Assessment of prognostic scores of brain metastases from lung adenocarcinoma with EGFR mutations.

The aim of this study was to analyze prognostic factors and evaluate the value of four prognostic scores including RPA, DS-GPA BS-BM, GGS for the EGFR mutant BM patients from lung adenocarcinoma treated with EGFR-TKI. Data of NSCLC were retrospectively reviewed from August 2010 to June 2015 using the medical database of Shanxi Provincial Cancer Hospital. Patients with BM from lung adenocarcinoma with mutant EGFR treated by EGFR-TKI or a combination of EGFR-TKI and WBRT were included. Potential prognostic factors were statistically examined. The C-index of each prognostic score was calculated. A total of 1063 BM patients with lung adenocarcinoma that had been identified with EGFR mutations were reviewed. A total of 104 patients that had been diagnosed with BM were confirmed to have mutant EGFR in primary tumors. These patients received treatment with EGFR-TKI or EGFR-TKI with WBRT to BM. The potential predictive factors in multivariable analysis included KPS (70 vs.70-80 vs. 90-100) and number of brain metastatic lesions. In the log-rank test, the indexes of RPA, DS-GPA BS-BM, and GGS were all significant predictors of OS. The C-indexes of each prognostic score were 0.79, 0.76, 0.77, and 0.74 in DS-GPA, RPA, GGS, and BS-BM, respectively. The indexes of RPA, DS-GPA BS-BM, GGS were applicable for asessing survival stratification in brain metastases from lung adenocarcinoma with presented EGFR mutations in our independent population. The DS-GPA appears to be the best predictive value. However, all four of the indexes could not evaluate the exact independent prognostic factors in multivariable analysis. A prognostic index specific for this group of patients was needed for targeted lung cancer therapy.

Exon 19 deletion of epidermal growth factor receptor is associated with prolonged survival in brain metastases from non-small-cell lung cancer.

Brain metastasis (BM) is a poor prognostic factor for non-small-cell lung cancer (NSCLC). Recent studies have shown that oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) were effective for BM from NSCLC with EGFR mutation. However, the relationship between EGFR mutations and prognosis of NSCLC BM patients remains to be determined. In this study, we investigated the impact of EGFR mutation status on the survival of BM patients from NSCLC. One hundred six patients with BM from NSCLC were retrospectively reviewed. Thirty-three subjects (24.3 %) were confirmed to have an exon 19 deletion, while another 33 had an exon 21 point mutation (L858R) (24.3 %). Log-rank test and Cox proportional hazards model were used to analyze the impact of variables on survival. The median survival of NSCLC with BM was 8 months. Log-rank test analysis showed that Eastern Cooperative Oncology Group Performance Status (ECOG-PS) at BM (p < 0.0001), control of primary tumor (p = 0.005), pathology (p = 0.01), EGFR mutations (p = 0.045), and 19 exon deletion (p = 0.007) were associated with a longer survival. In a Cox proportional hazards model, EGFR exon 19 deletion (p = 0.034), control of primary tumor (p = 0.024), and ECOG PS at BM (p = 0.006) were found to be independent prognostic factors. Moreover, there were prognostic differences between groups according to Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis (RPA) classification system (p < 0.0001). Exon 19 deletion is an independent prognostic factor in BM from NSCLC. It should be integrated into the prognostic scoring classification system for NSCLC.

Geometry engineering of a multiple resonance core via a phenyl-embedded strategy toward highly efficient narrowband blue OLEDs.

The geometry of the molecular skeleton is of importance for the property regulation of organic electronic materials. Herein, we present a phenyl-embedded molecular design strategy to adjust the molecular curvature and achieve the improvement of blue multiple resonance (MR)-emitters. The introduction of a bridged phenyl contributes to a highly twisted saddle skeleton and the separation of frontier molecular orbitals, which are beneficial for the increase of photoluminescence quantum yield (PLQY) as well as the decrease of singlet-triplet energy gap (ΔEST). Consequently, hp-BQAO features an accelerated reverse intersystem crossing rate and suppressed non-radiative decay rate simultaneously, which enables the assembly of high-performance narrowband blue OLEDs with a record-high external quantum efficiency (EQE) of 24.1% for the blue OLED devices exploiting nitrogen-carbonyl-containing MR-emitters without sensitizers.

