Identification of lung adenocarcinoma-specific exosome RNAs in peripheral blood by RNA-Seq analysis.

OBJECTIVE: Several plasma-derived exosome RNAs have been identified as key regulators in cancer development. They have been considered as potential biomarkers for a non-invasive "liquid biopsy" to diagnose and assess the progression of cancer. This study aimed to identify human lung adenocarcinoma-specific exosome RNAs in peripheral blood, while assessing the feasibility and efficiency of this recently developed deep-sequencing technology for transcriptome profiling. PATIENTS AND METHODS: Plasma-derived exosome RNAs were isolated from 13 lung adenocarcinoma patients, 3 patients with benign lung diseases, and 15 healthy volunteers. RNA-seq analysis of ribosomal RNA-depleted total RNA was performed. RNAs differentially expressed between lung adenocarcinoma and benign lung diseases or healthy volunteers were identified, followed by GO and KEGG pathway enrichment analyses for the identification of key exosome RNAs associated with lung adenocarcinomas. RESULTS: Significant differentially expressed RNAs, such as UDP glucuronosyltransferase family 1 member A1 (UGT1A1) and BAI1-associated protein 2 like 1 (BAIAP2L1), were identified as differentially expressed between lung adenocarcinoma patients and patients with benign lung diseases. Eight pseudogenes, including Tropomyosin 1 (Alpha) Pseudogene (LOC100129096), Prothymosin, Alpha Pseudogene 2 (PTMAP2), Cell Division Cycle 14C, Pseudogene (CDC14C), Tropomyosin 1 (Alpha) Pseudogene (LOC643634), Ferritin Heavy Chain 1 Pseudogene 2 (FTH1P2), Actin Related Protein 2/3 Complex Subunit 3 Pseudogene 3 (ARPC3P3), Ferritin Heavy Chain 1 Pseudogene 11 (FTH1P11), and Prothymosin Alpha Pseudogene 5 (PTMAP5) were identified from plasma-derived exosomes in lung adenocarcinoma patients, who were more abundant/detectable than healthy volunteers. CONCLUSIONS: Our data indicate that plasma-derived exosome RNAs, UGT1A1, and BAIAP2L1, as well as the eight isolated pseudogenes could serve as diagnostic and prognostic biomarkers for an effective non-invasive "liquid biopsy" of lung adenocarcinomas.

Immunotherapeutic maintenance treatment with toll-like receptor 9 agonist lefitolimod in patients with extensive-stage small-cell lung cancer: results from the exploratory, controlled, randomized, international phase II IMPULSE study.

Background: The immune surveillance reactivator lefitolimod (MGN1703), a DNA-based TLR9 agonist, might foster innate and adaptive immune response and thus improve immune-mediated control of residual cancer disease. The IMPULSE phase II study evaluated the efficacy and safety of lefitolimod as maintenance treatment in extensive-stage small-cell lung cancer (ES-SCLC) after objective response to first-line chemotherapy, an indication with a high unmet medical need and stagnant treatment improvement in the last decades. Patients and methods: 103 patients with ES-SCLC and objective tumor response (as per RECIST 1.1) following four cycles of platinum-based first-line induction therapy were randomized to receive either lefitolimod maintenance therapy or local standard of care at a ratio of 3 : 2 until progression or unacceptable toxicity. Results: From 103 patients enrolled, 62 were randomized to lefitolimod, 41 to the control arm. Patient demographics and response patterns to first-line therapy were balanced. Lefitolimod exhibited a favorable safety profile and pharmacodynamic assessment confirmed the mode-of-action showing a clear activation of monocytes and production of interferon-gamma-induced protein 10 (IP-10). While in the intent-to-treat (ITT) population no relevant effect of lefitolimod on progression-free and overall survival (OS) could be observed, two predefined patient subgroups indicated promising results, favoring lefitolimod with respect to OS: in patients with a low frequency of activated CD86+ B cells (hazard ratio, HR 0.53, 95% CI: 0.26-1.08; n = 38 of 88 analyzed) and in patients with reported chronic obstructive pulmonary disease (COPD) (HR 0.48, 95% CI: 0.20-1.17, n = 25 of 103). Conclusions: The IMPULSE study showed no relevant effect of lefitolimod on the main efficacy end point OS in the ITT, but (1) the expected pharmacodynamic response to lefitolimod, (2) positive OS efficacy signals in two predefined subgroups and (3) a favorable safety profile. These data support further exploration of lefitolimod in SCLC.

