbidity and longer hospital stay (median 29.5 vs 20 days, P.001), but the in-hospital mortality was not significantly different between the two groups (13% vs 8%, P=.37). Multivariate analysis showed that coexisting malignancy (odds ratio (OR)=10.33, P=.01), lack of fever higher than 38 degrees C (OR=17.97, P=.03), and isolation of fungi from pleural fluid (OR=32.66, P=.01) were independently and significantly associated with in-hospital deaths.The microbiology and mortality of community-acquired thoracic empyema were similar between the two age groups. Difference in chronological age did not explain in-hospital death. This finding highlights the importance of effective treatment to obtain better outcomes for older patients.

Central venous catheter-related Wangiella dermatitidis infection has been increasingly reported in the recent literature. We report a case of central venous catheter (Port-A-Cath)-related fungemia caused by W. dermatitidis in a 58-year-old woman with lung cancer. Two W. dermatitidis isolates were recovered from 2 blood samples drawn from a peripheral vein and the Port-A-Cath 4 months after its placement. The minimum inhibitory concentrations of 2 isolates to fluconazole and amphotericin B using the standard broth microdilution method were 48 microg/mL and 0.19 microg/mL, respectively. The Port-A-Cath was removed and the fungemia responded to amphotericin B treatment. W. dermatitidis should be categorized as a pathogen that can cause central venous catheter-associated fungemia, particularly in immunocompromised patients.

Introduction: The diaphragm plays a pivotal role in weaning and successful extubation. We hypothesized that ultrasonographic evaluation of the movements of the diaphragm by measuring liver/spleen displacement during spontaneous breathing trials is a good predictor for extubation outcome. Patients and methods: The studied subjects were intubated patients receiving mechanical ventilation who were scheduled to be extubated. The displacement of liver/spleen was measured by ultrasonography before extubation. The patients were classified into a success group (SG) or failure group according to the extubation outcome. The baseline data and organ displacements in these two groups were analyzed. The sensitivity and specificity for the mean organ displacements and weaning parameters to predict successful extubation were calculated. Results: We included 55 patients, 32 of whom (58%) were in the SG. The baseline data are similar for these two groups, but the mean values of liver and spleen displacements were higher in the SG. Using a cutoff value of 1.1 cm, the sensitivity and specificity to predict successful extubation were 84.4% and 82.6%, respectively, better than traditional weaning parameters in this study. Conclusion: The displacement of the liver/spleen, measured by ultrasonography, is a good predictor for extubation outcome.