An endothelial cell niche induces hepatic specification through dual repression of Wnt and Notch signaling.

Complex cross-talk between endoderm and the microenvironment is an absolute requirement to orchestrate hepatic specification and expansion. In the mouse, the septum transversum and cardiac mesoderm, through secreted bone morphogenetic proteins (BMP) and fibroblast growth factors (FGF), respectively, instruct the adjacent ventral endoderm to become hepatic endoderm. Consecutively, endothelial cells promote expansion of the specified hepatic endoderm. By using a mouse reporter embryonic stem cell line, in which hCD4 and hCD25 were targeted to the Foxa2 and Foxa3 loci, we reconstituted an in vitro culture system in which committed endoderm cells coexpressing hCD4-Foxa2 and hCD25-Foxa3 were isolated and cocultured with endothelial cells in the presence of BMP4 and bFGF. In this culture setting, we provide mechanistic evidence that endothelial cells function not only to promote hepatic endoderm expansion but are also required at an earlier step for hepatic specification, at least in part through regulation of the Wnt and Notch pathways. Activation of Wnt and Notch by chemical or genetic approaches increases endoderm cell numbers but inhibits hepatic specification, and conversely, chemical inhibition of both pathways enhances hepatic specification and reduces proliferation. By using identical coculture conditions, we defined a similar dependence of endoderm harvested from embryos on endothelial cells to support their growth and hepatic specification. Our findings (1) confirm a conserved role of Wnt repression for mouse hepatic specification, (2) uncover a novel role for Notch repression in the hepatic fate decision, and (3) demonstrate that repression of Wnt and Notch signaling in hepatic endoderm is controlled by the endothelial cell niche.

Clinical value of alveolar lavage supernatant specimens in the detection of the EGFR gene mutation in patients with non-small cell lung carcinoma.

Background: This study sought to compare the consistency of the epidermal growth factor receptor (EGFR) gene mutation detection results in the supernatant of alveolar lavage specimens to the tissue sample results, and the consistency of the blood EGFR gene mutation detection results to the tissue detection results. Methods: In total, 29 patients with non-small cell lung carcinoma (NSCLC) were selected, and their bronchoalveolar lavage fluid (BALF) was collected. The supernatant and precipitate were separated by centrifugation. Deoxyribonucleic acid (DNA) was extracted from the supernatant, and blood and tumor tissues were collected to detect patients' EGFR gene mutation status. Results: Of the 29 enrolled patients, 12 of the 23 tissue-biopsy patients (52.2%) were positive for EGFR mutations, 11 of the 28 blood-test patients (39.2%) were positive for EGFR mutations, and 13 of the 29 cases of the BALF-test patients (44.8%) were positive for EGFR mutations. The most common mutations were the exon 19 deletion mutation and the L858R point mutation. The EGFR gene mutation rate was higher in female, young, non-smoker, and stage IIIB patients (than stage IV patients), but the differences were not statistically significant (all P>0.05). Of the 29 NSCLC patients tested for the EGFR gene mutation, the BALF supernatant and blood results were the same for 27 patients (coincidence rate: 93.10%). Of the 23 of the 29 enrolled patients tested for the EGFR gene mutation, the BALF supernatant and tissue test results were the same for 21 patients (coincidence rate: 91.30%). Further, the blood-test and the tissue test results were the same for 20 patients (coincidence rate: 86.96%). Conclusions: The EGFR gene mutation rate was high in NSCLC patients. The coincidence rate of the EGFR gene mutation detection results between BALF supernatant and tumor tissues was slightly higher than that of the blood and tumor tissue EGFR gene mutation detection results.

Red fluorescent zwitterionic naphthalenediimides with di/mono-benzimidazolium and a negatively-charged oxygen substituent.

The C-H/C-X cross-coupling of a benzimidazolium salt with 2Br-NDI afforded two unprecedented zwitterionic NDIs with di/mono-benzimidazolium and an extra negatively-charged oxygen substituent. They exhibited intensified red fluorescence in polar solvents and negative solvatochromism due to an intramolecular charge transfer process, and could specifically label lysosomes and the endoplasmic reticulum in living A549 cells, respectively. They represent a rare case of NDI-derived ionic fluorophores.