ALK-Testing in non-small cell lung cancer (NSCLC): Immunohistochemistry (IHC) and/or fluorescence in-situ Hybridisation (FISH)?: Statement of the Germany Society for Pathology (DGP) and the Working Group Thoracic Oncology (AIO) of the German Cancer Society e.V. (Stellungnahme der Deutschen Gesellschaft für Pathologie und der AG Thorakale Onkologie der Arbeitsgemeinschaft Onkologie/Deutsche Krebsgesellschaft e.V.).

The EML4-ALK pathway plays an important role in a significant subset of non-small cell lung cancer patients. Treatment options such as ALK tyrosine kinase inhibitors lead to improved progression free survival and overall survival. These therapeutic options are chosen on the basis of the identification of the underlying genetic signature of the EML-ALK translocation. Efficient and easily accessible testing tools are required to identify eligible patients in a timely fashion. While FISH techniques are commonly used to detect this translocation, the broad implementation of this type of ALK testing into routine diagnostics is not optimal due to technical, structural and financial reasons. Immunohistochemical techniques to screen for EML4-ALK translocations may therefore play an important role in the near future. This consensus paper provides recommendations for the test algorithm and quality of the respective test approaches, which are discussed in the light of the current literature.

[ALK-Diagnostics in NSCLC - Immunohistochemistry (IHC) and/or Fluorescence-in-situ Hybridisation (FISH)]. / ALK-Testung beim nicht-kleinzelligen Lungenkarzinom (NSCLC): Immunhistochemie (IHC) und/oder Fluoreszenz-in-situ-Hybridisierung (FISH)?

The EML4-ALK pathway plays an important role in a significant subset of non-small cell lung cancer patients. Treatment options such as tyrosine kinase inhibitors directed against the EML4-ALK signalling pathway lead to improved progression free and overall survival. These therapeutic options are chosen on the basis of the identification of the underlying genetic signature of the EML-ALK translocation. Efficient and easily accessible testing tools are required to identify the patients in time. While FISH techniques have been implemented to characterize this translocation for some time, the implementation of this testing is hampered by its broad use of resources. Immunohistochemical techniques to identify and screen for EML4-ALK translocations may play an important role in the near future. This consensus paper offers recommendations of the sequence and quality of the respective test approaches which are validated on the basis of the current literature.

Improved diagnostics targeting c-MET in non-small cell lung cancer: expression, amplification and activation?

BACKGROUND: Several c-MET targeting inhibitory molecules have already shown promising results in the treatment of patients with Non-small Cell Lung Cancer (NSCLC). Combination of EGFR- and c-MET-specific molecules may overcome EGFR tyrosine kinase inhibitor (TKI) resistance. The aim of this study was to allow for the identification of patients who might benefit from TKI treatments targeting MET and to narrow in on the diagnostic assessment of MET. METHODS: 222 tumor tissues of patients with NSCLC were analyzed concerning c-MET expression and activation in terms of phosphorylation (Y1234/1235 and Y1349) using a microarray format employing immunohistochemistry (IHC). Furthermore, protein expression and MET activation was correlated with the amplification status by Fluorescence in Situ Hybridization (FISH). RESULTS: Correlation was observed between phosphorylation of c-MET at Y1234/1235 and Y1349 (spearman correlation coefficient rs = 0.41; p < 0.0001). No significant correlation was shown between MET expression and phosphorylation (p > 0.05). c-MET gene amplification was detected in eight of 214 patients (3.7%). No significant association was observed between c-MET amplification, c-MET protein expression and phosphorylation. CONCLUSION: Our data indicate, that neither expression of c-MET nor the gene amplification status might be the best way to select patients for MET targeting therapies, since no correlation with the activation status of MET was observed. We propose to take into account analyzing the phosphorylation status of MET by IHC to select patients for MET targeting therapies. Signaling of the receptor and the activation of downstream molecules might be more crucial for the benefit of therapeutics targeting MET receptor tyrosine kinases than expression levels alone.

Correlation between dihydropyrimidine dehydrogenase and efficacy and toxicity of fluoropyrimidine drugs.

At present, fluoropyrimidine, based on 5-fluorouracil (5-FU), remains one of the most frequently prescribed chemotherapeutics drugs for the treatment of cancer. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the catabolism of 5-FU, and DPD enzymatic activities are usually varied dramatically from individual to individual, including both the intrapatient differences and the interpatient variability. There is a certain correlation between the DPD activity and efficacy and toxicity following the administration of fluoropyrimidine drugs. Partial or complete loss of DPD activity can lead to serious or even lethal toxicity. In this article, we review the relationship between DPD activity and efficacy and toxicity following the administration of fluoropyrimidine drugs, and also the structure, function, and characteristics of DPD. We report here that measurement of DPD activity may become a strategy and be paid much attention to predict the efficacy and toxicity prior to starting a fluoropyrimidine-based therapy.

Constitutive Notch2 signaling in neural stem cells promotes tumorigenic features and astroglial lineage entry.

Recent studies identified a highly tumorigenic subpopulation of glioma stem cells (GSCs) within malignant gliomas. GSCs are proposed to originate from transformed neural stem cells (NSCs). Several pathways active in NSCs, including the Notch pathway, were shown to promote proliferation and tumorigenesis in GSCs. Notch2 is highly expressed in glioblastoma multiforme (GBM), a highly malignant astrocytoma. It is therefore conceivable that increased Notch2 signaling in NSCs contributes to the formation of GBM. Here, we demonstrate that mice constitutively expressing the activated intracellular domain of Notch2 in NSCs display a hyperplasia of the neurogenic niche and reduced neuronal lineage entry. Neurospheres derived from these mice show increased proliferation, survival and resistance to apoptosis. Moreover, they preferentially differentiate into astrocytes, which are the characteristic cellular population of astrocytoma. Likewise, we show that Notch2 signaling increases proliferation and resistance to apoptosis in human GBM cell lines. Gene expression profiling of GBM patient tumor samples reveals a positive correlation of Notch2 transcripts with gene transcripts controlling anti-apoptotic processes, stemness and astrocyte fate, and a negative correlation with gene transcripts controlling proapoptotic processes and oligodendrocyte fate. Our data show that Notch2 signaling in NSCs produces features of GSCs and induces astrocytic lineage entry, consistent with a possible role in astrocytoma formation.

Basal HIF-1α expression levels are not predictive for radiosensitivity of human cancer cell lines.

BACKGROUND AND PURPOSE: High levels of hypoxia inducible factor (HIF)-1α in tumors are reported to be associated with tumor progression and resistance to therapy. To examine the impact of HIF-1α on radioresistance under normoxia, the sensitivity towards irradiation was measured in human tumor cell lines that differ significantly in their basal HIF-1α levels. MATERIAL AND METHODS: HIF-1α levels were quantified in lysates of H1339, EPLC-272H, A549, SAS, XF354, FaDu, BHY, and CX- tumor cell lines by ELISA. Protein levels of HIF-1α, HIF-2α, carbonic anhydrase IX (CA IX), and GAPDH were assessed by Western blot analysis. Knock-down experiments were performed using HIF-1α siRNA. Clonogenic survival after irradiation was determined by the colony forming assay. RESULTS: According to their basal HIF-1α status, the tumor cell lines were divided into low (SAS, XF354, FaDu, A549, CX-), intermediate (EPLC-272H, BHY), and high (H1339) HIF-1α expressors. The functionality of the high basal HIF-1α expression in H1339 cells was proven by reduced CA IX expression after knocking-down HIF-1α. Linear regression analysis revealed no correlation between basal HIF-1α levels and the survival fraction at either 2 or 4 Gy in all tumor cell lines investigated. CONCLUSION: Our data suggest that basal HIF-1α levels in human tumor cell lines do not predict their radiosensitivity under normoxia.