Identifying Genomic Alterations in Small Cell Lung Cancer Using the Liquid Biopsy of Bronchial Washing Fluid.

Objective: With the rapid development of cancer genomics and immunomics, some new treatments of small cell lung cancer (SCLC) are emerging. However, there are limitations to the clinical use of tumor tissue. Our study aimed to evaluate the potential use of bronchial washing fluid (BWF) in the liquid biopsy of SCLC. Methods: Twenty-one extensive SCLC (ES-SCLC) patients were enrolled in this study. For all patients, four sample types, BWF supernatant (BWFs), BWF precipitate (BWFp), plasma and tumor tissue, were collected before receiving chemotherapy, and one type, plasma, was collected after chemotherapy. All samples were conducted to NGS using the 1021-gene panel. The concordance rates of genomic profiling using NGS in the four types of samples were evaluated. Multiple clinical information was analyzed for correlation. Results: We successfully tested 20 BWFs samples, 21 BWFp samples, 21 tumor tissue samples, 20 pre-treatment plasma, and 13 post-treatment plasma of these 21 patients. The detectability of somatic mutations was 100% for BWFs, BWFp, tumor tissues, and post-treatment plasma, and only one pre-treatment plasma was absent with any mutation. Matched tumor tissue, BWFs, BWFp, and pre-treatment plasma samples were subsistent for 19 patients. For these patients, 204 genomic alterations were identified in tissue samples, while 189 (92.6%), 175 (85.5%), and 163 (79.9%) alterations were detected in the matched BWFs, BWFp, and pre-treatment plasma, respectively. Moreover, we found that the three tumor markers associated with SCLC have a lower sensitivity than genomic alterations. The endocrine resistance pathway was found enriched in hyponatremia patients which may be related to the hyponatremia. The TMBs of BWF, BWFp, and pre-treatment plasma samples all had a strong correlation with that of tissue samples. Both the VAF and the MVAF of mutations in post-treatment plasma were less than those in pre-treatment plasma, which was in accordance with the evaluation of curative effect. Conclusions: For ES-SCLC patients, the liquid biopsy of BWF showed a highly potential advantage to identify DNA alterations, which suggested that genomic analysis of BWF liquid biopsy may have clinical value as a supplement for tissue and blood detection. Through the restricted validation, it can be widely used in routine clinical practice.

A study of the application of fiberoptic bronchoscopy combined with liquid-based cytology test in the early diagnosis of lung cancer.

Application value of fiberoptic bronchoscopy combined with liquid-based cytology test (LCT) in the early diagnosis of lung cancer was investigated. Clinical data of 901 patients who had suspicious lung space-occupying lesions and underwent bronchoscopy combined with LCT in Shanxi Provincial Cancer Hospital from June 2012 to June 2016 were retrospectively analyzed. Patients were divided into four groups to receive different fiberoptic bronchoscopies combined with LCT. Patients in Group A (n=276) received bronchoscopic washing cultures (BWC), Group B (n=204) received bronchoalveolar lavage (BAL), Group C (n=187) underwent endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and Group D (n=234) underwent transbronchial lung biopsy (TBLB). All patients received pathological biopsy to confirm the lesions. The diagnostic results of lung cancer and the incidence rates of postoperative adverse reactions/complications were analyzed and compared among the four groups. Among 901 patients, 741 cases were pathologically diagnosed with lung cancer. In Group A, there were 224 cases diagnosed with lung cancer, of which 193 cases were successfully detected with a detection rate of 86.17% and a κ-value of 0.426. In Group B, 171 cases were diagnosed with lung cancer, of which 149 cases were successfully detected with a detection rate of 87.13% and a κ-value of 0.430. In Group C, 154 cases were diagnosed with lung cancer, of which 146 cases were successfully detected with a detection rate of 94.81% and a κ-value of 0.769. In Group D, 192 cases were diagnosed with lung cancer, of which 170 cases were successfully detected with a detection rate of 88.54% and a κ-value of 0.440. Therefore, we conclude that fiberoptic bronchoscopy combined with LCT technique is safe and reliable for the diagnosis of the early-stage lung cancer.

Endothelial cells instruct liver specification of embryonic stem cell-derived endoderm through endothelial VEGFR2 signaling and endoderm epigenetic modifications.

Liver organogenesis requires complex cell-cell interactions between hepatic endoderm cells and adjacent cell niches. Endothelial cells are key players for endoderm hepatic fate decision. We previously demonstrated that the endothelial cell niche promotes hepatic specification of mouse embryonic stem cell(ESC)-derived endoderm through dual repression of Wnt and Notch pathways in endoderm cells. In the present study, we dissected further the mechanisms by which endothelial cells trigger endoderm hepatic specification. Using our previously established in vitro mouse ESC system mimicking the early hepatic specification process, endoderm cells were purified and co-cultured with endothelial cells to induce hepatic specification. The comparison of transcriptome profiles between hepatic endoderm cells isolated from co-cultures and endoderm cells cultured alone revealed that VEGF signaling instructs hepatic specification of endoderm cells through endothelial VEGFR2 activation. Additionally, epigenetic mark inhibition assays upon co-cultures uncovered that histone acetylation and DNA methylation promote hepatic specification while histone methylation inhibits it. This study provides an efficient 2D platform modelling the endothelial cell niche crosstalk with endoderm, and reveals mechanisms by which endothelial cells promote hepatic specification of mouse ESC-derived endoderm cells through endothelial VEGFR2 activation and endoderm epigenetic modifications.

HDAC inhibitors TSA and sodium butyrate enhanced the human IL-5 expression by altering histone acetylation status at its promoter region.

The expression of IL-5 correlated tightly with the maturation and differentiation of eosinophils, and is considered as a cytokine responsible for allergic inflammation. We report here that inhibition of HDAC activity by Trichostatin A (TSA) and sodium butyrate (NaBu), the two specific HDAC inhibitors, resulted in the elevation of both endogenous and exogenous activity of IL-5 promoter. We demonstrated that both the mRNA expression and protein production of IL-5 were stimulated by TSA and NaBu treatments. ChIP assays showed that treatments of TSA and NaBu caused hyperacetylation of histones H3 and H4 on IL-5 promoter in Jurkat cells, which consequently promoted the exogenous luciferase activity driven by this promoter. Moreover, site-directed mutagenesis studies showed that the binding sites for transcription factors NFAT, GATA3 and YY1 on IL-5 promoter were critical for the effects of TSA and NaBu, suggesting that the transcriptional activation of IL-5 gene by these inhibitors was achieved by affecting HDAC function on IL-5 promoter via transcription factors. These data will contribute to elucidating the unique mechanism of IL-5 transcriptional control and to the therapy of allergic disorders related to IL-5.

Recruitment of histone deacetylase 4 by transcription factors represses interleukin-5 transcription.

The critical role of IL-5 (interleukin-5) in eosinophilic inflammation implicates it as a therapeutic target for allergic diseases. The aim of the present study was to elucidate the molecular basis for the involvement of reversible histone acetylation in IL-5 transcriptional regulation. We provide evidence that HDAC4 (histone deacetylase 4) and p300, a known HAT (histone acetyltransferase), reversibly controlled the activity of the IL-5 promoter in vivo and in vitro, with a concurrent alteration of histone H3 acetylation status at the promoter regions. The nucleo-cytoplasmic shuttling of HDAC4 was shown to play an important role in the suppressive function of HDAC4 in IL-5 gene expression. Point mutation and reporter ChIP (chromatin immunoprecipitation) studies determined that the four transcription factors binding on the IL-5 promoter, i.e. C/EBPbeta (CAAT/enhancer-binding protein beta), GATA3 (GATA binding protein 3), NFAT (nuclear factor of activated T cells) and YY1 (Yin and Yang 1), were essential for the recruitment of HDAC4. Consistent with these observations, HDAC4 was found to form protein complexes with GATA3 and YY1, and to co-exist in the nuclei with GATA3. We propose that the unique regulatory mechanism of IL-5 gene transcription involves the reversible histone modification catalysed by HDAC4 and p300, which are recruited by the transcription factors. The dynamic balance in IL-5 transcriptional regulation is achieved through interactions among HATs/HDACs, histones and transcription factors. These data contribute to understanding the molecular mechanisms of IL-5 regulation, which is crucial to the development of new therapeutic strategies for IL-5-related allergic diseases.