Lung function in adult patients with cystic fibrosis after using the eFlow rapid for one year.

BACKGROUND: The new generation nebuliser PARI eFlow® rapid allows a highly efficient aerosol delivery at reduced inhalation time. However, lung function data during long-term use of this device are not available until now. METHODS: 70 clinically stable adult cystic fibrosis patients participated in this observation study. Lung function tests were performed prospectively 12 weeks after and again 9 to 12 months after switching the inhalation device from a conventional jet nebulizer to the PARI eFlow® rapid. Lung function data were collected retrospectively from the visits 1 year as well as 12 weeks prior to the switch-over. Lung function data for all time points were only available for 59 patients. Treatment time and patient's satification were recorded for both conventional and new nebuliser in all 70 patients. RESULTS: After 1 year of inhalation with eFlow® rapid, the mean change in FEV1% was - 1.4% (n = 59 patients). The decrease in FEV1 was smaller than the change in FEV1 after 1 year of inhalation with the conventional jet nebuliser (control period, -3.1%), although this difference was not statistically significant. The same effect was seen in MEF25(%) (-2.6% with conventional nebuliser compared to -1.6% after eFlow® rapid). Concerning the FVC, there was a greater improvement after 1 year of inhalation with the eFlow® rapid than with the jet nebuliser (+ 2.9% vs. +1.1%). For PEF%, there was an increase during the control period, whereas after inhalation with eFlow® rapid there was a decrease (+1.1% vs. -2.9%). All changes were not significantly different. The eFlow® rapid reduced total daily inhalation time by two-thirds (conventional nebuliser: 31.1 min/day; eFlow® rapid: 10.2 min/day, n = 70 patients). CONCLUSION: Inhalation with the new nebuliser eFlow rapid does not alter FEV1, FVC or PEF significantly after 1 year of inhalation. The treatment time could be reduced significantly by the eFlow® rapid.

Organ-cocultures (COCs) for long term in vitro studies of lung cancer using 2-photon microscopy.

Three-dimensional organ cultures allow performing research in vitro in a complex multi-cellular environment. We aimed at developing a long term coculture system (COC) for the study of lung cancer to repeatedly measure tumor volume. Organ cultures of bronchial mucosa with 1-2 mm diameter were embedded in agarose and bisected with a tissue slicer so that the organ culture within was cut into halves uncovering the connective tissue of the stroma of each half. A cell suspension of GFP-transfected EPLC 32M1 lung tumor cells was brought in contact with the connective tissue of the wounded surface. Adherent tumor cells grew invasively into the organ culture. Using 2-Photon microscopy, Z-stacks were recorded, reconstructed with appropriate analysis software, and the tumor volume was calcvulated. Tumor cells were identified by GFP-fluorescence. Repeated measurements of the same COC could be performed over up to 8 weeks. The tumor volume increased continually with the growth rate becoming slower towards the end of culture. A comparison of two clones of tumor cells which had shown different rates of proliferation in monolayer culture demonstrated that the clone with the higher rate of proliferation in monoculture produced tumors with more rapid growth in the COC model. In this study we present a coculture system for the study of lung cancer using 2-Photon microscopy. COCs are particularly appropriate for long term in vitro treatment studies.

[Interventional bronchoscopy in lung cancer]. / Bronchoskopische Therapieverfahren beim Lungenkarzinom.

Stenosis of central airways or hemoptysis are classical indications for interventional bronchoscopy in lung cancer. In the case of endoluminal tumor growth cryo-, laser- or brachytherapy are widely used. In the case of airway stenosis due to compression by extraluminal tumor masses balloon-dilatation and/or stenting and - with delayed effect - brachytherapy are first-choice therapies. Carcinoma in situ and early stage tumors can be treated curatively with brachytherapy or photodynamic therapy. Recently introduced bronchoscopic techniques like electro-magnetic navigation may result in new curative options for peripheral lung tumors.

Reduced expression of Bax in small cell lung cancer cells is not sufficient to induce cisplatin-resistance.

Resistance to cisplatin in the course of chemotherapy contributes to the poor prognosis of small cell lung cancer (SCLC). B cell lymphoma-2 is the founding member of a large family of proteins that either promote or inhibit apoptosis. We aimed at investigating if the pro-apoptotic members Bad, Bax, Bim and Bid are involved in cisplatin-resistance. - Cisplatin-resistance in the SCLC cell line H1339 was induced by repetitive exposure to cisplatin. Protein expression was quantified by Western Blot and immuno-fluorescence analysis. Protein expression was altered using siRNA interference. - Four cycles of 0.5 µg/ml cisplatin led to partial cisplatin-resistance in H1339 cells. The expression of Bad, Bim and Bid was comparable in naive and resistant cells while the expression of Bax was reduced in the resistant clone. But, reducing Bax expression in naive cells did not lead to altered cisplatin sensitivity neither in H1339 nor in H187 SCLC cells. - We conclude that the reduced Bax expression after exposure to cisplatin is not sufficient to induce cisplatin-resistance in SCLC cells.

[Multimodality therapy for lung cancer]. / Multimodale Therapie des Lungenkarzinoms.

The primary treatment of lung cancer depends on tumor stage. In case of lung cancer in clinical stage I to IIb and T3N1 surgical treatment is recommended. The use of adjuvant chemotherapy is indicated in stage II and IIIa. In case of limited N2-disease trimodality therapy with chemo- or radiochemotherapy followed by surgery and eventual adjuvant radiotherapy leads to five year survival rate of about 20-40. Non resectable or extended mediastinal lymph node metastases are an indication for definite combined radiochemotherapy. Secondary resection may be evaluated in experienced centers. If the tumor has infiltrated the mediastinum or the upper sulcus (T3/4) or in case of solitary metastasis an individual trimodal treatment plan has to be elaborated. Also for small cell lung cancer surgery combined with chemotherapy can be applied in stage I and II, else and especially in stage III radiochemotherapy should be applied. Additional prophylactic cranial irradiation is used. The majority of lung cancer patients suffers from metastatic disease. The value of systemic chemotherapy is limited with significant, but small improvement in overall survival. Also treatment with the new molecularly targeted drugs does not result in a breakthrough in unselected patient cohorts. Recently, substantial progress could be achieved by personalized treatment approaches for patients harbouring special genetic alterations.

Effects of cigarette smoke extract and nicotine on bronchial tone and acetylcholine-induced airway contraction in mouse lung slices.

BACKGROUND: Tobacco smoke is a key risk factor for chronic obstructive pulmonary disease, but it may also alter the pathophysiology of asthma. In the present study, we analyzed whether tobacco smoke has acute or chronic effects on bronchial tone and whether it alters bronchial reactivity in vitro. METHODS: Airways in murine lung slices were digitally recorded and the change in cross-sectional area with time was quantified. T-bet KO mice served as a model for bronchial hyperreactivity. T-bet KO mice show a shift towards type 2 helper T lymphocytes and display histological as well as functional characteristics of asthma. Cigarette smoke extract (CSE) was obtained using commercially available cigarettes (Gauloise Blondes) by drawing cigarette smoke slowly through a water pump into a tube containing 10 mL of DMEM culture medium. RESULTS: Acute exposure to CSE led to relaxation of the airway. Acute exposure to nicotine resulted in a minor relaxation of the airway in Balb/C mice and in nonsignificant relaxation of the airway in T-bet KO mice. The nicotinic acetylcholine-receptor hexamethonium partially inhibited CSE-induced airway relaxation. Airway contraction in response to acetylcholine was stronger in T-bet KO mice than in Balb/C mice. After exposure to CSE or nicotine for 48 hours, acetylcholine-induced airway contraction was no longer different between the 2 types of mice. CONCLUSIONS: Our data indicate that acute exposure to CSE leads to airway relaxation, which is partially mediated by nicotine. Chronic exposure to CSE reverses bronchial hyperreactivity in the airways of T-bet KO mice; this effect can be mimicked by chronic exposure to nicotine.