Applicability of graded prognostic assessment of lung cancer using molecular markers to lung adenocarcinoma patients with brain metastases.

Several scoring systems are available to estimate prognosis and assist in selecting treatment methods for non-small cell lung cancer (NSCLC) patients with brain metastasis, including recursive partitioning analysis (RPA), basic score for brain metastases (BS-BM), and diagnosis-specific graded prognostic assessment (DS-GPA). Lung-molGPA is an update of the DS-GPA that incorporates EGFR and/or ALK mutation status. The present study tested the applicability of these four indexes in 361 lung adenocarcinoma patients with brain metastasis. Potential predictive factors in our independent multivariate analysis included patient age, Karnofsky performance status, EGFR and ALK mutation status, and use of targeted therapy. In the log-rank test, all four systems predicted overall survival (OS) (P<0.001). Harrell's C indexes were 0.732, 0.724, 0.729, and 0.747 for RPA, BS-BM, DS-GPA, and Lung-molGPA, respectively. Our results confirmed that the Lung-molGPA index was useful for estimating OS in our patient cohort, and appeared to provide the most accurate predictions. However, the independent prognostic factors identified in our study were not entirely in agreement with the Lung-molGPA factors. In an era of targeted therapy, Lung-molGPA must be further updated to incorporate more specific prognostic factors based on additional patient data.

Foxa2 identifies a cardiac progenitor population with ventricular differentiation potential.

The recent identification of progenitor populations that contribute to the developing heart in a distinct spatial and temporal manner has fundamentally improved our understanding of cardiac development. However, the mechanisms that direct atrial versus ventricular specification remain largely unknown. Here we report the identification of a progenitor population that gives rise primarily to cardiovascular cells of the ventricles and only to few atrial cells (<5%) of the differentiated heart. These progenitors are specified during gastrulation, when they transiently express Foxa2, a gene not previously implicated in cardiac development. Importantly, Foxa2+ cells contribute to previously identified progenitor populations in a defined pattern and ratio. Lastly, we describe an analogous Foxa2+ population during differentiation of embryonic stem cells. Together, these findings provide insight into the developmental origin of ventricular and atrial cells, and may lead to the establishment of new strategies for generating chamber-specific cell types from pluripotent stem cells.

Efficacy and adequacy of conventional transbronchial needle aspiration of IASLC stations 4R, 4L and 7 using endobronchial landmarks provided by the Wang nodal mapping system in the staging of lung cancer.

BACKGROUND: The role of transbronchial needle aspiration (TBNA) in the diagnosis and staging of lung cancer has been well established. Recently, the efficacy of conventional TBNA in the staging of lung cancer has been enhanced by the use of endobronchial ultrasound (EBUS)-TBNA. Our study sought to evaluate the adequacy of TBNA of International Association for the Study of Lung Cancer (IASLC) stations 4R, 4L and 7 using endobronchial landmarks provided by the Wang nodal mapping system in the staging of lung cancer. METHODS: We retrospectively analyzed all bronchoscopic cases with conventional TBNA punctures positive for malignancy at our institution from 1 January to 31 October 2014. The endobronchial puncture site was guided by the Wang nodal mapping system. The Wang stations were correlated with the IASLC lymph node map. No endobronchial ultrasound or rapid on-site evaluation was used. Pathological analysis included cytological and histological examination. RESULTS: Diagnosis by histological analysis was obtained in 115 (55.3%) out of 208 puncture sites. The metastatic lymph nodes were distributed at IASLC stations 4R (W1, 3, 5) 46.6 %, 7 (W2, 8, 10) 19.7%, 4L (W4, 6) 11.5%, 11R (W7, W9) 11.1% 11L (W11) 9.6%, 2R (high station W3) 0.5%, and the proximal portion of station 8 (station W10 beyond the middle lobe orifice) 1%. No complications were observed. CONCLUSION: IASLC station 4R (W1, 3, 5), 7 (W2, 8, 10) and 4L (W4, 6) are adequate for the staging of lung cancer.

[Cloning and prokaryotic expression of yeast GCN5 and RPD3].

The yeast histone acetyltransferase (HAT) gene gcn5 and histone deacetylase (HDAC) gene rpd3 were cloned from yeast genomic DNA by PCR amplification. The two genes,both with additional 6xHis tag,were subcloned into pBV220 vector to construct expression plasmids pBVgcn5 and pBVrpd3,respectively. Both pBVgcn5 and pBVrpd3 were over-expressed in Escherichia coli upon temperature induction,as revealed by SDS-PAGE. The recombinant GCN5 and RPD3 were purified by using a 6xHis affinity column. The purified GCN5 was tested to possess the HAT activity by using a (14)C-labeling assay. This work has laid down the basis for further in vitro studies into roles of histone acetylation/deacetylation in modulating chromatin conformation and transcription activity.

Endoderm generates endothelial cells during liver development.

Organogenesis requires expansion of the embryonic vascular plexus that migrates into developing organs through a process called angiogenesis. Mesodermal progenitors are thought to derive endothelial cells (ECs) that contribute to both embryonic vasculogenesis and the subsequent organ angiogenesis. Here, we demonstrate that during development of the liver, which is an endoderm derivative, a subset of ECs is generated from FOXA2+ endoderm-derived fetal hepatoblast progenitor cells expressing KDR (VEGFR2/FLK-1). Using human and mouse embryonic stem cell models, we demonstrate that KDR+FOXA2+ endoderm cells developing in hepatic differentiation cultures generate functional ECs. This introduces the concept that ECs originate not exclusively from mesoderm but also from endoderm, supported in Foxa2 lineage-tracing mouse embryos by the identification of FOXA2+ cell-derived CD31+ ECs that integrate the vascular network of developing fetal livers.

KDR identifies a conserved human and murine hepatic progenitor and instructs early liver development.

Understanding the fetal hepatic niche is essential for optimizing the generation of functional hepatocyte-like cells (hepatic cells) from human embryonic stem cells (hESCs). Here, we show that KDR (VEGFR2/FLK-1), previously assumed to be mostly restricted to mesodermal lineages, marks a hESC-derived hepatic progenitor. hESC-derived endoderm cells do not express KDR but, when cultured in media supporting hepatic differentiation, generate KDR+ hepatic progenitors and KDR- hepatic cells. KDR+ progenitors require active KDR signaling both to instruct their own differentiation into hepatic cells and to non-cell-autonomously support the functional maturation of cocultured KDR- hepatic cells. Analysis of human fetal livers suggests that similar progenitors are present in human livers. Lineage tracing in mice provides in vivo evidence of a KDR+ hepatic progenitor for fetal hepatoblasts, adult hepatocytes, and adult cholangiocytes. Altogether, our findings reveal that KDR is a conserved marker for endoderm-derived hepatic progenitors and a functional receptor instructing early liver development.

Generation of functional hepatic cells from pluripotent stem cells.

Liver diseases affect millions of people worldwide, especially in developing country. According to the American Liver Foundation, nearly 1 in every 10 Americans suffers from some form of liver disease. Even though, the liver has great ability to self-repair, in end-stage liver diseases including fibrosis, cirrhosis, and liver cancer induced by viral hepatitis and drugs, the liver regenerative capacity is exhausted. The only successful treatment for chronic liver failure is the whole liver transplantation. More recently, some clinical trials using hepatocyte transplantation have shown some clinical improvement for metabolic liver diseases and acute liver failure. However, the shortage of donor livers remains a life-threatening challenge in liver disease patients. To overcome the scarcity of donor livers, hepatocytes generated from embryonic stem cell or induced pluripotent stem cell differentiation cultures could provide an unlimited supply of such cells for transplantation. This review provides an updated summary of hepatic differentiation protocols published so far, with a characterization of the hepatic cells generated in vitro and their ability to regenerate damaged livers in vivo following transplantation in pre-clinical liver deficient mouse models.